Biosci. Biotechnol. Biochem.

, 70 (3), 672676, 2006

Cinnamaldehyde Inhibits Phenylalanine Ammonia-Lyase

and Enzymatic Browning of Cut Lettuce
Narumi F UJITA,1;2 Eriko T ANAKA,1 and Masatsune M URATA1; y
1

Department of Nutrition and Food Science, Ochanomizu University, 2-1-1 Otsuka,

Bunkyo-ku, Tokyo 112-8610, Japan
2
Pickles Corporation, 1031-1 Kamitome, Miyosi-machi, Iruma-gun, Saitama 354-0045, Japan
Received September 14, 2005; Accepted November 14, 2005

Stored cut lettuce gradually turns brown on the cut

section after several days of storage, because cutting
induces phenylalanine ammonia-lyase (PAL) activity,
the biosynthesis of polyphenol is promoted, and the
polyphenols are oxidized by polyphenol oxidase. In this
study, we screened for inhibitors of PAL derived from
fermented broths of microbes and from foods and found
that a cinnamon extract denitely inhibited PLA of cut
lettuce. An active component was isolated by chromatographic procedures and was identied as trans-cinnamaldehyde. Browning of cut lettuce immersed in a
solution containing trans-cinnamaldehyde was denitely
repressed.
Key words:

lettuce (Lactuca sativa L.); enzymatic

browning; phenylalanine ammonia-lyase;
cinnamon; cinnamaldehyde

The shelf-time of cut vegetables and fruits such as

lettuce and apple is often limited by enzymatic browning. During the storage of cut vegetables and fruits, their
organoleptic characteristics are modied by the appearance of brown pigments. This browning is due to the
oxidation of polyphenols by polyphenol oxidase (PPO;
EC 1.14.18.1).1,2) After apples and bananas are cut, the
cut section usually turns brown within an hour. On the
other hand, it takes several days for the section of cut or
shredded vegetables such as lettuce and cabbage to turn
brown. This time lag is considered to be due to the de
novo biosynthesis of polyphenols. Mature apples contain
a sucient amount of polyphenols for rapid enzymatic
browning,3) while lettuce contains a far lower amount of
polyphenols than apples.4) The biosynthesis of polyphenols is hence considered to be a limiting factor in
enzymatic browning for cut vegetables such as lettuce.
Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5),
which converts L-phenylalanine to trans-cinnamic acid,
is the rate-limiting enzyme of the phenylpropanoid
pathway and is generally induced by wounding or
cutting of plant tissue.5) o-Diphenols such as dicaeoyl-

tartaric acid and 5-caeoylquinic acid formed along the

phenylpropanoid pathway are oxidized by PPO to form a
brown pigment when plant tissue is damaged by
wounding or parasitic invasion.
This metabolic pathway should operate during the
cold storage of cut lettuce. Several studies have described a relationship between PAL activity in lettuce leaves
and browning.68) Our group also showed that inhibitors
of the shikimic acid and phenylpropanoid pathways
repressed the browning of cut lettuce and that regulating
polyphenol biosynthesis is essential to repress the
browning.9) Heat shock treatment at 5060
C repressed
enzymatic browning of cut lettuce by suppressing the
induction of PAL due to cutting.10,11) The quality of cut
lettuce treated by heat shock is better than that of nontreated cut lettuce,12) but machinery instruments and
strict control of temperature are essential for heat-shock
treatment.
Chemical methods to prevent enzymatic browning of
cut lettuce have been reported.13,14) It is known that PAL
inhibitors such as 2-aminoindan-2-phosphonic acid15)
repress the browning of cut lettuce,9,16) but there is no
PAL inhibitor of natural products or foods. If there is a
PAL inhibitor derived from foods or substances generally regarded as safe (GRAS), it should suggest a new
method for the control of browning. Here we screened
for PAL inhibitors of fermented broths of microbes and
of foods, and found that cinnamon extract denitely
inhibited PAL. An active substance was isolated and
identied as cinnamaldehyde, which repressed the
browning of cut lettuce.

Material and Methods

Lettuce and storage. Commercially grown and harvested crisphead (iceberg) lettuce (Lactuca sativa L.)
specimens were purchased from a retail shop in Tokyo
in the 20032004 period and used for experiments
without further storage. After discarding two to four
leaves, the next 10 uninjured leaves were removed and

y
To whom correspondence should be addressed. Tel: +81-3-5978-5753; Fax: +81-3-5978-5755; E-mail: murata@cc.ocha.ac.jp
Abbreviations: PPO, polyphenol oxidase; PAL, phenylalanine ammonia-lyase; GRAS, generally regarded as safe

Cinnamaldehyde Inhibits PAL and Enzymatic Browning of Cut Lettuce

2 cm  4 cm midrib segments were excised starting 1 cm

from the base of the leaf. These segments were used as
cut lettuce. Cut lettuce (about 25 g) was washed with
water for 30 s, wrapped in clear food grade plastic
lm (Saran Wrap, Asahikasei, Osaka, Japan) and stored
at 4
C.
Enzyme extraction and assay. The procedure of
Siriphanich and Kader17) was adapted to the extraction
of PAL from lettuce. In a cold room (4
C) using a
TM90 mixer (Teskom, Tokyo), the lettuce segments
stored for 3 d were homogenized with three times
weights of a 0.2 M boric acid-NaOH buer (pH 8.8)
containing 5 mM 2-mercaptoethanol. Each homogenate
was ltered through four layers of cotton gauze, and the
resulting ltrate was centrifuged at 12;000  g for
30 min. The supernatant obtained was used as a crude
enzyme of PAL. PAL activity was measured by a
spectrometric method at 290 nm to detect the increase in
cinnamic acid as a product.17) The reaction solution
consisted of 2.35 ml of a 0.5 M boric acid-NaOH buer
(pH 8.8), 0.4 ml of 10 mM L-phenylalanine, 0.4 ml of the
crude enzyme solution, and 0.05 ml of distilled water or
ethanol. One unit of PAL activity was dened as the
amount of enzyme that produced 1 micromole of
cinnamic acid for 1 h at 30
C.
Screening for PAL inhibitors. Bacterial strains isolated from vegetables (112 strains: 40 Pseudomonas
spp., 11 Flavobacterium spp., 5 Bacillus spp., and 46
others) were incubated in a 3 ml of Brain Heart Infusion
(Beckton Dickinson, Sparks, MD, USA) at 37
C for
48 h with shaking. After 3 ml of ethanol was added to
each fermented broth and mixed well with a vortex
mixer, the broth was centrifuged at 3;000  g for
10 min. The supernatant obtained (0.05 ml) was added
to the assay solution for PAL activity. As a control,
0.05 ml of 50% ethanol solution was added.
Food samples (potato, cabbage, cucumber, sweet
pepper, rice bran, wheat our, broccoli, carrot, green
onion, ginger, Japanese horseradish, red pepper, nutmeg,
cinnamon, basil, pepper, thyme, black tea leaf, green tea
leaf, and oolong tea leaf) were purchased from a retail
shop in Tokyo. Each sample (5 g) was extracted with
30 ml of boiling water or ethanol for 15 min. After
centrifugation at 3;000  g for 15 min, 0.05 ml of the
supernatant obtained was added to the assay solution for
PAL activity instead of 0.05 ml of water or ethanol.
Isolation procedure of PAL inhibitor from cinnamon.
The PAL inhibitory activity of each fraction obtained by
the isolation procedures was checked. Cinnamon
(2.0 kg) was extracted with 12-liter of ethanol twice.
Distilled water (96-liter) was added to the extracts and
this solution was applied to a Diaion HP-20 column
(100 mm i.d.  400 mm; Mitsubishi Chemical, Tokyo).
The column was washed with 20% ethanol, the active
component being eluted with 100% ethanol. A peak of

673

fractions showing PAL inhibitory activity was collected

and evaporated. The crude paste obtained (29.2 g) was
applied to a silica gel column (100 mm i.d.  400 mm;
Silica gel 60, Merck, Darmstadt, Germany). The column
was developed with CH2 Cl2 and a peak of active
fractions was collected. After it was evaporated, it was
applied to a silica gel column again. The column was
developed with hexane, hexanedichloromethane (10:1,
5:1, 1:1, 1:2), and dichloromethane successively. A peak
of active fractions, which was eluted by hexane
dichloromethane (1:1), was collected and evaporated.
This fraction (124 mg) was further puried by HPLC.
The HPLC system was as follows: pump, Hitachi L6000 (Tokyo); column, YMC pack D-ODS (20 mm
i.d.  250 mm; Yamamura, Kyoto, Japan); eluent,
methanol-water (1:1); ow rate, 9.99 ml/min; detector,
Hitachi L-4200 (Tokyo); detection, 260 nm. A peak with
a retention time of 3135 min was collected and
concentrated in vacuo. Compound 1 (48 mg) was obtained as a pale yellowish paste.
Instrumental analyses. Spectroscopic measurements
were done using the following instruments: Hitachi 3310
(UV), JEOL JNM-EX400(NMR) and JEOL MStation
JMS-700 (EI-MS).
Reagents. Acetaldehyde, trans-cinnamaldehyde,
trans-cinnamic acid, trans-cinnamic alcohol, 3-phenylpropionaldehyde, 2-methoxycinnamaldehyde, and
methylcinnamaldehyde were purchased from Wako Pure
Chemical Industries (Osaka, Japan).
Physico-chemical properties of compound 1. Compound 1 was soluble in hexane, chloroform, and ethyl
acetate, slightly soluble in methanol, and insoluble
in water. UVmax (MeOH) nm: 280. EI-MS (m/z):
132 (M , 100), 103 (M-CHO), 77 (M-CHO-C2 H2 ), 51.
1
H-HMR  (ppm): 6.70 (1H, dd, J 8:0, 16.0), 7.41
(3H, m), 7.46 (1H, d, J 16:0), 7.56 (2H, m), 9.68 (1H,
d, J 8).
Treatment of cut lettuce. A crude fraction eluted from
a Diaion HP-20 column or trans-cinnamaldehyde was
used as a sample, and was dissolved in 1% ethanol. Cut
lettuce (about 25 g) was immersed in a 1% solution of
crude fraction, a 0.05% solution of trans-cinnamaldehyde, or a 1% ethanol solution (a control) for 30 min,
washed with water for 30 s, wrapped in clear food-grade
plastic lm (Saran Wrap), and stored at 4
C.
Evaluation of browning. The browning of the cut
edges of the lettuce segments was evaluated visually.
Scoring was in a range from 0 (no browning), 1 (slight
browning), 2 (denite browning), to 3 (extreme browning). At the same time, L , a , b values of cut edges of
lettuce segments were measured with a NF333 colorimeter (Nihon Denshoku Kogyo, Tokyo). Hue angle,
dened as tan1 (b /a ), was calculated.

674

N. F UJITA et al.

Results and Discussion

Eect of fermented broths of bacteria and food
extracts on PAL activity
The inhibitory eects of 112 kinds of fermented
broths and 20 kinds of food extracts on PAL activity
were examined. Only cinnamon extract by ethanol
inhibited PAL activity. We checked the reproducibility
of this inhibition using four kinds of retail cinnamon. All
the samples we purchased inhibited PAL activity
(Table 1). We then decided to identify an active
compound from cinnamon.
Isolation and identication of PAL inhibitor from
cinnamon
An active compound (compound 1) was isolated
using chromatographic procedures such as Diaion HP20, silica gel, and HPLC. Inhibitory activity was not
separated into more than two peaks at each step of
chromatographic procedure. Only one peak with about
32 min of retention time on HPLC showed PAL
inhibitory activity. Figure 1 shows a typical HPLC
pattern of the fraction from silica gel column chromatography and an isolated peak (compound 1) on HPLC.
Table 1. PAL Inhibition by Cinnamon Extract
Sample

PAL inhibition
(%)

Powdered cinnamon, brand A

Whole cinnamon, brand A
Powdered cinnamon, brand B
Powdered cinnamon, brand C

100
95
100
100

Cinnamon (5 g) was extracted with 30 ml ethanol.

Compound 1 showed UV absorption at 280 nm which

did not change with the addition of 1 N HCl or 1 N
NaOH solution. The 1 H-NMR spectrum of compound 1
showed the existence of an aldehyde group (H 9.68) and
two trans olenic protons (H 6.70 (1H, dd, J 8:0,
16.0) and H 7.46 (1H, d, J 16:0)) and a further ve
olenic protons. A decoupling experiment showed that a
proton of aldehyde coupled with an olenic proton (H
6.70). The MS of compound 1 showed that its molecular
weight was 132, and fragmentation peaks at 103 (MCHO) and 77 (M-CHO-C2 H2 ) showed the existence of
an aldehyde group connected to an olen. These results
strongly suggest that compound 1 is 3-phenyl-2(E)propenal, that is, trans-cinnamaldehyde. We then compared compound 1 with authentic trans-cinnamaldehyde. The retention times and UV spectra on 3D-HPLC
of these compounds were completely identical. The 1 HNMR data of these compounds were also identical.
From these results, compound 1 was identied as transcinnamaldehyde. The content of cinnamaldehyde in
cinnamon powder was estimated by HPLC analysis. The
cinnamon powder we used contained about 8 mg/g of
cinnamaldehyde. This result shows that the yield of
cinnamaldehyde was very low in the isolation procedure.
trans-Cinnamaldehyde is the major component in
cinnamon oil and is used as a popular food avoring. It
is used as a avoring ingredient in fruits and juices at the
levels as high as 6,400 ppm, 3,500 ppm in baked goods,
2,200 ppm in breakfast cereals, 2,000 ppm in baby food
and desserts, and 1,100 ppm in chewing gum.18) Oral
LD50 for cinnamaldehyde in rats is reported to be 2.2
3.4 g/kg, while that for guinea pigs and mice is 3.4 g/
kg,18) showing that the acute toxicity of trans-cinnamal-

Active peak

20

40
60
80
100
Retention time (min)

120

0
20
40
Retention time (min)

Fig. 1. Typical HPLC Patters of the Fraction from Silica Gel Chromatography (left) and of an Isolated Active Peak (right).
HPLC conditions are described in Materials and Methods.

Cinnamaldehyde Inhibits PAL and Enzymatic Browning of Cut Lettuce

dehyde is low. Cinnamaldehyde is regarded as a GRAS

avoring substance in the USA.19)
Table 2 shows the inhibitory activities of transcinnamaldehyde against lettuce PAL. PAL was dosedependently inhibited by trans-cinnamaldehyde, although a linear curve was not obtained. Its inhibitory
concentration of 50% was estimated to be 3.4 mg/ml. As
far as we know, there is no report that trans-cinnamalTable 2. Inhibitory Eect of trans-Cinnamaldehyde against Lettuce
PAL
Concentration of trans-cinnamaldehyde
(mg/ml)

Inhibition
(%)

7.5
5.0
2.5
1.25
0.25

91.6
66.8
41.9
31.7
13.3

(n 2)

Table 3. Inhibitory Activities of Cinnamaldehyde-Related Compounds against Lettuce PAL

PAL inhibition
(%)

Compound
Acetaldehyde
trans-Cinnamaldehyde
trans-Cinnamic acid
trans-Cinnamic alcohol
3-Phenylpropionealdehyde
2-Methoxy-cinnamaldehyde
(3-(2-Methoxyphenyl)-(2E)-propenal)
2-Methoxy-cinnamaldehyde
(2-Methyl-3-phenyl-(2E)-propenal)

dehyde or any food component inhibits PAL activity.

trans-Cinnamaldehyde did not inhibit PPO at all even at
10 mg/ml.
Eect of cinnamaldehyde and related compounds on
PAL activity
The PAL inhibitory activities of certain compounds
related to trans-cinnamaldehyde were examined
(Table 3). Acetaldehyde, cinnamic acid, and cinnamic
alcohol did not inhibit PAL at all. On the other hand,

-methyl-cinnamaldehyde and 2-methoxycinnamaldehyde, 3-phenylpropionaldehyde inhibited PAL. It appears that an aldehyde group connected to a phenylethyl
or a phenylvinyl group is essential to PAL inhibition.
Eect of a crude extract from cinnamon and transcinnamaldehyde on the browning of cut lettuce during
storage
Table 4 shows the eect of crude extract from
cinnamon on the browning of cut lettuce during cold
storage. Crude extract obtained with a Diaion HP-20

Table 4. Eect of Cinnamon Extract on the Browning of Cut Lettuce

during Storage
Storage time

0
100
0
0
67
82
100

Each compound (10 mg/ml) was added to a PAL assay solution (n 2).

675

Browning

(d)

Control

0
2
3
6

0
2
2.6
3

Crude extract
0
0
0
1

Browning was visually evaluated (0, no browning; 1, slight browning; 2,

denite browning; 3, extreme browning).  , crude fraction obtained by
Diaion HP-20.  , signicant dierence (p < 0:01) between the control (1%
ethanol) and lettuce treated with crude extract at the same storage time
(n 3).

Hue angle ( o )
120
cinnamaldehyde

100
80

Control

60
40
20
0 0

4
2
Storage time (d)

Fig. 2. Eects of Cinnamaldehyde Treatment on the Browning of Cut Lettuce during Cold Storage.
Cut lettuce was immersed in 0.05% trans-cinnamaldehyde solution (1% ethanol) for 30 min. After washing, it was stored at 4
C (n 7).
Control lettuce was similarly treated with 1% ethanol solution. Browning was estimated by hue angle.  , signicant dierence (p < 0:01)
between the control and treated lettuce at the same storage time.

676

N. F UJITA et al.

column was used. Sections of control cut lettuce

gradually turned brown. On the other hand, that treated
with a crude extract from cinnamon turned less brown.
This extract signicantly inhibited the browning. The
eect of trans-cinnamaldehyde on the browning of cut
lettuce during cold storage was then examined (Fig. 2).
Hue angle, dened as tan1 (b /a ), was used for the
estimation of browning. A lower value indicates
browning. Cut lettuce treated with trans-cinnamaldehyde showed signicantly higher values than the control
lettuce. These results clearly show that trans-cinnamaldehyde repressed the browning of cut lettuce during
storage. This is the rst report showing that a food or a
avor component represses the browning of cut lettuce
during storage.
In conclusion, a cinnamon extract and trans-cinnamaldehyde inhibited PAL activity and repressed the
browning of cut lettuce. We will further investigate
the eect of cinnamaldehyde on the quality of cut
lettuce and the mechanism of repression of browning by
trans-cinnamaldehyde.

9)

10)

11)

12)

13)

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