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Inhibitory Effects of Feeding with Carrots or (−)-Falcarinol on Development of Azoxymethane-Induced Preneoplastic Lesions in the Rat Colon

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Abstract

The effects of intake of dietary amounts of carrot or corresponding amounts of (-)-(3R)-falcarinol from carrots on development of azoxymethane (AOM)-induced colon preneoplastic lesions were examined in male BDIX rats. Three groups of eight AOM-treated rats were fed the standard rat feed Altromin supplemented with either 10% (w/w) freeze-dried carrots with a natural content of 35 mug falcarinol/g, 10% maize starch to which was added 35 mug falcarinol/g purified from carrots, or 10% maize starch (control). After 18 weeks, the animals were euthanized and the colon was examined for tumors and aberrant crypt foci (ACF), which were classified into four size classes. Although the number of small ACF was unaffected by the feeding treatments, the numbers of lesions as a function of increasing size class decreased significantly in the rats that received one of the two experimental treatments, as compared with the control treatment. This indicates that the dietary treatments with carrot and falcarinol delayed or retarded the development of large ACF and tumors. The present study provides a new perspective on the known epidemiological associations between high intake of carrots and reduced incidence of cancers.
Inhibitory Effects of Feeding with Carrots or (
)-Falcarinol on
Development of Azoxymethane-Induced Preneoplastic Lesions
in the Rat Colon
MORTEN KOBÆK-LARSEN,*,† LARS P. CHRISTENSEN,WERNER VACH,§
JELMERA RITSKES-HOITINGA,AND KIRSTEN BRANDT|
Biomedical Laboratory, University of Southern Denmark, Winsloewparken 23, DK-5000 Odense C,
Denmark, Department of Food Science, Danish Institute of Agricultural Sciences, Research Centre
Aarslev, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark, Department of Statistics, University of
Southern Denmark, Sdr. Boulevard 23A, DK-5000 Odense C, Denmark, and School of Agriculture,
Food and Rural Development, University of Newcastle upon Tyne, Agriculture Building,
Newcastle upon Tyne NE1 7RU, United Kingdom
The effects of intake of dietary amounts of carrot or corresponding amounts of (-)-(3
R
)-falcarinol
from carrots on development of azoxymethane (AOM)-induced colon preneoplastic lesions were
examined in male BDIX rats. Three groups of eight AOM-treated rats were fed the standard rat feed
Altromin supplemented with either 10% (w/w) freeze-dried carrots with a natural content of 35 µg
falcarinol/g, 10% maize starch to which was added 35 µg falcarinol/g purified from carrots, or 10%
maize starch (control). After 18 weeks, the animals were euthanized and the colon was examined
for tumors and aberrant crypt foci (ACF), which were classified into four size classes. Although the
number of small ACF was unaffected by the feeding treatments, the numbers of lesions as a function
of increasing size class decreased significantly in the rats that received one of the two experimental
treatments, as compared with the control treatment. This indicates that the dietary treatments with
carrot and falcarinol delayed or retarded the development of large ACF and tumors. The present
study provides a new perspective on the known epidemiological associations between high intake of
carrots and reduced incidence of cancers.
KEYWORDS:
Daucus carota
; aberrant crypt foci; (3
R
)-falcarinol; BDIX rats; natural toxicant; colon
carcinogenesis
INTRODUCTION
While it is well-known from epidemiological studies that a
high intake of vegetables and fruits reduces the risk of cancer
(1-4), knowledge is still very limited about which components
in these foods are primarily responsible for this reduction. Highly
bioactive plant secondary metabolites are mostly known for their
toxicities at high concentrations (5). However, one of the many
theories in this field predicts that these compounds could have
cancer-preventing effects at lower concentrations, corresponding
to the levels found naturally in food (6), as it has already been
shown for glucosinolates from Brassica vegetables (7).
Several epidemiological studies imply specifically the intake
of carrots as important for this preventive effect. Many studies
have been based on the hypothesis that the antioxidant β-car-
otene was responsible for this. However, subsequent intervention
studies ruled out this explanation, since supplementation with
β-carotene does not reduce cancer incidence and, in some cases,
even increases the risk for this disease (8-10). Even though
carrots are the major source of β-carotene in the diet in Northern
Europe and North America, they also contain a group of
bioactive polyacetylenes, of which falcarinol (Figure 1) clearly
is the most bioactive of the carrot polyacetylenes (11-14).
In the human diet, carrots are the almost exclusive dietary
source of falcarinol. A recent in vitro study aiming to screen
for potentially health promoting compounds from vegetables
showed that falcarinol could stimulate differentiation of primary
mammalian cells in concentrations between 1 and 50 ng/mL,
while toxic effects were found above 100 ng/mL (11). This
biphasic effect (hormesis) of falcarinol on cell proliferation is
fully in accordance with the hypothesis that most highly bio-
active compounds exhibit hormesis (15). Ingestion of carrot juice
containing 13 µg falcarinol/mL by human volunteers resulted
in a plasma concentration of falcarinol of 2 ng/mL for several
hours (16). This is within the range where the in vitro data
indicate that a potentially beneficial physiological effect would
be expected (11). Earlier studies showed that physiologically
* To whom correspondence should be addressed. Tel: +45 65 50 37
21. Fax: +45 65 90 68 21. E-mail: mkobaek@health.sdu.dk.
Biomedical Laboratory, University of Southern Denmark.
Danish Institute of Agricultural Sciences.
§Department of Statistics, University of Southern Denmark.
|University of Newcastle upon Tyne.
J. Agric. Food Chem.
2005,
53,
1823
1827 1823
10.1021/jf048519s CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/05/2005
relevant concentrations of falcarinol had a pronounced cytotoxic
effect on several human tumor cell lines in vitro (12-14).
Carrots and carrot extracts can also reduce the incidence of
hepatomas (17), although only minimal curative effects of fal-
carinol were found when a highly invasive form of brain cancer
in mice was studied (12). A more detailed account of the back-
ground for the present study has recently been described (6).
In the present study, we examined the possible cancer
preventive effects of realistic dietary amounts of carrot or
corresponding amounts of falcarinol from carrots on the
development of azoxymethane (AOM)-induced aberrant crypt
foci (ACF) and tumors in male BDIX rats. ACF are precan-
cerous lesions often used as biomarkers for colon carcinogenesis
(18), and we used the distribution among different size classes
as a measure of the extent of their progress toward actual tumors.
MATERIALS AND METHODS
Animals. Male rats from the BDIX/OrlIco strain with a certified
health report were purchased from IFFA CREDO (L’Abresle, France).
The animals were 8 weeks old at the time of the first injection with
AOM, as described in earlier studies (19). All animals were housed in
groups of two rats in Macrolon type III cages (Scanbur A/S, Køge,
Denmark). AOM-treated animals were housed inside an isolator [Isotec
type 13366 (M50), Harlan, The Netherlands] with negative pressure
(3 mm H2O) in order to protect the personnel and the environment
from this carcinogen and its metabolites. Two weeks after the final
AOM injection, the animals were moved outside the isolator and housed
in the same animal room. Rats that did not receive AOM injections
were housed outside the isolator in the same animal room throughout
the study. The animals were kept under standard laboratory condi-
tions: room temperature, 20-24 °C; relative humidity, 50-60%; and
12 h light/dark cycle (lights on from 6.00 to 18.00 h). The temperature
and relative humidity inside the isolator were not recorded separately.
The bedding consisted of irradiated aspen wood chips (Tapvei, Oy,
Kaavi, Suomi), and the cages were changed twice a week both inside
and outside the isolator. While outside the isolator, the rats were given
environmental enrichment as aspen wood shavings and/or wooden
blocks (Tapvei) twice a week. Animals in the isolator were allowed
free access to acidified tap water (acidified with HCl to pH 3 in order
to reduce bacterial growth) via water bottles, which were changed once
during their 8 week stay in the isolator (full details given in ref 19).
Outside the isolator, the rats had free access to nonacidified water in
water bottles, which were changed once a week. Food was available
ad libitum, both inside and outside the isolator. Fresh food was given
at least once a week on top of the remaining food.
Plant Material. Carrots (cv. Bolero) were grown organically at
Research Centre Aarslev in 2002. Tops and bottoms were removed
from fresh, washed carrots, which were then shredded, freeze-dried
below 50 °C (Danish Freeze-Dry, Kirke Hyllinge, Denmark), and
packed in sealed aluminum foil pouches until use.
Extraction, Isolation, and Quantification of (3R)-(9Z)-Heptadeca-
1,9-diene-4,6-diyne-3-ol [(-)-Falcarinol]. Falcarinol was isolated from
carrots according to the procedure described by Hansen et al. (11) with
a few modifications. Eight kilograms of freeze-dried carrots were
extracted twice with 12 L of ethyl acetate (99.9% HPLC grade, Aldrich-
Chemie, Steinheim, Germany) for 24 h at 8 °C. The combined extracts
were filtered and concentrated in vacuo (35 °C) under dim light. The
extract (26 g) was chromatographed on silica gel, eluting with n-hexane,
n-hexane-ethyl acetate (v/v) (9:1, 4:1, 7:3, 3:2, 1:1, 2:3, 3:7, 1:4),
ethyl acetate, and finally with CH3OH [99.9% high-performance liquid
chromatography (HPLC) grade, Aldrich-Chemie]. Fractions containing
crude falcarinol were further purified by preparative reversed phase
(RP) HPLC on a Develosil ODS-HG-5 (RP-18, 250 mm ×20 mm
i.d., Nomura Chemical Co., Seto, Japan) column using the following
stepwise gradient: CH3OH-H2O (v/v), 40:60, 50:50, 60:40, 70:30,
80:20, and 100:0, yielding 235 mg of (-)-falcarinol (purity of >99%,
as determined by analytical RP-HPLC). Monitoring was performed at
256 nm; flow rate, 6 mL/min. (-)-Falcarinol was obtained as a colorless
oil and identified by optical rotation, UV, mass spectrometry (MS) [gas
chromatography (GC)-MS (EI, 70 eV)], one-dimensional and two-
dimensional NMR (1H and 13C NMR and 1H-1H and 1H-13C
correlation spectroscopy), and the complete spectral data set cor-
responded fully with literature values (20-25). The optical rotation
for the isolated falcarinol was found to be levorotatory ([R]D20 -36.8°,
c0.88, CHCl3), which is consistent with literature values {[R]D-36.6°,
c0.92, CHCl3(24); [R]D-36.93°,c0.77, CHCl3(25)}for the 3R
configuration of falcarinol.
Falcarinol was quantified in carrot samples by analytical RP-HPLC.
Extraction of carrot samples for analytical RP-HPLC analysis was
performed by extracting 10 g of freeze-dried carrots twice with 100
mL of ethyl acetate according to the procedure of Hansen et al. (11).
Analytical RP-HPLC was performed on a Merck D-7000 Hitachi HPLC
system using diode array detection. Separations were performed on a
LiChrospher 100 RP-18 column (particle size 5 µm; 244 mm ×4mm
i.d.; Merck, Darmstadt, Germany) at 35 °C, by gradient elution with
solvent A [CH3OH-H2O-trifluoroacetic acid (TFA; Sigma Chemical
Co., St. Louis, MO) (v/v/v), 95:4.5:0.5], solvent B [H2O-CH3OH-
TFA (v/v/v), 94.5:5.0:0.5], and solvent C [CH3OH-tetrahydrofuran
(99.9% HPLC grade, Sigma Chemical Co.) (v/v), 50:50]. Elution
profile: 0 min 26% B and 19% C, 20 min 11% B and 19% C, 21-26
min 0% B and 0% C, 29 min 40% B and 0% C and 39 min 40% B and
0% C. All changes in the programmed gradient were linear; flow, 1.0
mL/min. The HPLC samples were cooled to 10 °C in the autosampler,
and 20 µL of sample was injected. Mobile phases were degassed with
ultrasound for 20 min before use. Falcarinol eluted at 26 min and had
the following UV-maxima: λmax 229, 243, 256 nm. Falcarinol was
identified by peak addition of an authentic standard and quantified in
carrot extract samples using falcarinol as external standard. The validity
of the HPLC method was checked with regard to accuracy, linearity,
and precision.
Rat Diet. Before the main experiment, four male BDIX rats were
given free access to both a standard rat feed (Altromin) and freeze-
dried carrots. The consumption of each type of feed was recorded daily,
and after a run-in period of 14 days, the voluntary intake of freeze-
dried carrots stabilized around 10-20% of the daily total feed intake.
For the main experiment, the standard rat feed (Altromin 1234, Chr.
Petersen Inc., Denmark) was pulverized before addition of the
supplementary materials, to ensure that the rats did not select one
component at the expense of another. Diet group 1 contained standard
rat feed supplemented with 10% pulverized freeze-dried carrots
containing 35 µg falcarinol/g; diet group 2 contained standard rat feed
supplemented with 10% maize starch and purified falcarinol corre-
sponding to 10% carrots; and diet group 3 (control group) contained
standard rat feed supplemented with 10% maize starch. Because the
isolated falcarinol for diet group 2 was applied to the diet in the form
of an ethanol solution, the diets for the two other groups were also
treated with the same amount of ethanol. Each time, portions of 20 kg
diet were prepared for each of the three groups and2Lof96%ethanol
or ethanol solution was applied to the diets using an atomizer, after
which the portion was allowed to dry at room temperature for
approximately1hindarkness before being packed in sealed aluminum
foil pouches. The Altromin and freeze-dried carrots were stored at -20
°C before preparations. The prepared diets were stored at room
temperature, mixed well before use, and used for approximately 1 month
before new diets were prepared. The content of falcarinol in the rat
diets (groups 1 and 2) was measured by analytical HPLC before use
and at approximately monthly intervals throughout the experiment. No
significant differences in the content of falcarinol in either diet were
observed during the experiment.
Design of the Rat Feeding Experiments. A total of 30 rats were
divided into three groups that received different diets, starting 10 days
before the first AOM injection. AOM purchased from Sigma Chemical
Co. was diluted with sterile 0.9% NaCl to a concentration of 5 mg/mL
at the Central Pharmacy of the Odense University Hospital (Odense,
Figure 1.
Chemical structure of (3
R
)-(9
Z
)-heptadeca-1,9-diene-4,6-diyne-
3-ol [(
)-falcarinol] isolated from carrots and tested in the present study.
1824
J. Agric. Food Chem.,
Vol. 53, No. 5, 2005 Kobæk-Larsen et al.
Denmark). The AOM solution was stored for about1hatroom
temperature before being injected. Eight of the 10 animals in each
treatment group were given weekly subcutaneous (s.c.) injections of
freshly prepared AOM at a dose of 15 mg/kg body weight for a period
of 2 ×2 weeks separated by a 1 week break (19). The range of the
injection volume used was 0.4 mL at the start and 1.0 mL at the end
of the AOM treatments. Two rats in each treatment group were injected
with a volume of sterile 0.9% NaCl related to the body weight.
Autopsy Procedures. The rats were euthanized after 18 weeks, when
the first symptoms of cancer (blood in stools) were observed in a few
animals, and autopsied to examine for macroscopic alterations. The
animals were killed in 100% CO2, after they had been anaesthetized
(duration max, 30 min) s.c. with a mixture of 0.3 mL of Hypnorm/kg
rat (0.095 mg/kg fentanyl citrate and 3 mg/kg fluanisone, JANSSEN
Animal Health, Beerse, Belgium) and 0.675 mL Dormicum/kg rat
(3.375 mg/kg midazolam, Dumex-Alpharma, Oslo, Norway). Im-
mediately after death, selected organs were fixed in 4% phosphate-
buffered formaldehyde, pH 7.4, for later histopathological examination.
The total length of the intestine was measured, and it was then cut
longitudinally, rinsed in 0.9% NaCl solution, cut into two equally sized
pieces, and pinned on a cork slab. Before fixation, the large intestine
was evaluated for macroscopic neoplasms, where diameter and location
in the intestine were registered.
Identification and Quantification of Tumors and ACF. After
fixation of the large intestine, Giemsa stain [6 mL of stock solution
(The Central Pharmacy at the Odense University Hospital) in 50 mL
of phosphate-buffered saline (PBS), pH 7.2, for 15 min] was used to
visualize the ACF, and excess stain was rinsed off with PBS. The tissue
was placed with the luminal side up in a Petri dish with enough PBS
to cover the tissue. The total numbers of ACF and tumors for each
section of the large intestinal were counted independently by two
persons, blinded to treatment modality, by using a stereomicroscope
at 40×magnification. The aberrant crypts were distinguished by their
increased size and thicker and deeply stained epithelial lining as
compared with normal crypts. An ACF may consist of one to several
crypts, and in the present study, the ACF were classified as small (1-3
crypts), medium (4-6 crypts), or large (more than seven crypts), while
neoplasms larger than 1 mm in diameter were classified as tumors.
Within each class of lesions, the variation coefficient of the single counts
between the two persons was less than 10%. The counts of the two
persons were averaged.
The tumors were fixed in 4% (v/v) formaldehyde buffered with 0.075
M sodium phosphate (pH 7) and embedded in paraffin. The tissues
were cut into 5 µm sections and were stained with haematoxylin and
eosin. Additional sections were cut until characterization of the
neoplasm was certain.
Statistical Analysis. The counts of the four different size classes of
(pre)neoplastic lesions were normalized in the carrot and falcarinol
treatment groups by dividing with the class specific mean count in the
control group. The four size classes were scored from one to four with
increasing lesion size. The effect of the class on the normalized counts
in the two treatment groups (carrot and falcarinol) was assessed by a
regression model using the size class score and the treatment group as
covariates. The correlation among the four size classes of ACF/tumors
within each individual rat has been taken into account in this analysis
by using robust variance estimates.
RESULTS
Although both (+)- and (-)-falcarinol have been isolated
from different plants and appear to occur regularly in plants of
the Araliaceae (12,14,25) and Apiaceae (11,21,23,26), the
optical rotation of falcarinol, and hence the absolute configu-
ration, when isolated from plants has often not been determined.
To the best of our knowledge, the absolute configuration of
falcarinol in carrots has not been determined. Falcarinol was
isolated from carrots by preparative HPLC and identified by
spectroscopic means to be levorotatory falcarinol (see Materials
and Methods). Thus, (-)-falcarinol (Figure 1) tested in the
present study on rats on development of AOM-induced colon
preneoplastic lesions possesses the 3Rconfiguration (24,25).
Animal weight gain was identical in all treatments (data not
shown), and no mortality, signs of distress, or disease nor gross
abnormalities were observed. The histopathological aspects of
the model using the same experimental design as the control
treatment have been reported previously (19). Neither tumors
nor ACF were observed in the two animals per dietary treatment
that were injected with 0.9% NaCl solution (data not shown).
The numbers of each size class of ACF and tumors found
are presented in Table 1. In every dietary treatment, while the
numbers of individual lesions decreased very much with
increasing size of the lesion, each size class still represented a
comparable amount of tissue with aberrant growth pattern. For
example, a tumor typically contained the same amount of tissue
as 50-200 small ACF.
All tumors identified macroscopically were subsequently
identified by histological examination as either adenomas or
carcinomas, with an approximately equal number of each type
in each treatment group (data not shown). All of the carcinomas
were restricted to invading the submucosa.
The number of animals was too small to determine whether
the treatments resulted in differences in the numbers of each of
the various lesion types found or in the sums of all types (Table
1). However, the carrot and falcarinol treatments showed a
significant (P)0.028) tendency to reduced numbers of (pre)-
cancerous lesions with an increasing size of lesions (Figure
2), from no difference relative to control for the smallest ACF,
to a one-third reduction for the fully developed tumors.
DISCUSSION
The relation between lesion size and treatment effect corre-
sponds to the trend expected if the bioactive compound is
capable of reducing the growth of precancerous lesions, under
conditions where the preventive effect is not sufficient to prevent
the initial proliferation (which takes place during the actual
period of challenge with the carcinogen). The observed results
could either be due to a decreased growth rate or survival of
the individual tumor precursor cells (cytotoxicity), to a decrease
in the rate of progression through the stages of carcinogenesis,
or to enhancement of one or more of the rats’ relevant immune
system components. Earlier studies of falcarinol, primarily in
vitro studies of its potential use as a drug, have already shown
effects corresponding to each of these mechanisms (5,27),
although their relative relevance in vivo is still unknown.
Our results thus indicate that consumption of carrot led to a
reduced risk of colorectal cancer in this rat model, which is in
line with the epidemiological data indicating that carrot
consumption provides a protective effect on the development
of cancers. They also show that the effect of falcarinol alone
was of the same magnitude as that of the entire carrots (Figure
Table 1.
Numbers of (Pre)Neoplastic Lesions of Different Sizes in
Colons of Rats Fed with Different Supplements; 10% Added to Their
Respective Diets, Results Expressed as Means
±
Standard Deviations
treatment (supplement used,
10% mixed in Altromin diet)
small
a
(1
3
crypts)
medium
a
(4
6
crypts) large
a
(>7 crypts)
tumors
a
(
g
1mm
diameter)
freeze-dried carrots containing
35
µ
g falcarinol/g 83
±
28 48
±
19 13
±
8 0.9
±
1.0
maize starch where 35
µ
g
falcarinol/g was added 101
±
43 59
±
35 18
±
12 0.8
±
0.7
maize starch (control) 98
±
17 67
±
16 21
±
9 1.4
±
1.1
a
None of the differences between the three diets were significant when analyzed
within one size class or on summations of total numbers across size classes.
Anticancer Effect of Carrots and Falcarinol
J. Agric. Food Chem.,
Vol. 53, No. 5, 2005 1825
2), which supports the hypothesis that falcarinol is an active
protective substance in carrots. Falcarindiol, which is the
quantitatively predominant polyacetylene in carrots (20), has
also been shown to possess cytotoxic (12,28) and antimutagenic
activity in vitro (29), although it appears to be much less
bioactive than falcarinol (11-14). The bioactivity of falcarin-
diol-3-acetate a further polyacetylene in carrots (20) has so far
not been investigated. The possible mode of action of falcarinol
may be related to its hydrophobicity and its ability to form an
extremely stable carbocation with the loss of water, thereby
acting as a very reactive alkylating agent toward proteins and
other biomolecules (30). This may also explain the highly
allergenic properties of falcarinol (30). A similar mode of action
may be possible for falcarindiol and falcarindiol-3-acetate, al-
though the possibility to generate two active centers for nucleo-
philic attack reduces the lipophilic character of these compounds
and hence their reactivity, in accordance with the observed
nonallergenic properties of falcarindiol (30). So the physiological
effects of falcarindiol and falcarindiol-3-acetate are expected
to be qualitatively similar but quantitatively less than those
found for falcarinol, and furthermore, they may even interact
with falcarinol in an antagonistic manner thereby affecting its
effectiveness. This could explain the possible, although not
significant, differences in the effect and the trend observed
between the treatments with falcarinol and the carrot diet
(Figure 1). Other relevant bioactive compounds in carrots that
have been considered in this context, but about which even less
information is available (6), are isocoumarins such as 6-meth-
oxymellein and a large number of mono- and sesquiterpenes.
As far as we know, these are the first results directly
indicating falcarinol as the primary cancer protective substance
from carrots. As a natural pesticide, falcarinol is best known
for its toxic properties, which are observed at high concentra-
tions, and falcarinol is listed as a toxicant in the Nettox database
(5). It is possible that falcarinol could be beneficial and act as
a cancer preventive substance in the relatively low amounts
found in food, even though it is harmful at high intake levels,
just as it has been shown for other highly bioactive food
components, such as ethanol (4).
To confirm the tendencies presented here in future studies,
higher numbers of animals and/or other models are needed. A
power calculation based on the reported data shows that to obtain
a 90% power for each of the medium and large ACFs, 25
animals per treatment group would be needed. For tumors, the
corresponding number would be 3-4 times higher, due to the
small number of tumors per animal.
The amount of carrot in the diet was chosen to correspond
to the voluntary intake when rats were given free choice between
freeze-dried carrots and Altromin. This ensured that the results
would be physiologically relevant, an issue that is particularly
important when studying food components. Imposing a nutri-
tionally deficient diet could by itself inhibit the development
of cancer (31), but because rats are known to be able to
efficiently detect and avoid nutritionally deficient diets (32),
the described procedure ensured that the carrot treatment was
nutritionally adequate. Still, if the use of starch as a substitute
for carrot in the feed for the other treatments caused so large a
difference in the nutritional value between treatments that the
formation of (pre)neoplastic lesions was affected, it would have
inhibited carcinogenesis in the starch-fed rats. This implies that
in our study we might be underestimating the difference between
the positive control (carrot) treatment and the negative control
(starch) treatment, so we have to regard the observed protective
effect as a minimum estimate. Ten percent of the dry weight of
the daily food intake corresponds to a daily human consumption
of 400-600 g fresh weight of carrot. This is the intake level of
fruit and vegetables recommended by many official agencies
responsible for food safety and quality, to decrease the risk of
cancer and other diseases (3).
The presence of equally high numbers of small ACF in all
treatments could be due to a saturation effect: that the intensive
AOM treatment might have induced the maximal number of
preneoplasms that are able to develop in this system, irrespective
of possible differences in susceptibility related to the feed
treatments. If this has been the case, reduction of the amounts
of AOM used in similar future studies may facilitate quantifica-
tion of effects of treatments on the total number of ACF formed.
If future studies confirm the present results, both in terms of
protection against cancer and absence of indications of toxicity,
it would indicate that the moderately increased falcarinol content
in food could reduce the risk of cancer. The present experiment
together with the experiments leading to it (6) thus highlights
Figure 2.
Effect of treatments with carrot or falcarinol on the average numbers per animal of four types of (pre)cancerous lesions in rat colons, each
size class representing increasingly advanced steps on the progression toward cancer. The size of ACF was measured as the number of crypts found
on a corresponding area of normal colon tissue. The smallest tumors correspond to an ACF size of approximately 20. The trend for reduced relative
numbers with increasing size of lesion was significant at
P
)
0.028.
1826
J. Agric. Food Chem.,
Vol. 53, No. 5, 2005 Kobæk-Larsen et al.
the need to include the many overlooked bioactive food
compounds such as falcarinol in ongoing and planned research
on improvement of food safety and quality, including basic
studies on the functioning of human cells during normal and
abnormal development.
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Received for review September 6, 2004. Revised manuscript received
December 20, 2004. Accepted December 22, 2004. The support of the
Danish Research Council for Agricultural and Veterinary Research,
Danish Food Technology Initiative (FØTEK 3) Grant 2011-00-0040,
and of the Development Centre Aarslev are gratefully acknowledged.
JF048519S
Anticancer Effect of Carrots and Falcarinol
J. Agric. Food Chem.,
Vol. 53, No. 5, 2005 1827
... A study examined colon preneoplastic lesions in AOM-treated rats that were fed carrots (10% freeze-dried carrot with a natural concentration of FaOH at 35 µg/g), FaOH (purified FaOH mixed at 3.5 µg/g in food) or a control for 18 weeks. The number and size of lesions decreased significantly in the rats that received either one of the two experimental treatments compared to the control group, indicating that carrots and FaOH slowed the growth of aberrant crypt foci (ACF) and tumours [117]. In a similar study, again using AOM-treated rats as a colon cancer model, feed containing a mixture of FaOH and FaDOH at concentrations four times higher than the previous study significantly reduced the number of tumours >1 mm, from 21 in controls to 12 in PA-treated rats [118]. ...
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Polyacetylene phytochemicals are emerging as potentially responsible for the chemoprotective effects of consuming apiaceous vegetables. There is some evidence suggesting that polyacetylenes (PAs) impact carcinogenesis by influencing a wide variety of signalling pathways, which are important in regulating inflammation, apoptosis, cell cycle regulation, etc. Studies have shown a correlation between human dietary intake of PA-rich vegetables with a reduced risk of inflammation and cancer. PA supplementation can influence cell growth, gene expression and immunological responses, and has been shown to reduce the tumour number in rat and mouse models. Cancer chemoprevention by dietary PAs involves several mechanisms, including effects on inflammatory cytokines, the NF-κB pathway, antioxidant response elements, unfolded protein response (UPR) pathway, growth factor signalling, cell cycle progression and apoptosis. This review summarises the published research on falcarinol-type PA compounds and their mechanisms of action regarding cancer chemoprevention and also identifies some gaps in our current understanding of the health benefits of these PAs.
... Polyacetylenes of the falcarinol-type are known as highly bioactive compounds exhibiting considerable biological effects in a wide range of organisms. Inter alia antifungal (Garrod et al., 1978;Harding and Heale, 1980), antimicrobial (Matsuura et al., 1996;Rollinger et al., 2003), anti-infl ammatory (Metzger et al., 2008), antituberculosis (Kobaisy et al., 1997), anticancer, and cytotoxic (Kobaek-Larsen et al., 2005;Matsunaga et al., 1990) properties have been reported. Furthermore, they are notorious contact allergens (Hansen and Boll, 1986), also possessing neurotoxic properties (Crosby and Aharonson, 1967) ...
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Polyacetylenes are known for their biofunctional properties in a wide range of organisms. In the present study, the most frequently occurring polyacetylenes, i.e. falcarinol, falcarindiol, and falcarindiol-3-acetate, were determined in six genera of the Apiaceae family. For this purpose, a straightforward and reliable method for the screening and quantification of the polyacetylenes using high-performance liquid chromatography coupled with diode array and mass spectrometric detection without tedious sample clean-up has been developed. Peak assignment was based on retention times, UV spectra, and mass spectral data. Quantification was carried out using calibration curves of authentic standards isolated from turnip-rooted parsley and Ligusticum mutellina, respectively. The references were unambiguously identified by Fourier transform-IR (FT-IR) spectroscopy, GC-MS, HPLC-MSn in the positive ionization mode, and ¹H NMR and ¹³C NMR spectroscopy. To the best of our knowledge, the occurrence of falcarindiol-3-acetate in Anthriscus sylvestris and Pastinaca sativa has been reported for the first time. The data revealed great differences in the polyacetylene contents and varying proportions of individual compounds in the storage roots of Apiaceous plants. The results of the present study may be used as a suitable tool for authenticity control and applied to identify novel sources devoid or particularly rich in polyacetylenes, thus facilitating breeding programs for the selective enrichment and depletion of these plant secondary metabolites, respectively.
... Compared to the control group, the number and size of lesions decreased significantly in rats that received either one of the two experimental treatments, compared to the control group. The study found that carrots and FaOH can slow the growth of aberrant crypt foci (ACF) and tumours [100]. In a similar study, again using AOM-treated rats as a colon cancer model, feed containing a mixture of FaOH and FaDOH at concentrations 4 times higher than the previous study, significantly reduced the number of tumours >1mm, from 21 in controls to 12 in polyacetylene-treated rats [101]. ...
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Full-text available
Polyacetylene phytochemicals are emerging as potentially responsible for the chemoprotective effects of consuming apiaceous vegetables. There is some evidence suggesting that polyacetylenes impact carcinogenesis by influencing a wide variety of signalling pathways, which are important in regulating inflammation, apoptosis, cell cycle regulation, etc. Studies have shown a correlation between human dietary intake of polyacetylene-rich vegetables with reduced risk of inflammation and cancer. Polyacetylene supplementation can influence cell growth, gene expression, and immunological responses, and reduce tumour number in rat and mouse models. Cancer chemoprevention by dietary polyacetylenes involves several mechanisms, including effects on inflammatory cytokines, the NF-κB pathway, antioxidant response elements, UPR pathway, growth factor signalling, cell cycle progression, and apoptosis. This review summarises the published research on falcarinol-type polyacetylene compounds and their mechanisms of action regarding cancer chemoprevention and treatment, and also identifies some gaps in our current understanding of the health benefits of these polyacetylenes.
... Carrots are the major dietary source of the falcarinols-falcarinol and falcarindiol-and their interaction with animals and human cancer cells and enzyme systems have been systematically investigated [14]. Purified falcarinols have been demonstrated to inhibit the growth of human cancer cell lines [15][16][17][18][19] and prevent neoplastic transformation in the large bowel in rats primed to develop bowel cancer [20][21][22]. The effect seen in rats can be reproduced when feeding the rats with raw carrots with purified falcarinols. ...
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A prospectively followed Danish cohort of 55,756 citizens with an observation time upwards of 25 years was investigated for association between eating raw carrots on a regular basis and developing various adenocarcinoma-dominant cancers and leukemia. Mean age at inclusion was 56.2 years (SD 4.4 years), and 52% were females. A dose-dependent reduction in incidence was seen for cancer of the lung (HR 0.76, CI95% 0.66; 0.87) and pancreas (HR 0.79, CI95% 0.61; 1.03), as well as leukemia (HR 0.91, CI95% 0.68; 1.21). Only for lung cancer was the association significant. In the case of pancreatic cancer, a possible type 1 error was present due to a low number of cancers. In cases of breast and prostate cancer, no association and no dose response were demonstrated. The association seen for lung and pancreatic cancer parallels that earlier demonstrated for large bowel cancer and indicates a cancer-protective effect from daily intake of raw carrots not limited to gastrointestinal adenocarcinomas. Processed carrots exhibited no effect. The preventive effect could be due to the polyacetylenic compounds falcarinol and falcarindiol in carrots, whereas carotene may not have an effect. The polyacetylenes are inactivated by heating, supporting our findings that only raw carrot intake has an effect. Indirect evidence for the cancer preventive effect of carrots in humans has reached a level where a prospective human trial is now timely.
... Czepa and Hofmann (2004) reported highest concentration of FaDOH in phloem and upper part of the root, falcarindiol 3-acetate in the upper part of the root, and FaOH uniformly distributed in the entire root. Polyacetylenes are reported for their beneficial effect on the human health (Brandt et al., 2004;Zidorn et al., 2005), reduced the number of neoplastic lesions, as well as growth rate of polyps suggesting a preventive effect of FaOH and FaDOH on colorectal cancer in rats (Kobaek-Larsen et al., 2005), antifungal activities (Garrod & Lewis, 1978;Mercier et al., 1990;Olsson & Svensson, 1996), and also offer resistance against various plant diseases and protection against many diseases. Malgorzata Baranska et al. (2005) reported that though the molecular structure of reported polyacetylenes is similar, but their spectra exhibit specific differences by the shift of their (triple bond) mode from 2258 to 2252 cm −1 . ...
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Processed carrot discards and graded carrots were mixed in different proportions (0, 25, 50, 75, 100%), with and without peels and crowns, and processed into carrot puree. A novel processing method consisting of multiple-pass ultrasonication with mechanical homogenization was used for the processing of puree from carrot rejects and waste (processed carrot discards). The samples were ultrasonicated for 9 min, followed by mechanical homogenization for 1 min subjected to three passes. The processed puree was evaluated for particle size distribution, β-carotene content, and color. Fourier transform infrared spectroscopy and Raman spectrophotometry data were collected for the molecular fingerprinting of the puree. Samples prepared by mixing processed carrot discards and graded carrots (50:50), with peels and crowns reported particle size distribution (PSD) for d (0.1) and d (0.5) as 125.13 and 295.25 μm, with a β-carotene content of (395.4 μg/g) and color value “a” 17.83. All the values were comparable to the responses recorded for puree samples processed without peels and crowns. The results confirm the sustainable utilization of processed carrot discards into puree, allowing the maximum retention of β-carotene and desirable particle size in the puree, reducing the influx of carrot rejects to landfills which are contributing to greenhouse gas emissions. Graphical Abstract
... Datura metel exerted antitumor effect againstA549 (lung), BGC-823 (gastric), K562 (leukemia) cancer cell lines, vero cell line, human lung carcinoma cells (A549), human colorectal adenocarcinoma cells (DLD-1), HepG-2, HeLa and SGC-7901 cell lines (274)(275)(276)(277)(278) . Daucus carota showed anticancer effect against wide range of cancer cells including lung, skin, breast, glioblastoma cancer cell, skin papilloma, myeloid leukemia (AML) cells, CaCo-2 cells, human lymphoid leukaemia cells, myeloid and lymphoid leukemia cells, human breast adenocarcinoma cells, MDA-MB-231, MCF-7, human colon adenocarcinoma cells (HT-29 and Caco-2) and HepG-2 cell lines (279)(280)(281)(282)(283)(284)(285)(286)(287)(288)(289)(290)(291) . Delphinium brunonianum inhibited he growth of Vero, MDCK cell lines and hepatoma cell line (292)(293) . ...
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Cancer is the second leading cause of death worldwide. The alternative natural therapies are required as they considered to have less toxic side effects compared to current chemotherapy. In the current review Web Science, PubMed, Scopus and Science Direct, were searched to provide information about medicinal plants that have shown anticancer activity against various forms of cancer.
... Experimental and clinical studies on carrots (powder or extract) and its active constituents (mainly carotenoids) revealed that they have hyperglycemic effect (Suzuki et al., 2002 andYlonen et al., 2003); anticancer activity due to the presence of alpha carotene and falcarinol (Michaud et al.,2002 andKobaek-Larsen et al., 2005) protective effect against coronary heart disease (Gaziano et al.,1995 andKrichevesky, 1999), hypocholesterolemic and hypolipidemic activities (Nicolle et al., 2003 andNicolle et al., 2004). ...
... Experimental and clinical studies on carrots (powder or extract) and its active constituents (mainly carotenoids) revealed that they have hyperglycemic effect [3,4]; anticancer activity due to the presence of alpha carotene and falcarinol [5,6] protective effect against coronary heart disease [7], hypocholesterolemic and hypolipidemic activities [8,9]. ...
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Plant products play a crucial role in the hepatoprotection through its antioxidants property. Therefore, search for modern medicine of plant origin with this property has become a central focus on hepatoprotection today. This study investigated to search a new hepatoprotective agent from natural sources, the methanol extract of nutitional plant, Daucus carota leaves was tested against liver damage of albino rats. Levels of serum marker enzymes i.e. SGOT, SGPT (aminotransferases), ALP (alkaline phosphatase) and TB (total bilirubin) in serum respectively. The histopathological changes of liver sections were also compared with the respective controls. 30% paracetamol induced significant (P<0.05) increase in liver enzymes alongwith the hepatic necrosis and other visible disarrangements in hepatic tissues. Simultaneously, oral treatment with kaempferol (KF) isolated from D. carota plant reversed to all the serum and liver parameters, dose-dependently, in 30% paracetamol treated rats. The biochemical results were also compared with the standard drug i.e. silymarin. These findings indicate the hepatoprotective potential of D. carota fruit against liver damage might be due to the presence of flavonoid like KF constituents.
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Falcarinol is a polyacetylene which is found in carrots and known to have anti-neoplastic properties in rodents. Research in the bioactivity of falcarinol in humans require methods for quantification of falcarinol in human serum. Here we report the development of an LC-MS/MS method and its use to measure serum falcarinol concentrations in humans following intake of a carrot product. Falcarinol was measured by LC-MS/MS using the m/z 268 to m/z 182 mass transition. Six calibrator levels (0.2-20 ng/mL) and 3 control levels (0.4, 2 and 8 ng/mL) were prepared by addition of falcarinol to human serum pools. Linearity of the developed method was good with a mean R² of 0.9942. Within-day, between-day and total coefficients of variation were 6.9-13.1%, 4.1-5.0% and 8.1-14.1%, respectively. The limits of detection and quantitation were 0.1 and 0.2 ng/mL, respectively, matrix effects 84.2%, recovery 101.4-105.4% and carry-over -0.24-0.07%. Serum falcarinol concentrations were measured in 18 healthy volunteers prior to and at 9 time-points following intake of a carrot product. Falcarinol concentrations peaked at the 1-hour time-point in 15 out of 18 volunteers after intake and declined according to a single exponential decay function with an aggregate t½ of 1.5 hours. In conclusion, an LC-MS/MS method for quantification of falcarinol in human serum with acceptable performance was developed and used to measure falcarinol concentrations following intake of a carrot product.
Chapter
Full-text available
Plant secondary metabolites (PSMs) are produced in the form of phytochemicals in various plant parts as a natural defense system against attack of various microorganisms and environmental stresses. The role of these compounds is beyond providing protection, as they are linked to many biochemical pathways inside and outside the plants and possess various well-known therapeutic applications. The extraordinary biological activities of plant secondary metabolites lead to their extensive use as an ingredient in medicines and for therapeutic and other culinary purposes since ages. The minimum effective concentration and effect (positive or negative) of plant secondary metabolites on particular metabolic pathways are the concerns which are still under a trial phase. They occur in very minute quantities within the plant cells, while the purity issues have resulted in manufacturing of their chemical derivatives and their industrial applications as well. Environmental, morphogenetic, and genetic factors and ultimately the processing affect the biosynthesis and the concentration of these PSM present. However, the plants are always in contact with changing conditions of light, water, temperature, pH, insect pest infestation, etc. which may adversely affect the accumulation of secondary metabolites. The present chapter has been compiled to give the readers an in-hand information about the plant secondary metabolites with primary objective of their food and functional repute. The overall contents will focus on broad classification of PSMs, various methods adopted for their extraction with their potential advantages and disadvantages, and effect of various methods of food processing on the bioavailability and bioactivity of the PSMs with proposed future research opportunities in their potential therapeutic applications.
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Background. Epidemiologic evidence indicates that diets high in carotenoid-rich fruits and vegetables, as well as high serum levels of vitamin E (alpha-tocopherol) and beta carotene, are associated with a reduced risk of lung cancer. Methods. We performed a randomized, double-blind, placebo-controlled primary-prevention trial to determine whether daily supplementation with alpha-tocopherol, beta carotene, or both would reduce the incidence of lung cancer and other cancers. A total of 29,133 male smokers 50 to 69 years of age from southwestern Finland were randomly assigned to one of four regimens: alpha-tocopherol (50 mg per day) alone, beta carotene (20 mg per day) alone, both alpha-tocopherol and beta carotene, or placebo. Follow-up continued for five to eight years. Results. Among the 876 new cases of lung cancer diagnosed during the trial, no reduction in incidence was observed among the men who received alpha-tocopherol (change in incidence as compared with those who did not, -2 percent; 95 percent confidence interval, -14 to 12 percent). Unexpectedly, we observed a higher incidence of lung cancer among the men who received beta carotene than among those who did not (change in incidence, 18 percent; 95 percent confidence interval, 3 to 36 percent). We found no evidence of an interaction between alpha-tocopherol and beta carotene with respect to the incidence of lung cancer. Fewer cases of prostate cancer were diagnosed among those who received alpha-tocopherol than among those who did not. Beta carotene had little or no effect on the incidence of cancer other than lung cancer. Alpha- tocopherol had no apparent effect on total mortality, although more deaths from hemorrhagic stroke were observed among the men who received this supplement than among those who did not. Total mortality was 8 percent higher (95 percent confidence interval, 1 to 16 percent) among the participants who received beta carotene than among those who did not, primarily because there were more deaths from lung cancer and ischemic heart disease. Conclusions. We found no reduction in the incidence of lung cancer among male smokers after five to eight years of dietary supplementation with alpha-tocopherol or beta carotene. In fact, this trial raises the possibility that these supplements may actually have harmful as well as beneficial effects.
Article
Objective. —To examine the relationship between beta carotene plasma concentration and beta carotene supplementation and risk of death from major disease causes.Design. —Cohort study of plasma concentrations; randomized, controlled clinical trial of supplementation.Setting. —Medical school-affiliated dermatology practices.Patients. —A total of 1188 men and 532 women with mean age of 63.2 years, who had enrolled in a randomized clinical trial of beta carotene supplementation to prevent nonmelanoma skin cancer.Intervention. —Oral beta carotene, 50 mg per day for a median of 4.3 years.Main Outcome Measures. —All-cause mortality and mortality from cardiovascular disease and cancer.Results. —During a median follow-up period of 8.2 years, there were 285 deaths. Persons whose initial plasma beta carotene concentrations were in the highest quartile (>0.52 μmol/L [27.7 μg/dL]) had a lower risk of death from all causes (adjusted relative rate [RR], 0.52; 95% confidence interval [CI] 0.44 to 0.87) and from cardiovascular diseases (adjusted RR, 0.57; 95% CI, 0.34 to 0.95) compared with persons with initial concentrations in the lowest quartile (<0.21 μmol/L [11.2 μg/dL]). Patients randomly assigned to beta carotene supplementation showed no reduction in relative mortality rates from all causes (adjusted RR, 1.03; 95% CI, 0.82 to 1.30) or from cardiovascular disease (adjusted RR, 1.16; 95% CI, 0.82 to 1.64). There was no evidence of lower mortality following supplementation among patients with initial beta carotene concentrations below the median for the study group.Conclusions. —These analyses provide no support for a strong effect of supplemental beta carotene in reducing mortality from cardiovascular disease or other causes. Although the possibility exists that beta carotene supplementation produces benefits that are too small or too delayed to have been detected in this study, noncausal explanations should be sought for the association between plasma concentrations of beta carotene and diminished risk of death.(JAMA 1996;275:699-703)
Article
Falcarinol [heptadeca-1,9(Z)-diene-4,6-diyn-3-ol] and falcarindiol [heptadeca-1,9(Z)-diene-4,6-diyne-3,8-diol] were extracted from edible parsley root and purified by means of multilayer coil countercurrent chromatography (MLCCC). The structures of the compounds were determined by spectroscopic and chemical derivatization methods. The quantities of falcarinol and falcarindiol were 30% and 9% of the extractable oil, respectively. Their influence on seed germination was examined.
Article
Three new acetylenic compounds named ginsenoynes I–K were isolated from a hexane extract of the roots of Panax ginseng. Their structures were determined by spectral and chemical methods.
Article
(−)-(9Z)-1,9-Heptadecadiene-4,6-diyn-3-ol was for the first time isolated in high yield (29%) from the resinous sap of Evodiopanax innovans Nakai, Araliaceae. This compound has been associated with the main polymerizable component of an ancient golden varnish. It has also been associated with a phytoalexin and a defense material which produces a water-insoluble coating film by photopolymerization under sunlight for a Takanotsume tree upon being wounded.
Article
A methanol extract from Peucedanum praeruptorum showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide). The methanol extract from P. praeruptorum was re-extracted with hexane, dichloromethane, n-butanol, and water, respectively. A suppressive compound in the hexane extract fraction was isolated by SiO2 column chromatography and identified as falcarindiol by EI-MS, IR, and 1H and 13C NMR spectroscopy. Falcarindiol exhibited an inhibition of the SOS-inducing activity of furylfuramide in the umu test. Gene expression was suppressed 75% at less than 0.15 μmol/mL, and the ID50 value was 0.10 μmol/mL. The diacetate compound of falcarindiol did not show any suppressive effect on the SOS induction of furylfuramide. Falcarindiol was also assayed with the mutagen 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which requires liver-metabolizing enzymes, and showed a suppressive effect similar to that with furylfuramide. The falcarindiol ID50 value versus Trp-P-1 was 0.096 μmol/mL. The antimutagenic activities of falcarindiol and falcarindiol diacetate against furylfuramide and Trp-P-1 were tested by an Ames test using S. typhimurium TA100, which indicated that falcarindiol suppressed the mutagenicity of furylfuramide and Trp-P-1 and falcarindiol diacetate suppressed the mutagenicity of Trp-P-1. Keywords: Umbelliferae; Peucedanum praeruptorum; falcarindiol; antimutagenic activity; umu test; Ames test
Article
The absolute configuration of falcarinol (1) was established by stereoselective total synthesis of the two enantiomers.
Article
The concentration-dependent activity of the polyacetylene falcarinol ((9Z)-heptadeca-1,9-dien-4,6-diyn-3-ol), isolated from carrots, was investigated in a bioassay with primary mammary epithelial cells in collagen gels and compared with that of β-carotene, the orange pigment in carrots. Falcarinol showed biphasic activity, having stimulatory effects between 0.01 and 0.05 µg ml−1 and inhibitory effects between 1 and 10 µg ml−1, whereas β-carotene showed no effect in the concentration range 0.001–100 µg ml−1. The results are discussed in relation to the health-promoting effects of carrots and related vegetables. Falcarinol was quantified in the carrot cultivars Bolero, Rodelika and Fancy by analytical reverse phase HPLC, subjected to various processing and storage conditions in order to study how long-term storage, blanching, freezing and boiling influence the content of falcarinol. Long-term storage of raw carrot cubes (1 cm3) reduced the falcarinol content by almost 35%. A similar reduction was found in steam-blanched carrot cubes (1 cm3). Long-term storage at −24 °C of steam blanched carrot cubes did not reduce the falcarinol content further. A reduction of almost 70% in the falcarinol content was found in carrot pieces boiled in water for 12 min compared with raw carrots. Copyright © 2003 Society of Chemical Industry
Article
Vegetables contain unknown compounds with important health promoting effect. The described project defined and tested a two-step screening procedure for identification of such compounds. Step 1 is initial screening according to three criteria: 1.1, chemically reactive functional groups; 1.2, toxicity at high concentrations or other bioactivity; and 1.3, presence in healthy foods. Step 2 is testing for minimum criteria defining health-promoting compounds: 2.1, positive or biphasic (“hormesis”) responses in bioassay; 2.2, human tissue concentrations corresponding to beneficial effects in bioassay; and 2.3, possibility to control content in food. Falcarinol from carrots fulfilled all 6 criteria and subsequently showed anticancer effect in rats.