Anal Bioanal Chem (2010) 398:13291338 DOI 10.

1007/s00216-010-4047-3

ORIGINAL PAPER

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

Sada El Abdellaoui & Emilie Destandau & Alix Toribio & Claire Elfakir & Michel Lafosse & Isabelle Renimel & Patrice Andr & 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. Bryophyllum pinnatum; family Crassulaceae) is a popular plant used in traditional medicine in many temperate regions of the world and particularly in South America. In Guyana, the leaves are traditionally used as an anti-inflammatory and antiseptic to treat coughs, ulcers, and sores. The purpose of this study was to implement a method for targeting and identifying molecules with antimicrobial activity, which could replace chemical preservatives in cosmetic applications. The leaves were extracted by a method based on pressurized liquid extraction (PLE), using different solvents. A study of antimicrobial activity and cytotoxicity tests were performed to select the most interesting extract. To isolate one or more active molecules, the selected crude extract was fractionated by centrifugal partition chromatography (CPC) and then antimicrobial activity and cytotoxicity of each fraction were tested under the same procedure. The last step consisted of identifying the main compounds in the most active fraction by LC-MS/MS.

Keywords Kalanchoe pinnata . Centrifugal partition chromatography . LC/MS/MS . Antimicrobial activity . Cytotoxicity

Introduction Kalanchoe pinnata (Lam.) Pers. (syn. Bryophyllum pinnatum; family Crassulaceae) is a popular plant that is used as a folk medicine in many temperate regions of the world and particularly in South America. Juice of the fresh leaves is used very effectively for the treatment of jaundice in folk medicines of the Bundelkhand region of India. In the Guianas, the leaves are traditionally used by the Guyana Patamona tribe as an anti-inflammatory and antiseptic for treating coughs, sores, wounds, and cuts. Previous studies have also reported antiulcer [1] antileishmanial [2], hepatoprotective [3], choleretic, antidiabetic, and antinociceptive [4] effects of leaf extracts. The antimicrobial activity of leaves was also studied by the agar-well diffusion method [5], but to date no relation between chemical structures and antimicrobial and cytotoxicity activities was proposed. The purpose of this study was to implement a bioassay screening of Kalanchoe pinnata from Guyana, in order to determine the molecules responsible for the antimicrobial activity, and which could be incorporated instead of chemical preservatives in cosmetic applications. Several steps were necessary to accomplish this screening. First, the plant leaves were extracted by a method based on pressurized liquid extraction (PLE), using two different solvents: methanol and ethyl acetate. Antimicrobial and cytotoxicity activities were then evaluated to select the most interesting crude extract. To isolate one or several active molecules, this selected extract was submitted to a fraction-

S. El Abdellaoui : E. Destandau (*) : A. Toribio : C. Elfakir : M. Lafosse Institut de Chimie Organique et Analytique, Universit dOrlansCNRS, UMR CNRS 6005, BP 67059, 45067 Orlans Cedex 2, France e-mail: emilie.destandau@univ-orleans.fr I. Renimel : P. Andr Dpartement Innovation Actifs, LVMH Recherche Parfums et Cosmtiques, 45800 Saint Jean de Braye, France P. Cancellieri : L. Landemarre GLYcoDIAG, Universit dOrlans, 45067 Orlans Cedex 2, France

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ation step using centrifugal partition chromatography (CPC). Then, each fraction was tested for its antimicrobial and cytotoxicity activities with the same tests. Finally, the major compounds present in the most active fraction were identified by using HPLC/MS/MS.

September 2006. The leaves were locally dried in the open air. After reception in France leaves were crushed in a grinder and then submitted to PLE. Methodology for analysis Extraction procedure

Material and methods Reagents and standards Organic solvents, ethyl acetate, methanol, acetonitrile, and ethanol used for extraction, CPC, and HPLC were of analytical grade from SDS Carlo Erba (Val-de-Reuil, France). Ultrapure water (resistance <18 M) was provided by an Elgastat UHQ II apparatus (Elga, Antony, France). Gallic acid and coumaric acid were purchased from Fluka (Marseille, France). Caffeic acid, ferrulic acid, cinnamic acid, maltose, and glucose were from Sigma-Aldrich (Saint Quentin Fallavier, France). Fructose and raffinose were purchased from Merck (Darmstadt, Germany). Apparatus Pressurized liquid extraction (PLE) was performed with an ASE 100 system from Dionex (Voisins le Bretonneux, France) equipped with 34-mL stainless steel cells. HPLC analyses were carried out with an Agilent HP 1100 chromatographic system (Waldbronn, Germany) equipped with a quaternary pump, a 20-L sample loop, a UV detector Kontron (Zurich, Switzerland), and an evaporative light scattering detector (ELSD) from Sedere (Alfortville, France) placed in series. The columns were a reversed-phase C18 (Alltima, 5 m, 150 4.6 mm) from Interchim (Fontenay-sous-Bois, France) and an NH2 polymer (Astec Aphera, 5 m, 1254 mm) from Astec (Whippany, NJ, USA). The preparative (CPC) instrument used in the present study was a semipreparative FCPC from Kromaton (Angers, France), equipped with a 200-mL rotor. The variable flow splitter (VFS) used to connect the CPC to the ELSD was provided by Rheodyne (Rohnert Park, CA, USA). The mass spectrometer (MS) was a Quattro Ultima triple quadrupole equipped with a z-spray dual orthogonal electrospray source from Waters. MassLynx1 4.1 software was used to process the data. The microvalve T-splitter used to connect the LC to the MS source was provided by Upchurch Scientific (Oak Harbor, USA). Plant material The leaves of K. pinnata (Lam.) Pers. were harvested from the natural site in the Kaw region of French Guyana, in Using solvent at high temperature and pressure, PLE [6, 7] accelerates extraction process and reduces the amount of solvent in comparison with conventional extraction methods such as maceration or Soxhlet extraction. Different extraction parameters were optimized as followed: temperature 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 avoid degradation of the molecules. Two different solvents were tested: methanol and ethyl acetate. LC fingerprints of extracts and fractions A global fingerprint analysis of the extracts and the fractions was performed by using an analytical reversedphase liquid chromatography (RPLC) procedure with double detection: UV and evaporative light scattering detector (ELSD). The separations were achieved on a C18 (Alltima, 5 m, 1504.6 mm) column, the UV was set at 254 nm, and ELSD conditions were nebulizer gas pressure, 2 bars; evaporative tube temperature, 50 C; gain, 9. A water/acetonitrile gradient was set as follows: 5 % ACN from 0 to 3 min, from 5 to 100% ACN from 3 to 18 min, and 100% ACN up to 30 min. Compounds were separated at 1 mL min1 at room temperature. LC-MS and LC-MS/MS of active fractions As the active fraction was polar, two orthogonal separation methods were tested: HILIC and RPLC. For further structural identification, a coupling of the LC system with MS spectrometry was necessary. The mass spectrometer needs a volatile mobile phase when LC-MS coupling is investigated. This is also a requirement for ELSD; as a consequence, LC methodology previously developed with ELSD was directly compatible with MS detection. HILIC-LC-MS To retain and separate the most polar compounds eluted in the void volume under RPLC conditions, an HILIC method was developed using a polymeric NH2 (Astec, 5 m, 4 mm125 mm). An isocratic elution was used for the separation. The mobile phase was composed of ACN/MeOH/aqueous solution of 0.2% formic acid (85:5:10 v/v/v) at 1 mL min1 and at room temperature.

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Mass spectra data were acquired in negative ion mode, and a splitter was used to send only 0.2 mL min1 to the ESI source. The mass spectrometry conditions were based first on literature [8] and then they were optimized by infusion of the phenolic acid standards dissolved in a mixture of MeOH/H2O (1:1, v/v) with 1% of acetic acid in order to improve the molecule ionization yield. Measurements were therefore performed in negative mode at 4 kV capillary voltage, 35 kV cone voltage, 80 C desolvation temperature, 120 C source temperature, and nitrogen gas was used for nebulization (148 L h1) and desolvation (61 L h1). RPLC-MS/MS Fraction analysis was then carried out using an RP C18 column (Alltima, 5 m, 1504.6 mm). Two different methods were used, one for the separation of phenolic acids and one for the flavonoids. For phenolic acids analysis, the method was optimized from a standard mixture of five phenolic acids (gallic, caffeic, coumaric, ferrulic, and cinnamic acids); the mobile phase consisted of aqueous solution of 1% acetic acid (solvent A) and methanol (solvent B) at a flow rate of 1 mL min1 under gradient elution mode. The gradient program was close to the conditions described in ref. [9]: 05 min, 5% B; 1015 min 10% B; 2025 min, 20% B; 40 60 min, 40% B, at room temperature. For MS detection, measurements were performed in ESI negative mode at 4 kV capillary voltage, 35 kV cone voltage, 80 C source temperature, 350 C desolvation temperature, and nitrogen gas was used for nebulization (88 L h1) and desolvation (64 L h1). For SRM experiments, argon gas was used for the fragmentation in the collision cell with an energy collision of 20 eV. The transition [M H] [M CO2] was selected for gallic acid, caffeic acid, coumaric acid, and cinnamic acid and the transition [M H] [M CH3 CO2] for ferrulic acid. Indeed, the product ion of ferrulic acid was due to the loss of both carbon dioxide and methyl group. For the flavonoids LC separation, the mobile phase consisted of aqueous solution of 1% formic acid (solvent A) and methanol (solvent B) at a flow rate of 1 mL min1 under gradient elution mode. The following gradient was used: 05 min, 5% B; 1015 min, 20% B; 2030 min, 50% B; 4045 min, 80% B; 5565 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 C source temperature, 350 C desolvation temperature, and nitrogen gas was used for nebulization (134 L h1) and desolvation (54 L h1). For SRM argon gas and 30 eV were used for fragmentation in the collision cell. These conditions were often employed for the identification of flavonoids [1013].

CPC method CPC, a separation method without solid stationary phase support, was chosen to fractionate the crude extract. Two immiscible liquids are used: the first is the stationary phase and the second is the mobile phase. The stationary phase is retained inside the column thanks to a centrifugal field, while the mobile phase is pumped through the stationary phase. The compounds are separated through the column according to their respective partition coefficients defined as the concentration of solute in the upper phase on the concentration of solute in the lower phase (KD = Cupper phase/ Clower phase). The compounds that have a stronger affinity for the stationary phase (KD >1) are retained inside the column for a longer period of time, whereas those that have a stronger affinity for the mobile phase (KD <1) are eluted faster out of the column. Many advantages characterize this technique such as no degradation of the isolated compounds on a solid support and the totality of the loaded extract can be recovered by pushing the liquid stationary phase out of the column at the end of the run [1418]. The elutionextrusion procedure was used to extrude compounds with too high retention volume. The method comprised two steps: the first was a regular chromatographic elution process in descending or ascending mode. Next, the stationary phase containing the most hydrophobic solutes was extruded out of the column continuously using fresh liquid stationary phase pumped in the same mode as in 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 upper organic stationary phase, the rotation speed was adjusted to 1200 rpm and the mobile phase flow rate was 4 mL min1. The stationary phase retention volume inside the column was 146 mL; the system was then stable with 73% retention of the organic phase. The CPC stream was divided by a VFS. One part was directly transferred to ELSD, whereas the second part was collected in test tubes. The VFS principle consists in using an active switching device that transfers a small aliquot of the CPC stream at discrete frequencies into a separate and independent auxiliary stream directed to ELSD [19]; 100 nL of the CPC stream were transferred at 1.667 Hz to an independent 300 L min1 stream consisting of acetonitrile/water (1:1, v/v) via a liquid chromatography pump. Approximately 350 mg of extract dissolved in 10 mL of the biphasic solvent system was loaded into the column. The lower aqueous mobile phase was then pumped at 4 mL min1 in the descending mode for 60 min. The whole contents of CPC column were then extruded by pumping the organic phase in descending mode at 4 mL min1 and 300 rpm. The experiment ended after 120 min.

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Biological assays Each extract was evaporated to dryness under N2 stream then diluted in DMSO before testing its antimicrobial activity and cytotoxicity. Antimicrobial activity evaluation The microorganisms used include all the strains recommended in the current regulatory method for cosmetic preservative efficacy testing (NF T75-611): Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Candida albicans ATCC 10231, and Aspergillus niger ATCC 16404. These microorganisms were grown for 2448 h at 35 C on tryptone soya broth (AES Chemunex, ref AEB122869) for the bacteria, YM broth (Difco, ref 268110) for Candida albicans, or Sabouraud broth (Difco, ref A3314D) for Aspergillus niger, before preparation of a pure saline inoculum containing 104 (fungus), 105 (yeast), or 106 (bacteria) cfu mL1. In order to evaluate the antimicrobial activity of a large number of samples, a micromethod derived from the preservative efficacy testing described by Orth et al. [20] was developed. Prior to the contamination, each sample was deposed at different concentrations in duplicate, in wells (2 mL) of a 96-deep-well microplate format. Concentrations were tested from 12 to 500 g mL1 for the extracts and from 1.25 to 160 g mL1 for the fractions. Each sample was then contaminated with 20 L of inoculum of each microorganism, the plates were covered with adhesive breathable film, and incubated at 28 C. Microbial counts: At each time (day 1, i.e., 24 h, days 7, 14, 21, and 28), 20-L samples were taken from the deep-well microplates. Counting was performed with a triphenyltetrazolium chloride (TTC) micromethod for the yeast and bacteria and with the conventional agar plating method on Sabouraud gelose (AES Chemunex, AEB522209) for Aspergillus niger. The TTC micromethod was performed as follow: In sterile 96-well (250 L) microplates, 20 L of each sample were tenfold serially diluted in 180 L 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 strain Candida albicans, yeast mold (YM) broth (Difco, ref 271120) was used instead of LB. The microplates were incubated for 48 h at 28 C and the microorganism growth was monitored as color change from colorless to pink/red. The reciprocal highest dilution indicating growth allows the determination of the log number of each microorganism at each time.

Cytotoxicity activity evaluation The determination of cellular proliferation and cell viability are keys areas in a wide variety of cell biological approaches. The assay is based on the cleavage of the yellow tetrazolium salt XTT to form an orange dye by metabolically active cells (Cell Proliferation Kit II, Roche, ref 11.465.015.001). The formazan dye formed is soluble in aqueous solutions and is directly quantified by using a scanning multiwall spectrophotometer. Cells, grown in a 96-well tissue culture plate, are incubated for 48 h in the presence or absence of Kalanchoe extract at different concentrations (50 to 0.3125 g mL1 of medium). After the incubation period, the treatment medium is removed and 100 L of XTT labeling mixture is added to each well, to obtain a final concentration of 0.3 mg mL1. The microplate is incubated for 3 h in a humidified atmosphere (37 C, 5% CO2). After this incubation period, orange formazan solution is formed. A decrease in number of living cells results in a decrease in the overall activity mitochondrial dehydrogenases in the sample. This decrease is directly correlated to the amount of formazan formed and monitored by the absorbance.

Results and discussion Crude extract Reversed-phase HPLC analysis of the MeOH and AcOEt The extraction yield of Kalanchoe leaves was different according to the solvent used. It was about 8% (w/w) with MeOH and 3% (w/w) with ethyl acetate. The chromatographic analyses of the methanolic crude extract and ethyl acetate crude extract were performed under RPLC conditions following the procedure described in LC fingerprints of extracts and fractions. Two different fingerprints (Fig. 1) were obtained with ELSD detection. The methanolic extract (Fig. 1a) was richer in contents than the ethyl acetate one (Fig. 1b) which confirmed the difference observed with the extraction yields. Both extracts contained compounds eluted around 12 min, but in very weak proportion in the ethyl acetate one. The methanolic extract presented other polar compounds eluted in the void volume. Antimicrobial activity evaluation The methanol and ethyl acetate extracts diluted at five concentrations ranging from 12 to 500 g mL1, but also a liquid preservative named Phenonip 0.5% used as standard reference, were contaminated with each of the five

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0.19 g/mL according to the methodology reported in Cytotoxicity activity evaluation. The basal activity was calculated versus medium containing DMSO, i.e., the kalanchoe extract solvent. Generally, an extract is considered as noncytotoxic if it can be used at concentrations from 50 to

a
Log (number of microorganisms)

8 6 4 2 0 0 10 20 30
methanol extract 500g/ml Ethyl acetate extract 500g/ml Phenonip 0,5%

b
Log (number of microorganisms)

Time (days)
8 6 4 2 0 0 10 20 30
methanol extract 500g/ml Ethyl acetate extract 500g/ml Phenonip 0,5%

c
Log (number of microorganisms)

Time (days)
8 6 4 2 0 0 10 20 30
methanol extract 500g/ml Ethyl acetate extract 500g/ml Phenonip 0,5%

d
Log (number of microorganisms)

Time (days)
8 6 4 2 0
methanol extract 500g/ml Ethyl acetate extract 500g/ml Phenonip 0,5%

Fig. 1 RPLC-ELSD chromatograms of Kalanchoe pinnata leaf extracts. Alltima C18 (150 mm4.6 mm, 5 m) column, room temperature, mobile phase water/acetonitrile in gradient elution, flow rate 1 mL min1, ELSD: nebulizer gas pressure, 2 bars; evaporative tube temperature, 50 C, gain 9. a Methanolic extract; b ethyl acetate extract

microbial strains and their antimicrobial activity was monitored over time (Fig. 2) as described in Antimicrobial activity evaluation. At the highest concentration, the methanol extract display a significant antimicrobial activity. Indeed, each fungus or bacterial population decreases from one to six log units in 7 days in the presence of the methanolic extract, whereas no decrease was observed with the ethyl acetate extract in the same period. Cytotoxicity evaluation As the methanolic extract displayed a significant antimicrobial activity, its cytotoxicity was then tested. Kalanchoe extract had been tested on keratinocytes for 48 h from 50 to

10

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e
Log (number of microorganisms)

Time (days)
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methanol extract 500g/ml Ethyl acetate extract 500g/ml Phenonip 0,5%

0 0 10

Time (days)

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30

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

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80 60 40 20 0 TDMSO 50 25 12.5 6.25 3.125 1.56 Concentration g/mL 0.78 0.39 0.19

gram. Three different peaks can be observed, well separated with a total baseline resolution. Thus, the different collected test tubes were regrouped in three fractions (denoted Fr1, Fr2, and Fr3) corresponding to these three peaks. Fr1 and Fr2 are eluted in the aqueous mobile phase and contained respectively polar and intermediate polar compounds. Fr3 collected after extrusion step (after 60 min of elution) corresponded to the more apolar compounds which were totally retained in the organic stationary phase. Antimicrobial susceptibility testing The three fractions obtained from the methanolic crude extract were diluted at five concentrations ranging from 10 to 160 g mL1. After contamination with each of the five microbial strains, the antimicrobial activity was monitored over time. A decrease of the microbial population was recorded only at the highest concentration (160 g mL1). At this concentration, the best antimicrobial activity was found for fraction Fr1 as it allows the decrease of four out of the five strains. Only the Aspergillus niger strain was not affected by the antimicrobial activity of the fraction Fr1 at this concentration. Fractions Fr2 and Fr3 are considered inactive as they induced no decrease of Escherichia coli, Pseudomonas aeruginosa, and Aspergillus niger even at 160 g mL1. Cytotoxicity evaluation The three fractions were tested on the cytotoxicity model and compared to the crude extract. All fractions were cytotoxic on our model. Fraction Fr3 showed a severe

Fig. 3 Cytotoxicity evaluation of Kalanchoe pinnata methanolic extract. Viability of keratinocytes according to the methanolic extract concentration

12.5 g mL1. Kalanchoe methanolic extract had a strong cytotoxicity activity as the viability of keratinocytes is totally recovered only for the weakest extract concentrations (0.78 g mL1) (Fig. 3). Other Kalanchoe species were tested and the same result was obtained. It can be supposed that the cytotoxicity activity is the consequence of one or more components of the Kalanchoe genus. A fractionation of the methanolic crude extract was then investigated in order to isolate and purify the active molecules. Isolation of active fraction CPC fractionation The first step in CPC separation is selection of the biphasic solvent system and determination of the partition coefficients (KD = Cupper phase/Clower phase) of the analytes. Under optimal conditions, where KD 1, the solutes are partitioned equally between the two phases and a satisfactory separation may occur. Crude extract (1 mg) was dissolved in a biphasic system (2 mL) in a test tube and the two phases were analyzed by RPLC-ELSD. The partition coefficient was calculated as the ratio of the major solute peak area in each phase. The various tested biphasic systems used solvents such as heptane, methanol, ethanol, ethyl acetate, acetonitrile, and water in different proportions. Among all these systems, the one that gave the best results was ethyl acetate/ethanol/water (4.5:1.5:4.5). Under these conditions, solutes eluted around 12 min corresponded to a KD value close to 1, which resulted in a reasonable CPC analysis time. The separation was performed in descending mode with organic stationary phase and aqueous mobile phase. Figure 4 depicts the corresponding CPC-ELSD chromato-

Viability %

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

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considered in order to retain and separate them. In HILIC, a hydrophilic stationary phase and an aqueous-organic phase with high organic solvent content (at least 60%) are used. The retention is mainly caused by partition of the analyte between water-enriched layer immobilized at the stationary phase surface and the relatively hydrophobic mobile phase. HILIC-LC-MS The preliminary development of the HILIC chromatographic system was carried out with a double UV/ELSD detection. Figure 5a shows the ELSD chromatogram and Fig. 5b the LC-UV one at 280 nm obtained under optimized HILIC conditions. Seven peaks could be detected by ELSD (Fig. 5a) in addition to the compounds eluted in void volume, whereas only three peaks (1, 2, and 5) were detected by UV (Fig. 5b). Solutes eluted in peaks 3, 4, 6, and 7 could be aliphatic compounds without chromophore groups, such as sugars, while solutes 1, 2, and 5 could belong to the phenolic acid family as described in literature [7]. To identify the compounds eluted in the seven peaks, a coupling to a mass spectrometer was investigated. First, the mass spectrometry conditions in the ESI source were optimized by direct infusion of sugar and phenolic acid standard solutions. Negative mode gave better results than positive mode. As described in Table 1, the Kalanchoe leaves contained fructose, glucose, and maltose; these sugars were identified thanks to their formate adduct ions [M HCOO] and their retention time in comparison with corresponding sugar standards. Unfortunately under HILICLC-MS conditions, no more precise identification of phenolic acids was obtained. RPLC-UV/MS/MS Structural identification The active fraction Fr1 contained polar compounds and the most polar of them were eluted in the void volume under RPLC conditions. The HILIC mode was first To identify solutes less polar than sugars, the fraction Fr1 was analyzed under RPLC conditions. A mixture of five phenolic acids (caffeic acid, ferrulic acid, gallic acid, coumaric acid, and cinnamic acid) was first analyzed by

Fig. 5 HILIC analysis of fraction Fr1: Astec polymeric NH2 (150 4.6 mm, 5 m) column, room temperature, mobile phase ACN/ MeOH/H2O (85:5:10) in isocratic elution, flow rate 1 mL min1. a ELSD: nebulizer gas pressure, 2 bars; evaporative tube temperature, 50 C, gain 9. b UV =280 nm

cytotoxic activity and produced a decrease of the keratinocytes population even with the lowest concentration (0.19 g mL1) tested. For fractions Fr1 and Fr2 the viability began to decrease respectively at only 1.56 g mL1 and 0.78 g mL1. Therefore Fr1 was the less cytotoxic fraction. According to the antimicrobial activities and the cytotoxicity of the different fractions, fraction Fr1 was the most interesting and, thus, it was analyzed by LC-MS/MS to get structural information on its bioactive molecules.

Table 1 Compounds identified in Kalanchoe pinnata leaves by HILIC-LC-MS

Peak number 1 2 3 4 5 6 7

Retention time (min) 2.93 3.30 4.75 5.32 7.51 7.81 17.5

max (nm) 229/276 225/278 No UV absorbance No UV absorbance 288 No UV absorbance No UV absorbance

m/z (amu) 297 330 197 225* 255 225* 387*

Tentative identification Phenolic derivative Phenolic derivative Unknown Fructose Phenolic derivative Glucose Maltose

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

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detected: gallic, caffeic, and coumaric acids in fraction Fr1. Figure 6 reports the corresponding extracted ion currents (XICs). The presence of other kinds of molecules such as flavonoids in Kalanchoe pinnata leaves [2] was described, so an optimization of RPLC-UV and MS/MS conditions was carried out as described in RPLC-MS/MS. Seven main compounds were detected by LC-UV (Fig. 7) and their identification by MS/MS revealed that they belong to the family of flavonol glycosides (compounds 17 in Table 2). By MS/MS another compound was detected at 33.6 min and identified as a flavanol oligomer without chromophore group absorbing at 366 nm (compound 8 in Table 2). The flavonol glycosides were derivates of quercetin, isorhamnetin, and kaempferol as described in Table 2. Components 1, 2, and 7 presented a fragment ion at m/z =301 which is typical of quercetin aglycon. For compounds 4 and 6, the fragmentation of their pseudomolecular ion led to a fragment ion at m/z =315 which is characteristic of isorhamnetin aglycon. Compounds 3 and 5 presented a fragment at m/z =285 which is characteristic of kaempferol aglycon. The MS/MS fragmentation also allowed us to identify the sugar moiety of the flavonoids. For instance, the fragmentation of compound 1 mainly gave an ion at m/z = 463 due to the loss of 146 from the pseudomolecular ion at m/z =609 which indicated the presence of a rhamnose moiety [M H 146]. The ion at m/z =301 was due to the loss of 162 from the ion at m/z =463 which indicated the presence of a glucose moiety [M H 146 162]. This fragmentation pathway suggested that the two sugars were linked to two different OH positions in the phenyl ring. In the same way,

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. Alltima C18 (1504.6 mm, 5 m) column, room temperature, mobile phase MeOH/H2O containing 1% acetic acid, flow rate 1 mL min1

RPLC-MS/MS using the SRM mode as described in RPLC-MS/MS. For each solute, a specific transition was selected; then, these five transitions were followed during the Fr1 analysis. Three phenolic acids were

Fig. 7 RPLC-UV =366 nm analysis of fraction Fr1. Alltima C18 (1504.6 mm, 5 m) column, room temperature, mobile phase MeOH/H2O containing 1% formic acid, flow rate 1 mL min1

Bioactive molecules in Kalanchoe pinnata leaves Table 2 Identification of flavonoids contained in Kalanchoe pinnata leaves by RPLC-MS/MS Peak number Retention time (min) Parent ions (m/z) [M H] 1 2 3 4 5 6 7 8 24.6 24.8 26.2 27.4 28.5 29.6 30.9 33.6 609 742 593 595 508 609 580 467 [M+HCOOH] 656 639 301/463 580/301/447/259 285 300/315 285/465 330/315/300/209 301/179/409 289 Quercetin glucose rhamnose Quercetin glucose arabinose rhamnose Kaempferol 3-O-rutinoside Isorhamnetin hexose pentose Kaempferol derivative Isorhamnetin derivative pentose, deoxyhexose Quercetin 3-O--l-arabinopyranosyl (1 2) -L-rhamnopyranoside Flavan-3-ol derivative Fragment ions (m/z) Tentative identification

1337

Ref.

[12] [12] [2] [2] [21]

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]. The pseudomolecular ion at m/z =593 of compound 3 gave a fragment ion m/z =447 by loss of 146 which indicated the presence of a rhamnose moiety [M H 146] and the fragment ion at m/z =285 due to the loss of 162 from the ion at m/z =447 indicated the presence of a glucose moiety [M H 146 162]. For compound 4, the ion at m/z =315 was probably due to the loss of a hexose and a pentose [M H 162 133]. The fragmentation of compound 5 caused the loss of 146 and 133 corresponding respectively to a deoxyhexose and a pentose moiety [M H 146 133]. As described in the literature [2], compound 6 is a quercetin diglycosyl and its fragmentation resulted in an ion at m/z =301 due to the loss of 146 and 133, which indicated the presence of rhamnose and arabinose moieties. The last compound corresponded probably to a flavan-3-ol derivative according to the literature [21], and its fragmentation led to an ion at m/z =289 which corresponded to catechin.

chemical preservatives. This study identified compounds already reported in the literature but also new ones.
Acknowledgement This project was supported by the French competitiveness cluster Cosmtique, Sciences de la Beaut et du Bien tre. The authors thank the Conseil Rgional du Centre (France), the Conseil Gnral du Loiret (France), the Communaut dagglomration dOrlans and the Ville dOrlans for their financial support. The authors acknowledge Jean-Marc Seigneuret and Emilie Dufour (Adonis, Alban Muller, France) for their help and providing the Kalanchoe pinnata leaves and, Marie-Pierre Papet for her advice.

References
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