Article pp.394-408 du Vol.26 n°5 (2006)

(1)

ARTICLE ORIGINAL ORIGINAL PAPER

Separation and Identification

of Phenolic Compounds in Jerusalem Artichoke (Helianthus tuberosus L.)

M. Tchoné

¹

, G. Bärwald

²

, G. Annemüller

³

, L.-G. Fleischer

^*

RÉSUMÉ

Séparation et identification des composés phénoliques du topinambour (Helianthus tuberosus L.)

Plusieurs méthodes chromatographiques telles que la colorimétrie, la chromatographie liquide à haute performance en phase inversée (RP-HPLC), le couplage de la chromatographie liquide et de la chromatographie en phase gaseuse à la spectrométrie de masse (LC-MS, GC-MS) ont été expérimentées et au besoin modifiées et adaptées pour la séparation, l’identification et la quantification des composés phénoliques dans différentes variétés de Topinambour. Vingt-deux composés phénoliques ont été identifiés. L’acide gallique (de 1 mg à 140 mg) ; l’acide protocatéchique (de 5 mg à 200 mg) ; l’esculine (de 4 mg à 270 mg) ; l’acide gentisique (de 30 mg à 3 g), la catéchine (de 1 mg à 300 mg), l’acide 4-hydroxybenzoïque (de 1 mg à 90 mg), l’acide chlorogénique (de 20 mg à 5 g), l’acide vanillique (de 1 mg à 520 mg), l’acide syringique (de 1 mg à 40 mg), l’acide caféique (de 1 mg à 240 mg), l’épicatéchine (de 4 mg à 800 mg), l‘acide 2-hydroxy-3-5-dinitrobenzoïque (de 2 mg à 140 mg), l’umbelliferone (de 2 mg à 110 mg), scopolétine (de 1 mg à 80 mg), l’acide p-coumarique (de 1 mg à 40 mg), l’acide coumarique-3-carbon (de 1 mg à 40 mg), l’acide ferulique (de 1 mg à 40 mg), l’acide sinapique (de 1 mg à 60 mg), l’acide 3-hydroxycinnami- que (trace) ; l’acide ellagique (de 2 mg à 40 mg), 4-hydroxycoumarine (de 4 mg à 300 mg) et l’acide salicylique (de 30 mg à 7 g). Toutes ces teneurs sont expri- mées pour 100 g de matière sèche. Parmi les composés phénoliques identifiés, vingt possèdent des vertus thérapeutiques.

Mots clés

topinambour, composés phénoliques, LC-ESI-MS, GC-CI-MS, HPLC, acide sali- cylique.

1. Dr.-Ing. Michel Tchoné – Technische Universität Berlin – Fakultät für Prozesswissenschaften – Sekr. ZI-3 – Amrumer Strasse 32, D-13353 Berlin – Email: tchonem@hotmail.com

2. Prof. Dr.-Ing. G. Bärwald – Technische Universität Berlin – Fakultät für Prozesswissenschaften – Sekr.

ACK 25 Ackerstrasse 76, D-13355 Berlin

3. Prof. Dr. sc. techn. G. Annemüller – Technische Universität Berlin – Fakultät für Prozesswissenschaften –

(2)

SUMMARY

Several methods such as colorimetry, reversed-phase high performance liquid chromatography (RP-HPLC), the coupling of liquid chromatography and gas chromatography with mass spectrometry (LC-MS, GC-MS) were examined for their suitability and applied to the separation, identification and quantification of phenolic compounds in different varieties of Jerusalem artichoke. Twenty two phenolic compounds have been separated, identified and quantitated as gallic acid (from 1 mg up to 140 mg), protocatechuic acid (from 5 mg up to 200 mg), esculin (from 4 mg up to 270 mg), gentisic acid (from 30 mg up to 3 g), catechin (from 1mg up to 300 mg), 4-hydroxybenzoic acid (from 1 mg up to 90 mg), chlorogenic acid (from 20 mg up to 5 g), vanillic acid (from 1 mg up to 520 mg), syringic acid (from 1 mg up to 40 mg), caffeic acid (from 1 mg up to 240 mg), epicatechin (from 4 mg up to 800 mg), 2-hydroxy-3-5-dinitrobenzoic acid (from 2 mg up to 140 mg), umbelliferon (from 2 mg up to 110 mg), scopoletin (from 1 mg up to 80 mg), p-cumaric acid (from 1 mg up to 40 mg), cumaric-3-carbon acid (from 1 mg up to 40 mg), ferulic acid (from 1 mg up to 40 mg), sinapic acid (from 1 mg up to 60 mg), 3-hydroxycinnamic acid (trace) ; ellagic acid (from 2 mg up to 40 mg), 4-hydroxycumarin (from 4 mg up to 300 mg) and salicylic acid (from 30 mg to 7 g). These phenolics contents are expressed for 100 g tubers or skins dry weight.

Twenty of the identified phenolic compounds are of interest in the medicine and with diets.

Keyworks

Jerusalem artichoke, phenolic compounds, GC-CI-MS, LC-ESI-MS, HPLC, salicylic acid.

1 – INTRODUCTION

Various functions and actions have been attributed to phenolic compounds, making determination of their concentration in foods highly desirable. They have been shown to play vital physiological roles. The suggested advantageous health effects (maintenance of health and protection from diseases such as cancer and coronary heart disease) of plant phenolics and the possibility to use antioxidant plant constituents as food ingredients has motivated plant phenolics research.

Several studies concerning phenolic compounds in wine, juice, fruit and vegetables have been published (FRANKEL et al. 1995, DAWES and KEENE, 1999, HÄKKINEN et al. 1999, KARADENIZ et al. 2000, OWEN et al. 2000, SCHLESIER et al. 2001, JEN-KUN et al.1998). Many analytical methods have been used for their determination. The classical method for determination of phenolic compounds is the colorimetric procedure, which uses the Folin-Ciocalteu reagent. However, this method is characterized by poor specificity, as other compounds present in the matrix such as Fe^II, ascorbic acid, glucose, urea, sulphite, nucleic acid fragments and amino acid such as cysteine, tryptophan and tyrosine, free SO₂ may contribute to the absorbance (SCHOLTEN and KACPROWSKI, 1993, MÖBIUS and GÖRTGES,1974). Moreover, it is not possible to quantify the individual phenols because the Folin-Ciocalteu procedure evaluates total phenols. Procedures that provide for the separation and quantitative

(3)

determination of individual phenolic compounds by either gas chromatography or liquid chromatography are much more satisfactory. Ultraviolet detection has been used extensively in the detection of phenolic compounds (ROMANI et al. 1999, HÄK- KINEN et al. 1999, HOHL et al. 2001, FRIEDMAN, 1997, CECCON et al. 2001, CZYZOWSKA and POGORZELSKI, 2002). Reverse-phase HPLC often achieves excellent separations of nonvolatile polar chemicals from complex mixtures under conditions that allow for the isolation of thermally labile compounds, but if unknown compounds are separated, their identification will be difficult becauce it does not provide informations about structure and molecular weight of the analyte. Gas chromatography is a fast, efficient and accurate technique, but it requires a derivation step due to the thermally labile compounds. The needs for alternative analytical methods are therefore obvious. The combination of liquid chromatography and gas chromatography with mass spectrometry is considered to be such alternative.

Applications of modern GC- and LC-MS include environmental analysis, foren- sics, drug testing, and pharmacological studies.

These techniques have been used successfully for the analysis of plant phenolics as well as several compounds isolated from biological mixtures (GELBMANN et al.

1997, HARTL and HUMPF, 1999, SCHLÖSSER et al. 1998, DERUITER et NOGGLE, 1998, SEWRAM et al. 1999).

This study reports for the first time the application of liquid chromatography- electrospray ionization (ESI) mass spectrometry and gas chromatography-chemical ionization (CI) mass spectrometry for the qualitative and quantitative determination of phenolic compounds in nine varieties of Jerusalem artichoke. The total phenolic contents as well as the effect of the harvest period on phenolics content are also monitored.

2 – MATERIAL AND METHODS

2.1 Reagents

The standards used throughout this investigation were catechin, chlorogenic acid, vanillic acid, epicatechin, umbelliferone, p-cumaric acid, ferulic acid, gallic acid monohydrate (from Roth, Karlsruhe, Germany), 4-hydroxibenzoic acid, 2-hydroxi-3- 5-dinitrobenzoic acid, (from Merck, Darmstadt, Germany), protocatechuic acid, esculin, gentisic acid, syringic acid, scopoletin, cumaric-3-carbon acid, sinapic acid, 3-hydroxicinnamic acid, ellagic acid, 4-hydroxicumarin and salicylic acid (from Fluka, Sigma-Aldrich Chemie GmbH, Germany) and caffeic acid (from Serva Feinbi- ochemica, Heidelberg, Germany).

(4)

3 – METHODS

3.1 Extraction of phenolic compounds

Fresh tubers of Jerusalem artichoke were cleaned under cold water with a scrub brush prior using. A sample of 50 g tubers or skins were homogenized in a Waring

“blendor” with 100 ml extracting solution (ethyl acetate-methanol 1:1 (v/v)) for 5 min.

The glass beaker of the “blendor” was rinsed with an additional 10 ml of extracting solution. The homogenate was heated with the blendor’s beaker in a ultra sound bath at 80°C for 10 min followed by pressing with a mechanical press after the homogenisation. The resulting residue was mixed with an additional 100 ml extracting solution, heated with the Blendor beaker in a ultra sound bath at 80°C for 10 min and pressed. The second residue was mixed with an additional 50 ml of the extracting solution and treated in the same way. The extracts were combined and the solution was allowed to cool before it was made up to final volume of 250 ml.

This extract was concentrated under vacuum and rinsed with methanol to 50 ml final volume prior determination of total phenolics or HPLC analyses.

3.2 Effect of the harvest period on phenolics content

Jerusalem artichoke tubers (Gigant, Gute Gelbe, Medius Brückmann, Medius Lindhoop, Large White, Petit Blanc, RoZo, Stamm and Waldspindel varieties, 2000 harvest) were purchased from German farmers and used throughout this investigation. The nine varieties of Jerusalem artichoke were kept after harvest in our laboratory under garden soil. For each variety tuber samples were picked at the beginning, after 12, 21, 33 and 52 days. The skins from each sample were subjected to the phenolic compounds extraction according to our developed method as described above.

4 – QUALITATIVE AND QUANTITATIVE ANALYSIS

4.1 LC-MS

A Hewlett-Packard model 1100 HPLC system equipped with G1312 A binary pump, G1313 A autosampler, G1322 A degasser and G1315 A diode array detector was used for the liquid chromatography. Chromatographic separation was performed on a Hypersil LichroCART^® 250-4 HPLC column and Nucleosil^® LichroCART^® 4x4 HPLC pre-colomn by scanning from 210 nm to 360 nm on Hewlett Packard system.

The mobile phase consisted of acetic acid (3.5 % in water) (A) and acetonitrile (B).

Concentration of the latter solvent was mixed to produce a flow rate of 1 ml/min with 16 min B, 5-25%; 5 min B, 25%; 10 min B, 25-5% and 15 min B, 5%.

Detection and quantification were carried out using external standards in methanolic solution.

ESI-MS (Electrospray ionization mass spectrometry) were recorded on a micro- mass platform LCZ system equipped with an electrospray ionization source and a Waters 2690 separation module.

(5)

4.2 GC-MS

GC analysis was performed on a Hewlett Packard gas chromatograph 5890 Series II equipped with a 30 m long, 0.25 mm id, 0.25 µm film thickness HP1-MS capillary column linked to a triple quadrupole Finnigan type TSQ 700 mass spec- trometer system equipped with an EI/CI combination ion source, digital alpha sta- tion, ICIS 8.3 data system and with a Finnigan ESI ion source. The column temperature setting was programmed to begin at 80°C for 2 min, then increase at the rate of 17°C/min to 280°C. The injection temperature was 250°C. Helium was used as a carrier gas with a constant flow at 1.5 ml/min. Samples were ionized by solvent-mediated chemical ionization (CI) induced by a discharge electrode held at a potential of 1000 V.

In GC-MS the TIC is registered instead of a conventional GC detector trace and one can obtain mass spectra for all GC peaks, thus allowing identification and/or quantitation of most components of a mixture. Further help to detect a specific component may be obtained by plotting of RICs of the expected molecular weight and/or of some fragment ions from the data.

The identification of individual phenolic compounds was carried out by co-injec- tions of references compounds with the samples and also by comparison of their mass spectra and retention times (RT) with those of standards. The standards and samples were derivatized using N, O-bis (trimethylsilyl)-trifluoroacetamide at room temperature prior gas chromatographic analysis.

5 – RESULTS AND DISCUSSION

Table 1 shows the highest total phenolics content of Jerusalem artichoke tubers from different varieties. Slight differences among the varieties were found in their total phenolics content (from 4 to 6 g phenolic compounds/100 g tuber dry weight and from 19 to 23 g phenolic compounds /100 g skins dry weight. The skins con- tained the greatest quantities of total phenolic compounds. The highest total phenolic contents occurred in the skin extract of the RoZo variety (23 g phenolic compounds/100 g skins dry weight) (table 1).

These results suggest that the phenolics compounds are localized in the external cell wall of Jerusalem artichoke. Quantitative data on the phenolics in Jerusalem artichoke are not available. Only qualitative statement have been found in the litera- ture (IBRAHIM et al. 1971, PAUPARDIN and GAUTHERET, 1965). Data about total content of phenolic compounds in fruits and vegetables are seldom published.

According to the investigations from Böhm and co-workers (BÖHM et al. 1999), the amounts of the total phenolics expressed as gallic acid equivalents, are 63 g phenolic compounds/ kg red grapes and 19 g phenolic compounds/kg black carrot. The content of phenolics compounds of Jerusalem artichoke, expressed as gallic acid equivalent (our own investigations) varies from 26 to 59 g phenolic compounds/ kg tubers dry weight.

(6)

Table 1

Highest contents of total phenolicsof Jerusalem artichoke from different varieties expressed as salicylic acid equivalent in methanol.

Data in g salicylic acid / 100 g skins or tubers dry weight.

Tableau 1

Teneur en composés phénoliques totaux de plusieurs variétés de Topinambour exprimées en g d’acide salicylique pour 100 g de matière sèche de tubercules

ou d’épluchures : les valeurs les plus élevées sont données.

5.1 Phenolic composition of Jerusalem artichoke

The aim of the experimental work was to know the phenolic composition of nine varieties of Jerusalem artichoke and the effect of the harvest period on their phenolic content. Figure 1 shows a representative LC-MS chromatogram of of the phenolic compounds in the methanolic mixture of 22 standards.

All the phenolic compounds separated in the standards mixture were also found in the skin extract of Jerusalen artichoke. In the skin extract of Jerusalem artichoke dominated protonated molecules [M+H]⁺. Some of them were not identified due to the complexity of the extract. All the standards yielded protonated molecules.

Figure 2 shows an example of GC-MS chromatogram (CI mode) from a mixture containing 22. Different molecular ions were present in the skin extracts of Jerusa- lem artichoke. The protonated molecules [M+H]⁺ were dominant but [M+4]⁺ ions (rest of reagent gas or gain of 4H), [M+15]⁺ ions (gain of CH₃), [M-15]⁺ ions (loss of CH₃) and [M-18]⁺ ions (loss of one molecule water) were also present in CI spectra due to the thermal decomposition and additional fragmentation.

The melting-point and solubility of the standards used are given in table 2. All the phenolic compounds used in this work as standards remained stable as shown in table 2 at the temperatures ranges over 100°C ; some are insoluble in water.

As shown in table 2, some standards used are decomposed at the temperature range over 202°C. The derivatization step has to be optimized to avoid fragmentation in the ion source and decomposition during the evaporation of the analyte prior to ionization. This evaporation should be done at the temperatures under the melting point.

PAUPARDIN and GAUTHERET, 1965 have found only seven phenolic compounds in Jerusalem artichoke tubers. The LC-MS and GC-MS techniques allowed the qualitative and quantitative determination of phenolic compounds listed in table 3.

Varieties Tubers extract Skins extract

RoZo 4.1 23.1

Stamm 4.8 20.6

Gigant (Topianka) 3.5 19.1

Medius Lindhoop 4.9 19.9

Medius Brückmann 8.0 19.9

Gute Gelbe 6.1 19.1

Petit Blanc 5.4 20.4

Large White 5.5 21.0

Waldspindel 5.4 21.1

(7)

3.26: gallic acid – 4.79: protocatechuic acid – 7.43: esculin – 8.21: gentisic acid – 8.48: 4-hydroxybenzoic acid 8.91: catechin – 9.46: chlorogenic acid – 11.64: caffeic acid – 11.84: epicatechin – 13.43: umbelliferon 13.78 scopoletin (7-hydroxy-6-methoxycumarin), p-cumaric acid

15.19: ferulic acid, sinapic acid, 3-hydroxycinnamic acid – 16.49: salicylic acid

Figure 1

LC-MS separation of individual phenolic compounds from a mixture of 22 standards in methanol.

Figure 1

Séparation par LC-MS des composés phénoliques d’un mélange méthanolique de 22 standards.

(8)

180: gallic acid – 194: cumarin-3-carbonic acid – 241: 4-hydroxycumarin – 276: 4-hydroxybenzoic acid 317: umbelliferon (7-hydroxycumarin) – 417: salicylic acid – 458: vanillic acid – 479: syringic acid 519: chlorogenic acid

Figure 2

Phenolic compounds in a methanolic mixture of 22 standards: GC-MS chromatogram in CI mode.

Figure 2

Composés phénoliques d’un mélange méthanolique de 22 standards : Chromatogramme GC-MS en mode CI.

(9)

Table 2

Melting-point and solubility of the standards used from Merck and Fluka catalog.

Tableau 2

Point de fusion et solubilité des substances de référence utilisées commercialisées par Merck et Fluka.

Standards Melting-point

in °C Observations

Solubility in water at 20 °C (g/ l)

Gallic acid 255-265 15

Gentisic acid 202-206 Soluble

Protocatechuic acid 202-204 Decomposition 20

4-hydroxybenzoic acid 212-215 Ignition temperature 8

Salicylic acid 158-161 2

Vanillic acid 208-210 Hardly soluble

Syringic acid 204-207 Insoluble

Ellagic acid > 350

Caffeic acid 234-237 Decomposition Hardly soluble

Ferulic acid 169-175

Sinapic acid 195-200 Decomposition, 202°C

Chlorogenic acid 203-205 Decomposition, 208-210°C

3-hydroxycinnamic acid 192-195 Hardly soluble

p-cumaric acid 219-222 Decomposition, 214-217°C Hardly soluble

4-hydroxycumarin 211-214 Hardly soluble

Umbelliferone (7-hydroxycumarin) 226-229 Hardly soluble

Cumarin-3-carbonic acid 188-191 13

Esculin (6,7-dihydroxycumarin-6 200 1.5

Beta-D-glucopyranosid) Scopoletin (7-hydroxy-6-

methoxicumarin) 204-206

2-hydroxi-3-5-dinitrobenzoic acid 170-174 Soluble

(3,5-dinitrosalicylic acid)

Catechin 200 Decomposition

Epicatechin 240-245 Decomposition

(10)

Table 3

Content of specific phenolic compounds in methanolic skin extracts of different Jerusalem artichoke varieties expressed as g phenolic compound/100 g skin dry weight:

highest values are given.

Tableau 3

Teneur en composés phénoliques individuels des extraits méthanoliques d’épluchures de différentes variétés de Topinambour exprimée en g de composés phénoliques pour 100 g de matière sèche d’épluchures : Les valeurs maximales sont données.

Twenty two phenolic compounds have been separated, identified and quantitated as gallic acid, protocatechuic acid, esculin, gentisic acid, catechin, 4-hydroxybenzoic acid, chlorogenic acid, vanillic acid, syringic acid, caffeic acid, epicatechin, 2-hydroxy-3-5-dinitrobenzoic acid, umbelliferon, scopoletin, p-cumaric acid, cumaric-3-carbonic acid, ferulic acid, sinapic acid, 3-hydroxycinnamic acid, ellagic acid, 4-hydroxycumarin and salicylic acid.

From the identified phenolic compounds 15 were determined for the first time in Jerusalem artichoke: gallic acid, protocatechuic acid, esculin, catechin, syringic acid, epicatechin, 2-hydroxy-3-5-dinitrobenzoic acid, umbelliferon, scopoletin, cumaric-3-carbonic acid, sinapic acid, 3-hydroxycinnamic acid, ellagic acid, 4- hydroxycumarin and salicylic acid. Salicylic acid was quantitatively the main phenolic compound found in all of varieties under investigation. Chlorogenic acid is the main phenolic compound in apple juice, apple wine and apple vinegar (MÜLLER and

Compounds Gigant Gute Gelbe Large White Medius B. Medius L. Petit

Blanc RoZo Stamm Waldspindel

Gallic acid 0,026 0,005 0,02 0,016 0,01 0,012 0,05 0,013 0,14 New

Protocatechuic acid 0,116 0,13 0,08 0,09 0,09 0,15 0,2 0,09 0,14 New

Esculin 0,093 0,11 0,15 0,18 0,12 0,15 0,14 0,25 0,27 New

Gentisic acid 0,95 0,52 0,33 2,78 0,4 1 0,7 0,75 0,63

Catechin 0,035 0,06 0,07 0,02 0,09 0,07 0,06 0,3 0,06 New

4-hydroxybenzoic acid 0,09 0,03 0,03 0,02 0,04 0,04 0,03 0,07

Chlorogenic acid 1,15 0,86 0,66 1 1 0,89 1,17 2,4 4,51

Vanillic acid 0,023 0,02 0,05 0,02 0,05 0,52 0,02 0,02 0,07

Syringic acid 0,03 0,008 0,02 0,04 0,009 0,02 New

Caffeic acid 0,019 0,02 0,04 0,02 0,07 0,24 0,06 0,16 0,05

Epicatechin 0,48 0,25 0,2 0,4 0,42 0,22 0,46 0,8 0,26 New

2-hydroxy-3-5-dinitrobenzoic acid 0,04 0,04 0,1 0,06 0,14 0,03 0,07 0,01 New

Umbelliferon (7-hydroxycumarin) 0,07 0,08 0,07 0,045 0,07 0,09 0,11 0,11 0,09 New

Scopoletin (7-hydroxy-6-methoxycumarin) 0,05 0,06 0,02 0,03 0,05 0,05 0,03 0,08 0,08 New

p-cumaric acid 0,03 0,02 0,02 0,02 0,01 0,02 0,03 0,04 0,022

Cumarin-3-carbonic acid 0,02 0,008 0,02 0,01 0,04 New

Ferulic acid 0,02 0,013 0,01 0,03 0,008 0,03 0,04 0,02

Sinapic acid 0,02 0,03 0,012 0,02 0,008 0,06 0,02 0,06 0,06 New

3-hydroxycinnamic acid 0,009 0,0001 trace 0,007 0,003 0,004 New

Ellagic acid 0,007 0,03 0,02 0,026 0,04 0,02 0,02 0,03 0,04 New

Salicylic acid 2,62 3,2 2,71 2,54 4,5 3,6 4,37 6,5 4,95 New

4-hydroxycumarin 0,22 0,15 0,12 0,12 0,2 0,15 0,21 0,27 0,3 New

(11)

TREUTTER, 2001). The neo-chlorogenic acid is abundant in cherry and blackcurrant wines (CZYZOWSKA and POGORZELSKI, 2002). Gallic acid is no more detectable in a finished wine from normal treated grapes (HENNIG and BURKHARDT, 1958).

These examples confirm that the reference compound depends on the substra- tum used.

Examining table 3, one can see that 4-hydroxybenzoic and 2-hydroxy-3-5-dinitrobenzoic acid were not found in the skin extract of the Jerusalem artichoke Stamm variety. Syringic acid was not found in Gigant, Medius Brückmann and Waldspindel varieties. Cumarin-3-Carbonic acid was not found in Gigant, RoZo, Medius Brück- mann and Waldspindel varieties. Ferulic acid was not found in the sort Gigant. 3- Hydroxycinnamic acid was not found in RoZo, Waldspindel and Gute Gelbe varieties. There are no reports on the specific phenolics content of Jerusalem artichoke.

5.2 Changes in phenolic compounds in Jerusalem artichoke grown in laboratory conditions

The study on the effect of the growth period on phenolics content in Jerusalem artichoke grown in laboratory has shown that the phenolic content varied qualita- tively and quantitatively with the growth period. Except for the cultivars RoZo and Waldspindel, it increased during the growing period, reached a maximum and decreased. This variation depends on growing conditions and stage of development of tubers. Table 4 shows the highest amount of total phenolics, expressed as sum of specific phenolic content.

Table 4

Effect of the growth period in laboratory on highest amount of total phenolics, expressed as sum of specific phenolic content (g phenolic compounds/100 g skins dry weight).

Tableau 4

Influence de la période de développement sur la teneur maximale exprimée comme somme des composés phénoliques individuels en g de composés phénoliques

pour 100 g d’épluchures, poids sec.

Great variations in phenolic content were observed among the varieties studied (table 4). The red varieties Stamm and Waldspindel yielded the highest phenolic contents. The changes in polyphenols were closely related to their chemical struc- tures, degree of the metabolism activation, variation of the moisture, specific electri- cal conductibility, and pH in the tubers.

Previous results have also shown a positive effect of the light intensity on phe-

0 day 12 days 33 days

RoZo (6) Gigant (8) Gute Gelbe (5)

Waldspindel (10) Large White (4) Petit Blanc (6)

Medius Brückmann (5) Medius Lindhoop (6) Stamm (10)

(12)

Flavonols and anthocyanins that are also present in fruits and vegetables skins could’t be determinated with the methods described and would need another method or column.

All the phenolic compounds found in Jerusalem artichoke are except cumarin-3- carbonic and 2-hydroxy-3-5-dinitrobenzoic acids antioxidants and potential bioac- tive compounds. The antioxidant activities of catechin, caffeic, ellagic, p-coumaric, ferulic and chlorogenic acids were determinated by Meyer and co-workers (MEYER et al. 1998 a, b). In addition, those of 4-hydroxycoumarin, umbelliferon and scopole- tin were reported by Foti and co-workers (FOTI et al. 1996). Tannins, particulary gallic acid and cathechin, exhibits an antioxydative activity on linoleic acid (CHUANG-YE et al. 1995, TAKUO et al. 1983, RIGO et al. 2000). Other investigations have shown that gallic, ferulic protocatechuic and caffeic acids exhibited a peroxy radical scavenging capacity (TAKAHASHI et al. 1999, WALTERS et al.1996). The radical scavenging capacity of caffeic and chlorogenic acids has been found to be stronger than those of α– tocopherol and buthylhydroxytoluen (CHEN and HO, 1997). Salicylic acid exhibits an antimicrobial effect on E. coli K12AM, Staphylococcus aureus IAM1011 and Bacillus substilis IAM 1521 (NGUYEN et al. 1982). The two methoxyl groups and the hydroxyl group in sinapic acid have been found to be effective for antibacterial activity against E. coli (TESAKI et al. 1998).

6 – CONCLUSION

A method for the extraction of phenolic compounds from vegetables was developed during this work. Up to 23 g phenolic compounds/100 g skins dry weight could be extracted from the skins of the Jerusalen artichoke sort RoZo. This result is a real progress compared to the process described by Paupardin and Gautheret (1965) till now. The method was with a variation coefficient of 6% reproducible.

Hereby is a suitable extractant to be used because some phenolic compounds such as vanillic, caffeic, 3-hydroxycinnamic, p-coumaric, 4-hydroxycoumaric acid and umbelliferone are either hardly soluble or insoluble in water.

The measurement of Jerusalem artichoke phenolic contents shows that this tuber is rich in phenolic compounds among them salicylic acid is the major component identified. The phenolics compounds in Jerusalem artichoke are mainly extracted from the skins. A new general problem to be resolve result in an efficient utilisation of distiller wash and waste water which are rich in the identified phenolics.

Distiller wash is for example used till now either in the agriculture as fertiliser or ani- mals food. The biological disintegration of phenolic compounds is certainly limited because a lot of them might exhibit an antimicrobial activity. Further more, phenolic antioxidants (up to 2 g/l) have been found in the waste water from an Italian olive oil mill (ROBLES, 2000). The manufacture of fruit and vegetable juices produces enor- mous quantities of residues (up to one third of the raw material quantity), which are rich in phenolic compounds and other precious nutrients. An additional option is to produce phenolic antioxidants from those residues. So the developed procedure of extraction can be integrated in an existing process. The solvent used should be evaporated under vacuum, adjusted and used again in order to avoid further environmental load. The phenolics produced either as concentrate or flour may serve in

“functional foods” as natural food ingredients (antioxidant or coloring sbstances).

(13)

Phenolic compounds that appear to have desirable antioxidant and medicinal properties were found in Jerusalem artichoke. Some have been reported to be anti- tumor agents and to exhibit antiviral and antimicrobial activities, hypotensive effects and antioxidant properties.

Among the identified 22 phenolic compounds, 15 were found for the first time in this plant. The use of these compounds as substances for food preservation should be profitable. Jerusalem artichoke powder could be used as antioxidants or supple- ment in other powders for example potatoe powder.

Chemical ionization is a relatively soft ionization method and in fact, it represents the first soft ionization introduced to mass spectrometry, but the derivatization and the evaporation of the analyte prior to ionization remain the critical steps.

7 – ABBREVATIONS USED

°C: Celsius degree CI: chemical ionization ESI: electrospray ionization

GC-MS: gas chromatography coupled with mass spectrometry LC-MS: liquid chromatography coupled with mass spectrometry M: molecular mass

min: minute nm: nanometer

m/z: mass-to-charge ratio

RIC: reconstructed ion chromatogram

RP-HPLC: reversed-phase-high performance liquid chromatography RT: retention time

TIC: total ion current or total ion chromatogram u: atom mass unit

UV: ultra violet V: volt

ACKNOWLEDGEMENT

The collaboration of the following is acknowledged: Dr. Martin Steiof, Dr. Buhr,

(14)

REFERENCES

BÖHM V., SCHLESIER K., BITSCH R., 2000. Kritis- che Betrachtung der protektiven Wirkung von Frucht- und Gemüsekonzentraten. Lebensmittel, 3, 280-282.

CECCON L., SACCÙ D., PROCIDA G., CARDINALI S., 2001. Liquid chromatographic determination of simple phenolic compounds in waste waters from olive oil production plants. Journal of the Association of Official Agricultural Chemists International, 84, 6, 1739-1744.

CHEN J. H., HO C. T., 1997. Antioxidant activities of caffeic acid and its related hydroxycinnamic compounds. Journal of Agricultural and Food Chemistry, 45, 2374-2378.

CHUANG-YE W., CHEIN-PING W., SHIAN-SUO H., FENG-LIN H., 1995. The inhibitory effect of tannins on lipid peroxidation of rat heart mitochondria. Journal of Pharmacy and Pharmacology, 47, 138-142.

CZYZOWSKA A., POGORZELSKI E., 2002. Changes to polyphenols in the process of production of must and wines from blackcurrants and cher- ries. Part I. Total polyphenols and phenolic acids. European Food Research and Techno- logy, 214, 2, 148-154.

DAWES H. M., KEENE J. B., 1999. Phenolic composition of kiwi juice. Journal of Agricultural and Food Chemistry, 47, 2398-2403.

DERUITER J., NOGGLE F. T., 1998. Gas chromatographic-mass spectrometric and high-performance liquid chromatographic analyses of the bromination products of the regiosomeric dimethoxyphenethylamine: differentiation of nexus from five positional isomers. Journal of Chromatographic Sciences, 36, 23-28.

FRANKEL E. N., WATERHOUSE A. L., TEISSEDRE P. L., 1995. Principal phenolic phytochemicals in selected california wines and their antioxidant activity in inhibition oxidation of Human Low- Density Lipoproteins (LDL). Journal of Agricultu- ral Food Chemistry, 43, 890-894.

FOTI M., PIATTELLI M., BARATA M. T., RUBERTO G., 1996. Flavonoids, coumarins, and cinnamic acids as antioxidants in a micellar system.

Structure-activity relationship. Journal of Agri- cultural and Food Chemistry, 44, 497-501.

FRIEDMAN M., 1997. Chemistry, biochemistry, and dietary role of potato polyphenols. Journal of Agricultural and Food Chemistry, 45, 1523-1540.

GELBMANN D., PRAECEPTOR A., SALZBRUNN W., EDER R., 1997. Quantitative Bestimmung flüchtiger Phenole in Rotweinen mittels Gas- chromatographie-Massenspektroskopie. Mittei- lungen Klosterneuburg, 47, 95-103.

HÄKKINEN S. H., KÄRENLAMPI S. O., HEINONEN I.

M., MYKKÄNEN, H. M., TÖRRÖNEN A. R., 1999. Content of the flavonols quercetin, myri-

cetin, and kaempferol in 25 edible berries. Jour- nal of Agricultural and Food Chemistry, 47, 2274-2279.

HARTL M., HUMPF H. U., 1999. Simultaneous determination of fumonisin B₁and hydrolyzed fumonisin B₁ in corn products by liquid chromatography-electrospray ionization mass spectrometry. Journal of Agricultural and Food Chemistry, 47, 5078-5083.

HENNIG K., BURKHARDT R., 1958. Der Nachweis phenolartiger Verbindungen und hydroaromatis- cher Oxycarbonsäure in Traubenbestandteilen, Wein und weinähnlichen Getränken II. Weinberg und Keller, 5, 593-600

HOHL U., NEUBERT B., PFORTE H., SCHONHOF I., B÷HM H., 2001. Flavonoid concentrations in the inner leaves of head lettuce genotypes. Euro- pean Food Research and Technology, 213, 205- 211.

IBRAHIM R. K., THAKUR M. L., PERMANAND B., 1971. Formation of anthocyanins in callus tissue cultures. Lloydia, 34, 2, 175-182.

JEN-KUN L., CHIH-LI L., YU-CHIH L., SHOEI-YN LIN-SHIAU I-MING J., 1998. Survey of catechins, gallic acid, and methylxanthines in green oolong, puerch, and black teas. Journal of Agricultural Food Chemistry, 46, 3635-3642.

KARADENIZ F., DURST R. W., WROLSTAD R. E., 2000. Polyphenolic composition of raisins. Jour- nal of Agricultural and Food Chemistry. 48, 5343-5350.

MEYER A. A., HEINONEN M., FRANKEL E. N., 1998 a.

Antioxidant interactions of catechin, cyanidin, caffeic acid, quercetin, and ellagic acid on human LDL oxidation. Food Chemistry, 61, 1/2, 71-75.

MEYER A. S., DONOVAN J. L., PEARSON D. A., WATERHOUSE A. L., FRANKEL E. N., 1998 b.

Fruit hydroycinnamic acids inhibit human low- density lipoprotein oxidation in vitro. Journal of Agricultural and Food Chemistry, 46, 1783-1787.

MÖBIUS C. H., GÖRTGES S., 1974. Polyphenolbes- timmung für die Praxis. Die Weinwissenschaft, 29 (5), 241-25.

MÜLLER C., TREUTTER D., 2001. Phenolische Ver- bindungen in Apfelsaft, Apfelwein und Apfeles- sig. Mitteilungen Klosterneuburg, 51, 138-147 NGUYEN, V. C., KURATA T., KATO H., FUJIMAKI M.,

1982. Antimicrobial activity of kumazasa (Sasa albo-marginita). Agricultural and Biological Che- mistry, 46, 4, 971-978.

OWEN R. W., MIER W., GIACOSA A., HULL W. E., SPIEGELHALDER B., BARTSCH H., 2000. Phe- nolic compounds and squalene in olive oils: the concentration and antioxidant potential of total phenols, simple phenols, scoiridoids, ligans and squalene. Food and Chemical Toxicology, 38, 647-659.

(15)

PAUPARDIN C., GAUTHERET R., 1965. Sur la nature des acides–phénols présents dans les tissus de tubercules de Topinambour ( Helianthus tubero- sus L., varieté Violet de Rennes) cultivés in vitro.

Comptes Rendus des Séances de l’Académie des Sciences, 261, 4206-4208.

RIGO A., VIANELLO F., CLEMENTI G., ROSSETTO M., SCARPA M., VRHOV≥EK U., MATTIVI F., 2000. Contribution of proanthocyanidins to the peroxy radical scavenging capacity of some italian red wines. Journal of Agricultural and Food Chemistry, 48, 1996-2002.

ROBLES A., 2000. “Phenoloxidase activity in strains of the hyphomycete Chalara paradoxa isolated from olive mill wastewater disposal ponds”. Enzyme and Microbial Technology, 26, 484-490.

ROMANI A., PINELLI P., MULINACCI N., VINCIERI F.F., TATTINI M., 1999. Identification and quantification of polyphenols in leaves of Myrtus com- munis L.. Chromatographia, 49, 1/2, 17-20.

SCHLESIER K., BÖHM V., BITSCH R., 2001. Unters- chiede im protektiven Potential von Grün- und Schwarztee. Ernährung im Fokus, 2, 2-4.

SCHLÖSSER J., MEHLICH A., BALLWANZ F., PETZ M., 1998. Preparation of doubly ¹³C-labelled benzylpenicillin as internal standard for residue analytical use with GC-MS and LC-MS. Frese- nius Journal of the Analytical Chemistry, 360, 498-501.

SCHOLTEN G., KACPROWSKI M., 1993. Zur Analy- tik von Polyphenolen in Wein. Die Weinwissens- chaft, 48, 33-38.

SEWRAM V., NIEUWOUDT T. W., MARASAS W. F.

O., SHEPHARD G. S., RITIENI A., 1999. Deter- mination of the Fusarium mycotoxins, fusaproli- ferin and beauvericin by high-performance liquid chromatography-electrospray ionisation mass spectrometry. Journal of Chromatography A, 858, 175-185.

TAKAHASHI H., IUCHI M., FUJITA Y., MINAMI H., FUKUYAMA Y., 1999. Cumaroyl triterpenes from Casuarina equisetifolia. Phytochemistry, 51, 543-550.

TAKUO O., YOSHIYUKI K., TAKASHI Y., TSUTOMU H., HIROMICHI O., SHIGERU A., 1983. Studies on the activities of tannins and related compounds from medical plants and drugs I. Inhibi- tory effect on lipid peroxidation in mitochondria and microsomes of liver. Chemical Pharmaceuti- cal Bulletin, 31, 5, 1625-1631.

TESAKI S., TANABE S., ONO H., FUKUSHI E., KAWABATA J., WATANABE M., 1998. 4- Hydroxy-3-nitrophenylacetic and sinapinsäure as antibacterial compounds from mustard seeds. Bioscience, Biotechnology and Bioche- mistry, 62, 5, 998-1000.

WALTERS M. T., HUGHES P. S., BAMFORTH C. W., 1996. The evaluation of natural antioxidants in beer and its raw materials. The Institute of Brewing-Asia Pacific Section: Proceedings of the Twenty-Fourth Convention, Singapore, 103- 109.