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MULTIVARIATE OPTIMISATION OF PARALLEL EXTRACTION OF CETRARIA ISLANDICA AND
ANALYSIS BY 1H NMR AND PCA
Anne-Cécile Le Lamer, Eric Hitti1, Joël Boustie, Sophie TomasiEA 4090 “Substances Lichéniques et Photoprotection”, UFR Sciences Pharmaceutiques et Biologiques, 35043 Rennes Cedex; 1Inserm U642, Laboratoire Traitement du Signal et de l’Image, 35042 Rennes Cedex, France ;
Phone: + 33 (0) 2 23 23 48 17. Fax: + 33 (0) 2 23 23 47 04. [email protected]
Context: Recently, there has been an increasing demand for new extraction techniques, amenable to automation with shortened extraction times and reduced organic solvent consumption. A challenge consists thus to the development of rapid and parallel extractive methods coupled to analytical methods which should be fast, reproducible and adapted for a wide array of compounds. An example of this approach has been developed with a lichen Cetraria islandica (Iceland moss).
1- Extraction of lichens (100 mg/mL) for 1 or 2 h with refluxing in various solvents using Büchi Syncore Reactor (20 mL) or Heidolph Synthesis apparatus (5 mL), respectively.
MATERIAL & METHODS
[1]. K. O. Vartia. Antibiotics from lichens. In: V Ahmadjan, MS Hale, eds. The lichens New York: Academic Press, 1973:547.[2]. H. M. Ogmundsdottir, G. M. Zoega, S. R. Gissurarson, K. Ingolfsdottir, J. Pharm. Pharmacol. 1998, 50(1), 107-115.[3]. G. E. Pereira, J. P. Gaudillere, C. Van Leeuwen, G. Hilbert, M. Maucourt, C. Deborde, A. Moing, D. Rolin, Anal. Chim. Acta. 2006, 563(1-2), 346-352.
TLC (elution: Hex/Et2O/Formic acid 65:40:10, v/v/v)
1H NMR: Samples of the extracts were dissolved at 3.34 mg/mL in DMSO-d6. Spectra were recorded at 298 K on a Jeol GSX 270 MHz . A typical proton NMR consists of 128 scans requiring 16 min acquisition time with a spectral width of 15 ppm a pulse width (PW) = 45° (5 µs) and inter-pulse delay of 3.96 s. The data were Fourier-transformed to 128 k data points using line broadening factor of 0.12 Hz. The spectra were referenced to internal TMS.
After removing the peak solvent (at 2.5 and 3.34 ppm), principal components analysis (PCA) of the pre-processed NMR data were conducted within MATLAB 6.5.1 using mean-centered scaling.
2- Analysis
INTRODUCTION
Various conditions of extraction were compared and results analyzed with TLC, 1H NMR and PCA (Principal Components Analysis) [3]. Three batches
of C. Islandica have been studied.
Lichen :
Fruticose lichen
Common on northern countries and montainous areas elsewhere European Pharmacopoeia [1]
• demulcent• anti-inflammatory
Major compounds [2] : Polysaccharides, fumarprotocetraric acid (FPA) and protolichesterinic acid (PLA)
The Iceland moss
0.30.80.8
11.31.3
2.51.25
32.7
1.3
(%)Extraction rate
n-heptanen-hexanePetroleum etherIsopropyl etherCHCl3, CH2Cl2
EtOAc
THFAcetoneEthanolMethanolAcetonitrile (ACN)
O
CO O
CH2OH
OH
MeCHO
HO
Me
COOHO
CO O
CH2-O-CO-CH=CH-COOH
OH
MeCHO
HO
Me
COOHO
CO O
CH2-O-Me
OH
MeCHO
HO
Me
COOHO
CO O
CH2-O-C2H5
OH
MeCHO
HO
Me
COOH
Chloro-phylls
Polyols
n-C13H27 OO
HOOC
n-C13H27 OO
HOOC
Table 1: Quantitative (extraction rate) and qualitative analyses (TLC) of various extracts (2 h) on a lichen from France (JB/04/08c). LA: (+)-lichesterinic acid, PLA: (+)-protolichesterinic acid, EPA: 9’-O-ethylprotocetraric acid, MPA: 9’-O-methylprotocetraric acid, FPA: fumarprotocetraric acid, PA: protocetraric acid
Amount
Figure 3: Representative 1H NMR spectral profiles of french C. islandica sample: (a) n-Heptane, (b) THF, (c) ACN
Figure 1: Representation of the PCA of various C. islandica extracts. Each class was defined by ellipse. Class 1:(♦) n- hexane, n-heptane, petroleum ether, isopropyl ether, dichloromethane, ethyl acetate; class 2: (●) acetone, THF, petroleum ether then THF, chloroform then THF; class 3: (■) EtOH, MeOH, ACN, n-hexane then MeOH, petroleum ether then MeOH, CHCl3 then
MeOH, THF then MeOH, n-heptane then ACN, isopropyl ether then ACN, CHCl3 then ACN, EtOAc then ACN.
(1) 1 hr, n-Heptane, (2) 1 hr, THF, (3) 1 hr, ACN (4) 1 hr EtOH ; (a) danish batch, Cailleau, (b) batch from Lozère (France), (c) danish batch n°15759, Cailleau
LA PLA EPA MPA FPA PA
Successive extractions have been also carried out. 1H NMR spectrum was recorded on the last extract.
RESULTS
LA
PLA
FPA, PA
EPA
Figure 2: 1H NMR spectral profiles of all studied extracts on lichen from France. Blue spectra for class 1, red spectra for class 2, green spectra for class 3
a
b
EtOAc
c
Isop. Ether
Conclusion: With three different extracts, the metabolite fingerprinting of a lichen could be assessed. Chemometric analysis applied in appropriate NMR data will be performed for lichen chemotaxonomic classification.
The best extraction rates (~ 3%) were obtained with aprotic and protic polar solvents (THF, EtOH and MeOH) (Table 1).
PCA (Figure 1) applied on the 1H NMR data (Figures 2 and 3) to visualize clustering of samples and to detect the metabolites responsible for the discrimination of sample groups. Satisfactory separation between apolar (class 1, ♦ label), and polar extracts (class 2, ● label and class 3, ■ label) was achieved.
LA and PLA lactones were present alone in solvents of class 1 (Figure 3) except for ethyl acetate and isopropyl ether which extract also a very low amount of depsidones (FPA, PA, characteristic signal at d 10.6 ppm) (Table 1). 1H NMR data treatment did not distinguish these two extracts due to this too low amount (Figure 2).
Solvents of class 2 differed from those of class 3 on the most positive side of axis 2 of PCA. The observation of 1H NMR spectra suggested that the characteristic signals between 4 and 5 ppm (e.g. presence of EPA) and the less extracted amount of FPA, PA are responsible for this separation (Figure 3). Solvents of class 3 hydrolyze FPA into MPA or EPA, even if successive extracts using apolar solvent were performed (e.g. EtOAc then ACN). Moreover, no polyol was extracted with THF (class 2) (Table 1).
No qualitative difference has been shown between batches of different geographical origin which are correctly classified, indicating their same metabolite profiling (Figure 1). Only quantitative differences led to the distinction of the three batches.
