Pyrrolizidine-Derived Alkaloids: Highly Toxic Components in the Seeds of Crotalaria Cleomifolia Used in Popular Beverages in Madagascar

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Communication Pyrrolizidine-Derived Alkaloids: Highly Toxic Components in the Seeds of Crotalaria cleomifolia Used in Popular Beverages in Madagascar

Anjaramampionona Henintsoa Duvale Solofomalala 1, Clara Fredeline Rajemiarimoelisoa 2, Randriamampianina Lovarintsoa Judicael 1, Hanitra Ranjana Randrianarivo 1, Danielle Aurore Doll Rakoto 1, Victor Louis Jeannoda 1 and Ahcène Boumendjel 3,4,*

1 Fundamental and Applied Biochemistry Department, Faculty of Sciences, University of Antananarivo, P.O. Box 906, Antananarivo 101, Madagascar; [email protected] (A.H.D.S.); [email protected] (R.L.J.); [email protected] (H.R.R.); [email protected] (D.A.D.R.); [email protected] (V.L.J.) 2 Pharmacy Department, Faculty of Medicine, University of Antananarivo, P.O. Box 375, Antananarivo 101, Madagascar; [email protected] 3 Univ. Grenoble Alpes, INSERM, LRB, 38000 Grenoble, France 4 Univ. Grenoble Alpes, CNRS, DPM, 38000 Grenoble, France * Correspondence: [email protected]

Abstract: Seeds of Crotalaria cleomifolia (Fabaceae) are consumed in Madagascar in preparation of popular beverages. The investigation of extracts from the seeds of this species revealed the presence Citation: Solofomalala, A.H.D.; of high amounts of alkaloids from which two pyrrolizidine-derived alkaloids were isolated. One Rajemiarimoelisoa, C.F.; Judicael, of them was fully characterized by spectroscopic and spectrometric methods, which was found R.L.; Randrianarivo, H.R.; Rakoto, to be usaramine. Owing to the high toxicity of these alkaloids, issuing a strong warning among D.A.D.; Jeannoda, V.L.; Boumendjel, populations consuming the seeds of Crotalaria cleomifolia must be considered. A. Pyrrolizidine-Derived Alkaloids: Highly Toxic Components in the Seeds of Crotalaria cleomifolia Used in Keywords: Crotalaria; Fabaceae; traditional use; pyrrolizidine; alkaloids; usaramine; toxicity Popular Beverages in Madagascar. Molecules 2021, 26, 3464. https:// doi.org/10.3390/molecules26113464 1. Introduction Academic Editors: Roberto Fabiani Crotalaria is a genus of flowering plants that belong to the Fabaceae family that is and Eliana Pereira composed of about 600 species distributed across all continents, among which 400 are distributed in Africa [1]. Crotalaria is also known under the name rattlebox. This name Received: 19 May 2021 takes its origin from the rattle sound made by seeds when the dry pods are shaken. The Accepted: 4 June 2021 genus Crotalaria is used in traditional medicine as deworming, in malaria treatment, and Published: 7 June 2021 in management of fever [2,3]. In agriculture, the crotalaria species are used as a pre-plant cover for soybean crops to avoid the spread of parasites [4]. However, Crotalaria genus Publisher’s Note: MDPI stays neutral is known for its toxicity. In pastoral regions, several species are known to induce fatal with regard to jurisdictional claims in toxicity among grazing cows, horses, and sheep, mainly due to the presence of toxic published maps and institutional affil- alkaloids [5,6]. Indeed, according to several reports, the seeds of certain Crotalaria genus iations. may contain up to 5% of toxic alkaloids, mostly pyrrolizidine-derived alkaloids known for their severe hepatotoxicity and renal failure, following bio-activation by cytochrome P450 (CYP) enzymes in the liver [7,8]. In Madagascar, Crotalaria cleomifolia is an introduced species from the African continent. Copyright: © 2021 by the authors. It is known by two other names—Crotalaria longibracteata and Crotalaria shumaniana [9]. Licensee MDPI, Basel, Switzerland. Crotalaria cleomifolia is a shrub with simple or composed leaves, with yellow or white flowers This article is an open access article and fruits in pods (Figure1)[ 10]. A field survey revealed that in the Manjakandriana region, distributed under the terms and near the capital Antananarivo, the leaves and the stems of Crotalaria cleomifolia are used conditions of the Creative Commons as green fertilizers, whereas the crushed seeds are used in the preparation of a coffee Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ substitute [11]. To date, the seeds of C. cleomifolia have never been investigated for their 4.0/). phytochemical composition. In this context, we were interested in the seeds that are used in

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their phytochemical composition. In this context, we were interested in the seeds that are theused traditional in the traditional beverage beverage preparations. preparations. Based on Based the well-established on the well-established literature, literature, we focused we ourfocused attention our attention on the potential on the presencepotential ofpresen toxicce pyrrolizidine-derived of toxic pyrrolizidine-derived alkaloids thatalkaloids may seriouslythat may compromiseseriously compromise the traditional the traditional use of the seedsuse of of theC. seeds cleomifolia of C.. cleomifolia Herewith,. weHerewith, report thewe extraction,report the extraction, puriﬁcation, purification, and structural and st identiﬁcationructural identification of alkaloids of fromalkaloids the seedsfrom the of C.seeds cleomifolia of C. cleomifolia..

Figure 1. Crotalaria cleomifolia. From left to right—aerial part, leaves, flowers, and pods. Figure 1. Crotalaria cleomifolia. From left to right—aerial part, leaves, flowers, and pods. 2. Results 2. Results 2.1.2.1. ExtractionExtraction andand PurificationPurification ofof AlkaloidsAlkaloids TwoTwo parallelparallel extractions extractions (starting (starting from from 50 50 g ofg groundedof grounded seeds) seeds) were were realized, realized, follow- fol- inglowing classic classic procedures procedures that relythat onrely the on basic the basic character character of alkaloids of alkaloids (see Section (see Materials4)[ 12]. Theand extractionMethods) in[12]. acidic The conditions extraction providedin acidic cond almostitions a twofold provided higher almost quantity a twofold of crude higher alkaloids quan- astity compared of crude toalkaloids the extraction as compared in basic to conditions the extraction (136 in mg basic versus conditions 66 mg, respectively), (136 mg versus with 66 roughlymg, respectively), the same chemicalwith roughly composition the same accordingchemical composition to the TLC andaccording HPLC to analyses. the TLC Theand purificationHPLC analyses. of the The crude purification extracts wasof the performed crude extracts on a silicawas performed gel chromatography on a silica gel column, chro- usingmatography a gradient column, of dichloromethane:methanol using a gradient of dichloromethane:methanol to provide two pure fractions to provide (44 mg two and pure 39 mg),fractions each (44 containing mg and one39 mg), pure each compound, containing namely, one pure compounds compou1nd,and namely,2. compounds 1 and 2. 2.2. Structural Identification 2.2. StructuralCompound Identification1 (44 mg) was analyzed by IR, mass spectrometry, and NMR (1D and 2D). The IRCompound spectrum showed1 (44 mg) the was presence analyzed of hydroxyl by IR, mass (3466 spectrometry, cm−1) and carbonyl and NMR (1716 (1D cm and−1) groups2D). The (see IR experimental spectrum showed section the and presence Supplementary of hydroxyl Materials (3466 forcm the−1) and IR spectrum). carbonyl (1716 The compoundcm−1) groups was (see assigned experimental the molecular section and weight Suppl andementary molecular Materials formula for of the 351 IR g/mol spectrum). and CThe18H compound25NO6, respectively, was assigned as determined the molecular by HRMSweight (seeand Supplementarymolecular formula Materials). of 351 g/mol The 13 andC NMR C18H25 showedNO6, respectively, 2 carbonyls as (176.1 determined and 170.5 by ppm), HRMS 4 (see olefinic Supplementary carbons (133.6–136.6 Materials). ppm), The 413 saturatedC NMR showed carbons 2 carbonyls linked to oxygen(176.1 and (68.2–82.5 170.5 ppm), ppm), 4 olefinic and two carbons methyl (133.6–136.6 groups (12.4 ppm), and 14.34 saturated ppm) (13 carbonsC NMR linked data are to providedoxygen (68.2–82.5 in the experimental ppm), and section two methyl and in groups Supplementary (12.4 and 1 Materials).14.3 ppm) (13 TheC NMRH NMR data are data provided were in in agreement the experimental with the section information and in Supplementary obtained from 13 theMaterials).C NMR The spectrum 1H NMR and data showed were in a agreement profile similar with to the those information of pyrrolizidine-alkaloids obtained from the 1 (13HC NMR dataspectrum are provided and showed in the a experimental profile similar section to those and of in pyrrolizidine-alkaloids Supplementary Materials). (1H HomoNMR data and hetero-2Dare provided NMR in broughtthe experimental more evidence section and and confirmed in Supplementary that the compoundMaterials). wasHomo in agreementand hetero-2D with NMR the known brought macrocyclic more evidence pyrrolizidine and confirmed alkaloid, that usaramine, the compound which waswas reportedin agreement in Crotalaria with thespecies known (Figure macrocyclic2)[ 13– 15pyrrolizidine]. Being aware alkaloid, that retrosine usaramine,(Figure which2) iswas frequently reported isolated in Crotalaria from speciesCrotalaria (Figureand differs2) [13–15]. from Being usaramine aware bythat the retrosine configuration (Figure of 2) 1 theis frequently double bond( isolatedE for from usaramine Crotalaria and andZ for differs retrosine), from usaramine the H NMR by the data configuration of compound of 1the—and double especially bond(E the for chemical usaramine shift and of theZ for olefinic retrosine), proton the bearing 1H NMR the data methyl of compound group—were 1— comparedand especially to those the ofchemical usaramine shift and of the retrosine. olefinic The proton chemical bearing shift the of methyl the indicated group—were proton incompared compound to those1 appears of usaramine at 6.5 ppm, and which retrosine. is in full The synchronization chemical shift of with the the indicated configuration proton ofin usaraminecompound (~6.51 appears ppm at in 6.5 usaramine ppm, which versus is in ~5.8 full synchronization pp in retrosine). with [13,16 the]. Compoundconfiguration2 wasof usaramine analyzed and(~6.5 found ppm toin belongusaramine to pyrrolizidine-derived versus ~5.8 pp in retrosine). alkaloids and[13,16]. to be Compound structurally 2 close to compound 1 (MS and 1H NMR are provided in Supplementary Materials) [13]. The

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was analyzed and found to belong to pyrrolizidine-derived alkaloids and to be structur- 1 allycompound close to wascompound assigned 1 the(MS molecular and H NMR weight are and provided molecular in Supplementary formula of 335 g/molMaterials) and [13]. The compound was assigned the molecular weight and molecular formula of 335 C18H25NO5, respectively, as determined by HRMS.. Unfortunately, the full structure was g/molnot determined, and C18H25 dueNO5 to, respectively, the instability as of determined the compound by HRMS.. in solution Unfortunately, at room temperature. the full structureIndeed, duringwas not the determined, 2D experiments, due to the which instability take longer of the thancompound1H NMR, in solution the compound at room temperature.decomposes Indeed, as evidenced during by the the 2D appearance experiments, of signals which thattake werelonger not than present 1H NMR, in the the1H compoundNMR spectrum. decomposes According as evidenced to the molecular by the weightappearance and the of molecularsignals that formula, were not compound present in2 couldthe 1H beNMR senecionine spectrum. and According integerrimine, to the molecular however, weight the 1H NMRand the proﬁle molecular did not formula, match compoundwith either 2 structures, could be senecionine based on the and literature integerrimine, [13] (Figure however,2). the 1H NMR profile did not match with either structures, based on the literature [13] (Figure 2).

N N N N

H H H H H H O O O O H O O H O O CH3 O CH3 O CH3 O CH3 O

H3C H3C O O O O HO HO H3C H3C CH3 CH3 OH OH OH OH usaramine senecionine retrosine integerrimine FigureFigure 2. 2. StructureStructure of of compound compound 11 (usaramine),(usaramine), retrosine, retrosine, in integerrimine,tegerrimine, and and senecionine. senecionine.

3.3. Discussion Discussion TheThe use use of of plants plants in in preparation preparation of of popular popular herbal herbal drinks drinks is is frequent frequent in in many many regions regions ofof the the world. world. Beverages Beverages include include infusion infusion or or de decoctioncoction of of herbals, herbals, spices, spices, fruits, fruits, or or other other plantplant materials materials [17]. [17]. It It is is known known that that seeds seeds of of CrotalariaCrotalaria cleomifolia cleomifolia areare used used in in Madagascar Madagascar inin remedies remedies in in folk folk medicine medicine in indifferent different forms forms of preparations, of preparations, such such as infusions as infusions and andde- coctions.decoctions. The Themost most popular popular use is use its isconsumption its consumption as a coffee as a coffee beans beans regardless regardless of the ofpres- the encepresence of potential of potential toxic toxiccomponents. components. Due to Due the tolack the of lack studies of studies regarding regarding the chemical the chemical com- positioncomposition of the of seeds the seeds from from CrotalariaCrotalaria cleomifolia cleomifolia andand the the strong strong assumption assumption regarding regarding the the presencepresence of of toxic toxic pyrrolizidine-derived pyrrolizidine-derived alkaloids, alkaloids, we we undertook undertook the the present present study study to to shed shed lightlight on on the alkaloidalalkaloidal composition.composition. To To ensure ensure that that the the popular popular drink drink contains contains pyrrolizidine- pyrroliz- idine-derivedderived alkaloids, alkaloids, we preparedwe prepared drinks drinks according according to standardto standard preparation preparation (about (about 10 10 g gof of powdered powdered seedsseeds in 200 200 mL mL of of boiled boiled wa waterter for for 10 10min). min). A sample A sample was waslyophilized lyophilized and analyzedand analyzed by 1H byNMR,1H NMR,showing showing the presence the presence of typical of signals typical for signals pyrrolizidine-derived for pyrrolizidine- alkaloids.derived alkaloids. The latter The observation latter observation was confirmed was confirmed following following the specific the specific extraction extraction of alka- of loidsalkaloids that allowed that allowed the isolation the isolation and andcharacterization characterization of usaramine of usaramine (compound (compound 1) and1) and its closeits close analog analog (compound (compound 2). 2). BasedBased on thethe quantitiesquantities of of pure pure compounds compounds isolated, isolated, the the seeds seeds may may contain contain over 0.16%over 0.16%of toxic of compounds.toxic compounds. The quantityThe quantity of recovered of recovered pyrrolizidine-derived pyrrolizidine-derived alkaloids alkaloids in thein thedrinks drinks are are dependent dependent on differenton different factors—the factors— quantitythe quantity of seeds of seeds used, used, the volume the volume of water of waterand its and temperature, its temperature, and the and time the oftime boiling. of boiling. Moreover, Moreover, it was it evident was evident that the that observed the ob- servedyield of yield 0.16% of was0.16% the was most the pessimistic, most pessimistic, since certain since compounds certain compounds were not extractedwere not andex- tractedconsiderable and considerable loss may occur loss may during occur the during extraction the extraction and purification and purification steps. According steps. Ac- to cordingthe late to regulatory the late regulatory requirement requirement (May 2021) (M establisheday 2021) established by the Herbal by the Medicinal Herbal Medicinal Products ProductsCommittee, Committee, the tolerated the tolerated amount of amount pyrrolizidine of pyrrolizidine alkaloids inalkaloids herbal medicinal in herbal plants/permedicinal plants/perday should day not should exceed not 1.0 exceed mg [18 ].1.0 Based mg [18]. on thisBased recommendation, on this recommendation, the observed the yield ob- servedof 0.16% yield (160 of mg/g) 0.16%was (160 extremely mg/g) was high extremely and potentially high and highly potentially toxic with highly regards toxic towith the regardsquantities to the used quantities to make dailyused beveragesto make daily (at least beverages few grams (at least of seeds few pergrams beverage). of seeds per beverage).Pyrrolizidine-derived alkaloids are frequently isolated as macrocyclic dilactones, using a combinationPyrrolizidine-derived of a pyrrolizidine alkaloids (necine are frequently base) with necicisolated acid as to macrocyclic produce macrocyclic dilactones, rings us- ingwith a combination a range of sizes of a pyrrolizidine (Figure3)[ 19 (necine]. In addition base) with to macrocyclic necic acid to dilactones, produce macrocyclic mono and diesters of necine as open chains, such as lycopsamine and echimidine, were reported [19].

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Molecules 2021, 26, 3464 4 of 6 rings with a range of sizes (Figure 3) [19]. In addition to macrocyclic dilactones, mono and diesters of necine as open chains, such as lycopsamine and echimidine, were reported [19].

FigureFigure 3. 3.Scaffolds Scaffolds involved involved in in the the biosynthesis biosynthesis of of macrocyclic macrocyclic pyrrolizidine-derived pyrrolizidine-derived alkaloids. alkaloids. The starsThe (*)stars indicate (*) indicate that both that enantiomers both enantiomers may exist. may Theexist. carbon–carbon The carbon–carbon double double bond ofbond necic of acid necic can beacid found can inbe the foundZ and in Etheconﬁguration. Z and E configuration.

MacrocyclicMacrocyclic alkaloids alkaloids derived derived from from pyrrolizidine pyrrolizidine are producedare produced by the by plants the plants as defense as de- toolsfense against tools herbivores.against herbivores. No therapeutic No therapeu applicationstic applications were linked were to these linked compounds to these com- but ratherpounds their but toxicity rather is their often toxicity put in theis often spotlight. put in They the arespotlight. considered They to are be phytotoxinsconsidered thatto be presentphytotoxins a high that risk present for humans a high and risk grazing for huma livestockns and grazing [5,6,20, 21livestock]. In mammals, [5,6,20,21]. they In mam- may causemals, severe they may hepatotoxicity cause severe [22 ,hepatotoxicity23], pneumotoxicity [22,23], [24 pneumotoxicity], and mutagenic [24], effect and [25 mutagenic,26]. The toxicityeffect [25,26]. was mostly The toxicity due to thewas bio-activation mostly due to by the cytochrome bio-activation P450 by (CYP) cytochrome enzymes P450 in liver,(CYP) toenzymes achieve thein liver, hydrolysis to achieve of dilactones the hydrolysis and release of dilactones the necine and bases release [27,28 ].the necine bases [27,28].It was reported that livestock fed with pyrrolizidine alkaloid-containing plants, un- dergoIt severe was reported lung, liver, that and livestock kidney fed tissue with damage pyrrolizidine [20]. One alkaloid-containing relevant case was plants, recently un- reporteddergo severe from lung, Brazilian liver, farmers and kidney showing tissue that damage horses [20]. fed One with relevant oats contaminated case was recently with Crotalariareportedseeds from diedBrazilian due tofarmers severe showing liver failure that [26 horses]. The fed high with level oats of hepatotoxicity contaminated andwith carcinogenicCrotalaria seeds effect died associated due to withsevere this liver class failure of compounds [26]. The presentshigh level a serious of hepatotoxicity human health and problem.carcinogenic Hence, effect any associated consumption with of this seeds class of Crotalaria of compounds cleomifolia presentsshould a beserious banned, human as suchhealth use problem. could be associatedHence, an withy consumption a high degree of ofseeds toxic of effect. Crotalaria cleomifolia should be banned, as such use could be associated with a high degree of toxic effect. 4. Materials and Methods 4.1.4. Materials Materials and Methods 4.1. PodsMaterials of Crotalaria cleomifolia were harvested in May 2014 at Manjakandriana, near Antananarivo (geographical coordinates, south: 18◦54006.900, East: 047◦45008.100). Seeds Pods of Crotalaria cleomifolia were harvested in May 2014 at Manjakandriana, near were separated from pods then dried at room temperature in the shadow. After drying, they Antananarivo (geographical coordinates, south: 18°54′06.9′’, East: 047°45′08.1′’). Seeds were crushed in a fine powder using an electric grinder. Solvents used for the extraction werewere purchased separated fromfrom Cooperpods then (Paris, dried France) at room and temperature were used asin the received. shadow. Reagents After drying, used forthey the were qualitative crushed studies in a fine were powder purchased using an from electric Sigma-Aldrich grinder. Solvents (Saint Quentinused for Fallavier,the extrac- France).tion were purchased from Cooper (Paris, France) and were used as received. Reagents used1H for and the13 Cqualitative NMR (1D andstudies 2D) were spectra purchased were measured from Sigma-Aldrich on a Bruker Avance (Saint 400 Quentin MHz Fallavier, France). (Mannheim, Germany) NMR spectrometer in CDCl3 or CD3OD. Chemical shifts are re- 1 13 portedH in and parts C per NMR million (1D and (δ) and 2D) couplingspectra were constants measured (J) in on Hz. a Bruker The residual Avance CDCl 400 3MHzor 1 13 CD(Mannheim,3OD were usedGermany) as internal NMR standards spectrometer for H in and CDClC3 NMR, or CD respectively.3OD. Chemical IR spectrashifts are were re- measuredported in on parts a Perkin per million Elmer 1600(δ) and spectrophotometer. coupling constants Optical (J) in rotations Hz. The were residual measured CDCl3 at or roomCD3OD temperature were used using as internal a Perkin standards Elmer 343 for Plus 1H and polarimeter. 13C NMR, Electrospray respectively. ionization IR spectra (ESI) were massmeasured spectra on were a Perkin recorded Elmer at 1600 the Institut spectrophoto de Chimiemeter. Mol Opticaléculaire rotations de Grenoble were onmeasured a Waters at Xevoroom G2-S temperature Q TOF instrument using a Perkin (Guyancourt, Elmer France)343 Plus with polarimeter. a nanospray Electrospray inlet. ionization (ESI) mass spectra were recorded at the Institut de Chimie Moléculaire de Grenoble on a 4.2.Waters Methods Xevo G2-S Q TOF instrument (Guyancourt, France) with a nanospray inlet. 4.2.1. Extraction of Alkaloids 4.2. ExtractionMethods in acidic media. A total of 30 g of the seeds powder were mixed with hydrochloric4.2.1. Extraction acid of (1M, Alkaloids 30 mL), suspended in methanol (100 mL) and stirred for 24 h at room temperature. After filtration under vacuum, methanol was evaporated under reduced Extraction◦ in acidic media. A total of 30 g of the seeds powder were mixed with hy- pressuredrochloric at 30acidC. (1M, The 30 aqueous mL), suspended solution in was methanol treated (100 with mL) ammonium and stirred hydroxide for 24 h at (20%) room until pH 10. The solution was extracted with dichloromethane (3 × 20 mL). The organic temperature. After filtration under vacuum, methanol was evaporated under reduced phase was dried over sodium sulfate, filtered, and evaporated to dryness, to afford the pressure at 30 °C. The aqueous solution was treated with ammonium hydroxide (20%) crude extract (136 mg). Extraction in basic media. A total of 20 g of the seeds powder were treated with 20 mL of ammonium hydroxide (20%) and mixed with dichloromethane (300 mL), then stirred at

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room temperature for 24 h. After ﬁltration, the solution was evaporated under reduced pressure, the obtained residue was dissolved in 20 mL of distilled water, and sulfuric acid (10%) was added until reaching pH = 2. The solution was extracted with diethyl ether. The aqueous solution was treated with ammonium hydroxide (20%) until pH = 10. The basic solution was extracted with dichloromethane (3 × 20 mL). The organic solution was washed with water, dried over sodium sulfate, and evaporated to dryness to afford the crude residue (66 mg). A comparative TLC (silica gel, eluent: CH2Cl2/CH3OH 9:1) and revelation under UV light at 254 nm showed that the two extracts presented the same proﬁle. Hence, the two extracts were mixed.

4.2.2. Determination of the Presence of Alkaloids in the Extracts The two extracts highlighted above were subjected to qualitative chemical screening for identiﬁcation of alkaloids. To this end, we used Wagner, Dragendorff, and Mayer tests [26]. Both extracts tested were positive, indicating the presence of alkaloids in crude extracts.

4.2.3. Puriﬁcation of the Extract The crude extract (200 mg) was puriﬁed on a silica gel chromatography column, using a gradient of dichloromethane:methanol: 98:2; 95:5; 90:10, and 0:100. In total, 35 fractions (10 mL each) were collected, analyzed by TLC using dichloromethane/methanol—92/8 as eluent then concentrated. Fractions 5 (44 mg) and 8 (39 mg) showed the presence of two compounds in pure forms, here compounds 1 and 2. 25 ◦ Spectral characterization of compound 1. [C]D +6.7 (c 0.009, MeOH); IR (CHCl3) −1 1 3466, 3059, 2971, 2845, 1716, 1443, 1273, 1213, 1158 cm ; H NMR (CD3OD): δ 6.50 (q, J = 7.2 Hz, 1H), 6.22 (s, 1H), 5.70 (d, J = 11.5 Hz, 1H), 5.15 (bs, 1H), 4.30 (bs,1H), 4.21 (d, J = 11.4 Hz, 1H), 3.96 (d, J = 16 Hz, 1H), 3.61 (m, 2H), 3.40 (dd, J = 5, 15.2 Hz, 1H), 3.25 (t, J = 8 Hz, 1H), 2.61 (m, 2H), 2.48–2.0 (m, 3H), 1.70 (m, 1H), 1.80 (m, 1H), 1.83 (m, 1H), 1.74 13 (d, J = 7 Hz, 3H), 0.82 (d, J = 7.1 Hz, 3H). C NMR (CD3OD) δ 176.1, 170.5, 136.6, 136.5, 135.1, 133.6, 82.5, 77.9, 76.9, 68.2, 63.4, 61.2, 53.9, 38.1, 34.4, 30.4, 14.3, 12.4; MS: HRMS calcd. for C18H26NO6 352.1660 (M + 1), obsd. 352.1755.

5. Conclusions Speciﬁc extractions were conducted on the seeds of Crotalaria cleomifolia and revealed the presence of two pyrrolizidine-derived alkaloids, from which one was identiﬁed as usaramine. The latter is known for its high hepatotoxicity, as evidenced by in vivo studies. Hence, any consumption of the seeds of Crotalaria cleomifolia should be banned, as such use could be associated with a high degree of intoxication.

Supplementary Materials: The following are available online. Figure S1. MS, 1D, and 2D NMR data of compound 1 and 1H NMR of compound 2. Author Contributions: A.H.D.S. collected the plant and performed the phytochemical study. C.F.R., D.A.D.R., R.L.J., H.R.R., and V.L.J. collected and conceived the study, writing, review, and field survey. A.B. supervised the work, realized a part of the phytochemical work, wrote the manuscript, and managed the work. All authors have read and agreed to the published version of the manuscript. Funding: The authors acknowledge the financial support from Grenoble Alpes University, University of Antananarivo. A.H.D.S. was the recipient of a three months fellowship (BGF) granted by the French Embassy in Madagascar. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no known competing financial interests or personal relationships that could influence this work. Molecules 2021, 26, 3464 6 of 6

Sample Availability: Samples of the seeds of Crotalaria cleomifolia are availbale from authors.

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