Screening of plant cell cultures for their capacity to dimerize eugenol and isoeugenol: Preparation of dehydrodieugenol

Article abstract of DOI:10.1016/j.molcatb.2011.05.005

Nine plant cell cultures were used to catalyze the oxidative coupling reaction of eugenol and isoeugenol. All the evaluated plant cell cultures carried out the oxidative reaction. The calli of Medicago sativa and the cell suspension of Coriandrum sativum

Full text of DOI:10.1016/j.molcatb.2011.05.005

Journal of Molecular Catalysis B: Enzymatic 72 (2011) 102–106
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Screening of plant cell cultures for their capacity to dimerize eugenol
and isoeugenol: Preparation of dehydrodieugenol
Liliana Hernández-Vázqueza, Ma. Teresa de Jesús Olivera-Floresb,
Francisco Ruíz-Teránc, Ivon Ayalac, Arturo Navarro-Ocan˜ac,∗
a Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana, Unidad Xochimilco, A.P. 23/181 México, D.F., Mexico
b Departamento de Bioquímica, Laboratorio de Cultivos Vegetales, Facultad de Química, UNAM, Ciudad Universitaria, México, D.F. 04510, Mexico
c Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, México, D.F. 04510, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Nine plant cell cultures were used to catalyze the oxidative coupling reaction of eugenol and isoeugenol.
All the evaluated plant cell cultures carried out the oxidative reaction. The calli of Medicago sativa and
the cell suspension of Coriandrum sativum produced the highest yield of dehydrodieugenol.
© 2011 Elsevier B.V. All rights reserved.
Received 1 December 2010
Received in revised form 5 May 2011
Accepted 16 May 2011
Available online 7 June 2011
Keywords:
Biotransformation
Oxidative coupling
Plant cell culture
Eugenol
Dehydrodieugenol
1. Introduction
used for the enantioselective oxidation of 2-naphthols to the 1,1-
binaphthyl-2,2-diols [8]. In addition, this enzyme has been used
Plant cell cultures exhibit a vast biochemical potential for the
production of specific secondary metabolites. The plant cell culture
may also retain the ability to transform exogenous substrates into
products of interest [1]. Biotransformation, in which the cell culture
acts as a bioreactor [2], is considered to be an important method to
turn cheap and simple substrates into rare and expensive products
[1–3]. Reactions catalyzed by cell cultures include: hydroxylation,
oxidation, reduction, hydrogenation, glycosylation, and hydrolysis
[3].
The development of biocatalysts for oxidation reactions is a ver-
satile and very important area of research, with enzymes and cell
cultures playing a particularly important role. This approach con-
stitutes a cleaner and greener alternative to traditional chemical
methods in which catalysts such as FeCl3, K3Fe(CN)6 and Cu(OH)Cl
are used for the oxidative coupling of phenolic compounds [4,5].
in the biotransformation of phenolic compounds, abundant in
essential oils, such as eugenol and isoeugenol [9], leading to the for-
mation of coupled products linked through the aromatic ring, that
are interest for the biogenesis of neolignans. However, the addi-
tion of H2O2 to the reaction mixture decreases the chemical yield
in some cases [6]. An alternative is the use of plant cell cultures in
which cell wall peroxidases rapidly metabolize a huge amount of
the H2O2 produced by the addition of foreign substrates.
Recently, Takemoto et al. have found that Camellia sinensis cell
culture is an efficient source of peroxidase (POD) [10]. This cell
culture has been used for the oxidative coupling of dibenzylbutano-
lides and for the enantioselective oxidative coupling of 2-naphthol
derivatives, resulting in moderate to good ee values [11].
O-Methoxyphenols, such as eugenol (4-allyl-2-methoxy phe-
nol) (1), isoeugenol (4-propenyl-2-methoxyphenol) (3) and the
dimeric compounds are constituents of essential oils in a great
diversity of medicinal plants (Fig. 1) [12]. Consequently, these com-
pounds have attracted considerable attention in the flavour and
food industry.
On the other hand eugenol (1, Fig. 1), the main compo-
nent in clove oil, is a valuable starting compound for several
drugs. It has been used in cosmetics and food products as a
flavouring additive, antimicrobial and antioxidant agent [13],
and in dentistry, for instance, in combination with zinc oxide
Horseradish peroxidase (HRP),
a commercially available
enzyme, has been established as an effective biocatalyst for organic
and inorganic oxidation reactions [6]. Kutney et al. [7] reported
HRP catalyzed carbon–carbon bond formation of suitable diben-
zylbutanolides for biotransformation to lignans. HRP has also been
∗
Corresponding author. Tel.: +52 55 56225346; fax: +52 55 56225309.
E-mail address: arturono@servidor.unam.mx (A. Navarro-Ocan˜a).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.05.005

L. Hernández-Vázquez et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 102–106
103
OH
OH
procedure [4b]. 1H NMR (CDCl3/TMS): ı 1.37 (d, 3H, J = 6.8 Hz),
1.86 (dd, 3H, J = 6.8, 1.6 Hz, 3.40–3.50 (m, 1H), 3.84 (s, 3H), 3.88
(s, 3H), 5.09 (d, 1H, J = 9.6 Hz), 5.80 (s, 1H), 6.11 (dq, 1H, J = 15.6,
6.4 Hz), 6.36 (dd, 1H, J = 15.6, 1.2 Hz), 6.76 (s, 1H), 6.78 (s, 1H),
6.86–6.91 (m, 2H), 6.96 (s, 1H), spectroscopy data were consistent
with those reported in the literature [4]. TLC and HPLC analysis used
the dimeric compounds 2 as reference samples for determinations.
The dimeric compound 4 was used as reference sample for mon-
itoring the biotransformation on TLC, and this was isolated from
biotransformation with Bouvardia ternifolia.
OH
MeO
OMe
MeO
Dihydrodieugenol (2)
Eugenol (1)
OH
MeO
2.3. Biological material
OCH3
O
Nine plant species were used: alfalfa (Medicago sativa
L.), bean (Phaseolus vulgaris), coriander (Coriandrum sativum),
matarique (Psacalium peltatum), melon (Cucumis melo), carrot
(Dacus carota) capulín (Prunus serotina), mammillaria (Mammillaria
huitzilopochtli) and B. ternifolia.
HO
OCH3
Isoeugenol (3)
Dehydrodiisoeugenol (4)
Fig. 1. Structure of monomer and dimers.
2.3.1. Calli
The calli of alfalfa, bean, coriander, matarique, melon and
carrot were obtained from using a method previously reported
[20]. The alfalfa and bean calli were maintained in Schenk and
Hildebrant (SH) medium [21] supplemented with sucrose (3%), 2,4-
dichlorophenoxyacetic acid (2,4-D), 2 and 3 mg L−1 respectively,
glycine (20 mg L−1), kinetine (0.2 and 1 mg L−1 for each respec-
it is used as a pulp capping agent, temporary filling and root
conduit sealer [14]. However, eugenol causes allergenic con-
tact dermatitis [15] and high concentrations of this compound
have been reported to have some cytotoxic properties [16].
Dehydrodieugenol (2, Fig. 1) (biseugenol, 3,3ꢀ-dimethoxy-5,5ꢀ-
di-2-propenyl-1,1ꢀ-biphenyl-2,2ꢀdiol) the symmetrical dimer of
eugenol, is a natural O,Oꢀ-dihydroxy biphenyl [17], which shows
biological activity comparable with that observed in eugenol. This
dimer has shown greater antioxidant [4] and anti-inflammatory
activity than eugenol [18] and less cytotoxicity [19].
tively), and vitamins (myo-inositol 100 mg L−1
, thiamine–HCl
5 mg L−1, nicotinic acid 5.0 mg L−1, pyridoxine–HCl 5 mg L−1). In
the case of bean calli, ascorbic acid (0.75 mg L−1)–citric acid
(0.75 mg L−1) were also added as antioxidants.
The calli of melon and carrot were maintained in Murashige
and Skoog (MS) medium [21] supplemented with sucrose (3%), 2,4-
D (3 mg L−1), glycine (20 mg L−1), kinetine (6 mg L−1), and ascorbic
acid (0.4 mg L−1)–citric acid (0.4 mg L−1). Coriander and matarique
calli were subcultured in MS medium supplemented with sucrose
(3%), benzylaminopurine (BA), 0.3 mg L−1. In the case of corian-
der 2,4-D (3 mg L−1) and ascorbic acid (1.5 mg L−1)–citric acid
(1.5 mg L−1) were used, and for matarique, indol-3-acetic acid (IAA),
1 mg L−1 and ascorbic acid (100 mg L−1)–citric acid (100 mg L−1).
For all media 2.5 g L−1 of gelzanTM CM were added as a setting agent
and the pH fixed at 5.7. All calli were incubated at 25 ◦C with a pho-
toperiod of 16 h of light and 8 h of darkness, except for matarique
calli that were incubated in darkness at the same temperature.
The mammillaria (M. huitzilopochtli) calli was obtained from
in vitro grown plants which were cut in 1 cm sections and five
of these explants were introduced to MS medium supplemented
with sucrose (3%), 2,4-D (1 mg L−1), glycine (10 mg L−1), kine-
tine (1 mg L−1), vitamins (myo-inositol 100 mg L−1, thiamine–HCl
(B1) 10 mg L−1, nicotinic acid (B3) 1 mg L−1, pyridoxine–HCl (B6)
1 mg L−1 and glycine 2 mg L−1), and gelzanTM CM (2.5 g L−1), at pH
5.7 to develop the calli [22].
For capulin P. serotina seeds were obtained from the fruit of a
wild tree, which were germinated in the following manner, the
seeds were scarified and surface-sterilized by immersion for 10 min
in a 30% aqueous solution of a commercial bleach with 0.1% of
tween and 10 ␮L of mycrodyn®, followed by five washings with
sterile water. The seeds were germinated on Murashige and Skoog
(MS) medium and after three weeks the plants were dissected.
Explants from petioles were cut in 0.5 cm sections and transferred
to MS medium supplemented with sucrose (3%), 2,4-D (2 mg L−1),
ascorbic acid (10 mg L−1)–citric acid (10 mg L−1), and gelzanTM CM
(2.5 g L−1), at pH 5.7 to develop the calli. The conditions for the incu-
bation were similar to those described above. The first subculture
took place after three weeks; the calli were maintained on solid MS
medium and subcultured every three weeks [23].
The aim of this work was to evaluate the capacity of nine plant
cell cultures to catalyze the dimerization of eugenol and isoeugenol
via the oxidative coupling reaction, as well as to develop an alter-
native to chemical methods for the synthesis of the bioactive
compound biseugenol.
2. Materials and methods
2.1. General
Reagents and solvents were purchased from Organic Research,
Baker or Aldrich, and were used without any additional purification.
1H NMR spectra were recorded on a Varian 400 MHz instrument
in CDCl3, using tetramethylsilane (TMS) as an internal reference.
Thin Layer Chromatography (TLC) was used for a preliminary qual-
itative analysis of the biotransformation products and this was
performed on silica gel plates. Alugram® SIL G/UV 254 0.2 mm
(Macherey-Nagel) and hexane–AcOEt (4:6) was used as the elu-
ent. The quantitative analysis of dehydrodieugenol produced after
the biotransformation was performed on a Waters-1525 High Pres-
sure Liquid Chromatography (HPLC) equipment with a UV detector
®
(Waters 2487) under the following conditions: Symmetry C-18
column at 30 ◦C, a flow rate of 1 mL/min, UV wave length at 230
and 280 nm, and a H2O–MeOH (70:30) solvent system with 0.1%
trifluoroacetic acid.
2.2. Substrate and products
Dehydrodieugenol (2) was prepared from eugenol following a
procedure previously reported [5]. 1H NMR (CDCl3/TMS): ı 3.35 (d,
4H, J = 6.8 Hz), 3.91 (s, 6H), 5.04–5.14 (m, 4H), 5.91–6.04 (m, 2H),
6.72 (d, 2H, J = 2.1 Hz), 6.75 (d, 1H, J = 2.1 Hz), spectroscopy data
were consistent with those reported in the literature [5,4b]. Dehy-
drodiisoeugenol (4) was obtained from isoeugenol (3) by a reported

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L. Hernández-Vázquez et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 102–106
Calli of B. ternifolia was established from young leaves of a wild
3. Results and discussion
plant of B. ternifolia (personal communication). The cultures were
maintained in MS medium supplemented with sucrose (3%), 2,4-
D (1 mg L−1), glycine (2 mg L−1), kinetine (0.05 mg L−1), vitamins
(myo-inositol 100 mg L−1, thiamine–HCl (B1) 10 mg L−1, nicotinic
acid (B3) 1 mg L−1, pyridoxine–HCl (B6) 1 mg L−1) and gelzanTM
(2.5 g L−1). The conditions of the incubation were similar to those
described above.
3.1. Cell culture
All the evaluated calli were friable, except that of mamillaria,
which was compact. The cell suspension cultures of B. ternifolia,
Medicago sativa, Psacalium peltratum, P. vulgaris and D. carota were
easily disaggregated, whereas P. serotina, C. sativum, and C. melo
were less disaggregated. All showed a low oxidation during the
propagation of them.
2.3.2. Production of plant cell suspensions
3.2. Dimerization of 1 and 3 catalyzed by calli and cell
suspensions monitored by TLC
The medium used was the same as that used to obtain calli from
the different species but without gelzanTM .Ten grams of calli were
added to an Erlenmeyer flask containing 250 mL of medium and
then subcultured at 14 days intervals and kept in a rotatory shaker
at 25 ◦C and 125 rpm, with a photoperiod of 16 h of light and 8 h of
darkness.
TLC analysis of the mixture of compounds, in each biotransfor-
mation reaction of eugenol (1) using calli, showed the presence
of a compound more polar than the substrate (Fig. 2). This was
identified as dehydrodieugenol (2) by comparison with a reference
sample, which confirmed the biotransformation of eugenol by the
biocatalyst.
For capulin (C), matarique (G) and bean (E) the biotransforma-
tion of 1 seemed to be complete because eugenol was not observed.
In the plant cell culture, used as control, where the substrate was
not added (Fig. 2), some compounds were observed in the con-
trols (Cꢀ and Eꢀ), these did not interfere on the biotransformation
reaction. In this work, we focused on determine which plant cell
culture could be used to transform eugenol no attention was done
on the isolation of the products formed in the controls of capulin
and bean (Cꢀ, Eꢀ). Since the TLC analysis was performed on samples
of similar concentration, the size of the spots observed was a reli-
able indication of the progress on the transformation of eugenol to
product.
The results for the biotransformation of 1 with cell suspension
(data not shown) were similar to those obtained for calli. However,
the total transformation of the substrate was not observed in the
bean cell suspension and for capulin 2 was not the sole product.
The TLC analysis of the reaction mixtures from the biotransfor-
mation of isoeugenol using calli presented a mixture of products
(Fig. 3). In all cases, at least three reaction products were observed,
the main product was identified as dehydrodiisoeugenol (4) by
comparing the colour and Rf spot values observed with the ref-
erence compound previously obtained by the chemical method.
In all cases a complete transformation of the substrate was
observed, except for M. hutzilopozchtli calli (F). We may say, from
the spot intensity observed in TLC, that the highest transformation
was obtained with the calli of B. ternifolia and P. vulgaris, taking into
consideration that the reaction mixtures analyzed were at similar
concentration. The mixture of compounds obtained from the bio-
transformation using B. ternifolia were chosen in order to isolate
the main product from which 23% of yield was obtained. The spec-
troscopy data are in concordance to those previously described for
dehydrodiisoeugenol (4) obtained from 3.
2.4. Screening of the biocatalytic capacity of the plant cell
cultures to dimerize 1 and 3 (oxidative coupling)
2.4.1. Dimerization of 1 and 3 using calli monitored by TLC
analysis
Ten grams of a 14-day-old calli of the different plants were dis-
rupted (with a vortex) in 50 mL of phosphate buffer, at pH 6.5 and
8000 rpm. The substrates, 1 and 3 (50 mg), were dissolved in 1.0 mL
of acetone and then added to each flask containing the homoge-
nized calli. The flasks were incubated in the dark at 25 ◦C for five
days on a rotator shaker (120 rpm). As a control the same amount
of calli without the substrate but with the same amount of ace-
tone was set under the same conditions. After five days, the cells
were filtered under reduced pressure. The filtrate was collected,
extracted with ethyl acetate (3 × 25 mL) and concentrated at vac-
uum. The residues were dissolved in acetone (1 mL) and spotted
onto TLC plates. A mixture of hexane–AcOEt (4:6) was used as the
eluent. Samples of eugenol (1), isoeugenol (3), dehydrodieugenol
(2) were used as references.
When B. ternifolia was used to perform the biotransforma-
tion the mixture of compounds, obtained during the reaction, was
purified using TLC using as eluent hexane–methanol–ethylacetate
(7:1:2, Rf 0.6). The yield recovered was 23% and the spectroscopy
data coincided with those previously reported for dehydrodi-
isoeugenol (4) obtained from isoeugenol (3).
2.4.2. Dimerization of compounds 1 and 3 using cell suspensions
monitored by TLC analysis
The substrates 1 and 3 (50 mg) were dissolved in 1.0 mL of ace-
tone and then these were added to each flask containing a cell
culture suspension of 14 days old. The mixtures were incubated for
additional five days at 25 ◦C and 125 rpm, with a photoperiod of 16 h
of light and 8 h of darkness. The analysis of the biotransformation
showed a similar composition of product as the calli.
When cell suspensions were used as a biocatalyst (data not
shown), the results were similar to those obtained with, calli except
in the case of melon, where the substrate was not totally trans-
formed and capulin, where dehydrodiisoeugenol (4) was observed
as the sole product.
In the case of isoeugenol no less than three compounds were
observed in the reaction where dehydrodiisoeugenol was obtained
in higher amount than the other compounds (Fig. 3). The quan-
titative analysis on eugenol (1) transformation was done leaving
the compounds obtained on the transformation of isoeugenol for
further analysis.
2.5. Preparation of dehydrodieugenol for further analysis by HPLC
Dehydrodieugenol (2) was prepared under similar conditions
to those described for the calli and cell culture suspensions, using
150 mg of eugenol distributed in three flasks with calli or a cell
suspension. The incubation conditions and product extraction
were similar to those described above. The extract was dissolved
in methanol (2 mL) and analyzed by HPLC. The yield of dehy-
drodieugenol was calculated from a calibration curve.
The calli and cell suspensions evaluated in this work catalyzed
the dimerization of eugenol and isoeugenol via oxidative coupling.
However, under the conditions studied, the reactivity was different

L. Hernández-Vázquez et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 102–106
105
Fig. 2. TLC chromatogram of the biotransformation of eugenol biocatalyzed by calli. The letters refer to the plants listed in Table 1. *, Eugenol; A, M. sativa; B, B. ternifolia; C,
P. serotina; D, C. sativum; E, P. vulgaris; F, M. hutzilopochtli; G, Matarique; H; C. melo; I, D. carota; **, Dihydrodieugenol. The letters Aꢀ–Iꢀ are the control reactions for the same
plants (without substrate).
Fig. 3. TLC chromatogram of the biotransformation of isoeugenol biocatalyzed by calli. The letters refer to the plants listed in Table 1. *, isoeugenol; A, M. sativa; B, B. ternifolia;
C, P. serotina; D, C. sativum; E, P. vulgaris; F, M. hutzilopochtli; G, Matarique; H, C. melo; I, D. carota. The letters Aꢀ–Iꢀ are the control reactions for the same plants (without
substrates).
Table 1
for each culture. All the assayed cell cultures would be suitable for
Yields of dehydrodieugenol and eugenol recovered after the cell culture reaction.
carrying out oxidative coupling of other phenols.
Plant
Recovered eugenol (%)
Yield of
dehydrodieugenol (%)a
3.3. Dimerization of eugenol (1): preparation of
dihydrodieugenol (2)
Calli
Cell suspension Calli
Cell suspension
Medicago sativa
Bouvardia ternifolia
Prunus serótina
Coriandrum sativum
Phaseolus vulgaris
Mammillaria hutzilopochtli 59.75
Psacalium peltratum
Cucumis melo
Dacus carota
18.28
36.38
19.33
32.02
0.44
44.79
35.18
0.86
15.24
15.28
78.09
1.29
35.81
24.45
3.22
16.79
23.21
4.39
5.53
8.29
5.83
15.02
30.35
0.24
33.14
27.61
0.27
3.50
15.99
10.17
Fig. 4 shows the obtained yield from the biotransformations of
eugenol (1) to dehydrodieugenol (2) with calli and cell suspen-
sions of the evaluated plants and Table 1 shows the percentage
of recovered eugenol in each experiment.
The dehydrodieugenol obtained using calli ranged from 3.2 to
35.8% yield. The highest yield was obtained from the reaction with
alfalfa calli (35.8%), from which 18.28% of eugenol was recovered.
This result was an improvement on that reported for the reaction
catalyzed by HRPO, which yielded only 22% of 2 [9]. The lowest
yield was observed with capulin calli, which produced only 3.2% of
2 and 19.3% recovery of 1.
0.29
40.14
34.15
37.14
63.13
a
HPLC: Symmetry® C-18 column at 30 ◦C, a flow rate of 1 mL/min, UV wave
length at 230 and 280 nm, and a H2O–MeOH (70:30) solvent system with 0.1%
trifluoroacetic acid.
When cell suspensions were used, the highest yield was
observed with coriander (33.1%), with a 15.2% recovery of eugenol.
The lowest conversion was observed using capulin, whose yield
was less than 1%, with a recovered eugenol being less than 1%.
Although Bovardia and P. vulgaris produced lower yields than
alfalfa (30.3 and 27.6% respectively), the amount of biseugenol
obtained did not differ substantially between calli and cell suspen-
sions. Liquid or solid media used in the growth of the cells was not
an important factor in the yield of these plants.
The lowest yields were obtained with both calli or cell suspen-
sions of capulin, mammillaria and matarique, in each case being
less than 10%. In the case of coriander, melon and carrot, the highest
yields were obtained with cell suspensions.
In summary, the yield of dehydrodieugenol appears to depend
on the plant species and the type of the plant cell culture used,
whether callus or cell suspension. Although the yields of 2 were
not very high, it is important to pointing out that they could be
Fig. 4. Yield of dehydrodieugenol from the cell culture reactions.

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L. Hernández-Vázquez et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 102–106
improved by modifying the reaction conditions and optimization.
Chemical methodology produces higher yields, employing oxidiz-
ing agents such as the iron compounds K3Fe(CN)6 (98%), CuCl(OH)
and triethylenediamine (72%) [4,5] however, the methodology used
in this work presents a cleaner and greener approach compared to
traditional methods of oxidative coupling of eugenol.
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4. Conclusion
TLC analysis showed that the nine plant species tested as bio-
catalysts were able to successfully biotransform isoeugenol and
eugenol to their dimers. Consequently, these cultures can be used
for oxidative coupling of phenolic compounds. HPLC analysis of the
biotransformation of eugenol showed that the highest yield of com-
pound 2 was obtained with the calli of alfalfa (35.8%). However, this
yield was lower when cell suspensions were used.
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