Target-Guided Isolation of O-Tigloylcyclovirobuxeine-B from Buxus Sempervirens L

molecules

Article Target-Guided Isolation of O-tigloylcyclovirobuxeine-B from Buxus sempervirens L. by Centrifugal Partition Chromatography

Lara U. Szabó and Thomas J. Schmidt *

Institute of Pharmaceutical Biology and Phytochemistry (IPBP), University of Münster, Pharma Campus Correnstraße 48, D-48149 Münster, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-251-83-33378

Academic Editor: Derek J. McPhee Received: 7 September 2020; Accepted: 16 October 2020; Published: 19 October 2020

Abstract: The increasing drug resistance of malaria parasites challenges the treatment of this life-threatening disease. Consequently, the development of innovative and effective antimalarial drugs is inevitable. O-tigloylcyclovirobuxeine-B, a nor-cycloartane alkaloid from Buxus sempervirens L., has shown promising and selective in vitro activity in previous studies against Plasmodium falciparum (Pf ), causative agent of Malaria tropica. For further investigations, it is indispensable to develop an advanced and efficient isolation procedure of this valuable natural product. Accordingly, we used liquid–liquid chromatography including centrifugal partition chromatography (CPC) to obtain the pure alkaloid on a semi-preparative scale. Identification and characterization of the target compound was accomplished by UHPLC/+ESI-QqTOF-MS/MS, 1H NMR and 13C NMR. In conclusion, this work provides a new and efficient method to obtain O-tigloylcyclovirobuxeine-B, a valuable natural product, as a promising antiplasmodial lead structure for the development of innovative and safe medicinal agents.

Keywords: Buxus sempervirens L.; nor-cycloartane alkaloids; O-tigloylcyclovirobuxeine-B; antimalarial activity; target-guided isolation; centrifugal partition chromatography

1. Introduction Buxus sempervirens L. (European Box; Buxaceae) is an indeciduous shrub, which represents a rich source for nor-triterpene alkaloids of the nor-cycloartane type. Decoctions of the leaves are well known in folk medicine for a variety of indications, including malaria [1,2]. Malaria is a poverty-related infectious disease caused by protozoans of the genus Plasmodium and transmitted by infected female Anopheles mosquitoes. Worldwide there were 228 million estimated malaria cases in 2018 [3]. The development of new eﬀective agents against this life-threatening disease is imperative because of increasing drug resistance. Extracts of the leaves from B. sempervirens L. have shown selective in vitro activity against Plasmodium falciparum that causes the majority of malaria deaths [4–6]. In 2014, Althaus et al. obtained a small amount of O-tigloylcyclovirobuxeine-B from an alkaloid-enriched fraction of B. sempervirens leaf extract by bioactivity-guided isolation. The natural product yielded an IC50 value against Pf of 0.46 µg/mL (0.92 µM) vs. 9.4 µg/mL (18.9 µM) for cytotoxicity against L6 rat cells [5]. Consequently, O-tigloylcyclovirobuxeine-B is a promising lead compound for the development of novel and safe medicinal agents against malaria. For further investigations, including in vivo studies, it is required to establish a simple, reproducible and productive isolation procedure for this valuable compound. For this purpose, we describe a new eﬃcient semi-preparative scale isolation of

Molecules 2020, 25, 4804; doi:10.3390/molecules25204804 www.mdpi.com/journal/molecules Molecules 2020, 25, x 2 of 10 tigloylcyclovirobuxeine-BMolecules 2020, 25, 4804 using only liquid–liquid partition chromatography. The authenticity of2 ofthe 10 isolated O-tigloylcyclovirobuxeine-B was proven by 1H NMR, 13C NMR and +ESI-QqTOF-MS/MS. O-tigloylcyclovirobuxeine-B using only liquid–liquid partition chromatography. The authenticity of 2.the Results isolated andO-tigloylcyclovirobuxeine-B Discussion was proven by 1H NMR, 13C NMR and +ESI-QqTOF-MS/MS. We established an advanced and improved isolation method of O-tigloylcyclovirobuxeine-B (1) 2. Results and Discussion without using a solid stationary phase. After extraction of about 1.5 kg of dried plant material, only three Westeps established of liquid–liquid an advanced partition and (one improved acid/base isolation partitioning method step of andO-tigloylcyclovirobuxeine-B two steps of centrifugal partition(1) without chromatography using a solid stationary(CPC)) were phase. required After in extractionorder to obtain of about 35 mg 1.5 of kg target of dried compound. plant material, onlyThe three advantages steps of liquid–liquid in using this partition all-liquid (one separation acid/base scheme partitioning are diverse, step and including two steps a high of centrifugal recovery ofpartition injected chromatography sample, a low solvent (CPC)) consumption were required (less in orderwaste to and obtain expense), 35 mg minimized of target compound. tailing of eluted peaks,The no advantagesirreversible inloss using of sample this all-liquid due to chemisorption, separation scheme high arereproducibility diverse, including and high a high purification recovery levelsof injected [7–9].sample, a low solvent consumption (less waste and expense), minimized tailing of eluted peaks, no irreversible loss of sample due to chemisorption, high reproducibility and high puriﬁcation 2.1.levels Target-Guided [7–9]. Isolation of O-tigloylcyclovirobuxeine-B (1)

2.1.1.2.1. Target-Guided Extraction and Isolation Alkaloid-Enrichment of O-tigloylcyclovirobuxeine-B of B. sempervirens (1) L. Leaves

2.1.1.The Extraction discovery and of Alkaloid-Enrichment the target compound of asB. antipl sempervirensasmodialL. Leavesprinciple of B. sempervirens leaves followed the observation of an increased antiplasmodial activity of an alkaloid-enriched fraction in comparisonThe discovery to the crude of the dichloromethane target compound (DCM) as antiplasmodial extract [5]. The principle alkaloid-enriched of B. sempervirens fraction leaves was obtainedfollowed by the acid/base observation extraction of an increased of the crude antiplasmodial DCM extract. activity In the ofpresent an alkaloid-enriched study, we obtained fraction 4.63 ing ofcomparison the alkaloid to fraction the crude (ALOF) dichloromethane from 112 g (DCM)of crude extract DCM [ 5extract]. The (GBUS) alkaloid-enriched from 1.47 fractionkg of plant was material.obtained Compared by acid/base to extractionthe crude extract, of the crude a 19-fold DCM enrichment extract. In of the O-tigloylcyclovirobuxeine-B present study, we obtained 4.63(1) was g of observedthe alkaloid in UHPLC/+ESI-QqTOF-MS fraction (ALOF) from 112 (hencefort g of crudeh DCM termed extract “LC/MS”) (GBUS) analysis from 1.47 (Figure kg of 1). plant material. Compared to the crude extract, a 19-fold enrichment of O-tigloylcyclovirobuxeine-B (1) was observed in UHPLC /+ESI-QqTOF-MS (henceforth termed “LC/MS”) analysis (Figure1).

FigureFigure 1. UHPLCUHPLC/+ESI-QqTOF-MS/+ESI-QqTOF-MS chromatograms: chromatograms: Base Base peak peak chromatogram chromatogram of m /zof200–1000 m/z 200–1000 (black) , (black),Extracted Extracted ion chromatogram ion chromatogram of m of/z m/z249 249 [M [M+ +2H] 2H]22++ (pink),(pink), Extracted Extracted ion ion chromatogram chromatogram of m/z of 497m/z [M497 + [M H]++ H](blue);+ (blue); (A) (dichloromethaneA) dichloromethane extract extract (GBUS); (GBUS); (B) ( Balkaloid) alkaloid fraction fraction (ALOF). (ALOF). The The target target compoundcompound was was enriched enriched from from GBUS GBUS to to ALOF ALOF by by factor factor 19. 19.

2.1.2. Selection of Suitable Two-Phase Solvent Systems

Molecules 2020, 25, 4804 3 of 10 Molecules 2020, 25, x 3 of 10

2.1.2.InSelection order to ofseparate Suitable a Two-Phasesample successfully Solvent Systems by CPC the biphasic solvent system should satisfy some crucial requirements: For an adequate retention of the stationary phase, the settling time should be shorterIn order than to 30 separate s [7]. The a sample distribution successfully coefficient by CPC (termed the biphasic KC according solvent to system [10]) shouldof the target satisfy compound(s)some crucial requirements:between the two For phases an adequate should retention be close of to the one stationary (0.5 ≤ K phase,≤ 1.0) [8]. the A settling smaller time KC shouldvalue tendsbe shorter to give than a less 30 efficient s [7]. Thepeak distribution resolution while coeffi acient larger (termed KC value KC mayaccording result in to broader [10]) of peaks the target due compound(s) between the two phases should be close to one (0.5 K 1.0) [8]. A smaller K value to protracted elution time. Hence the target compound(s) should show≤ ≤ a close to equal distributionC betweentends to the give upper a less and effi cientlower peak phases resolution [8,9]. while a larger KC value may result in broader peaks due to protractedTo choose elution an appropriate time. Hence biphasic the target solvent compound(s) system for shouldthe separation, show a closethe required to equal distribution distribution ofbetween the alkaloids the upper was andevaluated lower phasesby thin [layer8,9]. chromatography (TLC) and LC/MS analysis (ALOF and fractionTo 2). choose In addition, an appropriate the phase biphasic systems solvent were systemassessed for with the separation,regard to a thestable required and rapid distribution (<30 s) phaseof the separation. alkaloids was evaluated by thin layer chromatography (TLC) and LC/MS analysis (ALOF and fractionWe observed 2). In addition, that the the eluent phase system systems Hexane:EtOAc/MeOH:H were assessed with regard2O (7:3/7:3) to a stable(v/v/v/ andv) can rapid provide (<30 a s) partitionphase separation. appropriate for the separation of the alkaloid fraction (KC value 1.16, determined by LC/MS). This two-phaseWe observed system that belongs the eluent to the system HEMWat Hexane:EtOAc family (n-Hexane-Ethyl/MeOH:H2O (7:3 acetate-Methanol-Water)/7:3) (v/v/v/v) can provide [7, a 11]partition and is appropriatesuitable to separate for the separation compounds of theover alkaloid a wide fraction range of (K polarityC value 1.16,by varying determined proportions by LC/MS). of theThis four two-phase components. system belongs to the HEMWat family (n-Hexane-Ethyl acetate-Methanol-Water) [7,11] andFor is suitable the subfractionation to separate compounds of fraction 2, over the asolvent wide range system of Hexane/CH polarity by3 varyingCN:CH2Cl proportions2 (10/7:3) (v of/v/ thev) wasfour used components. and, with a KC value of 0.63, ensured a partition coefficient well suitable for the target compound.For the subfractionation of fraction 2, the solvent system Hexane/CH3CN:CH2Cl2 (10/7:3) (v/v/v) was used and, with a KC value of 0.63, ensured a partition coefficient well suitable for 2.1.3.the target Separation compound. of the Alkaloid-Enriched Fraction by Centrifugal Partition Chromatography

2.1.3.The Separation alkaloid offraction the Alkaloid-Enriched was separated into Fraction 20 fractions by Centrifugal by centrifugal Partition partition Chromatography chromatography (CPC). During elution mode, fractions 1–15 were collected and, subsequently, the extrusion phase yieldedThe fractions alkaloid fraction16–20. wasAll separated fractions into were 20 fractions analysed by centrifugalby LC/MS. partition Fraction chromatography 2 contained (CPC). O- tigloylcyclovirobuxeine-BDuring elution mode, fractions (1) as a 1–15main were constituent collected (Figure and, 2). subsequently, The major part the of extrusion the target phase compound, yielded enrichedfractions by 16–20. factor All 10 fractions over the were ALOF, analysed was byconcentr LC/MS.ated Fraction in this 2 contained fraction whileO-tigloylcyclovirobuxeine-B lower amounts were present(1) as a also main in constituent fraction 1. (Figure2). The major part of the target compound, enriched by factor 10 over the ALOF, was concentrated in this fraction while lower amounts were present also in fraction 1.

Intens . x105 5

0 0 2 4 6 8 10 12 Time [min] Figure 2. UHPLC/+ESI-QqTOF-MS chromatogram of fraction 2: Base peak chromatogram of

m/z 200–1000 (black), Extracted ion chromatogram of m/z 249 [M + 2H]2+ (pink), Extracted ion Figurechromatogram 2. UHPLC/+ESI-QqTOF-MS of m/z 497 [M + H] chromatogram+ (blue). Enrichment of fraction of 2: theBase target peak chromatogram compound from of m/z ALOF 200– to 1000fraction (black), 2 was Extracted 10-fold. ion chromatogram of m/z 249 [M + 2H]2+ (pink), Extracted ion chromatogram of m/z 497 [M + H]+ (blue). Enrichment of the target compound from ALOF to fraction 2 was 10-fold. 2.1.4. Separation of Fraction 2 by Centrifugal Partition Chromatography 2.1.4. ASeparation second CPC of Fraction separation 2 by was Centrifugal performed Partition to purify ChromatographyO-tigloylcyclovirobuxeine-B (1) from fraction 2. TheA target second compound CPC separation appeared was almost performed quantitatively to purify in O test-tigloylcyclovirobuxeine-B tubes 16 and 17. Following (1) from crystallization fraction 2.from The acetonitrile, target compound 35 mg ﬁne appeared white needles almost of O -tigloylcyclovirobuxeine-Bquantitatively in test tubes (1) were 16 obtained.and 17. Following The LC/MS crystallizationanalysis showed from that acetonitrile, the target compound35 mg fine was white>90% needles pure, of accompanied O-tigloylcyclovirobuxeine-B only by two small (1 peaks) were of obtained.congeneric The alkaloids LC/MS (seeanalysis Figure showed3 and Materialsthat the target and Methods,compound Section was > 3.3.390%). pure, accompanied only by two small peaks of congeneric alkaloids (see Figure 3 and Materials and Methods, section 3.3.3.).

Molecules 2020, 25, 4804 4 of 10 MoleculesMolecules 2020 2020, ,25 25, ,x x 44 of of 10 10

Intens . Intens . x105 x105

4 4

3 3

2 2

1 1

0 0 0 2 4 6 8 10 12 Time [min] 0 2 4 6 8 10 12 Time [min] FigureFigure 3. 3.3. UHPLC/+ESI-QqTOF-MSUHPLC/+ESI-QqTOF-MSUHPLC/+ESI-QqTOF-MS chromatogramchromatogram chromatogram ofof of OO-tigloylcyclovirobuxeine-B-tigloylcyclovirobuxeine-B (1( 1):): BaseBase Base peak peak 2+ chromatogramchromatogram of of m/z mm/z/z 200–1000 200–1000200–1000 (black), (black),(black), Extracted ExtractedExtracted ion ionion chromatogram chromatogramchromatogram of ofof m/z mm/z/z 249 249249 [M [M + + 2H] 2H]2H]2+2+ (pink), (pink),(pink), + ExtractedExtracted ion ion chromatogram chromatogram of ofof m/z mm/z/z 497497 497 [M [M[M ++ + H] H]H]++ (blue). (blue).(blue). Enrichment Enrichment Enrichment of of of 1 1 1from fromfrom fraction fraction fraction 2 2 was was 7.8-fold. 7.8-fold.

2.2. Identiﬁcation of O-Tigloylcyclovirobuxeine-B (1) 2.2.2.2. Identification Identification of of O-Ti O-Tigloylcyclovirobuxeine-Bgloylcyclovirobuxeine-B (1) (1) The identity of the isolated compound with O-tigloylcyclovirobuxeine-B (1) (Figure4) was TheThe identityidentity ofof thethe isolatedisolated compoundcompound withwith OO-tigloylcyclovirobuxeine-B-tigloylcyclovirobuxeine-B (1(1) ) (Figure(Figure 4)4) waswas 1 13 conﬁrmed by comparison of the 1H NMR, 13 C NMR and +ESI-QqTOF-MS/MS spectra (Figures S2–S8, confirmedconfirmed by by comparison comparison of of the the 1 HH NMR, NMR, 13 CC NMR NMR and and +ESI-QqTOF-MS/MS +ESI-QqTOF-MS/MS spectra spectra (Figures (Figures S2-S8, S2-S8, Supplementary Materials) which were in full agreement with reports in literature [5]. SupplementarySupplementary Materials) Materials) which which were were in in full full agreement agreement with with reports reports in in literature literature [5]. [5].

Figure 4. Chemical structure of O-tigloylcyclovirobuxeine-B (1). FigureFigure 4. 4. Chemical Chemical structure structure of of O O-tigloylcyclovirobuxeine-B-tigloylcyclovirobuxeine-B ( 1(1).). 2.3. +ESI-QqTOF MS/MS fragmentation of O-tigloylcyclovirobuxeine-B (1) 2.3.2.3. +ESI-QqTOF +ESI-QqTOF MS/MS MS/MS fragmentation fragmentation of of O-tigloylcyclovirobuxeine-B O-tigloylcyclovirobuxeine-B (1) (1) In addition to the analytical data reported previously [5], it was of interest to characterize the MS In addition to the analytical data reported previously [5], it was of interest to characterize the fragmentationIn addition of to the the target analytical compound data reported in order previously to allow unambiguous [5], it was of dereplication interest to characterize in future work the MS fragmentation of the target compound in order to allow unambiguous dereplication in future MS(e.g., fragmentation when investigating of the othertargetBuxus compoundspecies in for order related to compounds).allow unambiguous Therefore, dereplication MS/MS fragmentation in future workwork (e.g.(e.g. whenwhen investigatinginvestigating otherother BuxusBuxus speciesspecies+ forfor relatedrelated compounds).compounds). Therefore,Therefore, MS/MSMS/MS of the singly protonated molecular ion [M + H] (m/z 497) was+ studied. The resulting mass spectrum fragmentation of the singly protonated molecular ion [M + H]+ (m/z 497) was studied. The resulting fragmentation(Figure5A) clearly of the showed singly protonated a very characteristic molecular fragmentation ion [M + H] ( patternm/z 497) comprising was studied. all ionsThe resulting expected mass spectrum (Figure 5A) clearly showed a very characteristic fragmentation pattern comprising all massfor the spectrum loss of (Figure the two 5A) amino clearly substituents showed a asvery well ch asaracteristic the tiglic fragmentation acid moiety. Furthermore,pattern comprising a neutral all ionsions expected expected for for the the loss loss of of the the two two amino amino substituen substituentsts as as well well as as the the tiglic tiglic acid acid moiety. moiety. Furthermore, Furthermore, loss of 26 Da from some fragments, corresponding to C2H2 (=ethyne), appears to result from a a neutral loss of 26 Da from some fragments, corresponding to C2H2 (= ethyne), appears to result from aretro-Diels-Alder-related neutral loss of 26 Da from decyclization some fragments, with corresponding bond cleavage to between C2H2 (= ethyne), C-5 and appears C-6 as well to result as C-7 from and a retro-Diels-Alder-related decyclization with bond cleavage between C-5 and C-6 as well as C-7 and aC-8. retro-Diels-Alder-related The fragmentation pathway decyclization along with with bond the theoretical cleavage betweenm/z values C-5 andand massC-6 as deviations well as C-7 of and the C-8. The fragmentation pathway along with the theoretical m/z values and mass deviations of the C-8.experimental The fragmentation signals is shownpathway in Figurealong with5B. the theoretical m/z values and mass deviations of the experimentalexperimental signals signals is is shown shown in in Figure Figure 5B. 5B.

Molecules 2020, 25, 4804 5 of 10 Molecules 2020, 25, x 5 of 10

Intens. 1. +MS2(497.4200), 40.0eV, 4.331-5.239min #772-934 A 500

1+ 400 497.4133

1+ 300 366.3196

1+ 1+ 1+ 200 295.2446 321.2567 1+ 352.2959 466.3695 1+ 397.3599 100 1+ +1 452.3526 +1 340.2988 421.3134 0 275 300 325 350 375 400 425 450 475 500 m/z

FigureFigure 5. (A5. )A:+ESI +ESI MS MS/MS/MS spectrum spectrum (CID (CID 40 eV;40 onlyeV; only the regionthe region for Figure for Figure5B is shown; 5 B is theshown; full spectrumthe full is presentedspectrum inis Figure presented S8, Supplementary in Figure S8, Materials). Supplementary (B) Fragments Materials) and. possibleB: Fragments fragmentation and possible pathway + of thefragmentation [M + H]+ ion pathway of O-tigloylcyclovirobuxeine-B of the [M + H] ion of O-tigloylcyclovirobuxeine-B (1). (1).

Molecules 2020, 25, 4804 6 of 10

3. Materials and Methods

3.1. Plant Material The leaves (along with a low amount of small twigs) of B. sempervirens L. were collected in February 2019 on a private property in Havixbeck, Germany, from the same plants as in the previous study [5]. The identity of the plant was conﬁrmed by T. J. Schmidt and a voucher specimen of the collection was deposited at the herbarium of the Institute of Pharmaceutical Biology and Phytochemistry, University of Münster (voucher No.: LS_BS_01 and LS_BS_02). The air-dried leaves were separated from the branches and ground with an IKA MF basic mill (IKA, Staufen, Germany) to 1 mm mesh size.

3.2. Extraction and Preparation of the Alklaloid-Enriched Fraction The powdered plant material (1467 g) was extracted exhaustively in a Soxhlet-apparatus in portions of approximately 240 g with 1.5 L dichloromethane (DCM) for 36 h until the supernatant was colorless. After rotary evaporation under reduced pressure at 40 ◦C, a total of 112 g of crude extract (GBUS) was obtained. An acid–base extraction was carried out to enrich the alkaloids from 103 g of the crude extract. For each extraction batch, 5 g of the extract was dissolved in 60 mL of DCM and extracted six times with 25 mL of diluted sulfuric acid R (1M, European Pharmacopoeia Reagent) in a separating funnel. Due to their increased hydrophilicity, the protonated alkaloids concentrate in the aqueous phase, while lipophilic and neutral compounds mainly accumulate in the lipophilic DCM phase. After evaporation, the DCM phase yielded 103.98 g of the lipophilic and neutral fraction (LNB). The collected acidic water phase was alkalized to a pH value of 10 with solid sodium hydroxide ≈ and subsequently extracted six times with 200 mL of DCM. The deprotonated alkaloids accumulate in the lipophilic phase, while hydrophilic impurities remain in the aqueous phase. Of the alkaloid fraction (ALOF), 4.63 g was obtained from the evaporated DCM phase. According to LC/MS quantiﬁcation, GBUS and ALOF contained 0.06% (63.43 mg) and 1.14% (52.89 mg) of the target compound 1, respectively, corresponding to an enrichment factor of 19. The recovery rate was 90.6% (note that only 103 of the 112 g GBUS were used).

3.3. Isolation of O-Tigloylcyclovirobuxeine-B (1)

3.3.1. Selection of a Two-Phase Solvent System Preliminary small-scale tests were carried out to determine a suitable phase system for the fractionation. For this purpose, 3 mg of ALOF was each mixed with 2 mL of each, the upper and lower phase, in a snap-cap glass and shaken out. The distribution of the alkaloids (see Table S1, Supplementary Materials) was visualized with Liebermann-Burchard (acetic anhydride (5 mL):sulfuric acid (5 mL):ethanol (50 mL), European Pharmacopoeia Reagent) and Anisaldehyde spray reagents (4-Methoxybenzaldehyde (0.5 mL):acetic acid (10 mL):methanol (85 mL):sulfuric acid (5 mL), European Pharmacopoeia Reagent) on a TLC plate in case of ALOF (TLC plate silica gel 60 F254, Merck KGaA, Darmstadt, Germany; mobile phase: butan-1-ol:H2O:CH3COOH (10:3:1) (v/v/v)[12]; see Figure S1, Supplementary Materials) and evaluated quantitatively by LC/MS analysis. The distribution coeﬃcient (KC = 1.16) of the target compound was determined according to [10] as the concentration of 1 in the lower stationary phase divided by that of the upper mobile phase.

3.3.2. Fractionation of the Alkaloid Fraction (ALOF) by Centrifugal Partition Chromatography The separation of the alkaloid fraction (4 g, sample concentration: 250 mg/9 mL) was carried out on a CPC-250 (Gilson, Limburg, Germany) chromatography system. The eluent system Hexane: EtOAc/MeOH:H2O (7:3/7:3) (v/v/v/v) was equilibrated in a separating funnel before application. In ascending mode (1200 rpm, 3 mL/min) portions of 6 mL were collected into test tubes, monitored by TLC and visualized with Dragendorﬀ’s spray reagents (bismuth subnitrate (0.85 g):H2O (40 mL):acetic Molecules 2020, 25, 4804 7 of 10 acid (10 mL):potassium iodide solution (400 g L 1; 20 mL), European Pharmacopoeia Reagent). · − After termination of the elution mode (720 mL) the extrusion phase (400 mL) was also separated and recovered in test tubes by stopping the rotation and increasing the ﬂow rate (5 mL/min) at the same time. The elution phase yielded 15 fractions and O-tigloylyclovirobuxeine-B was detected in the elution volume range from 54 to 96 mL (fraction 2; KC range 0.1–0.29; note that the lower KC value in comparison with the selection test above is likely due to the much higher concentration of 1 under these conditions, which may lead, e.g., to dimerization of the analyte [10]) by comparison with an authentic reference sample [5]. The content of 1 in fraction 2 was 11.7% (39.6 mg 1 in 337.7 mg of fraction 2 obtained from 4 g ALOF). The recovery rate was thus 86.8% and the enrichment factor of this separation step was 10.

3.3.3. Subfractionation of Fraction 2 by Centrifugal Partition Chromatography The following modifications were made to the fractionation of fraction 2 in contrast to 3.3.2. The biphasic solvent system Hexane/CH3CN:CH2Cl2 (10/7:3) (v/v/v) was used. This separation system had been used during our previous purification of 1 [13]. In the present work, the distribution coefficient (KC = 0.63) in this solvent system was determined by LC/MS as above (2.4 mg of fraction 2 equilibrated in 2 mL of each of the two phases; see Figure S2, Supplementary Materials) and found suitable. The flow rate for the elution mode was set to 2.5 mL/min and rotation to 1300 rpm. The elution phase yielded 130 eluates (6.25 mL each). 310 mg of fraction 2 were separated in this way. O-tigloylcyclovirobuxeine-B (1) was present almost quantitatively in pure form in the elution volume range from 93.75 to 106.25 mL (test tubes 16 and 17; KC range 0.28–0.33; again, a much higher analyte concentration than in the previous experiment was the likely cause for the lower KC values). Following crystallization from acetonitrile, 35 mg fine white needles of O-tigloylcyclovirobuxeine-B (1) were obtained. The purity was 91.1% as estimated from the LC/MS analysis (compare Figure3). The recovery rate of this last purification step was 96.2% and the enrichment factor 7.8. The overall enrichment of the three purification steps was thus 1482-fold with a total recovery rate of 76%. The target compound (tR 5.39 min, [M + H]+: m/z 497.4116, [M + 2H]2+: m/z 249.2087) was accompanied by small amounts of two congeneric alkaloids with [M + H]+ at m/z 415 and 485 constituting about 3.9 and 4.9% (tR 3.77 and 5.13 min, Figure3), as estimated by integration of extracted ion chromatograms for the respective [M + 2H]2+ ions. The former of these impurities was unambiguously identified as unesterified cyclovirobuxeine-B by its exact mass ([M + H]+: m/z 415.3731, + 2+ calcd for C27H47N2O 415.3738; [M + 2H] : m/z 208.1923) and by comparison to authentic reference compound [13,14] The formation of this compound in small quantities by hydrolysis of 1 during the workup and isolation process cannot be excluded. The latter displayed an [M + H]+ at m/z 485.4179 + 2+ corresponding to C31H53N2O2 ([M + 2H] at 243.2134). From the occurrence of the very intense doubly protonated ion; however, it can be expected to be a diaminocycloartanoid related to 1, which cannot be characterized in more detail at present.

3.4. Spectroscopic Analysis of O-tigloylcyclovirobuxeine-B (1)

3.4.1. NMR Spectroscopy NMR spectra were recorded on Agilent DD2 600 MHz spectrometer (Agilent, Santa Clara, CA, 1 USA) at 25 ◦C in CDCl3. Spectra were referenced to the solvent signals ( H: 7.260 ppm; 13C: 77.160 ppm) and were evaluated with MestReNova version 11.0 software (Mestrelab Research, Santiago de Compostela, Spain).

3.4.2. UHPLC/+ESI-QqTOF-mass Spectrometry UHPLC/+ESI-QqTOF-MS and MS/MS measurements were performed as described previously [5] with minor modifications. The solvent flow rate was set at 0.4 mL/min. Water (with 0.1% formic Molecules 2020, 25, 4804 8 of 10 acid; A) and acetonitrile (with 0.1% formic acid; B) were used as mobile phase. The binary gradient adopted was as follows: 0–1.88 min: linear from 15% B to 30% B; 1.88–7.88 min: linear from 30% B to 33% B; 7.88–9.9 min: linear from 33% B to 50% B; 9.9–9.93 min: linear from 50% B to 100% B; 9.93–15.88: isocratic 100% B; 15.88–15.98 min: linear from 100% B to 15% B; 15.98–20.0 min: isocratic 15% B. The injection volume of pure compound was 1 µL. Sample concentrations: GBUS (10 mg/mL), ALOF (10 mg/mL), fraction 2 (1 mg/mL), O-tigloylcyclovirobuxeine-B (1) (0.1 mg/mL). Quantification of the target compound was carried out based on peak integration in extracted ion chromatograms of the intense [M + 2H]2+ ion. A calibration line (see Figure S9, Supplementary Materials, R2 = 0.9958) was obtained from six different concentrations of the purified compound (10, 8, 6, 4, 2 and 1 µg/mL), each analysed three times. Samples of GBUS, ALOF, fraction 2 and the samples for determination of K values during selection of CPC solvent systems were diluted to appropriate concentration within the range of the calibration line. All calculated concentrations were corrected by the purity factor of 0.91. To study the fragmentation of 1, a separate experiment in MRM mode was carried out with the purified compound, pre-selecting the [M + H]+ ion at m/z 497.42 10 and a collision energy of 40 eV. ± 3.5. Spectral Data of O-tigloylcyclovirobuxeine-B (1)

1 O-tigloylcyclovirobuxeine-B (1): white crystals; H NMR (600 MHz, CDCl3; δ (ppm), intensity, mult., J (Hz)): 6.84 (1H, qq, 7.1, 1.5, H-3‘), 5.60 (1H, ddd, 10.7, 1.4, 1.3, H-6), 5.37 (1H, ddd, 10.3, 6.1, 3.1, H-7), 5.10 (1H, ddd, 8.5, 6.0, 1.1, H-16), 2.67 (1H, m, H-20), 2.57 (1H, dd, 6.2, 2.5, H-8), 2.35 (3H, s, H-33), 2.30 (6H, s, H-31/32), 2.14 (1H, m, H-15), 2.12 (1H, m, H-17), 2.04 (1H, m, H-3), 1.84 (1H, m, H-11), 1.83 (1H, m, H-5), 1.81 (3H, dq, 1.4, H-5‘), 1.76 (3H, dq, 7.1, 1.3, H-4‘), 1.76 (1H, m, H-2), 1.74 (1H, m, H-12), 1.54 (2H, m, H-1), 1.54 (1H, m, H-2), 1.49 (1H, m, H-12), 1.44 (1H, m, H-11), 1.24 (1H, d(d), 14.2, (<1), H-15), 1.10 (3H, d, 6.2, H-21), 1.04 (3H, s, H-29), 1.00 (3H, s, H-18), 0.94 (3H, s, H-28), 0.79 (3H, s, H-30), 0.72 (1H, d, 4.1, H-19), 0.18 (1H, d, 4.1, H-19); 13 − C NMR (150 MHz, CDCl3; δ (ppm)): 168.08 (qC, C-1‘), 137.82 (qC, C-2‘), 128.87 (CH, C-3‘), 128.17 (CH, C-7), 127.98 (CH, C-6), 80.41 (CH, C-16), 71.55 (CH, C-3), 57.37 (CH, C-20), 57.03 (CH, C-17), 49.52 (qC, C-14), 48.85 (CH, C-5), 46.18 (qC, C-13), 44.31 (CH3, C-31/32), 43.13 (CH, C-8), 42.77 (CH2, C-15), 41.55 (qC, C-4), 33.24 (CH3, C-33), 32.28 (CH2, C-12), 31.13 (CH2, C-1), 28.73 (qC, C-10), 26.18 (CH3, C-29), 25.11 (CH2, C-11), 20.73 (qC, C-9), 20.06 (CH2, C-2), 18.51 (CH3, C-21), 18.18 (CH2, C-19), 17.57 (CH3, C-28), 16.58 (CH3, C-30), 15.98 (CH3, C-18), 14.56 (CH3, C-4‘), 12.17 (CH3, C-5‘). + 2+ + +ESI-QqTOF-MS (m/z): 497.4116 [M + H] , 249.2134 [M + 2H] (calcd for C32H53N2O2 : 497.4102, 2+ + for C32H54N2O2 : 249.2087). MS/MS data for [M + H] are reported in Figure5. The spectral data were in full agreement with ﬁndings reported in literature [5].

4. Conclusions The present work reveals a state-of-the-art semi-preparative scale isolation method of O-tigloylcyclovirobuxeine-B (1), a compound with promising antiplasmodial activity. In comparison to previously described extractions of this natural compound [5,15] we have not used any solid packing material. Thereby we achieved a high recovery and purification of our sample during isolation. Our findings demonstrate that CPC is an advantageous chromatographic technique for natural compound isolation also in case of amino nor-cycloartane-type alkaloids from Buxus. The target compound of our isolation, O-tigloylcyclovirobuxeine-B (1), has the potential to serve as a lead compound against Plasmodium falciparum. Since B. sempervirens is a very popular ornamental plant that requires periodic trimming, its leaves represent a very sustainable source of this valuable compound. Studies on temporal variability of the content of 1 in B. sempervirens leaves are in progress, in order to identify an optimal harvesting time to further increase the yield. Consequently, the isolation method presented here is an important step in obtaining sufficient quantities of this interesting Molecules 2020, 25, 4804 9 of 10

alkaloid for further in-depth studies with the aim of designing analogues with improved activity and toxicity proﬁles.

Supplementary Materials: The following are available online, Table S1: Solvent system selection process for the alkaloid fraction (ALOF). Figure S1: Determination of a suitable phase system for the fractionation by TLC. Figure S2: Determination of a suitable phase system for the fractionation of fraction 2 by UHPLC/+ESI-QqTOF-MS. Figure S3: 1 1 H NMR spectrum of O-tigloylcyclovirobuxeine-B (1) (600 MHz, CDCl3). Figure S4: Detail of the H NMR spectrum 13 of O-tigloylcyclovirobuxeine-B (1) (600 MHz, CDCl3). Figure S5: C NMR spectrum of O-tigloylcyclovirobuxeine-B 13 (1) (150 MHz, CDCl3). Figure S6: Detail of the C NMR spectrum of O-tigloylcyclovirobuxeine-B (1) (150 MHz, CDCl3). Figure S7: +ESI/QqTOF mass spectrum (full scan) of O-tigloylcyclovirobuxeine-B (1). Figure S8: +ESI/QqTOF MS/MS spectrum of the [M + H]+ ion of O-tigloylcyclovirobuxeine-B (1). Figure S9: Calibration line for the quantitive determination of O-tigloylcyclovirobuxeine-B (1). Author Contributions: Conceptualization, T.J.S.; methodology, L.U.S. and T.J.S.; investigation, L.U.S.; resources, T.J.S.; writing—original draft preparation, L.U.S. and T.J.S.; visualization, L.U.S. and T.J.S.; supervision, T.J.S.; project administration, T.J.S; funding acquisition, L.U.S. and T.J.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: L.U.S. is most grateful for financial support by Apothekerstiftung Westfalen-Lippe in the form of a doctoral fellowship. Thanks are due to J. Sendker and F. Jürgens (Münster) for support with the UHPLC/+ESI-QqTOF-MS/MS analyses, to J. Köhler (Münster) for NMR measurements and F. Petereit (Münster) for an introduction into CPC methodology to L.U.S. The authors are very grateful to G. Jerz, Technical University of Braunschweig, Germany, for valuable discussions. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Neves, J.M.; Matos, C.M.; Moutinho, C.; Queiroz, G.; Gomes, L.R. Ethnopharmacological Notes about Ancient Uses of Medicinal Plants in Trás-Os-Montes (northern of Portugal). J. Ethnopharmacol. 2009, 124, 270–283. [CrossRef][PubMed] 2. Leporatti, M.L.; Pavesi, A.; Posocco, E. Phytotherapy in the Valnerina Marche (Central Italy). J. Ethnopharmacol. 1985, 14, 53–63. [CrossRef] 3. World Health Organization. World-Malaria-Report-2019. Available online: https://www.who.int/ publications/i/item/9789241565721 (accessed on 13 August 2020). 4. Orhan, I.E.; Erdem, S.A.; Senol, F.S.; Kartal, M.; ¸Sener, B. Exploration of Cholinesterase and Tyrosinase Inhibitory, Antiprotozoal and Antioxidant Effects of Buxus Sempervirens L. (boxwood). Ind. Crop. Prod. 2012, 40, 116–121. [CrossRef] 5. Althaus, J.B.; Jerz, G.; Winterhalter, P.; Kaiser, M.; Brun, R.; Schmidt, T.J. Antiprotozoal Activity of Buxus Sempervirens and Activity-Guided Isolation of O-Tigloylcyclovirobuxeine-B As the Main Constituent Active Against Plasmodium Falciparum. Molecules 2014, 19, 6184–6201. [CrossRef][PubMed] 6. Cai, S.; Risinger, A.L.; Nair, S.; Peng, J.; Anderson, T.J.C.; Du, L.; Powell, D.R.; Mooberry, S.L.; Cichewicz, R.H. Identification of Compounds With Efficacy Against Malaria Parasites from Common North American Plants. J. Nat. Prod. 2015, 79, 490–498. [CrossRef][PubMed] 7. Oka, F.; Oka, H.; Ito, Y. Systematic Search for Suitable Two-Phase Solvent Systems for High-Speed Counter-Current Chromatography. J. Chromatogr. A 1991, 538, 99–108. [CrossRef] 8. Ito, Y. Golden Rules and Pitfalls in Selecting Optimum Conditions for High-Speed Counter-Current Chromatography. J. Chromatogr. A 2005, 1065, 145–168. [CrossRef][PubMed] 9. Marston, A.; Hostettmann, K. Counter-Current Chromatography As a Preparative Tool—Applications and Perspectives. J. Chromatogr. A 1994, 658, 315–341. [CrossRef] 10. Conway, W.D. Counter-Current Chromatography: Simple Process and Confusing Terminology. J. Chromatogr. A 2011, 121, 6015–6023. [CrossRef][PubMed] 11. Li, Z.; Zhou, Y.; Chen, F.; Zhang, L.; Yang, Y. Property Calculation and Prediction for Selecting Solvent Systems in CCC. J. Liq. Chromatogr. Relat. Technol. 2003, 26, 1397–1415. [CrossRef] 12. Khodzhaev, B.U.; Shakirov, R.; Aripov, K.N.; Shakirov, T.T.; Yunusov, S.Y. Polybuffer Distribution of the Combined Alkaloids Of Buxus Sempervirens. Chem. Nat. Compd. 1975, 11, 126. [CrossRef] Molecules 2020, 25, 4804 10 of 10

13. Althaus, J.B. Naturstoﬀe Mit Antiprotozoaler Wirkung: Alkamide ausgewählter Asteraceae Und Triterpenalkaloide Aus Buxus Sempervirens L. Ph.D. Thesis, Westfälische Wilhelms-Universität Münster, Münster, Germany, 2015. 14. Nnadi, C.; Althaus, J.B.; Nwodo, N.J.; Schmidt, T.J. A 3D-QSAR Study on the Antitrypanosomal and Cytotoxic Activities of Steroid Alkaloids by Comparative Molecular Field Analysis. Molecules 2018, 23, 1113. [CrossRef] [PubMed] 15. Kupchan, S.; Kennedy, R.; Schleigh, W.; Ohta, G. Buxus alkaloids—XII. Tetrahedron 1967, 23, 4563–4586. [CrossRef]

Sample Availability: Samples of compound 1 are available from the authors.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional aﬃliations.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).