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Synthesis of @-Iononein an Aldehyde/Xmthine Oxidase/@-Carotene System Involving Free Radical Formation Aur6lie Bossert and Jean Marc Belin'J Laboratoire Recherche-DBvelogpement,Metayer Aromatiques Ind., 9 rue Gambetta, F-94500 Champigny S/Marne, France, and Laboratoire de Biotechnologie, Universit.4 de Bourgogne, ENS.BANA, Campus Universitaire Montmuzard, 1 Esplanade Erasme, 21000 Dijon, France The enzymic cooxidation of @-carotene(BC) by xanthine oxidase (XO) in aqueous solutions leads to @-ionone(BI) and derivatives: epoxy-@-ionone(EPBI), dihydroactinidiolide, 8-cyclocitral, pseudoionone, etc. We demonstrate, in this article, that this formation during the first hour of stirring and the use of is due to free radical (R) aldehydes as substrate. The bleaching of BC does not occur when the common substrate of XO, xanthine, is used; this proves that the superoxide anion 02'-alone is not active on BC. BI formation in this case is not observed.
1. Introduction
Ionones and their derivatives are widely distributed in nature as they are important constituentsof many essential oils ( I ) . However,the presence of b-ionone (BI)' in plants (raspberries, carrots, tomatoes, etc.) is too small to allow its extraction, and the flavor industry only uses synthetic BI at this point. Iononesare thought to originate in nature from carotenes by complex enzymatic degradations (2), but all reactions happening in vivo or post mortem are not well elucidated. BC might be oxidized directly by activated species of oxygen ('02,OH', free radicals) during photosensitivereactions (3,4),aerobic fermentation during tea processing (5),or on storage of carrots in air (6).In all of these cases, BI was detected. In vitro, cooxidative pathways from BC have been reproduced with different enzymes in aqueous solutions: phenoloxidase (5), lactoperoxidase (7),lipoxygenase (8), and xanthine oxidase (10). All of these reactions involve free radical and active oxygen species generation (7,9). Superoxide anion, 02*-, is generated by XO (EC 1.1.3.22) and its oxidative power has already been tested on BC (10) and lignin ( I I ) , but without success. No degradation products of these polymers were observed. The improvement of this system for BI production seemed interesting. The present study deals with this improvement and the characterization of the radical speciesgenerated by ESR spin-trap methodology. Indeed, many authors have identified radicals formed in a xanthine/XO system (13, 14), but only one report discusses the nature of radicals generated when aldehydes are substituted for xanthine (12). By dissolving BC in the presence of a detergent, it is possible to obtain a stabilized water solution directly accessible in the enzymic reaction phase. Author to whom correspondence should be addressed. + Metayer Aromatiques Ind. t Universite de Bourgogne.
Abbreviations: BC, 8-carotene; BI, @-ionone;EPBI, epoxy8-ionone; EDTA, ethylenediaminetetraacetic acid; R' alkyl r a d i d Osc,superoxide anion; OW, hydroxyl radical, DMPO, 5,6-dimethyl-l-pyrrolineN-oxide; XO, xanthine oxidase; SOD, superoxidedismutase; THF,tetrahydrofuran; MeOH,methanol; CH&la, dichloromethane; GC, gas chromatography; MS, mass spectrometry; ESR, electron spin resonance; HF'LC, highperformance liquid chromatography; SE, standard error. 875&7938/94/3010-0129$04.50/0
2. Materials a n d Methods 2.1. Chemicals. Acetaldehyde butanal, propanal, xanthine, and allopurinol were obtained from Sigma Chemical Co. (St. Louis, MO). BC (synthetic, 97%)was purchased from Fluka Biochemika (Buchs, Switzerland) and stored at -18 OC. All other chemicalswere of analytical grade, and distilled water was used in all solutions. Deferroxamine and doubly distilled DMPO were directly supplied by the ESR laboratory. 2.2. Enzymes. XO (grade I11 from buttermilk) and SOD (from bovine liver) were obtained from Sigma. The purity of XO was checked following the method of Beauchamp and Fridovich (15). 2.3. Aqueous Solution of BC. BC (250 mg) and 3.6 mL of Tween 80 were dissolved in 200 mL of chloroform and evaporated to dryness under vacuum. The residue was immediately dissolved in 50 mL of 0.25% EDTA solution and filtered through filter paper. Thereafter, 100 mL of 0.01 M sodium acetate buffer (pH 4.6) was added. The BC solutionwas prepared on the day of the experiment (16). This solutionwas then diluted for our different testa after its concentration was determined by HPLC. 2.4. Cooxidation Experiments. All tests were monitored in a 2-L capacity enzymatic reactor at 37 OC and 750 rpm stirring. The total reaction volume was 300 mL, composed of 10-90 mg/L BC in aqueous solution, 27 X 1o-S units/mL XO, 48 mM acetaldehyde (or butanal) or 10.1M xanthine, 1V M EDTA, and 0.05 M phoaphate buffer, pH 8.0; the reaction was started with the addition of XO. Samples (30 mL) were taken every hour to follow BC degradation (HPLC, 10 mL) and ionone appearance (20 mL for volatile5 extraction). The reaction in the sampleswas stopped by the addition of 50 rM allopurinol. For each new system tested, three independent experiments were realized. 2.5. HPLC Method for the Determination of BC. Measurement of the amount of unreacted BC was carried out on a Merck HPLC system: column, Merck Lichrucart 250-4, Lichrospher 100RP18,5 pm; pump, L-6200 Merck; solvents,acetonitrile(90-35% ), chloroform (10-45%),and acetic acid (10%)in THF (10%);flow rate, 0.8 mL/min; injected volume, 10 pL; detector, photodiode Merck L-3ooO; absorbance mode at X = 450 nm. 2.6. Extraction and Identification of Volatiles. Samples (20 mL) were extracted with CHzClz after the
@ 1994 American Chemlcal Society and American Insmute of Chemical Engineers
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Wbtechnol. Rug.., 1994, Vol. 10, No. 2
A AC.BC10 0 BUT.BC10
SO
R E F . BC IO
A AC.BC90 0 BUT.BC90 REF. BC
eo
50 40 V
0
I
2
3
4
6
e
Time ( h l
Figure 1. Degradation of BC in 48 mM aldehyde/27 X 10-8 unit/ mL XO/BC systems: AC, acetaldehyde; BUT butanal; REF, control without XO. Initial concentration of B C was set at 10 and 90 mg/L. Experiments and analyses were performed BB described in Materials and Methods. Data are mean f SE from three independent experiments.
addition of internal standard methylisoeugenoland then concentrated under Nz to a final volume of 3 mL. Concentrates were kept at -18 "C until their injection into a GC or GUMS. Quantitative determination of BI and EPBI was carried out on a Varian 3400 gas chromatograph temperature program, 90 "C, 4 "C/min to 220 OC, hold 20 min; injector, 250 OC;detector, 250 "C; 1 pL injected splitless; column, CP Wax-58CB; i.d., 0.25 mm; film thickness, 2 rm; length, 50 m; vector gas, Hz, Qualitative identification of all volatiles was realized by GC/MS with the ion-trap system Finnigan Mat ITS 40. 2.7. ESR Experiments. The spin-trapping studies were performed by using the spin-trap DMPO at a final concentration of 50 mM. EPR spectra were recorded at room temperature using a Bruker 300E apparatus. The settings were as follows: modulation frequency,100 KHz; modulation amplitude, 0.506 G; scan time, 1 min; microwave power, 20 mW; microwave frequency, 9.742 GHz, 2.8. Controls. Controls without XO addition were tested and analyzed under the same conditions. Aroma production was calculated after subtraction of the quantities found in the controls (autooxidation of BC).
3. Results 3.1. Degradation of BC. The bleaching of BC (Figure 1) is the first visible proof of BC oxidation. In 2-4 h, the orange solutions turn to pale yellow, almost colorless solutions when low initial concentrations are used. The tendency of BC to autooxidize (no XO addition) in the presence of aldehydes is given as the reference. When 10 mg/L of BC is placed in solution initially, almost 100% of this BC ie degraded after 2 h of stirring, and the curves obtained with acetaldehyde and butanal are identical. To know whether the system was limited by its initial concentration of BC, we used 90 mg/L BC solutions; the addition of XO has a very significant effect during the first hour: on average, 75.5% of the BC is degraded in 1 h in the presence of butanal and 55% is degraded with acetaldehyde. After 1.5 h, the bleaching of BC seems to follow the same kinetics as the reference. This means that enzymaticaction only occuts for 1.5hand the reaction in the presence of butanal is more violent on BC. In the presenceof xanthine as substrate, no bleachingis observed except the normal oxidation at the air of BC. However, the activity of XO on xanthine was checked before cooxidation experiments, and the formation of uric acid
Table 1. Summary of Substances Identified in the Different Sysfams after 1 h of Egeynutie Degradation of BC by XO in the Presence of Acetaldehyde (AC) or Butand (BUT). type substances identification* aldehyde hydrocarbon limonene MS AC naphthalene MS AC alcohol benzyl alcohol MS AC, BUT aldehyde benzaldehyde Ms AC geranial* MS,RT AC,BUT nerd* MS,RT AC,BUT ketone &ionone* MS,RT AC,BUT epoxy-@-ionone* MS,RT AC,BUT dihydroactinidiolide* MS, AC, BUT others @-cyclocitral* MS,RT AC,BUT y-octalactone MS BUT y -decalactone MS BUT y-undecalactone MS BUT pseudoionone* MS AC
An asterisk (*) indicates the m a t important volatilee found in the solutions. MS, maas spectrometry. RT, retention time.
*
was observed. Hence, the absence of bleaching in this case is not due to enzyme inactivity. 3.2. Production of &Ionone and Derivatives. GC/ MS analysis allowed us to identify many volatiles formed in the solutions. After the first hour of incubation, we were already able to detect the substances presented in Table 1. It is not possible to identify all of the peaks visualized on the chromatograms, but those unidentified peaks represent only a small fraction of all volatiles. Many of them seem to be epoxides of degraded BC. No further investigation to identify them exactly is necessary, as the focus of this work was essentially the identification of BI and ita derivatives. BI (principal fragments obtained by MS were m + l/e = 177,135,149,90,and 193), EPBI (135, 123, 147,94, and 2091, and dihydroactinidiolide (56,88, 111, 137, and 181) are identified in all of the oxidizing systems. Their amounts depend on the time of reaction after the mixing of all constituents and on the substrate used for the XO. Figure 2 shows the time course of the appearance of BI and EPBI in the media when two different aldehydes are used. In the presence of acetaldehyde, the maximum for BI (350 pg/L) is reached after 3 h, vs. 2 h when butanal is used, but in this w e , the maximum is barely smaller (300 rg/L), and this difference is not significant. In both experiments, BI and EPBI production follow the same time course, showing a 1-h shift between the two. EPBI is produced more slowlyand hence seems to be an oxidized product of BI. After 3 h, both degrade very quickly, slowing after 5 h of stirring, In order to improve these systems for the production of BI, we worked with higher initial concentrations of BC (150200 mg/L), but the maximum amount of BI did not significantly increase. When the system with 90 mg/L initial BC is compared with the maximum amounts obtained from the cooxidation of 10 mg/L BC (BI = 250 Fg/L and EPBI = 260 rg/L in a AC/XO system), we do not see a factor of 9 difference. However, the increase in initial BC decreases the yield of BI in the systems, which are not limitedby the initial concentrationof BC. A model with sequential charges of BC was tested with 10 mg/mL initial BC. The addition of BC (10 mg/L) after 2 h has no bleaching effect nor does it produce BI. In any case, the enzymic system seems to be saturated, inhibited even before the second hour after mixing the chemicals. Control systems without BC are tested the activity measurement of XO in cooxidizing conditions indeed shows a total inhibition
w.,1994, Vol. I O , No. 2
&"/.
191 DMW-OH
0
AC.81
A
8UT.BI
AC.EPBI
A
BUT.EPEI
I
DMPO-R I
0
S
1s
10
20
Time 111)
Figure 2. Aroma production during the cooxidation of BC (90 mg/L) with aldehyde/XO systems: AC,48 mM acetaldehyde; BUT, 48 mM butanal; BI, @-ionone;EPBI, epoxy-&ionone; 27 X 10-9 unit/mL XO. The reaction was performed as described in section 2.4. Extraction and analysis of volatile8 was carried out as in section 2.6. Dataare mean f SE from three independent
I
I
DMPO-OH I
XO+Pmpmd+DMPO
experiments.
of the enzyme after 18 min of reaction time. Further investigations demonstrated an inhibition by both the substrate and the products of the reaction. Hence, enzyme activity, bleaching of BC, and BI production are wellcorrelated phenomena: specific enzymatic action occurs over 1 or 2 h, after which the BC bleaching stops, but the BI and EPBI produced are still degraded. This can only be the result of some radical-involving mechanism. 3.3. Free Radicals Involved in the Systems. In order to understand why aldehyde/XO systems are active on BC but the xanthine/XO one is not, ESR studies using spin-trap DMPO are useful. Radicals trapped by DMPO give typical spectra in magnetic fields. The EPR spectra obtained 2 min after the mixing of XO with the different substrates are shown in Figure 3. In the presence of xanthine (part A), the spectrum of the DMPO adduct consists of a typical 1:2:2:1 quartet originating from the hydroxyl adduct of DMPO, DMPO-OH. This main signal could originate from the reaction of OH' with DMPO, but also may be the decomposition product of the unstable DMPO-OOH adduct (half-time 50 s) (17,19).When 3% MeOH is added to the xanthine/XO system (Figure 4D), the spectrum obtained after 2 min still corresponds to the formation of the DMPO-OH adduct plus a small signal from a DMPO-Me adduct. DMPO-Me adducts indeed appear when Me' radicals (obtained by reaction of OH' on MeOH) react with DMPO. Thus, it is evident that OH.radicals are also present in the system. The addition of SOD to this system resulted in no signal with DMPO. This confirms that the presence of OH*radical formation is induced via the first enzymatic generation of 02'radicals,probably followingFenton's reaction (20)because of iron traces in the buffer. Hence, XO with xanthine first generates 02'-radicals in aqueous solutions. With acetaldehyde as substrate (Figure 3B), the spectrum is very different: a sextet from the alkyl adduct of DMPO,DMPO-R, and a quartet from the hydroxyl radical can be identified, but an important sextet signal is visible. As shown in Figure 4A, it is not the result of a reaction of the aldehyde alone on DMPO. The addition of 1 mM deferroxamine (Figure4B) does not modify the spectrum obtained with the standard system (Figure3B): the nature of the EPR signal is not iron-dependent. Anyway, the first radical generated is the superoxide ion, as shown in Figure 4C: the quenching of all 02'-by the addition of 10
,
I
DMPO-R I
I
L
I
DMPO-WH
I 3
0
3380
3400
3420
3440
I61
Figure 3. EPR spectra of the DMPO adducta formed in the presence of 27 X 10-9unit/mL XO, 50 mM DMPO and (A)10-4 M xanthine, (B) 48 mM acetaldehyde, (C)48 m M propanal, or (D) 48 mM butanal. Each spectrum was obtained 2 min after mixing substratesand XO. Data acquisition: microwavepower, 20 m W modulation amplitude,0.506 G; microwave frequency, approximately 9.74 GHz.
units/mL SOD does not produce a signal. However, the identification of the main sextet signal is under further investigation. This signal is specific to the use of acetaldehyde,whose reactivity in aqueoussolution could induce secondary radical generation. As with xanthine + 3% MeOH, the same control is conducted with acetaldehyde (not shown), and we can conclude that the DMPO-OH adduct essentially corresponds to the decomposition of the DMPO-OOH adduct. In the presence of propanal (Figure 3 0 , a major DMPO-R spectrum and a smaller DMPO-OH signal are observed. With butanal (Figure 3D), the same DMPO-R adduct is easy to recognize, but no DMPO-OH is detected. Nevertheless,a small DMPOOOH signal is observed. With these two less reactive aldehydes, the sextet signal is not present. Thus, with propanal, XO produces R' and 02'-radicals. In the w e of butanal, the same radicals are generated, but the DMPO-OOH adduct can be directly observed. Butanal is indeed more difficult to dissolve, and when in contact with XO, the kinetics of radical formation is slower, so that after 2 min it is possible to observe the trapping of 02'- by DMPO. The production of DMPO-R adducts when aldehyde are used as substrates leads us to conclude that aldehydes themselvesreact with their oxygenatedradicals and induce the R' radical generation. Thus, we can now affm that
6khChd. Rog., 1994, VOl. 10, No. 2
132
sufficient for ita quenching by BC. The bleaching of BC in our experiments is due to the secondarily formed radicals. Although direct oxidation of BC during photosensitization (26) and by direct molecular oxygen (27) may not be influenced by a steric phenomenon, the attack on the ninth position on BC molecules seems to be favored, leading to the appearance of C1&1 compounds (28). The action of R' radicals in our systems seems to follow the same course and induces BI and EPBI as main products. This leads us to the conclusion that their enzymatic industrial production might be possible.
A
I
DMPOOH
I
,
I
DMPO-R
DMPOOH
I
I
1
I '
I
I
DMPO-R
I 3360
I 3380
3400
3420
3440
IO1
Figure 4. EPR spectra of DMPO adducts obtained from 27 X l P unit/mL XO in the presence of 50 mM DMPO, 48 mM acetaldehyde (A-C), and 1W M xanthine (D): (A) Control without X O (B) system + 1 mM deferroxamine; (C)system + 10 unita/mL SOD,(D) xanthine + 3% MeOH. Spectra were acquired aa described in Figure 3.
the most water-soluble aldehydes, when in the presence of XO, produce alkyl radicals whose productionis initiated by the superoxide radical. 4. Discussion
In this article, we stated that the formation of BI and derivativesfrom the enzymic cooxidation of BC is initiated by a radical phenomenon. It has been demonstrated by other authors (5,9) that free radical formation and the production of BI are correlated during the processing of green tea,but no quantificationwas made. They described EPBI as an oxidized product from BI, as we conclude too. This is confiimed in a work dealing with the cooxidation of BC by a linoleic acid/lipoxygenase system (8). In this system,the time courses of the appearance of aromaswere very similar to those in our experimenta,aswere the limited yields obtained, but no inhibition of the enzyme was mentioned. One paper (IO)reported the use of the same acetaldehyde/XO system, but BC was not prepared for ita perfect diesolution in the aqueousphase, and no enzymatic degradation occurred. We compared the particle size of such a preparation (simple emuleion of BC/Tween So/ water) with those of our aqueous BC solution using a laser granulometer. The first presented an average diameter of 11.5 pm v8 0.7 pm in our preparation! The accessibility of BC for its enzymatic cooxidation is an important factor too.
Some authors have specified the inhibition of XO when aldehydes are used as substrates (21,24). Moreover, no paper deals with the identification of radicals produced in an aldehyde/XO system. However, the saturation of XO by its own substrate/producta seems to be difficult to eliminate in a single reaction phase, where all chemicals and the enzyme remain in contact. We also point out in this report that the superoxide radical is inactive on BC for inducing ita bleaching, even though hydroxyl radical and singlet oxygen are very reactive specieson C-C double bonds. Thiswas suprising for a radical which usually damages tissues (25). The oxidizing potential of superoxide anion is probably not
Acknowledgment This work was supported by Metayer AromatiquesInd., 9-11 av. de la Liberation, BP 31, F-94100 St. Maur lee Fossh, France. The authors thank VBronique Maupoil from the Laboratoire de Phyaiopathologie et Pharmacologie Cardiovasculaire Experimentale, Facult4 de Medecine de Dijon, for all ESR studies.
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