molecules Article Cyanogenic Glycoside Analysis in American Elderberry Michael K. Appenteng 1 , Ritter Krueger 1, Mitch C. Johnson 1, Harrison Ingold 1, Richard Bell 2, Andrew L. Thomas 3 and C. Michael Greenlief 1,* 1 Department of Chemistry, University of Missouri, Columbia, MO 65211, USA; [email protected] (M.K.A.); [email protected] (R.K.); [email protected] (M.C.J.); [email protected] (H.I.) 2 Department of Chemistry, Truman State University, Kirksville, MO 63501, USA; [email protected] 3 Division of Plant Sciences, Southwest Research Center, University of Missouri, Columbia, MO 65211, USA; [email protected] * Correspondence: [email protected]; Tel.: +01-573-882-3288 Abstract: Cyanogenic glycosides (CNGs) are naturally occurring plant molecules (nitrogenous plant secondary metabolites) which consist of an aglycone and a sugar moiety. Hydrogen cyanide (HCN) is released from these compounds following enzymatic hydrolysis causing potential toxicity issues. The presence of CNGs in American elderberry (AE) fruit, Sambucus nigra (subsp. canadensis), is uncertain. A sensitive, reproducible and robust LC-MS/MS method was developed and optimized for accurate identification and quantification of the intact glycoside. A complimentary picrate paper test method was modified to determine the total cyanogenic potential (TCP). TCP analysis was performed using a camera-phone and UV-Vis spectrophotometry. A method validation was conducted and the developed methods were successfully applied to the assessment of TCP and quantification of intact CNGs in different tissues of AE samples. Results showed no quantifiable trace of CNGs in commercial AE juice. Levels of CNGs found in various fruit tissues of AE cultivars Citation: Appenteng, M.K.; Krueger, studied ranged from between 0.12 and 6.38 µg/g. In pressed juice samples, the concentration range R.; Johnson, M.C.; Ingold, H.; Bell, R.; measured was 0.29–2.36 µg/mL and in seeds the levels were 0.12–2.38 µg/g. TCP was highest in the Thomas, A.L.; Greenlief, C.M. stems and green berries. Concentration levels in all tissues were generally low and at a level that Cyanogenic Glycoside Analysis in poses no threat to consumers of fresh and processed AE products. American Elderberry. Molecules 2021, 26, 1384. https://doi.org/10.3390/ Keywords: American elderberry; total cyanogenic potential; cyanogenic glycosides; picrate method; molecules26051384 solid phase extraction; UHPLC-MS/MS Academic Editor: Wilfried Rozhon Received: 28 January 2021 1. Introduction Accepted: 1 March 2021 Published: 4 March 2021 American elderberry (AE), Sambucus nigra (subsp. canadensis) is a rapidly growing specialty crop in the United States [1]. Native to eastern and midwestern North America, Publisher’s Note: MDPI stays neutral AE is increasingly cultivated for its fruits and flowers that are used in a variety of foods, with regard to jurisdictional claims in jellies, syrups, wines, and more importantly, dietary supplement products [2]. Elderberry published maps and institutional affil- is known for its nutritional and medicinal health benefits [3–5]. The fruit is rich in carbohy- iations. drates, fatty acids, organic acids, minerals, vitamins (A, B6 and C), essential oils, and is high in fiber [6,7]. Researchers have linked elderberry products to anti-inflammatory, anti- oxidant, anti-carcinogenic, anti-viral, anti-influenza, and antibacterial activities [3,8–13]. Whereas little scientific research has been conducted on AE as compared to its close relative, the European elderberry (EE), Sambucus nigra (subsp. nigra), both species are excellent Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. sources of flavonoids, polyphenols and anthocyanins [3,8,9,14,15]. The elderberry industry This article is an open access article is poised for major expansion and has increased significantly in sales (~0.8 to 107.6 M dol- distributed under the terms and lars) between 2011 and 2019 [16]. However, its competitiveness with other herbal dietary conditions of the Creative Commons supplements [16] may by hampered in part due to uncertainty regarding the presence of Attribution (CC BY) license (https:// cyanogenic glycosides (CNGs) and/or their putative toxicity. creativecommons.org/licenses/by/ Cyanogenic glycosides are naturally occurring molecules in plants (nitrogenous sec- 4.0/). ondary plant metabolites) which consist of aglycone and a sugar moiety [17,18]. A gen- Molecules 2021, 26, 1384. https://doi.org/10.3390/molecules26051384 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 18 Molecules 2021, 26, 1384 Cyanogenic glycosides are naturally occurring molecules in plants (nitrogenous2 of 17 secondary plant metabolites) which consist of aglycone and a sugar moiety [17,18]. A generalized structure is shown in Figure 1. There are about 60 CNGs widely distributed eralizedin the plant structure kingdom, is shown occurring in Figure in over1. There 2600 areplant about species 60 CNGs representing widely distributedmore than 130 in thefamilies plant [19 kingdom,–22]. CNGs occurring are stored in over in 2600vacuoles plant within species plant representing cells, separating more than them 130 fami-from liesplant [19 hydrolyzing–22]. CNGs areendogenous stored in enzymes vacuoles within(β‐1,6‐glycosidases plant cells, separating and hydroxynitrile them from lyases) plant hydrolyzing[17,23]. Although endogenous intact CNGs enzymes are (nontoxic,β-1,6-glycosidases endogenous and plant hydroxynitrile enzymes can lyases) react [17 with,23]. AlthoughCNGs, and intact release CNGs hydrogen are nontoxic, cyanide endogenous (HCN) causing plant enzymes potential can toxicity react withissues CNGs, [17– and19,24,25]. release Most hydrogen CNG‐containing cyanide (HCN) plants causing also produce potential these toxicity endogenous issues [17 enzymes–19,24,25 so]. when Most CNG-containingtheir tissues are disrupted, plants also for produce example these by crushing, endogenous herbivory, enzymes or so disease, when theirCNGs tissues can react are disrupted,with endogenous for example enzymes by crushing,resulting herbivory,in the release or disease,of HCN CNGs[23,26]. can In plants, react with CNGs endoge- serve nousas important enzymes chemical resulting defense in the release compounds of HCN against [23,26 ].herbivores In plants, and CNGs pathogens serve as important[19,21,27]. chemicalClinical trials defense have compounds shown mixed against results herbivores regarding and the pathogens potential [19 of,21 amygdalin,27]. Clinical (a trialsCNG havefound shown in Sambucus mixed results) in regardingcancer treatment the potential and of as amygdalin a cough (a suppressant CNG found in inSambucus various) inpreparations cancer treatment [28,29]. and In ashumans, a cough consumption suppressant inof various cyanogenic preparations plants can [28 cause,29]. In sub humans,‐acute consumptioncyanide poisoning of cyanogenic (depending plants on can dose) cause with sub-acute symptoms cyanide including poisoning anxiety, (depending headache, on dose)vomiting, with symptomsnausea, abdominal including anxiety,cramps headache,diarrhea, vomiting, dizziness, nausea, weakness abdominal and crampsmental diarrhea,confusion. dizziness, Acute cyanide weakness toxicity and in mental humans confusion. (0.5–3.5 Acutemg kg cyanide−1 body weight) toxicity in[18,19] humans can −1 (0.5–3.5result in mg decreased kg body consciousness, weight) [18,19 hypotension,] can result in paralysis, decreased consciousness,coma and even hypotension, death [17– paralysis,19,24,25,30,31]. coma Acute and even cyanide death poisoning [17–19,24 has,25 ,been30,31 reported]. Acute from cyanide the poisoningingestion of has apricot been reportedkernels [32], from bitter the ingestionalmonds of[33], apricot and cassava kernels [34]. [32], bitter almonds [33], and cassava [34]. Figure 1. Generic structure for a cyanogenic glycoside,glycoside, wherewhere RR11 isis often methyl or aa protonproton andand RR22 is a variable organic group. The only studystudy previouslypreviously mademade on onSambucus Sambucus canadensis canadensis(American (American elderberry) elderberry) is by is Buhrmesterby Buhrmester et al.et al. [20 [20].]. They They examined examined the the presence presence or or absence absence of of cyanogenic cyanogenic glycosides glycosides for individualsindividuals from from nine nine populations populations of ofSambucus Sambucus canadensis canadensisL. (elderberry) L. (elderberry) in east-central in east‐ Illinois.central Illinois. The study The tested study for tested cyanogenic for cyanogenic glycosides glycosides in the leaves. in the Of leaves. the nine Of elderberry the nine populationselderberry populations examined, only examined, one population only one had population a test positive had for a HCN test productionpositive for each HCN of theproduction three times each tested. of the In three another times population tested. In theanother production population of HCN the wasproduction highly variable.of HCN Thewas cyanogenichighly variable. glycoside The cyanogenic was determined glycoside to be was (S)-sambunigrin determined to by be gas (S) chromatographic‐sambunigrin by separationgas chromatographic of the TMS-derivative. separation of the TMS‐derivative. A review ofof thethe medical medical literature literature revealed revealed no no reports reports of of elderberry elderberry juice juice poisoning poisoning in thein the past past 30 years. 30 years. The CentersThe Centers for Disease for