FR2732112A1 - METHOD FOR IDENTIFYING PLANT SPECIES AND THEIR HYBRIDS - Google Patents

Abstract

According to the method of the invention, an extract of polyphenolic compounds, derived from a plant material of a plant species or one of its hybrids or their lower rank taxa, or from an elaborated product, is subjected to from this material, to at least one multidimensional NMR analysis, followed by a quantitative analysis of the signals and the statistical processing of the integration data, leading to the obtaining of characteristic data of the plant species, of the hybrid and lower rank taxa. Application to the identification of a plant species of its hybrids or lower rank taxa and use for purposes of control of their origin.

Description

METHOD OF IDENTIFYING SPECIES

VEGETALES AND THEIR HYBRIDS

  The subject of the invention is a method for identifying plant species and their hybrids, and lower rank taxa based on

the use of a repository.

  Lower-order taxa means in particular subspecies, varieties, sub-varieties, forms and sub-forms, as well as cultivars or grape varieties and clones,

  without being bound by strict definitions of these.

  Taking as an example Vitis vinifera, Y. labrusCA, V. riaria or M. rupeftars which are vines as a plant species, it is commonly considered that Viti corresponds to the genus, vinifera, rIparla, labguaRa, or rupestr i correspond to the species. , that these species include several cultivars or grape varieties, among

which various clones.

  The expressions "plant species" and

  "hybrids" as used in the description and

  the claims include, unless otherwise indicated

  contrary, the taxa of lower rank which they contain. In the field of vines, for example, a great deal of research is being done to develop methods of grape recognition and, among these, clones, allowing control services to ensure that a vine, even a bottle of wine, corresponds well to the indications given. Their recognition by the only ampelographic observation is difficult and makes it necessary to resort to

laboratory methods.

  Chromatography separation techniques have thus been proposed. However, they do not allow an overall and reproducible analysis of the products, some of the tannins being retained on the column. Moreover, these techniques do not allow

differentiate the clones between them.

  The use of NMR has also been reported

  or genetic engineering techniques.

  However, the work described, with regard to NMR, focuses on wines and

not on the organs of the vine.

  It is more specifically studies on products resulting from the fermentation of yeasts in white wines, such as glycerol, or fermentable products, such as sugars, which are therefore not

proper to the vineyard.

  With regard to NMR, these are one-dimensional techniques (1H and 13c) for which the spectral lines overlap, which

  induces errors in the integration data.

  As for genetic engineering techniques, they have the disadvantage of being difficult to implement. In addition, they require highly qualified personnel and laboratory equipment meeting strict standards, which makes it impossible to envisage applications on an industrial scale, taking into account

resulting costs.

  The search for reliable means of characterization led the inventors to take into account a type of compounds present in the vine and many other plant species, and to subject it to a succession of analyzes carried out under specified conditions. The work carried out has thus shown that, unexpectedly, the polyphenolic compounds have characteristic qualitative and quantitative constants of a plant species and its hybrids and, within a plant species and its hybrids, characteristic of their rank taxa

  as their subspecies, varieties, sub-species

  varieties, forms and subforms as well as cultivars or

  grape varieties and mime among them, clones.

  The invention is based on the use of these phenolic compounds to obtain information constituting an identity card of a plant species or

  of a given hybrid and their lower rank taxa.

  The purpose of the invention is therefore to provide a new method for recognizing plant species and their hybrids, their lower rank taxons based on the use of a reference system enabling a

  discrimination of great reliability.

  The invention thus aims to provide more specifically a reference system, or database, for a plant species, or its hybrids and their lower rank taxa, developed from characteristics of the phenolic compounds they contain. In another aspect, the invention aims to provide means for verifying the nature of the rank of a

  taxon of a plant species or a hybrid to be controlled.

  It also relates to means for controlling from a product derived from a plant material, the belonging of the latter to a taxon of lower rank

  of a plant species or a hybrid.

  The method, according to the invention, for identifying a plant species, or its hybrids, or a lower-ranking taxon of this species or its hybrids, is characterized by subjecting an essentially constituted extract polyphenolic compounds, derived from a given plant material or hybrid, from their lower rank taxa, or from a product made from these materials, to at least one multidimensional NMR analysis, followed by a quantitative analysis of the signals and the statistical processing of the integration data, leading to the obtaining of characteristic data of the plant species,

  the hybrid, or their lower rank taxa.

  The exploitation of the data of the NMR maps obtained according to the invention, concerning the polyphenolic extracts of the materials and products studied, makes it possible to have a high reliability recognition technique of the lower rank taxa to which these materials belong, or from from which these products are obtained. This reliability implies carrying out the extractions and analyzes in the

same conditions.

  Advantageously, these data prove to be constant for a given material, which allows their use to develop a reference system or database constituting a signature of the genetic potential of rank taxa

lower studied.

  According to a preferred embodiment of the invention, the polyphenol extracts are subjected to 1H-13C heteronuclear NMR analysis, providing information on the structure of the polyphenols present.

in the extracts.

  More specifically, an analysis is

two-dimensional (2D).

  By means of proton detection, advantageously using a so-called inverted 1H-13C detection probe, thus of high sensitivity 1H, the correlations between 1H and 13C (correlation spots) are established by means of their

mutual coupling.

  Advantageously, the HMBC (Heteronuclear Multiple Bond Connectivity) analysis, which establishes long-range heteronuclear IH-13C correlations, is especially useful. We thus see the coupling between the protons and the distant carbons 13

two or three links.

  The pulse sequence used is more specifically that of a HMBCGAS (Gradient Accelerated Spectroscopy). Pulses of magnetic field gradients are sent during the experiment, which allows a satisfactory suppression of the central peaks, that is to say horizontally on the medium of the spectrum containing no information, and this with a time of experience in the conditions of implementation

  process compared to HMBC without gradients.

  In addition, HMBC-GAS cards have the advantage of greater readability. Indeed, they do not contain vertical streaks ("tl noise) located at the frequency of each signal and coming from the imperfection of the system of elimination of such signals in the case of" quadrature detection "in the

experiences without gradients.

  Interferograms (FID) are first multiplied by a square sinusoidal function to increase the signal-to-noise ratio (S / N) and undergo a

  Fourier transform in magnitude.

  Alternatively, or in addition to HMBC-GAS analysis, the polyphenol extract is subjected to HMQC (Heteronuclear Multiple Quantum Correlation) analysis. This

  analysis makes it possible to study the heterogeneous correlations

  between 1H and 13C at a single link. The interferograms (FID) are first multiplied by a square sinusoidal function to increase the S / B ratio, and undergo a Fourier Transform, but in this case in amplitude, then they are phases. In another variant, used alone or in combination with HMBC-GAS and / or HMQC analysis, the extract to be studied is subjected to HMQC-HOHAHA analysis (HOmonuclear HArtman HAhn). This analysis makes it possible to see not only the coupling of a carbon 13 with the proton it bears directly, but also with all the protons that are coupled to the latter. The main experimental parameters correspond to those of the HMQC

  to which is added a "spin-lock time".

  To carry out these experiments, it is advantageous to use a spectrometer equipped with a very stable temperature control system in order to

  to avoid disturbances on the NMR charts.

  The correlation spots of the NMR analyzes are then integrated and the data obtained are

  multidimensional statistical analysis.

  The integration corresponds to the calculation of the volume of each correlation peak by adding all the points present in the sector delimited around each correlation task, each correlation task being

  digitized in points of variable intensity.

  All integration data is submitted

to a multidimensional analysis.

  In particular, analysis of variance (ANOVA) and / or principal components analysis (PCA) and / or analysis

discriminant factorial (AFD).

  ANOVA allows you to select correlation (variable) tasks that provide the most information. PCA represents individuals (clones) in a two-dimensional space, individuals being

  initially present in a space with N dimensions (N -

  number of selected variables). This analysis makes it possible to study the individuals in the space of the principal components, so as to be able to determine the existence

possible groups and anomalies.

  AFD is used to discriminate

  optimal predefined groups of clones.

  The use of ANOVA makes it possible to select the most informative variables. It is recalled that this technique is based on the calculation of a factor F proportional to the ratio of the variance calculated for all groups together on the sum of the variances calculated within each pre-defined group. In the case of

  the vine, these groups are the grape varieties, or the clones.

  The variables for which F is the highest are retained for the ACP and the AFD. Reducing this number of variables before performing the multidimensional analysis ensures that reliable results are obtained. Such results are obtained with a ratio of number of samples / number of variables

greater than 3.

  Once the number of variables is reduced, a principal component analysis, ACP, is performed on the data. This technique calculates new variables (orthogonal principal components) by linear combination of the original variables, from the set of individuals (grape varieties, clones) with which quantitative characters are associated. These main components make it possible to distinguish individuals as best as possible, without presupposing the existence of

groupings among individuals.

  The analysis is performed on raw data.

  We obtain a representation of the individuals in a two-dimensional plane where the first calculated axis has the greatest variance leading to the greatest dispersion of individuals on this axis. This analysis makes it possible to detect the existence of atypical groups or individuals. To discriminate optimally individuals with defined subgroups (varieties, cultivars, clones ...), these distinctions are not exhaustive or limiting, by the results of the PCR, it is advantageously carried out an AFD. For this purpose, new variables, linear combinations of the original variables, are calculated by searching for a

  severity for each group as far away as possible.

  AFD is then used for the assignment of individuals

  additional to one of the previous groups.

  The foregoing steps are advantageously carried out on the total polyphenol extracts of a plant material of the plant species or of the hybrid to be studied, or of a product produced from this plant species or from this hybrid, this material and this product

  hereinafter referred to as raw materials.

  These raw materials are subjected to one and, advantageously, several extractions carried out under identical conditions. Organic solvents are used, if necessary added with water. Suitable solvents include acetone, acetate

  of ethyl, ether, alone or as a mixture.

  For convenience of handling, it is advantageous to use these extracts in freeze-dried form. Raw materials come from plant species or their hybrids with plant organs rich in polyphenolic compounds. Of particular note is the vine, the scope of the invention extending however to the recognition of other tannins than those of the vine, as the oenological tannins of

pine, china, or walnut.

  The differentiation technique of the invention is also of great interest for the recognition of different varieties of fruit trees (for example of apple trees), or of flower varieties.

  (including roses), or medicinal plants.

  The plant organs used to obtain the extracts are chosen, advantageously, taking into account their richness in polyphenolic compounds and their easy treatment. This includes, for example, leaves, seeds, pips or, where appropriate, bark or

flower petals.

  Products made from the species or plant organ include fruit juice, or products derived from their fermentation, such as wine or beer. The invention thus provides the means, from the polyphenolic content of a plant extract, to establish a set of characteristic data of a plant species or its hybrids and, according to an aspect of great interest, rank taxa lower than this

  plant species or its hybrids.

  Given the constant nature obtained for these data, under the conditions set out above, the latter are used according to the invention for

  establish a bank as a repository.

  This reference is established, by accumulation of results, by operating under identical conditions, for a plant species and its hybrids, or for taxons of lower rank, according to the plant organ or the product made from the plant organ , from where

  the polyphenolic compounds are extracted.

  The invention relates to a process for the identification of a plant species or its hybrids and their lower rank taxa, characterized in that an extract originating from a plant organ or from a product produced from from this organ or plant species, its lower rank hybrids and taxa, to multidimensional NMR analysis and to the processing of NMR data as indicated above, and the results obtained are compared with the data of a reference

corresponding to the species.

  The invention thus provides the means of controlling with great precision an origin and, hence, a set of qualities of a plant material or a product made from this material, such as fruit juices or products derived from of their fermentation as

wine or beer.

  Other characteristics and advantages of the invention are given in the examples which follow at

for illustration purposes.

  In these examples, reference is made to FIGS. 1 to 17, which represent respectively: FIGS. 1 to 3 show respectively the maps of HMBC-GAS, HMQC and HiMQC-HOHAHA of seed extracts of grapes of the Black Merlot clone 343, - Figure 4, the results of the ANOVA on the HMBC of seed extracts of different grape varieties, - Figure 5, the hierarchical classification for these grape varieties (on HMBC-GAS), FIGS. 6 and 7 show the results of ACP and AFD respectively on the HMBC-GAS of these grape varieties, FIGS. 8, 9 and 10, the results of ANOVA on HMBC-GAS. respectively, of Merlot Noir, Cabernet Franc and Cabernet Sauvignon, - Figures 11, 12 and 13, the results of the PCA on HMBC-GAS, respectively Merlot Noir, Cabernet Franc and Cabernet Sauvignon, - Figures 14, 15 and 16, the corresponding results of AFD, FIG. 17, map of HMBC-GAS grape leaves gradients of Cabernet Sauvignon 337, and FIG. 18, the results of the NANOVA on

HMBC-GAS of vine leaves.

  paJ_: Study of grape seed extracts. An operating protocol applied is reported

  grape seed clones of three grape varieties.

  These are Cabernet Franc (abbreviated CF), Cabernet Sauvignon (abbreviated as CS), and Merlot Noir (abbreviated as MN) and clones: N 312, 332, 331, 330, 187 from Blanquefort and, N 312, 332, 331 of Mountain, for CF; N 335, 337, 339, 341, 191 for CS and

N 181, 343, 346, 347, 348 for MN.

  This operating procedure comprises: I. the extraction of polyphenols; II. NMR analysis of the extracts obtained;

  III. integration of NMR analysis data; and IV.

  a statistical analysis of these results.

  Each of these steps is detailed below.

  I. Extraction of polyphenols Three extracts of each clone were prepared by proceeding as follows: 50 g of seed of raisins were macerated for 24 hours in the dark from 60 ml of

acetone / water mixture (2/3).

  The mixture is subjected to a leaching step

  for 15 minutes. We recover a first fraction.

  310 ml of the same solvent mixture was added to the macerated pips and leached for

  at 12 o'clock. We recover a second fraction.

  We combine the two fractions and evaporate

  acetone under reduced pressure at 30 C.

  The remaining solution is extracted with ethyl acetate (AcOEt), according to the following scheme: AcOEt volume (ml) Agitation 10 min 5 min 5 min 50 5 min The two layers are then centrifuged for

eliminate the insoluble interface.

  After evaporation of the ethyl acetate,

  dissolve the extract in water and freeze-dry.

  The extraction yield is 2 g / 100 g of

dried seeds about.

  The polyphenolic extracts of pips contain mainly flavanols, catechin and epicatechin, corresponding to the following formulas, as well as

their polymers.

OH OHO HO

HO O

II .. v _-- OH

f OH 01-

  OH epicatechin catuchin II. NMR Analysis An identical amount of extract for each clone (100 mg) is dissolved in deuterated dimethyl sulphoxide (DMSO-d6) and analyzed by 2D NMR. Three experiments are recorded for each extract: A so-called inverted detection probe 1H-13C is used, which has the advantage of a very large

sensitivity 1H.

  We obtain - the proton spectrum by projection on the horizontal axis, the carbon 13 spectrum by projection on the vertical axis, - the correlations between these two nuclei (correlation spots of the 2D map) by means of

their mutual coupling.

  Experiment i: HMBC analysis (ref 1) giving long heteronuclear (1H-13C) correlations

distances (2JC-H and 3JC-H).

  The numerical references "ref.1" and following are specified in the "Bibliography" section at the end of

description.

  The pulse sequence used is the one

of an HMBC -GAS.

  The acquisition parameters are as follows.

  The excitation frequency in the dimension 1H is

  500.14021 MHz and 125.7728 MHz in the 13C dimension.

  The spectral window is 5319.15 Hz in the dimension

  1H and 27670.02 Hz in the 13C dimension.

  Each free precession signal (FID) is digitized on 2048 points and 512 experiments are recorded in the 13C dimension. The acquisition data is first multiplied by a square sinusoid to increase the signal-to-noise ratio (S / N) and undergoes a Fourier Transform in

  magnitude. For each experiment, 16 FIDs are accumulated.

  The experience time is 4 hours. Experiment 2: HMQC analysis (ref 2) giving the heteronuclear correlations (1H-13C) direct (1JC-H). The main acquisition parameters of

data are as follows.

  The excitation frequency in the 1H dimension is 500.139658 MHz and 125.7672 MHz in the 13C dimension. The spectral width is 3787.88 Hz

  in dimension 1H and 16520.27 Hz in that of 13C.

  Each FID is digitized on 2048 points and 256 experiments are recorded in the 13C dimension against 512 experiments for the HMBCGAS. This reduction in the number of experiments deteriorates the resolution of the correlation spots in the 13C dimension, but makes it possible to maintain an experiment time identical to that of the HMBC. For each experiment, 32 FIDs are accumulated to achieve a proper S / N ratio. The experience time is 5 hours. The acquisition data is first multiplied by a square sinusoidal function to increase the S / N ratio and undergo a Transform of

  Fourier in amplitude, then are phased.

  Experiment 3: HMQC-HOHAHA (ref.3) giving the heteronuclear correlations (1H-13C) relayed to

coupled protons.

  The main experimental parameters correspond to those of the HMQC to which is added "a

mixing time "(spin-lock).

  The three experiments are recorded at 303 K. The spectrometer is equipped with a very stable temperature control system, which avoids disturbances on the NMR maps, such as streaks at the correlation spots. The NMR maps obtained at the end of each of these three experiments are respectively given

Figures i to 3.

  III. The information contained in the 2D NMR maps of each clone is translated into numerical data by calculating the volume of each peak of correlation.

using a computerized system.

  For this purpose, we define sectors around each correlation task and the volume is calculated by adding all the points present in the sector (each correlation task is digitized in points

variable intensity).

  The data obtained is then subjected to a

  multidimensional statistical analysis.

  IV. statistical analysis - mltid ninlle.

  data obtained. with the HiBC-GASg IV.A. analysis of cepas: a) analysis of variance (ANOVA) By analysis of variance, we select the correlation spots (variables) that bring the most

information (ref 4 and 5).

  For the data obtained with the HMBC-GAS, which are represented in FIG. 4, the most discriminating variables (F higher than 150) are the following:

  N 22, 25, 27, 28, 31, 32, 34, 35, 37, 38, 42 and 44.

  These variables correspond mainly to 1H correlation spots between 2 and 3 ppm of a

  and between 6 and 7 ppm on the other hand.

  b) hierarchical analysis of the individuals Then a hierarchical classification of the individuals is carried out so that the distances between them depend on their similarity and

  this without presupposing the existence of groups.

  As shown in Figure 5, which represents the cluster analysis, the grape varieties are quite well differentiated since only the samples marked with a

asterisk are misclassified.

  These samples correspond to clones 191 of CS and 343 of MN. The integration data show that they have characteristics

  different clones from the same grape varieties.

  c) principal components analysis (PCA) This analysis (refs 6, 7 and 8) allows an analysis of the individuals (clones) in a two-dimensional space, the individuals being initially present in a space with N dimensions, N being the number selected variables. We study the structure of individuals in such a way as to be able to determine the existence

possible groups and anomalies.

  The results obtained represented in FIG. 6, for F greater than 30, show that the grape varieties are

  overall well differentiated.

  The clones inside the CS grape are also well differentiated, with a more or less

great similarity of the clones.

  This is not the case for the other grape varieties, which probably results from the choice of variables according to the grape variety criterion, which are not

  discriminant for clones of CF and MN varieties.

  d) discriminant factor analysis (DFA) This analysis places the samples in a two-dimensional space so as to have the barycenters of the pre-defined groups (ref 6, 7 and 8) as far as possible, by constructing discriminating axes that are linear combinations of the starting variables. Figure 7 shows the results of the AFD obtained from the data of

HMBC-GAS (F greater than 150).

  We see that overall the grape varieties

are well separated.

  Etuda statistical modules To validate the discrimination technique of grape varieties and clones, the robustness of the models was tested by statistical tests (Test

  of Jacknife and Cross Validation).

  Each individual was removed successively, handed over to the original population and then AFD was performed and the coordinates of the abducted individual were calculated from the discriminating equations. We then check if the individual is affected in the right group. It is considered that the technique is validated when stable discriminant equations are obtained irrespective of the individual removed and the percentage

  of high ranked individuals is high.

  At each operation, new discriminant axes were obtained: XlalliVarl + al2Var2 + ... + alpVarp X2 = a2lVarl + a22Var2 + ... + a2pVarp Xn-anlVarl + an2Var2 + ... + anpVarp Xn: discriminating axes n: number of individuals anp: coefficients of the discriminant axes Varp: pth variable selected by ANOVA From these data, several calculations were carried out: To know the importance of the coefficients of the models and their stability, the average (m) and, the standard deviation (EC)

  of each coefficient were calculated.

  The center of gravity of each group was also calculated for each operation and each individual assigned to the group whose center of gravity was closest to him. We can calculate the percentage of well-ranked individuals each time and study the rate of good ranking and the stability of the ranking for each

  group and for all groups together.

  The means (m) and standard deviation (EC) of the coefficients of axes i and 2 are given below (number

of individuals: 45 raw data).

Avg. Axis 1 Coef. m Axis 2 CoeLf.

EC Var. EC Var.

  COCF. 1 24.2 3.51 14.5 28.8 356 12.4 Cocf. 2 20.5 2.28 11.1 2.75 1.64 59.6 Cocf. 3 13.7 4.17 30.4 42 3.89 9.25 Coef. 4 20.9 2.51 12 21 2.35 11.2 Coef. 5 2.94 2. 36 80.3 2.11 22 105

22 0

  COCF. 6 32.7 2.56 7.82 30.4 2.07 6.83 Cocf. 7 11.4 3.75 33 23.3 2.54 10.9 Coef. 8 11.4 3.7 32.5 18.2 3.95 21.6 Cocf. 9 13.9 2.15 15.5 5.79 2.19 37.8 Coef.10 23.7 2. 17 9.17 5.56 2.68 48.2 Cocf.ll 4.23 1.34 31.7 12.8 1.84 14.3 Coef.12 4.36 1.44 32. 9 5.84 2.08 35.7 _ The calculations made for the HMBC-GAS show that for axis 1, the coefficients 1, 2, 4, 6 and 10 are the most important in the model and have good stability (low standard deviations). Given the values of the coefficients, we deduce that it is the variables 22, 25, 28, 32, 37 and 38 that are the most influential

for the construction of axis 1.

  For axis 2, the coefficients 1, 3, 4, 6 and 7 are the most important in the model and have good stability (low standard deviations). Given the values of the coefficients, we deduce that it is the variables 22, 27, 28, 32 and 34 that are the most influential for the

construction of axis 2.

  Percentages of highly ranked individuals are 99.85 for CF and MN and 100 for CS, CF312mg and CF331Mg clones are responsible for

  percentage of CFs because of their proximity to NDs.

  Discrimination of grape vines using the HMBC-GAS NMR data is therefore

  reliable technique taking into account the statistical results.

  IV.2 alyse dem clones For the recognition of clones, the grape varieties

were analyzed separately.

  ANOVA analysis Figures 8, 9 and 10 show the values of F in the analysis of variance, obtained from the HMBC-GAS NMR spectra, respectively for the clones.

MN, CF and CS grape varieties.

  For each variety, the value of F selected and the variables chosen are indicated below: Merlot Black: F> 10; chosen variables: 3,

4, 6, 9, 12, 34, 37;

  Cabernet Franc: F> 30.5; selected variables:

  7, 22, 23, 25, 34, 36, 37, 38, 39, 43;

  Cabernet Sauvignon: F> 55; variables

chosen: 3, 6, 7, 9, 39, 45, 47.

  PCR Analysis The results of the PCR analysis on HMBC-GAS of the polyphenolic extracts of seeds for MN, CF and CS, are respectively reported in FIGS. 11, 12 and 13. There is a tendency for black Merlot to group by clone except individuals MN 181 bt and

MN 346 bt 194.

  For Cabernet Franc, clones tend to form groups with more or less similarities

between them.

  Clones differing only in terroir

  (312, 331, 332) are very different.

  In the case of Cabernet Sauvignon, we notice

good separation of the clones.

  AFD analysis The results of the discriminant factor analysis on HMBC-GAS of grape seed extracts are given in FIGS. 14, 15 and 16 respectively for MN (F> 10), CF (F> 30.5). ) and CS (F>

55).

We obtain in all 3 cases a good

differentiation of clones.

  The differentiation of grape varieties and clones by integration of HMBC-GAS correlations constitutes

* so a reliable technique.

  V - Statistical analysis and identification with HMOC on the one hand, with HMOC - HOHAHa. other Pmart. The techniques reported above are applied to the spectral data of HMQC and HMQC-HOHAHA.

for grape varieties and clones.

  In both cases, good discrimination is obtained. & Ex1. 2: Study of vine leaf extracts. Polyphenolic extracts of grapevine leaves are prepared from the following grape varieties and clones: Merlot Noir MN grapes clones: 343 and 347 Cabernet Sauvignon CS clones: 335 and 337 Cabernet Franc CF clones: 312 and 187 I. Extraction of polyphenols Macerated 50 g of dry leaves for 24 hours protected from light in 500 ml of mixture

acetone / water (2/3).

  The mixture is subjected to a leaching step

slow for 1 to 2 hours.

  The acetone is evaporated under reduced pressure at 30 ° C. A green precipitate composed mainly

  chlorophyll and waxes are formed.

  To eliminate waxes and chlorophyll, a centrifugation step is used and a

extraction with ether.

  The mixture is subjected to centrifugation to

separate the two phases.

  The remaining solution is extracted with ethyl ether (Et20) according to the following scheme: Et2O (ml) Agitation 10 min 5 min 100 5 min The remaining phase is subjected to an evaporation step to remove the residual ether, then the remaining solution is extracted with ethyl acetate, proceeding as follows: V AcOEt (ml) Stirring 10 min 5 min 5 min 100 5 min 5 min 5 min 5 min 5 min The acetate is then evaporated under reduced pressure and the solute is taken up with water to lyophilize. The polyphenolic extracts obtained are essentially composed of flavonoids, quercetin and isoquercitrin, these compounds corresponding to the following formulas:

OH OH

OH OH

UH O-Glucos

HO O HO 0

  isoquercitin quercetin The extraction yield is 3g / 100g of leaves

dry around.

  II NMR analysis of the extract are dissolved in 0.5 ml of DMSO-d6. For the first snippet of each clone, three HMBC-GAS are recorded, and for the second snippet, only one experiment is recorded to check the

  reproducibility of the extraction technique.

  The experimental parameters are as follows: excitation frequency of 1H: 500,1412769 MHz and 13C: 125,7728 MHz; 10. spectral window of 1H: 14.489 ppm and

13C: 220 ppm.

  The number of accumulations per experiment is 12. Each interferogram (FID) is digitized on 2048 points and 512 experiments are recorded in the 13C dimension. The duration of the experiment is 2 hours. The data is multiplied by a square sinusoidal function and undergoes a Fourier Transform in magnitude mode. Figure 17 shows the map of

NMR obtained.

  III Statistical Analysis a) ANOVA Figure 18 shows the results of

  ANOVA analysis of the HMBC-GAS spectra data.

  The most discriminating variables are the

  following (F> 60): Nos. 113, 115, 117 and 118.

  These variables correspond to correlation spots of 1H at = -12.5 ppm with carbons & i = 99 ppm,

  S = 145 ppm, S = 161 ppm and = 163 ppm.

  These characteristics are those of a proton of a hydroxyl which is linked by the establishment of a hydrogen bond with a carbonyl, this type of proton is found in the flavonoids which are the majority compounds of the extracts of vine leaves.

b) Hierarchical classification.

  By operating as indicated for the extracts of grape seeds, a differentiation of

  grape varieties, in particular the CS grape variety.

  c) ACP and AFD analyzes In these two experiments, carried out as indicated above, the clones are generally

well separated.

  The results show that the leaf extracts also make it possible to detect a specificity of the polyphenolic content of each grape variety, or of each clone, since differences appear between them.

clones and grape varieties.

Claims (15)

  1. A method for identifying a plant species or its hybrids and lower rank taxa of this plant species or this hybrid, characterized in that an extract consisting essentially of polyphenolic compounds, originating from a plant material or said hybrid and their lower rank taxa, or a product made from this material, to at least one multidimensional NMR analysis, followed by a quantitative analysis of the signals and the statistical processing of the integration data, leading to the obtaining of characteristic data of the plant species,   the hybrid and lower rank taxa.   2. Method according to claim 1, characterized in that the polyphenol extracts are subjected to a two-dimensional 1H-13C heteronuclear analysis. 3. Method according to claim 2, characterized in that an HMBC analysis is carried out, plus especially an HMBC-GAS.   4. Method according to claim 2 or 3,   characterized in that HMQC analysis is performed.   5. Method according to one of claims 2 to   4, characterized in that an HMQC- HOHAHA.   6. Method according to one of the claims   previous ones, characterized in that the acquisition parameters, excitation frequency in the 1H dimension, excitation frequency in the 13C dimension and the spectral windows in the two dimensions are determined specifically for each type of analysis and that each interferogram (FID) is multiplied by a square sinusoidal function to increase the signal / noise ratio and undergoes a Fourier transform in magnitude mode in the case of the HMBC-GAS and in amplitude mode,   then phase, in the case of HMQC or HMQC-HOHAHA.   7. Method according to one of the claims   in which the correlation spots of the NMR analyzes are then integrated and the data obtained are analyzed. multidimensional statistics.   8. Method according to one of the claims   precedence, characterized by an analysis of variance (ANOVA), and / or principal component analysis (PCA), and / or a factor analysis discriminant (AFD).   9. Method according to one of the claims   previous, characterized in that it is carried out on the total polyphenolic extracts of a plant material of the species to be studied, or a product made from   this species or a plant organ.   10. Method according to claim 9, characterized in that the plant material comes from species with plant organs rich in polyphenolic compounds or tannins, such as vine, pine, oak, walnut, fruit trees , from   floral varieties, or medicinal plants.   11. The method of claim 10, characterized in that the plant organs are leaves, seeds, seeds and, where appropriate, barks, or petals.   12. Method according to claim 9, characterized in that the product made from the plant material is fruit juice, or products   from their fermentation, such as wine or beer.   13. Method according to one of the claims   preceding, characterized in that it is used to establish a repository by accumulation of results of analyzes performed, operating according to the provisions   defined in the foregoing claims, on a   material or a product of a plant species or a given hybrid, or lower-ranking taxa of that plant species or hybrid, the materials or   products being treated under identical conditions.   14. Process for the control of the origin and / or the quality of a plant species, a hybrid, or lower-ranking taxa of this plant species or of this hybrid, characterized in that an extract is submitted from a plant organ, or a product made from it, or from the plant species or hybrid, or lower-ranking taxa, to NMR analysis, followed by processing of NMR as   defined in one of claims 1 to 12, and the   comparison of the results with the data of a reference system corresponding to the plant species, the hybrid, or the lower-ranking taxa of this plant species or this hybrid, to which the element to be analyzed is supposed to belong.   15. Process according to any one of   preceding claims, characterized in that it is   applied to the identification of grape varieties and vine clones.