bioactive molecules in kalanchoe pinnata leaves extraction, purification, and identification

ORIGINAL PAPER

Bioactive molecules in Kalanchoe pinnata leaves: extraction,purification, and identification

Saïda El Abdellaoui & Emilie Destandau & Alix Toribio & Claire Elfakir &

Michel Lafosse & Isabelle Renimel & Patrice André & Perrine Cancellieri &Ludovic Landemarre

Received: 23 May 2010 /Revised: 15 July 2010 /Accepted: 19 July 2010 /Published online: 18 August 2010# Springer-Verlag 2010

Abstract Kalanchoe pinnata (Lam.) Pers. (syn. Bryophyl-lum pinnatum; family Crassulaceae) is a popular plant usedin traditional medicine in many temperate regions of theworld and particularly in South America. In Guyana, theleaves are traditionally used as an anti-inflammatory andantiseptic to treat coughs, ulcers, and sores. The purpose ofthis study was to implement a method for targeting andidentifying molecules with antimicrobial activity, whichcould replace chemical preservatives in cosmetic appli-cations. The leaves were extracted by a method based onpressurized liquid extraction (PLE), using differentsolvents. A study of antimicrobial activity and cytotoxicitytests were performed to select the most interestingextract. To isolate one or more active molecules, theselected crude extract was fractionated by centrifugalpartition chromatography (CPC) and then antimicrobialactivity and cytotoxicity of each fraction were testedunder the same procedure. The last step consisted ofidentifying the main compounds in the most activefraction by LC-MS/MS.

Keywords Kalanchoe pinnata . Centrifugal partitionchromatography . LC/MS/MS . Antimicrobial activity .

Cytotoxicity

Introduction

Kalanchoe pinnata (Lam.) Pers. (syn. Bryophyllum pinna-tum; family Crassulaceae) is a popular plant that is used asa folk medicine in many temperate regions of the world andparticularly in South America. Juice of the fresh leaves isused very effectively for the treatment of jaundice in folkmedicines of the Bundelkhand region of India. In theGuianas, the leaves are traditionally used by the GuyanaPatamona tribe as an anti-inflammatory and antiseptic fortreating coughs, sores, wounds, and cuts. Previous studieshave also reported antiulcer [1] antileishmanial [2], hepato-protective [3], choleretic, antidiabetic, and antinociceptive[4] effects of leaf extracts.

The antimicrobial activity of leaves was also studiedby the agar-well diffusion method [5], but to date norelation between chemical structures and antimicrobial andcytotoxicity activities was proposed.

The purpose of this study was to implement a bioassayscreening of Kalanchoe pinnata from Guyana, in order todetermine the molecules responsible for the antimicrobialactivity, and which could be incorporated instead ofchemical preservatives in cosmetic applications. Severalsteps were necessary to accomplish this screening. First, theplant leaves were extracted by a method based onpressurized liquid extraction (PLE), using two differentsolvents: methanol and ethyl acetate. Antimicrobial andcytotoxicity activities were then evaluated to select the mostinteresting crude extract. To isolate one or several activemolecules, this selected extract was submitted to a fraction-

S. El Abdellaoui : E. Destandau (*) :A. Toribio : C. Elfakir :M. LafosseInstitut de Chimie Organique et Analytique,Université d’Orléans–CNRS, UMR CNRS 6005,BP 67059, 45067 Orléans Cedex 2, Francee-mail: [email protected]

I. Renimel : P. AndréDépartement Innovation Actifs,LVMH Recherche Parfums et Cosmétiques,45800 Saint Jean de Braye, France

P. Cancellieri : L. LandemarreGLYcoDIAG, Université d’Orléans,45067 Orléans Cedex 2, France

Anal Bioanal Chem (2010) 398:1329–1338DOI 10.1007/s00216-010-4047-3

ation step using centrifugal partition chromatography (CPC).Then, each fraction was tested for its antimicrobial andcytotoxicity activities with the same tests. Finally, the majorcompounds present in the most active fraction wereidentified by using HPLC/MS/MS.

Material and methods

Reagents and standards

Organic solvents, ethyl acetate, methanol, acetonitrile, andethanol used for extraction, CPC, and HPLC were ofanalytical grade from SDS Carlo Erba (Val-de-Reuil,France). Ultrapure water (resistance <18 MΩ) was providedby an Elgastat UHQ II apparatus (Elga, Antony, France).Gallic acid and coumaric acid were purchased from Fluka(Marseille, France). Caffeic acid, ferrulic acid, cinnamicacid, maltose, and glucose were from Sigma-Aldrich (SaintQuentin Fallavier, France). Fructose and raffinose werepurchased from Merck (Darmstadt, Germany).

Apparatus

Pressurized liquid extraction (PLE) was performed with anASE 100 system from Dionex (Voisins le Bretonneux,France) equipped with 34-mL stainless steel cells.

HPLC analyses were carried out with an Agilent HP 1100chromatographic system (Waldbronn, Germany) equippedwith a quaternary pump, a 20-μL sample loop, a UV detectorKontron (Zurich, Switzerland), and an evaporative lightscattering detector (ELSD) from Sedere (Alfortville, France)placed in series.

The columns were a reversed-phase C18 (Alltima, 5 μm,150×4.6 mm) from Interchim (Fontenay-sous-Bois,France) and an NH2 polymer (Astec Aphera™, 5 μm,125×4 mm) from Astec (Whippany, NJ, USA).

The preparative (CPC) instrument used in the present studywas a semipreparative FCPC from Kromaton (Angers,France), equipped with a 200-mL rotor. The variable flowsplitter (VFS) used to connect the CPC to the ELSD wasprovided by Rheodyne (Rohnert Park, CA, USA).

The mass spectrometer (MS) was a Quattro Ultima triplequadrupole equipped with a z-spray dual orthogonalelectrospray source from Waters. MassLynx1 4.1 softwarewas used to process the data. The microvalve T-splitter usedto connect the LC to the MS source was provided byUpchurch Scientific (Oak Harbor, USA).

Plant material

The leaves of K. pinnata (Lam.) Pers. were harvested fromthe natural site in the Kaw region of French Guyana, in

September 2006. The leaves were locally dried in the openair. After reception in France leaves were crushed in agrinder and then submitted to PLE.

Methodology for analysis

Extraction procedure

Using solvent at high temperature and pressure, PLE [6,7] accelerates extraction process and reduces the amountof solvent in comparison with conventional extractionmethods such as maceration or Soxhlet extraction. Differentextraction parameters were optimized as followed: tempera-ture of the extraction cell, 40 °C; static solvent extraction time,5 min; flushed volume, 60%; three static cycles; purge time,100 s. The temperature was limited to 40 °C to avoiddegradation of the molecules. Two different solvents weretested: methanol and ethyl acetate.

LC fingerprints of extracts and fractions

A global fingerprint analysis of the extracts and thefractions was performed by using an analytical reversed-phase liquid chromatography (RPLC) procedure withdouble detection: UV and evaporative light scatteringdetector (ELSD). The separations were achieved on a C18(Alltima, 5 μm, 150×4.6 mm) column, the UV was set at254 nm, and ELSD conditions were nebulizer gas pressure,2 bars; evaporative tube temperature, 50 °C; gain, 9. Awater/acetonitrile gradient was set as follows: 5 % ACNfrom 0 to 3 min, from 5 to 100% ACN from 3 to 18 min,and 100% ACN up to 30 min. Compounds were separatedat 1 mL min−1 at room temperature.

LC-MS and LC-MS/MS of active fractions

As the active fraction was polar, two orthogonal separationmethods were tested: HILIC and RPLC. For furtherstructural identification, a coupling of the LC system withMS spectrometry was necessary. The mass spectrometerneeds a volatile mobile phase when LC-MS coupling isinvestigated. This is also a requirement for ELSD; as aconsequence, LC methodology previously developed withELSD was directly compatible with MS detection.

HILIC-LC-MS To retain and separate the most polarcompounds eluted in the void volume under RPLCconditions, an HILIC method was developed using apolymeric NH2 (Astec, 5 μm, 4 mm×125 mm). Anisocratic elution was used for the separation. The mobilephase was composed of ACN/MeOH/aqueous solution of0.2% formic acid (85:5:10 v/v/v) at 1 mL min−1 and atroom temperature.

1330 S. El Abdellaoui et al.

Mass spectra data were acquired in negative ion mode,and a splitter was used to send only 0.2 mL min−1 to theESI source. The mass spectrometry conditions were basedfirst on literature [8] and then they were optimized byinfusion of the phenolic acid standards dissolved in amixture of MeOH/H2O (1:1, v/v) with 1% of acetic acid inorder to improve the molecule ionization yield. Measure-ments were therefore performed in negative mode at −4 kVcapillary voltage, −35 kV cone voltage, 80 °C desolva-tion temperature, 120 °C source temperature, andnitrogen gas was used for nebulization (148 L h−1) anddesolvation (61 L h−1).

RPLC-MS/MS Fraction analysis was then carried out usingan RP C18 column (Alltima, 5 μm, 150×4.6 mm). Twodifferent methods were used, one for the separation ofphenolic acids and one for the flavonoids.

For phenolic acids analysis, the method was optimizedfrom a standard mixture of five phenolic acids (gallic,caffeic, coumaric, ferrulic, and cinnamic acids); themobile phase consisted of aqueous solution of 1% aceticacid (solvent A) and methanol (solvent B) at a flow rateof 1 mL min−1 under gradient elution mode. The gradientprogram was close to the conditions described in ref. [9]:0–5 min, 5% B; 10–15 min 10% B; 20–25 min, 20% B;40–60 min, 40% B, at room temperature. For MSdetection, measurements were performed in ESI negativemode at −4 kV capillary voltage, −35 kV cone voltage,80 °C source temperature, 350 °C desolvation tempera-ture, and nitrogen gas was used for nebulization (88 L h−1)and desolvation (64 L h−1). For SRM experiments, argongas was used for the fragmentation in the collision cellwith an energy collision of 20 eV. The transition [M−H]−→ [M−CO2]

− was selected for gallic acid, caffeicacid, coumaric acid, and cinnamic acid and the transition[M−H]−→ [M−CH3−CO2]

− for ferrulic acid. Indeed, theproduct ion of ferrulic acid was due to the loss of bothcarbon dioxide and methyl group.

For the flavonoids LC separation, the mobile phaseconsisted of aqueous solution of 1% formic acid(solvent A) and methanol (solvent B) at a flow rate of1 mL min−1 under gradient elution mode. The followinggradient was used: 0–5 min, 5% B; 10–15 min, 20% B;20–30 min, 50% B; 40–45 min, 80% B; 55–65 min,100% B at room temperature. For MS detection,measurements were performed in ESI negative mode at−4 kV capillary voltage, −35 kV cone voltage, 120 °Csource temperature, 350 °C desolvation temperature, andnitrogen gas was used for nebulization (134 L h−1) anddesolvation (54 L h−1). For SRM argon gas and 30 eVwere used for fragmentation in the collision cell. Theseconditions were often employed for the identification offlavonoids [10–13].

CPC method

CPC, a separation method without solid stationary phasesupport, was chosen to fractionate the crude extract. Twoimmiscible liquids are used: the first is the stationary phaseand the second is the mobile phase. The stationary phase isretained inside the column thanks to a centrifugal field,while the mobile phase is pumped through the stationaryphase. The compounds are separated through the columnaccording to their respective partition coefficients definedas the concentration of solute in the upper phase on theconcentration of solute in the lower phase (KD=Cupper phase/Clower phase). The compounds that have a stronger affinityfor the stationary phase (KD>1) are retained inside thecolumn for a longer period of time, whereas those that havea stronger affinity for the mobile phase (KD<1) are elutedfaster out of the column. Many advantages characterize thistechnique such as no degradation of the isolated com-pounds on a solid support and the totality of the loadedextract can be recovered by pushing the liquid stationaryphase out of the column at the end of the run [14–18]. Theelution–extrusion procedure was used to extrude com-pounds with too high retention volume. The methodcomprised two steps: the first was a regular chromato-graphic elution process in descending or ascending mode.Next, the stationary phase containing the most hydrophobicsolutes was extruded out of the column continuously usingfresh liquid stationary phase pumped in the same mode asin the first step.

A biphasic solvent system composed of ethyl acetate/ethanol/water (4.5:1.5:4.5, v/v/v) was used for the K.pinnata leaf extract. After filling the column with the upperorganic stationary phase, the rotation speed was adjusted to1200 rpm and the mobile phase flow rate was 4 mL min−1.The stationary phase retention volume inside the columnwas 146 mL; the system was then stable with 73% retentionof the organic phase. The CPC stream was divided by aVFS. One part was directly transferred to ELSD, whereasthe second part was collected in test tubes. The VFSprinciple consists in using an active switching device thattransfers a small aliquot of the CPC stream at discretefrequencies into a separate and independent auxiliarystream directed to ELSD [19]; 100 nL of the CPC streamwere transferred at 1.667 Hz to an independent300 μL min−1 stream consisting of acetonitrile/water (1:1,v/v) via a liquid chromatography pump.

Approximately 350 mg of extract dissolved in 10 mL ofthe biphasic solvent system was loaded into the column.The lower aqueous mobile phase was then pumped at4 mL min−1 in the descending mode for 60 min. The wholecontents of CPC column were then extruded by pumpingthe organic phase in descending mode at 4 mL min−1 and300 rpm. The experiment ended after 120 min.

Bioactive molecules in Kalanchoe pinnata leaves 1331

Biological assays

Each extract was evaporated to dryness under N2 streamthen diluted in DMSO before testing its antimicrobialactivity and cytotoxicity.

Antimicrobial activity evaluation

The microorganisms used include all the strains recom-mended in the current regulatory method for cosmeticpreservative efficacy testing (NF T75-611): Pseudomonasaeruginosa ATCC 9027, Staphylococcus aureus ATCC6538, Escherichia coli ATCC 8739, Candida albicansATCC 10231, and Aspergillus niger ATCC 16404. Thesemicroorganisms were grown for 24–48 h at 35 °C ontryptone soya broth (AES Chemunex, ref AEB122869) forthe bacteria, YM broth (Difco, ref 268110) for Candidaalbicans, or Sabouraud broth (Difco, ref A3314D) forAspergillus niger, before preparation of a pure salineinoculum containing 104 (fungus), 105 (yeast), or 106

(bacteria) cfu mL−1.In order to evaluate the antimicrobial activity of a

large number of samples, a micromethod derived fromthe preservative efficacy testing described by Orth et al.[20] was developed. Prior to the contamination, eachsample was deposed at different concentrations in dupli-cate, in wells (2 mL) of a 96-deep-well microplate format.Concentrations were tested from 12 to 500 μg mL−1 forthe extracts and from 1.25 to 160 μg mL−1 for thefractions.

Each sample was then contaminated with 20 μL ofinoculum of each microorganism, the plates werecovered with adhesive breathable film, and incubated at28 °C.

Microbial counts: At each time (day 1, i.e., 24 h, days7, 14, 21, and 28), 20-μL samples were taken from thedeep-well microplates. Counting was performed with atriphenyltetrazolium chloride (TTC) micromethod for theyeast and bacteria and with the conventional agar platingmethod on Sabouraud gelose (AES Chemunex,AEB522209) for Aspergillus niger. The TTC micromethodwas performed as follow: In sterile 96-well (250 μL)microplates, 20 μL of each sample were tenfold seriallydiluted in 180 μL of Letheen broth (LB) (Difco, ref 268110)containing 1.5% Tween 80 (Sigma, ref P1754) and 0.001%TTC (Sigma, ref T8877) (six dilutions). For the strainCandida albicans, yeast mold (YM) broth (Difco, ref271120) was used instead of LB. The microplates wereincubated for 48 h at 28 °C and the microorganism growthwas monitored as color change from colorless to pink/red.The reciprocal highest dilution indicating growth allows thedetermination of the log number of each microorganism ateach time.

Cytotoxicity activity evaluation

The determination of cellular proliferation and cell viabilityare keys areas in a wide variety of cell biologicalapproaches. The assay is based on the cleavage of theyellow tetrazolium salt XTT to form an orange dye bymetabolically active cells (Cell Proliferation Kit II, Roche,ref 11.465.015.001). The formazan dye formed is soluble inaqueous solutions and is directly quantified by using ascanning multiwall spectrophotometer. Cells, grown in a96-well tissue culture plate, are incubated for 48 h in thepresence or absence of Kalanchoe extract at differentconcentrations (50 to 0.3125 μg mL−1 of medium). Afterthe incubation period, the treatment medium is removedand 100 μL of XTT labeling mixture is added to each well,to obtain a final concentration of 0.3 mg mL−1. Themicroplate is incubated for 3 h in a humidified atmosphere(37 °C, 5% CO2). After this incubation period, orangeformazan solution is formed. A decrease in number ofliving cells results in a decrease in the overall activitymitochondrial dehydrogenases in the sample. This decreaseis directly correlated to the amount of formazan formed andmonitored by the absorbance.

Results and discussion

Crude extract

Reversed-phase HPLC analysis of the MeOH and AcOEt

The extraction yield of Kalanchoe leaves was differentaccording to the solvent used. It was about 8% (w/w) withMeOH and 3% (w/w) with ethyl acetate.

The chromatographic analyses of the methanolic crudeextract and ethyl acetate crude extract were performedunder RPLC conditions following the procedure describedin “LC fingerprints of extracts and fractions”. Two differentfingerprints (Fig. 1) were obtained with ELSD detection.The methanolic extract (Fig. 1a) was richer in contents thanthe ethyl acetate one (Fig. 1b) which confirmed thedifference observed with the extraction yields. Both extractscontained compounds eluted around 12 min, but in veryweak proportion in the ethyl acetate one. The methanolicextract presented other polar compounds eluted in the voidvolume.

Antimicrobial activity evaluation

The methanol and ethyl acetate extracts diluted at fiveconcentrations ranging from 12 to 500 μg mL−1, but also aliquid preservative named Phenonip 0.5% used as standardreference, were contaminated with each of the five


microbial strains and their antimicrobial activity wasmonitored over time (Fig. 2) as described in “Antimicrobialactivity evaluation”. At the highest concentration, themethanol extract display a significant antimicrobial activity.Indeed, each fungus or bacterial population decreases fromone to six log units in 7 days in the presence of themethanolic extract, whereas no decrease was observed withthe ethyl acetate extract in the same period.

Cytotoxicity evaluation

As the methanolic extract displayed a significant antimicro-bial activity, its cytotoxicity was then tested. Kalanchoeextract had been tested on keratinocytes for 48 h from 50 to

0.19 μg/mL according to the methodology reported in“Cytotoxicity activity evaluation”. The basal activity wascalculated versus medium containing DMSO, i.e., thekalanchoe extract solvent. Generally, an extract is consideredas noncytotoxic if it can be used at concentrations from 50 to

Fig. 1 RPLC-ELSD chromatograms of Kalanchoe pinnata leafextracts. Alltima C18 (150 mm×4.6 mm, 5 μm) column, roomtemperature, mobile phase water/acetonitrile in gradient elution, flowrate 1 mL min−1, ELSD: nebulizer gas pressure, 2 bars; evaporative tubetemperature, 50 °C, gain 9. a Methanolic extract; b ethyl acetate extract

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Fig. 2 Antimicrobial activity of methanolic (red) and ethyl acetate(green) extracts of Kalanchoe pinnata leaves. Residual population ofa Candida albicans; b Escherichia coli; c Staphylococcus aureus; dPseudomonas aeruginosa; and e Aspergillus niger is measured at eachtime (days 1, 7, 14, 21, and 28) ) after initial contamination


12.5 μg mL−1. Kalanchoe methanolic extract had a strongcytotoxicity activity as the viability of keratinocytes is totallyrecovered only for the weakest extract concentrations(≤0.78 μg mL−1) (Fig. 3).

Other Kalanchoe species were tested and the same resultwas obtained. It can be supposed that the cytotoxicityactivity is the consequence of one or more components ofthe Kalanchoe genus.

A fractionation of the methanolic crude extract was theninvestigated in order to isolate and purify the activemolecules.

Isolation of active fraction

CPC fractionation

The first step in CPC separation is selection of the biphasicsolvent system and determination of the partition coeffi-cients (KD=Cupper phase/Clower phase) of the analytes. Underoptimal conditions, where KD≈1, the solutes are partitionedequally between the two phases and a satisfactory separa-tion may occur. Crude extract (1 mg) was dissolved in abiphasic system (2 mL) in a test tube and the two phaseswere analyzed by RPLC-ELSD. The partition coefficientwas calculated as the ratio of the major solute peak area ineach phase. The various tested biphasic systems usedsolvents such as heptane, methanol, ethanol, ethyl acetate,acetonitrile, and water in different proportions. Among allthese systems, the one that gave the best results was ethylacetate/ethanol/water (4.5:1.5:4.5). Under these conditions,solutes eluted around 12 min corresponded to a KD valueclose to 1, which resulted in a reasonable CPC analysistime. The separation was performed in descending modewith organic stationary phase and aqueous mobile phase.Figure 4 depicts the corresponding CPC-ELSD chromato-

gram. Three different peaks can be observed, well separatedwith a total baseline resolution. Thus, the different collectedtest tubes were regrouped in three fractions (denoted Fr1,Fr2, and Fr3) corresponding to these three peaks. Fr1 andFr2 are eluted in the aqueous mobile phase and containedrespectively polar and intermediate polar compounds. Fr3collected after extrusion step (after 60 min of elution)corresponded to the more apolar compounds which weretotally retained in the organic stationary phase.

Antimicrobial susceptibility testing

The three fractions obtained from the methanolic crudeextract were diluted at five concentrations ranging from 10to 160 μg mL−1. After contamination with each of the fivemicrobial strains, the antimicrobial activity was monitoredover time. A decrease of the microbial population wasrecorded only at the highest concentration (160 μg mL−1).At this concentration, the best antimicrobial activity wasfound for fraction Fr1 as it allows the decrease of four outof the five strains. Only the Aspergillus niger strain was notaffected by the antimicrobial activity of the fraction Fr1 atthis concentration. Fractions Fr2 and Fr3 are consideredinactive as they induced no decrease of Escherichia coli,Pseudomonas aeruginosa, and Aspergillus niger even at160 μg mL−1.

Cytotoxicity evaluation

The three fractions were tested on the cytotoxicity modeland compared to the crude extract. All fractions werecytotoxic on our model. Fraction Fr3 showed a severe

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Fig. 3 Cytotoxicity evaluation of Kalanchoe pinnata methanolicextract. Viability of keratinocytes according to the methanolic extractconcentration

Fig. 4 CPC-ELSD chromatogram obtained for the fractionation of theKalanchoe pinnata methanolic extract. Three fractions were collected:Fr.1, Fr.2, and Fr.3


cytotoxic activity and produced a decrease of the keratino-cytes population even with the lowest concentration(0.19 μg mL−1) tested. For fractions Fr1 and Fr2 the viabilitybegan to decrease respectively at only 1.56 μg mL−1 and0.78 μg mL−1. Therefore Fr1 was the less cytotoxicfraction.

According to the antimicrobial activities and the cyto-toxicity of the different fractions, fraction Fr1 was the mostinteresting and, thus, it was analyzed by LC-MS/MS to getstructural information on its bioactive molecules.

Structural identification

The active fraction Fr1 contained polar compounds andthe most polar of them were eluted in the void volumeunder RPLC conditions. The HILIC mode was first

considered in order to retain and separate them. In HILIC,a hydrophilic stationary phase and an aqueous-organicphase with high organic solvent content (at least 60%) areused. The retention is mainly caused by partition of theanalyte between water-enriched layer immobilized at thestationary phase surface and the relatively hydrophobicmobile phase.

HILIC-LC-MS

The preliminary development of the HILIC chromatograph-ic system was carried out with a double UV/ELSDdetection. Figure 5a shows the ELSD chromatogram andFig. 5b the LC-UVone at 280 nm obtained under optimizedHILIC conditions. Seven peaks could be detected by ELSD(Fig. 5a) in addition to the compounds eluted in voidvolume, whereas only three peaks (1, 2, and 5) weredetected by UV (Fig. 5b). Solutes eluted in peaks 3, 4, 6,and 7 could be aliphatic compounds without chromophoregroups, such as sugars, while solutes 1, 2, and 5 couldbelong to the phenolic acid family as described in literature[7]. To identify the compounds eluted in the seven peaks, acoupling to a mass spectrometer was investigated. First, themass spectrometry conditions in the ESI source wereoptimized by direct infusion of sugar and phenolic acidstandard solutions. Negative mode gave better results thanpositive mode. As described in Table 1, the Kalanchoeleaves contained fructose, glucose, and maltose; thesesugars were identified thanks to their formate adduct ions[M−HCOO]− and their retention time in comparison withcorresponding sugar standards. Unfortunately under HILIC-LC-MS conditions, no more precise identification ofphenolic acids was obtained.

RPLC-UV/MS/MS

To identify solutes less polar than sugars, the fraction Fr1was analyzed under RPLC conditions. A mixture of fivephenolic acids (caffeic acid, ferrulic acid, gallic acid,coumaric acid, and cinnamic acid) was first analyzed by

Peak number Retention time (min) λ max (nm) m/z (amu) Tentative identification

1 2.93 229/276 297 Phenolic derivative

2 3.30 225/278 330 Phenolic derivative

3 4.75 No UV absorbance 197 Unknown

4 5.32 No UV absorbance 225* Fructose

5 7.51 288 255 Phenolic derivative

6 7.81 No UV absorbance 225* Glucose

7 17.5 No UV absorbance 387* Maltose

Table 1 Compounds identifiedin Kalanchoe pinnata leaves byHILIC-LC-MS

*m/z values corresponding to[M+HCOOH]−

Fig. 5 HILIC analysis of fraction Fr1: Astec polymeric NH2 (150×4.6 mm, 5 μm) column, room temperature, mobile phase ACN/MeOH/H2O (85:5:10) in isocratic elution, flow rate 1 mL min−1. aELSD: nebulizer gas pressure, 2 bars; evaporative tube temperature,50 °C, gain 9. b UV λ=280 nm


RPLC-MS/MS using the SRM mode as described in“RPLC-MS/MS”. For each solute, a specific transitionwas selected; then, these five transitions were followedduring the Fr1 analysis. Three phenolic acids were

detected: gallic, caffeic, and coumaric acids in fractionFr1. Figure 6 reports the corresponding extracted ioncurrents (XICs).

The presence of other kinds of molecules such asflavonoids in Kalanchoe pinnata leaves [2] was described,so an optimization of RPLC-UV and MS/MS conditionswas carried out as described in “RPLC-MS/MS”. Sevenmain compounds were detected by LC-UV (Fig. 7) andtheir identification by MS/MS revealed that they belong tothe family of flavonol glycosides (compounds 1–7 inTable 2). By MS/MS another compound was detected at33.6 min and identified as a flavanol oligomer withoutchromophore group absorbing at 366 nm (compound 8 inTable 2). The flavonol glycosides were derivates ofquercetin, isorhamnetin, and kaempferol as described inTable 2. Components 1, 2, and 7 presented a fragment ionat m/z=301 which is typical of quercetin aglycon. Forcompounds 4 and 6, the fragmentation of their pseudomo-lecular ion led to a fragment ion at m/z=315 which ischaracteristic of isorhamnetin aglycon. Compounds 3 and 5presented a fragment at m/z=285 which is characteristic ofkaempferol aglycon.

The MS/MS fragmentation also allowed us to identifythe sugar moiety of the flavonoids. For instance, thefragmentation of compound 1 mainly gave an ion at m/z=463 due to the loss of 146 from the pseudomolecular ion atm/z=609 which indicated the presence of a rhamnose moiety[M−H−146]−. The ion at m/z=301 was due to the loss of162 from the ion at m/z=463 which indicated the presence ofa glucose moiety [M−H−146−162]−. This fragmentationpathway suggested that the two sugars were linked to twodifferent –OH positions in the phenyl ring. In the same way,

Fig. 7 RPLC-UV λ=366 nm analysis of fraction Fr1. Alltima C18(150×4.6 mm, 5 μm) column, room temperature, mobile phaseMeOH/H2O containing 1% formic acid, flow rate 1 mL min−1

Fig. 6 RPLC-MS/MS analysis of fraction Fr1 using SRM mode.XICs of a m/z 169→125; b m/z 179→135; c m/z 163→119. AlltimaC18 (150×4.6 mm, 5 μm) column, room temperature, mobile phaseMeOH/H2O containing 1% acetic acid, flow rate 1 mL min−1


for compound 2, the pseudomolecular ion first lost a hexose,then a pentose, and a deoxyhexose [M−H−162−133−146]−, which led to the ion at m/z=301 [13]. Thepseudomolecular ion at m/z=593 of compound 3 gave afragment ion m/z=447 by loss of 146 which indicated thepresence of a rhamnose moiety [M−H−146]− and thefragment ion at m/z=285 due to the loss of 162 from theion at m/z=447 indicated the presence of a glucose moiety[M−H−146−162]−. For compound 4, the ion at m/z=315was probably due to the loss of a hexose and a pentose[M−H−162−133]−. The fragmentation of compound 5caused the loss of 146 and 133 corresponding respectivelyto a deoxyhexose and a pentose moiety [M−H−146−133]−. As described in the literature [2], compound 6 is aquercetin diglycosyl and its fragmentation resulted in anion at m/z=301 due to the loss of 146 and 133, whichindicated the presence of rhamnose and arabinose moieties.The last compound corresponded probably to a flavan-3-olderivative according to the literature [21], and its fragmenta-tion led to an ion at m/z=289 which corresponded tocatechin.

Conclusion

This paper presents a methodology to identify molecules withantimicrobial activity from plant extracts. The differenttechniques of extraction (PLE), purification (CPC), structuralidentification (LC-MS/MS), and biological tests can be usedas a bioguided screening for many plants. Our study allowedus to obtain an active fraction of Kalanchoe pinnata leaves,with low cytotoxicity, which could be advantageously addedin cosmetic formulations to decrease the concentration of

chemical preservatives. This study identified compoundsalready reported in the literature but also new ones.

Acknowledgement This project was supported by the Frenchcompetitiveness cluster “Cosmétique, Sciences de la Beauté et duBien être”. The authors thank the Conseil Régional du Centre(France), the Conseil Général du Loiret (France), the Communautéd’agglomération d’Orléans and the Ville d’Orléans for their financialsupport. The authors acknowledge Jean-Marc Seigneuret and EmilieDufour (Adonis, Alban Muller, France) for their help and providingthe Kalanchoe pinnata leaves and, Marie-Pierre Papet for heradvice.

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Table 2 Identification of flavonoids contained in Kalanchoe pinnata leaves by RPLC-MS/MS

Peak number Retention time (min) Parent ions (m/z) Fragment ions (m/z) Tentative identification Ref.

[M−H]− [M+HCOOH]−

1 24.6 609 656 301/463 Quercetin glucose rhamnose [12]

2 24.8 742 – 580/301/447/259 Quercetin glucose arabinose rhamnose –

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–

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[2]

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bioactive molecules in kalanchoe pinnata leaves extraction, purification, and identification

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