Abstract
BACKGROUND:
Sambucus species have been used for various purposes. Many people know that some of the Sambucus species display high antioxidant activity, but it is much less well-known that they also contain harmful compounds – cyanogenic glycosides.
OBJECTIVE:
Different cyanogenic glycosides and phenolics were determined in three different Sambucus species (Sambucus nigra - black elderberry, Sambucus ebulus – dwarf elder and Sambucus racemosa - red elderberry) and their plant parts.
METHODS:
Their contents were quantified with the aid of high-performance liquid chromatography (HPLC) and mass spectrophotometry (MS).
RESULTS:
The highest values of harmful compounds accumulated in the leaves of black elderberry (1.03 mg/g DW) and the lowest in the leaves of red elderberry (0.001 mg/g DW). In contrast, dwarf elder (Sambucus ebulus) contained the highest levels of beneficial phenolics in flowers (31.10 mg/g DW) and red elderberry berries the lowest, with only 0.58 mg per g DW.
CONCLUSIONS:
Dwarf elder was the richest in phenolics and in scope in cyanogenic glycosides. Its flowers and berries contained 40% higher total analyzed phenolic contents than black elderberry, which is considered to be rich in phenolic and antioxidant contents. It should be noted that dwarf elder additionally contained some other harmful compounds, for what further pharmacological studies should be carried out.
Keywords
Introduction
Sambucus species have been used for various purposes. They can be planted as an ornamental bush, with some of them also being used for erosion control [1, 2]. Of most importance is that the flowers and berries of some species have been greatly used for medicinal purposes since ancient times. They have been used as food colorants, processed into concentrates, syrups, various extracts, juices and also wines [2–8]. In addition to flowers and berries, used for processing into different products, the bark and roots can also be used to make toys, tools and musical instruments [9].
Elder flowers and berries contain not only beneficial but also some potentially toxic compounds, cyanogenic glycosides such as sambunigrin, and contact can lead to cyanide poisoning in humans [10, 11]. Cyanide poisoning generally causes diarrhea, vomiting and weakness. In some cases tachycardia can also occur, so people with heart and blood pressure disorders must use elder plant parts cautiously [10]. Their main function in the plant is protection against herbivores [12]. In addition to cyanide poisoning, some allergies to the flower pollen and some berry extracts can also appear, including with children playing with toys made from fresh elder stems [10]. Dwarf elder (Sambucus ebulus) also contains (allergenic) toxic lectins (ebulin), which exhibit ribosomal inactivating activity and its leaves can cause contact dermatitis [11, 13–15].
The Sambucus genus, from the Adoxaceae family, includes from 5 to 30 species and many more cultivars and hybrids [1, 16–18]. They grow naturally in northern hemisphere and prefer light to medium and heavy soils, mainly in semi-shade woodlands [6]. They are mainly tree-like shrubs with odd-pinnate leaves composed of 3–15 serrate leaflets and fragrant flowers arranged in corymbs. The fruits are berries and a favorite food for many birds, which rapidly spread a variety widely around the country. The main species are Sambucus ebulus, S. nigra, S. racemosa, S. africana and S. palmensis [14, 19]. The best known is black elderberry (BE) or common elder (Sambucus nigra), native to Europe, Asia and North Africa. In addition to this elder, dwarf elder (Sambucus ebulus) and red elderberry (Sambucus racemosa) additionally grow in Europe. Dwarf elder (DE) is a perennial herb with 0.5–2 m high, erect, usually unbranched stems growing in large groups from an extensive perennial underground rhizome. All plant parts have a specific foetid smell [14, 20]. Red elder (RE) is a low growing shrub, about 2 to 6 m tall, which blooms and ripens before dwarf and black elder and produces bright white to yellow green flowers, which latter ripen into bright-red berries. Black and dwarf elder make white (dwarf occasionally pink) hermaphroditic flowers in large corymbs, which develop over the summer into violet-black drupes in clusters holding hundreds of berries [21, 22]. The main difference between the clusters of berries is that in dwarf elder they face skywards and in black elder hang upside down. Black and dwarf elder have up to 2000 berries in a single cluster, while red elder has about 800 berries in a cluster.
Such black elderberry berries and flowers have been used for thousands of years to prevent or control many different diseases [4, 22]. The different polysaccharides and polyphenolics have a synergistic effect, which might explain the traditional use of some Sambucus species against various health disorders or diseases [11, 18]. It has been reported that black and dwarf elder contain various levels of sugars and organic acids and are a rich source of secondary metabolites, particularly quercetin derivatives, hydroxycinamic acids and important anthocyanin derivatives, including cyanidin-3-glucoside, cyanidin-3-sambubioside, cyanidin-3-sambubioside-5-glucoside and cyanidin 3,5-diglucoside [1, 23], which act as a potent agent against oxidative stress in the human body and are additionally responsible for the orange, red and blue colours of the fruits [24, 25]. Anthocyanins are mainly responsible for anti-inflammatory and anti-oxidative activity, while flavonoids and chlorogenic acid additionally have an anti-microbial and anti-viral effect on human health [20, 25].
Dwarf elder (Sambucus ebulus) was also used in Persia, Iran, Turkey, Romania and Bulgarian in folk medicine for the treatment of various diseases, mainly rheumatism, fever, infections and edemas. In ancient oriental medicine, its leaves and roots were used to treat bee, snake and nettle bites, arthritis and sore throats [20]. The berries have also been used for coloring, for red juice or to dye hair and the leaves are said to repel mice and moles [26–29].
Consumers are showing ever greater interests in natural remedies, including those using Sambucus species. Many studies have been performed on the polyphenolic contents and some primary metabolites in black elder [4, 31] and dwarf elder [20]. Far fewer studies have been done on cyanogenic glycosides in black elderberry [31, 32]. Almost nothing has been done on elder species such as dwarf elder and red elderberry and their cyanogenic glycosides contents. The aim of this study was to determine the relationship between beneficial (phenolics) and potentially harmful (cyanogenic glycosides) compounds in different plant parts (flowers, leaves and berries) of three different elderberry species (S. nigra - black elderberry, S. ebulus – dwarf elder and S. racemosa – red elderberry), growing native in central Europe. Last but not least, this study is also important for breeders to make hybrid species with the highest contents of phenolics and the lowest contents of cyanogenic glycosides.
Materials and methods
Plant material
The investigation included three different wild-growing elderberry species: black or European or common elderberry (Sambucus nigra) (BE), dwarf elder (Sambucus ebulus) (DE) and red elderberry (Sambucus racemosa) (RE), which were growing under the same ecological conditions. All plants were growing in similar, semi-shade situations. The flowers and leaves of each species were harvested at the same stage – the time of full flowering. All samples were collected at the same location, from several shrubs (Vojsko – Slovenia; 46°01’24.1”N; 13°53’08.2”E; altitude 1077 m) in year 2017. For RE sampling, the date was 13 May 2017, for BE 21 June 2017, and for DE 31 July 2017. Fruits were harvested when entire umbels were ripe. For RE, the harvesting was done on 21 June, BE on 3 September and DE on 20 September. Approximately half a kg of each plant part was picked.
Chemicals
The following chemicals were obtained from Sigma Aldricht Chemie (Steinheim, Germany): chlorogenic acid (5-caffeoylquinic acid), neochlorogenic acid (3-caffeoylquinic acid), cryptochlorogenic acid (4-caffeoylquinic acid), cyanidin-3-O-glucoside were standards for phenolics. Amygdalin and prunasin were standards for cyanogenic glycosides. Additionally from Sigma we purchased methanol and acetonitrile. From Fluka Chemie (Buchs, Switzerland) we obtained standards as (+)Catechin, (–)Epicatechin, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-O-rutinoside, p-coumaric acid, procyanidin B2 and kaempferol-3-O-glucoside. From Apin Chemicals (Abingdon, UK) was purchased isorhamnetin-3-O-rutinoside and peonidin-3-glucoside from Extrasynthese (Genay, Frence). Water for extraction and the mobile phase was double distilled and purified with the Milli-Q system (Millipore, Bedford, MA, USA).
Extraction of cyanogenic glycosides and phenolics from plant material
For biochemical analysis, the selected plant parts of the three elder species were crushed in a mortar and 1 g of the plant paste was put into a 10 ml screw-cap tube. Seven independent repetitions for each plant part of each Sambucus species were performed. The solid sample was extracted with 5 ml of methanol/water (70:30, v/v, MeOH/H2O) for 30 min at 30°C according to the method of Senica et al. [32]. The macerates were then centrifuged for 7 min at 4°C and 10 000 rpm and the supernatant was filtered through a polyamide filter (Macherey-Nagel; Düren, Germany) into vials prior to HPLC and MS analysis.
HPLC – DAD-MSn analysis of phenolic compounds
Individual phenolics were identified using the Accela HPLC system (Thermo Scientific, San Jose, CA) with a diode array detector (DAD) at 280 nm, 350 nm and 530 nm, which was controlled by CromQuest 4.0 chromatography workstation software. The column used was a Gemini C18 (150×4.6 mm 3μm; Phenomenex, Torrance, USA) operated at 25°C. Mobile phase A was 0.1% formic acid with 3% acetonitrile (ACN) in double distilled water (v/v/v) and mobile phase B was 0.1% formic acid with 3% double distilled water in ACN (v/v/v). Samples were eluted according to the linear gradient described by Wang et al. [33]. The injection quantity was 20μl and the flow rate was maintained at 0.6 ml per min.
All phenolic compounds were identified using a mass spectrometer (Thermo Scientific, LCQ Deca XP MAX) with electrospray ionization (ESI) operating in negative ion mode. The analyses were carried out using full scan data-dependent MSn scanning from m/z 110 to 1600. The injection volume was 10μl and the flow rate was maintained at 0.6 ml per min. Other technical characteristics are described in Mikulic-Petkovsek et al. [17]. The contents of selected phenolics were expressed in μg per gram of dry weight (DW).
MS analysis of cyanogenic glycosides
Separation of cyanogenic glycosides was performed on a HYPERSIL GOLD aQ column (Thermo Scientific) at 25°C and flow rate 0.8 ml/min. The sample injection volume was the same as for phenolics. Mobile phase A was 3% methanol in double distilled water (v/v/v) and mobile phase B was 3% double distilled water in methanol (v/v/v), with gradient elution: 0–1 min, 80% A; 1–8 min, 80–0% A; 8–10 min, 0% A; 10–14 min, 80% A.
The presence of cyanogenic glycosides was confirmed on a TSQ Quantum Access Max quadrupole mass spectrometer. The MS instrument was operated using an (ESI) source in positive ion mode. The ESI parameters were as follows: capillary temperature 275°C, corona voltage 4.7 kV, sheat gas 60 L per/h, auxiliary gas 10 L per/h. Mass spectra were scanned in range from m/z 70 to 650. Collision-induced dissociation was achieved using argon as the collision gas in the collision cell. Cyanogenic glycosides were analyzed in selected reaction monitoring (SRM) mode. Data acquisition was performed using Xcalibur 2.2.Software. Contents of cyanogenic glycosides were expressed in μg per gram of dry weight (DW).
Statistical analysis
Results are presented as mean±standard deviation of seven replications. Statistical program R-Commander was used to test the differences among different Sambucus species and their plant parts. Significant differences among different treatments were calculated by one-way analysis of variance (ANOVA). The contents of individual phenolics and cyanogenic glycosides as their sum were tested using the Duncan test, with the significant difference level 0.05.
Results and discussion
Three (3) different Sambucus species were examined in this study. There were significant differences in both harmful and beneficial contents in all plant parts (leaves, flowers and berries) of the three different elderberry species. All plant parts of all three investigated species contained much higher contents of beneficial phenolics than harmful cyanogenic glycosides (Table 2). Beneficial phenolics ranged from 0.58 mg/g (berries of red elderberry) to 31.10 mg/g (flowers of dwarf elder), while harmful cyanogenic glycosides from 0.01 (leaves of red elderberry) to 1.03 mg/g DW (leaves of black elderberry), which was more than a 90% distance.
Cyanogenic glycosides
Cyanogenic glycosides found in Sambucus species, have been claimed to have a toxic effect on human health. Koss-Mikołajczyk et al. (2016) [34] suggested in their study that Sambucus berries, even at the not fully ripe stage, are safe for food and pharmaceutical products and have no adverse effect on human health. In our study, the contents of cyanogenic glycosides significantly differed between different plant parts within the three different elderberry species (Table 1, 2 and Fig. 1). Cyanogenic glycosides reached the highest levels in leaves of BE (Sambucus nigra) (1033.20μg/g), while with DE (Sambucus ebulus) and RE (Sambucus racemosa), flower parts accumulated the highest levels (58.19μg/g and 4.45μg/g, respectively) (Table 2, and Fig. 2). In contrast, the lowest contents occurred in BE in berries (54.88μg/g), while DE and RE had the lowest contents in leaves, 8.76 and 1.05μg/g, respectively. In general, BE contained the highest contents of total analyzed cyanogenic glycosides of all three species. RE had the lowest contents. In all three Sambucus species, we detected three different cyanogenic glycosides (Fig. 1). Delagrecca (2000) also found that elder species contain several types of cyanogenic glycosides [35]. Specifically, sambunigrin, its epimer prunasin and in some species also holocalin and its epimer zierin. We determined by mass spectrofotometry only peaks with M + m/z 295 and daughter ions 291 and 185 as prunasin, sambunigrin M + m/z 295 and daughter ions 163 and 133 and M + m/z 457, with daughter ions 453 and 347 as a single amygdalin isomer. Holocalin and zierin – cyanogenic glycosides found in the study of Delagrecca (2000) [35], with detected mass 311, were not confirmed in our study for any studied Sambucus specie. Berries of BE had all three peaks, while flowers of DE had just two peaks. Sambunigrin was only identified in traces. One amygdalin isomer (M+ at m/z 457) was the highest contributor in DE (dwarf elder), while mainly sambunigrin and also prunasin were higher contributors in black elderberry (Table 2, and Fig. 1). Sambunigrin represented more than 90% of total cyanogenic glycosides in the leaves and flowers of BE (black elderberry), while the ratio between prunasin and sambunigrin in berries was 1:1. Koss-Mikołajczyk and coauthors [34] also found out the highest sambunigrin levels in leaves of black elderberry. In DE, the highest contributor to total cyanogenic compounds was amygdalin isomer, with about 70 % contribution, prunasin about 24%, while the presence of sambunigrin was less than 6%. Red elderberry (RE) had the lowest contents of cyanogenic glycosides in all three species. The contribution of selected cyanogenic glycosides was in the same proportion. On average their content was approximately 99% lower from black elderberry and 72% from dwarf elderberry.
Presence of individual phenolic compounds in different plant parts of three Sambucus species
Presence of individual phenolic compounds in different plant parts of three Sambucus species
The content of some phenolics, their groups and some cyanogenic glycosides as their sum of different plant parts of three Sambucus species (μg/g dry weight)
The average content±standard error, in μg/g, different letters (a–o) in the same row indicate significant differences in phenolics and cynogenic glycosides contents between different Sambucus species and their plant parts (p < 0,05) by Duncan’s multiple range test.
TIC from mass spectrophotometer of different detected cyanogenic glycosides in two Sambucus species.
The contents of total analyzed cyanogenic glycosides (CGG) in different plant parts of three Sambucus species.
There was a total of 59 different phenolics identified in all three elderberry species. Of these, there were 47 different phenolics in BE (black elderberry), 38 in DE (dwarf elder) and 36 in RE (red elderberry) (Table 1). The proportion of phenolics present altered between species as well as their plant parts. The most extensive group was that of flavonols, which consist of 10 different quercetin glycosides, 8 kaempferol glycosides, and 8 isorhamnetin glycosides for all three Sambucus species. Flowers from all three species had the highest phenolic contents, while leaves had the lowest contents with BE and DE and berries with RE (Table 2). The main contributors to total analyzed phenolics were caffeic acid derivatives, isorhamnetin-, kaempferol- and quercetin- glycosides, and in berries, anthocyanins. The group of anthocyanins, flavanols and caffeic acid derivatives had the highest proportion in berries of BE, DE and RE, respectively. The richest source of phenolics in this study was the flowers of DE, which have already been used in traditional medicine for more than a hundred years. Flowers had 31.1 mg/g of total analyzed phenolics per dry weight, which was 60% more than in BE berries and 80% more than in RE berries. In contrast, RE was very poor in phenolics compared to the other two, with the exception of leaves, which contained 62% and 47% higher phenolics contents, respectively, than BE and DE (Table 2). Some previous studies also confirmed RE and their hybrids to be a poor source of phenolics [17, 23]. The highest contributors to total analyzed phenolics contents in DE berries were flavanols, followed by caffeic acid derivatives (mainly chlorogenic acid) and kaempferol glycosides (Table 2). Quercetin glycosides and caffeic acid derivatives were also higher contributors in all three Sambucus species and their plant parts. Flavanones, including different naringenin glycosides, appeared only in flowers and berries of BE (Tables 1 and 2).
Derivatives of caffeic acid
Of total analyzed phenolics, we detected 8 different caffeic acid derivatives (Table 1). The highest values were contributed by three cinnamic acids (3-, 4- and 5-caffeoylquinic acid). Mikulic-Petkovsek and coauthors found the same contribution [17]. The group of caffeic acid derivatives was the main contributor to total analyzed phenolics in all plant parts of RE, and flowers and leaves of BE (Table 2). Among three higher contributors, chlorogenic acid (5-caffeoylquinic acid) with a mass M– m/z 353, was the highest contributor to this phenolic group in all plant parts of RE and BE and the berries of DE, while in the leaves and flowers of DE, cryptochlorogenic acid (4-caffeoylquinic acid) was the highest contributor to hydroxycinnamic acids (Table 2). Of all three studied species, leaves of RE had the highest contents of chlorogenic acid had leaves of RE elderberry (7.54 mg/g), followed by berries of DE (5.88 mg/g) and flowers of RE (5.67 mg/g). Chlorogenic acid was five-times higher in DE than in BE berries, which is in accordance with the study of Dulf et al. [15], in which the contents of chlorogenic acid was also a few times higher in DE than in BE. Mikulic-Petkovsek et al. [17] also found that the berries of DE were a highly rich source of chlorogenic acid. Dwarf elder (DE) berries did not contain only the highest levels of chlorogenic acid but also the highest levels of both 4-caffeoylquinic acid and 3-caffeoylquinic acid in all plant parts of the different Sambucus species. It has been reported that chlorogenic acid has an anti-microbial and anti-viral effect on human health [20, 25]. On that basis, DE has been used in folk and traditional medicine for the treatment of various infections and edemas.
Derivatives of p-coumaric acid
This group had lower contents than the group of caffeic acid derivatives, with the exception of DE leaves and flowers, which had higher values (Table 2). Derivatives of p-coumaric acid from all investigated plant material were highest in all plant parts of DE. On average, their contents were 3-times higher than in BE and 8-times higher than in RE (Table 2). The highest contributors were p-coumaric acid hexoside with M– m/z 325, and 3-p-coumaroylquinic acid with molecular ion M– m/z 337 present in all three species. Additionally, 5-p-coumaroylquinic acid 1 and 2 were also present in all investigated species.
Isorhamnetin glycosides
They belong to the group of flavonols. We found 8 different isorhamnetin glycosides in this study. Of all plant parts flowers of RE (red elderberry) had the highest values (38.67 mg/g), followed by flowers of BE (black elderberry) (18.85 mg/g) and leaves and berries of DE (dwarf elder) (17.06 and 17.81 mg/g, respectively) (Table 2). This is in agreement with the study of Vrchotova et al. [1], who reported that flowers were a rich source of isorhamnetin derivatives. In contrast, leaves of RE and BE had very low values of isorhamnetin glycosides, more than 50-times lower than in their flowers. Low levels of isorhamnetin glycosides in BE berries were also found by Lee and Finn [6]. They compared three Sambucus species, DE (dwarf elder), BE (black elderberry) and American elderberry and reported that berries of DE reached higher levels of isorhamnetin glycosides than those of American elderberry, while the contents of total isorhamnetin derivatives were smallest in BE berries, similar to the results of our study. The highest contributor to total isorhamnetin derivatives in RE in our study was isorhamnetin hexoside rhamnoside with molecular ion m/z 477. Isorhamnetin-3-rutinoside with M– m/z 623 was detected only in BE (Table 1).
Kaempferol glycosides
The second major contributor to total flavonol content was kaempferol glycosides and the third most prevalent phenolics in DE. We detected a total of 8 different kaempferol glycosides (Table 1). Flowers contained the highest levels of kaempferol glycosides in all three elderberries, BE (1.52 mg/g), DE (28.04 mg/g) and RE (2.04 mg/g). Dwarf elder (DE) was richest in kaempferol glycosides in all three species. The average kaempferol contents were 18-times higher than in BE and 14-times higher than in RE. The highest values were reached by kaempferol-3-glucoside with molecular ion m/z 447, which decayed to fragment ion m/z 285. Kaempferol–acetylhexoside, kaempferol-3-glucoside and kaempferol-3-rutinoside were the main contributors to total kaempferol glycosides in the flowers of BE and RE.
Quercetin glycosides
Quercetin glycosides, in addition to caffeic acid derivatives, were the second most abundant phenolics in BE and RE. The main quercetin in BE and DE was quercetin-3-rutinoside (rutin) with molecular ion m/z 609, which decayed to fragment ion m/z 301. Rutin was also reported to be the main contributor in elderberries in many other studies [1, 34]. In addition to rutin, other quercetin derivatives were also identified, in considerably lower amounts, including quercetin-acetylhexoside and quercetin-hexoside pentoside. Quercetin-galactoside with molecular ion m/z 463, was only detected in all plant parts of DE, quercetin-rhamnoside only in leaves and quercetin-3-xyloside only in fruits of BE (Table 1). The highest quercetin-3-rutinoside contents were detected in all plant parts of BE, 2.03 mg/g in leaves, 2.67 mg/g in flowers and 3.53 mg/g in berries. Total quercetin contents in our study ranged from 0.29 mg/g (in berries of red elderberry) to 3.58 mg/g (in flowers of black elderberry). Rutin was present in lower concentrations in leaves than in flowers and berries. This is in agreement with a number of studies [1, 36]. In berries of BE, quercetin derivatives reached 35.31 mg/g, in dwarf elderberry 26.76 mg/g and in red elderberry 0.03 mg/g. Quercetin-3-rutinoside was more than two times higher in the berries of BE than in those of DE. The same was found in the study of Dulf et al. [15], in which DE contained a 3-times higher content of rutin than in BE. The lowest proportion of quercetin glycosides, as well as quercetin-3-rutinoside, was determined in our study in fruits of RE, in comparison with the study of Mikulic-Petkovsek et al. [17], in which a RE hybrid (S. racemosa var miquelli) had the lowest levels of all investigated Sambucus species and cultivars [17]. In a study by Lee and Finn [6], some BE hybrids had higher contents of quercetin glycosides than American elder (Sambucus canadensis). Additionally, they found in their study [6] that berries of BE had 9.56 mg/g of quercetin-3-rutinoside and American elder had even lower contents. It appears that elderberries from Europe contain higher levels of quercetin glycosides.
Flavanols
The following compounds belong in this phenolic group: catechin, epicatechin and procyanidin dimers, which were the major compound in berries of DE. The most abundant in flavanol contents was DE (9.46 mg/g), followed by BE (0.62 mg/g) and RE (2.51 mg/g) with the poorest contents. They were present only in berries of all three studied species and RE flowers. Dwarf elder (DE) berries had 21-times higher levels than RE flowers and 15-times higher than BE berries. Procyanidins dimers have important functions as an anti-oxidative, anti-mutagenic, anti-cytotoxic and cardio protective agents for human health [37]. This is perhaps why DE has been used in so many countries in folk medicine.
Anthocyanins
Anthocyanins, with a highly beneficial effect on human health, were present in the fruits of all three studied elderberry species. It is the predominant polyphenolics in dark colored black elderberries [17, 38]. Anthocyanins, in combination with flavonols, have the highest antioxidant activity [15]. Furthermore, elderberry is thought to be much better in total antioxidant capacity than blueberry, mulberry, raspberry and strawberry [3]. A total of 9 different anthocyanins were detected in the three studied elderberry species. Cyanidin-based anthocyanins were also the major anthocyanins in some other studies [15, 23]. BE contained the highest values of total anthocyanins (5.30 mg/g), which was 3-times higher than in berries of DE (1.75 mg/g) and 29-times higher than in berries of RE (0.18 mg/g) (Table 2). The major anthocyanins in BE were cyanidin-3-sambubioside (molecular mass m/z 581) and cyanidin-3-glucoside (m/z 449), with a 50% and 43% share, respectively. Cyanidin-3-sambubioside and cyanidin-3-glucoside have also been confirmed as the main contributors to BE berries in many other studies [4, 34]. Both anthocyanins contribute greatly to high antioxidant activity [10, 34]. Lee and Finn [6] found that BE berries contain much higher levels of cyanidin-3-glucoside and cyanidin-3-sambubioside than berries of American elder (Sambucus canadensis). In our study, too, their values in BE berries were much higher than in the other two species. The sum of total anthocyanins ranged from 3.9–8 mg/g in a study by Lee and Finn [6] and in our study the sum of total anthocyanins for BE was 3 mg/g. Both major anthocyanins were present in the other two studied elderberries (DE, RE) in much lower amounts. In DE, the highest contributors to total anthocyanins were cyanidin- pentoside hexoside (63%), malvidin pentoside (10%) and a derivative of petunidin (15%). In RE, the main contributor to anthocyanins was cyanidin glycoside, with an 83% share (data not shown). For dwarf elder (DE), the content of total anthocyanins in our study was 1.7 mg/g, which is in agreement with the results of Jimenez et al. [11], who reported values from 0.22 to 2.118 mg/g. They also reported that DE does not contain only antioxidant compounds but also high levels of lectins [11]. It needs to be born in mind that bioactive compounds differ along the stage of ripeness [34] and it is important to ensure the stage for picking up the berries with the highest antioxidant activity.
Conclusion
Elderberry flowers and berries, especially from BE, are predominantly used for processing and in medicine. Dwarf elder (DE) has also been used a lot for medicinal proposes in many countries. In this study, we found that both DE flowers and berries contained a 40% higher total analyzed phenolic contents than black elderberry (BE), which is considered to be rich in phenolic and antioxidant contents. Red elderberry (RE) contained much lower phenolic contents than both DE and BE. In contrast, BE also had the highest content of cyanogenic glycosides, while the lowest were found in RE. Medium contents of cyanogenic glycosides were measured in dwarf elder. The richest plant parts in phenolics with all Sambucus species were flowers. BE and DE contained 13% and 9% more phenolics in flowers than in berries, respectively. There was a much higher difference between leaves and flowers. The phenolic contents in RE leaves were very close to those in flowers. RE berries contained for 96% lower phenolic contents than did in their flowers. In conclusion, DE was the richest in phenolics and in scope in cyanogenic glycosides. It should be noted that DE additionally contained some lectins, which have some harmful effects on human. Further pharmacological study should be carried out, to confirm the absence of any other harmful compound in any of the elderberry species, despite the high presence of beneficial compounds. After such studies, dwarf elder (DE) can be proposed as being beneficial for human health. Based on this study and the results on cyanogenic glycosides contents, the poisoning risk with toxic HCN can be excluded if fully ripe berries of any of the studied Sambucus species are eaten. But, caferully, because of other harmful compounds is needed much more studies to prove that. We proposed anyway to treated raw berries with heating to loss some doubtful components. There are many previously reports of health disorders after a ingesting of Sambucus raw berries.
Funding
The authors report no funding.
Conflicts of interest
The authors have no conflict of interest to report.
Footnotes
Acknowledgments
The research is part of Horticulture program No. P4-0013-0481 funded by the Slovenian Research Agency (ARRS).