Biotransformation of Aromatic Aldehydes by Cell Cultures of Peganum harmala L. and Silybum marianum (L.) Gaertn
|Iranian Journal of
Pharmaceutical Research (2004) 2: 127-130
Received: March 2004
Accepted: August 2004
Copyright ? 2004 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services
Biotransformation of Aromatic Aldehydes by
Cell Cultures of Peganum
harmala L. and Silybum marianum (L.)
Gholamreza Asghari*a, Gholamali Saidfarb , Shohreh Mahmudib
Sciences Research Center, School of Pharmacy, Isfahan
University of Medical Sciences, Isfahan, I.R.Iran.
Pharmacognosy, School of Pharmacy, Isfahan University of
Medical Sciences, Isfahan, I.R.Iran.
Many aldehydes are important components of
natural flavours. They are used in food, cosmetic, and
biomedical industries in large amounts. Plant cells or
microorganisms carry out their production by biotransformation,
which is one of the biotechnological methods that allow them to
be defined as 'natural'.
Cell cultures of
Silybum marianum and
have been studied with a view to investigat their abilities to
produce flavonolignans and b-carboline alkaloids respectively. However, we
have isolated S. marianum and P. harmala culture strain, which are able to metabolise
several aromatic aldehydes. Ten culture strains derived from
S. marianum and
P. harmala were
examined for their ability to biotransform exogenous aromatic
aldehyde compounds, including benzaldehyde,
cinnamaldehyde and 3-methoxy, 4-hydroxy benzaldehyde.
Callus cultures of Silybum
Peganum harmala were
established from seedlings, and healthy suspensions were grown
using the Murashige and Skoog medium. Exogenous aromatic
aldehydes were fed to S.
P. harmala cell
suspension cultures. Biotransformation reactions were detected
over 24 h of incubation. The cultures then extracted with
dichloromethane and extracts subjected to GC and GC-MS
analysis. The S. marianum cultured cells in this study exhibit greater
selectivity in the reduction of aromatic aldehydes than
P. harmala cultured
cells. The ability of cultured plant cells to biotransform
substrate appears to be dependent on the culture strains as
well as the nature and position of the substituent on the
Biotransformation; Aromatic aldehydes;
Several aromatic aldehydes are among the
most important aromatic flavour compounds used in the food and
perfume industries. They are also used in cosmetic and
biomedical industries in large amounts.
Silybum marianum (L.) Gaertn. (Asteraceae) is an annual or biennial
plant, native to the Mediterranean area, which has now spread
to other warm and dry regions. Cultivation of S. marianum has
been carried out for production of flavonolignans silymarin,
which is a component of herbal remedies for the treatment of
bile and liver diseases (1).
Cell cultures of Silybum marianum have been
studied in order to investigate their abilities to produce
phenols and flavonolignans (2-4). The growth and flavonolignan
production of suspensions were tested using different
concentrations of KNO3, KH PO4, iron and calcium (5).
Flavonolignan production from Silybum
marianum root cultures has
also been reported (6).
Peganums are 30-90 cm high bushy herbs
which are widely distributed in the Irano-Turanion region with
extensions into the dry Mediterranean regions of Europe and
Africa. The seeds and roots of Peganum
harmala L. (Zygophyllaceae)
contain alkaloids such as harmine, harmaline, harmol, and
harmalol (7). The seed extract has antispasmodic,
antihistaminic (8), and vasorelaxant effects (9).
Cell cultures of Peganum harmala L. have
been widely studied with a view to investigate their abilities
to produce amines and b-carboline alkaloids (10, 11).
Biotransformations are increasingly being
used in the manufacture of specific chemicals, especially
flavours and fragrances (12). Several reports on
biotransformation of aromatic aldehydes as precursors for the
production of aromatic alcohols, using plant cells and
microalgae, have been published in the literature (13, 14). So
far, there is no report on using S.
marianum cultures in
biotransformation studies for the production of aromatic
Seeds of Silybum
marianum and Peganum harmala were
surface sterilized in 30% w/v hydrogen peroxide containing 1% Tween 80
for 2 min, then germinated on wet filter paper in Petri
dishes in the dark at 25 C. The cotyledons were
then transferred onto the Murashige and Skoog media containing
5 ppm ascorbic acid, 2 ppm 2,4-dichlorophenoxyacetic acid
and 0.1 ppm kinetin (15). Calli were maintained by subculturing
every 4 weeks, and suspension cultures were formed by agitation
5 g callus to liquid medium until a suspension of free cells
was formed. The suspensions were then placed on a rotary shaker
running at 100 rpm, and maintained by subsequent subculturing,
using a dilution of 1 to 2, into new fresh liquid media.
The callus and suspensions were
maintained in a 12 h light / dark cycle at 27°C and
subcultured every 4 weeks. Suspension cultures grown over more
than six generations were used for substrate feeding and
Substrate feeding and product extraction. The compounds chosen for study were a
series of aromatic aldehydes including the parent aldehyde
benzaldehyde, the 2- methoxybenzaldehyde,
4-methoxybenzaldehyde, cinnamaldehyde and
The substrates were obtained from Sigma. Chemical purity
(greater than 98%) was determined by capillary gas
chromatography (GC). Substrates were dissolved in a
water-miscible solvent (ethanol 70%), which resulted in good
mixing of the substrate upon addition to the aqueous medium.
The substrates were added to suspension cultures to make a
final concentration of 100 ppm cell volume (50% p.v). Control
readings were made without the addition of substrate to
cultures and with addition of substrate to cell-free medium.
The cultures were incubated under the conditions mentioned
above. After the incubation period, the flask was swirled to
ensure good mixing and two samples were removed with a 10-ml
pre-sterilized, glass-tipples pipette. A new pipette was used
for each sample.
After 24 h both the cells and the media
were extracted using dichloromethane, followed by
centrifugation (1000 g for 5 min). The extract was reduced to a
volume of 100 l under nitrogen, then 0.1 l was analyzed
by gas liquid chromatography (GLC).
Gas chromatography analysis was carried
out on a Perkin-Elmer 8500 gas chromatograph with FID detector
and a BP-1 capillary column (39 m x 0.25 mm; film thickness
0.25 m). The carrier gas was helium with a flow rate of 2
ml/min. The oven temperature for the first 4 min was kept at
60°C and then increased at a rate of 4°C /min until
reached a temperature of 280°C. Injector and detector
temperatures were set at 280°C.
Confirmation of peak identity was
effected by co-chromatography with standards and GC-MS. The
mass spectra were recorded on a Hewlett Packard 6890 MS
detector coupled with Hewlett Packard 6890 gas chromatograph
equipped with a HP-5MS capillary column (30 m x 0.25 mm;
film thickness 0.25 m). The gas chromatography condition was as
mentioned previously. Mass spectrometer condition was as
follows: ionized potential 70 eV, source temperature 200 C (16,
Results and Discussion
The biotransformation of aromatic
aldehydes by S. marianum and P. harmala is summarised in Table 1. Using aromatic
aldehydes the relationship between structure and
biotransformation ability of the test cultured plant cells was
Benzaldehyde was readily reduced to
benzyl alcohol by both culture strains derived from different
plant species. This transformation was completed within a 24 h
A selective reduction of
methoxybenzaldehyde was catalysed by both culture strains. Only
4-methoxybenzaldehyde was transformed to its corresponding
alcohol. In fact no conversion was observed after four days
when 2-methoxybenzaldehyde was fed to both culture strains. The
S. marianum reduced the 4-methoxybenzaldehyde as effectively
(>95%) as the P. harmala. Almost a total biotransformation of
4-methoxybenzaldehyde occurred within the one day period.
However, Lappin et al. demonstrated that suspension
cultures of Lavandula angustifolia reduced monoterpenoid aldehydes to their
corresponding primary alcohols, but octanal was not reduced
Of the cultures under investigation, none
exhibited the ability to biotransform
3-methoxy,4-hydroxybenzaldehyde. Compounds such as vanillin and
ethylvanillin which have hydroxy groups at the ortho or para
position were similarly reported to be difficult to transform
or were not transformed at all by Dunaliella
tertiolecta cultures (19).
These results give further evidence regarding the importance of
the nature of functional group in the substrate administered,
and the structural moieties in the vicinity of the functional
The interesting point to note is that
when cinnamaldehyde was added to both suspension cultures, a
reduction was only observed in the cultured cells of P. harmala. The
biotransformation of cinnamldehyde in P. harmala was more rapid
than that of benzaldehyde (Figure 1). It was reported
that P. harmala cultures had large capacities for biotransformation
and great potentials for the selective structural modification
on chiral molecule (20). No conversion detected when
cinnamaldehyde was introduced into the cultured suspension
cells of S. marianum. This result may indicate that the enzymes
involved in reduction reactions of cinnamldehyde are
specific to a particular culture strain, since cinnamldehyde
cannot be transformed by the S.
marianum culture. Similarly,
Tabata et al have shown that from cell suspension cultures of
ten different plant species, only six were able to glucosylate
salicyl alcohol (21).
In conclusion, it appears that the
aromatic aldehydes reduction reaction may vary with culture
strains as well as the chemical structure of substrates.
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