Food Sci. Technol. Res., +- (-), ,.

1ῌ,/,, ,**1

Technical paper

Quantitation of Carotenoids in Commonly Consumed Vegetables in Japan

Koichi AIZAWA῍ and Takahiro INAKUMA

Research Institute, Kagome Co., Ltd., +1 Nishitomiyama, Nasushiobara-shi, Tochigi -,3ῌ,10,, Japan

Received February +, ,**1 ; Accepted May +*, ,**1

The carotenoid content, potentially an antioxidant, of 1* vegetables in Japan was determined by

reversed-phase HPLC. a-Carotene was detected in // of 1* vegetable samples ; b-carotene, in all 1* ;
lycopene, in four types of tomatoes and Kintoki carrot ; capsanthin, in only two types of sweet peppers ;
lutein, in 02 of the 1* ; zeaxanthin, in -* of the 1* ; and b-cryptoxanthin in +, of the 1* samples. All leafy
vegetables contained relatively high levels of b-carotene and lutein. In contrast, fruits showed variable
carotenoid compositions. A high content of lycopene was quantified in tomatoes and capsanthin was found
only in red sweet peppers. In root crops, orange carrots contained high levels of a- and b-carotene, and a
high level of lycopene was found in the Kintoki carrot. In other crops, such as head, beans, pods,
inflorescences, and spears, carotenoid content was not found to be high in this study.

Keywords : vegetable, carotene, lycopene, capsanthin, lutein, zeaxanthin, b-cryptoxanthin

Introduction conditions. Yano et al. (,**/) reported that the caro-

Carotenoids, present in fruit and vegetables, are widely
believed to provide many health benefits. They are known
as superior quenchers of singlet oxygen (Di Mascio et al.,
+323), and some epidemiologic studies have shown an
inverse relationship between carotenoid consumption and
the risk of many cancers and other chronic diseases
(Ziegler, +33+ ; Sies et al., +33, ; Gerster, +33- ; Kritchevsky,
+333).
More than 0** natural carotenoids have been identified.
Chemically, they are classified as carotenes (those without
any oxygen molecules) or xanthophylls (those with one or
more oxygen molecules). However, only about ,* caro-
tenoids, including their metabolites, have been identified
in human plasma (Khachick et al., +33/). Lutein, zeaxan-
thin, b-cryptoxanthin, a-carotene, b-carotene, and lycopene
are the most abundant in plasma. Carotenoids are bio-
synthesized by bacteria, algae, fungi, and plants (Armstrong
et al., +330), but not by animals, which must obtain them
from their food.
With the increased interest in the possible link between
carotenoid intake from food and health, the need for
reliable data on the individual carotenoid content of those
foods has become increasingly important. For vegeta-
bles, Mangles et al. (+33-) compiled a carotenoid database
of +,* foods from published data to which they attach
confidence levels. The USDA-NCC Carotenoid Database
(Holden et al. +333) is also an extremely valuable resource.
However, there may be some di#erence in the carotenoid
composition of vegetables grown in Japan due to the
di#ering characteristics of cultivars, climate, and growing
῍ cted with hexane : ethyl acetate (3 : + [v/v]) several times
To whom correspondence should be addressed.
E-mail : Koichi_Aizawa@kagome.co.jp
tenoid levels in some fruits obtained in Japan were slight-
ly di#erent from those listed in the USDA-NCC Carote-
noid Database. Thus, the objective of this study was to
determine the carotenoid content in commonly consumed
vegetables in Japan.
Materials and Methods
Carotenoids Capsanthin, lutein, zeaxanthin, and b-
cryptoxanthin were obtained from Extrasynthese (Genay,
France). a-Carotene, b-carotene and lycopene were ob-
tained from Wako (Tokyo, Japan).
Sampling and Sample Preparation All samples were
purchased from local Japanese markets in the Nasu-
shiobara city of Tochigi area. For each sample, , or .
vegetables were selected from di#erent production areas
and seasons. Each vegetable was homogenized with -ῌ
pyrogallol and +*-g samples were removed for analysis.
Carotenoid analysis was performed in duplicate.
Carotenoid quantification The AOAC procedure (AOAC,
+32*) for measuring carotenoids in feeds was modified and
used to extract the samples.
Approximately /.*-g samples were first added to a +*-
mL hexane : ethanol : acetone : toluene (+* : 1 : 0 : 1 [v/v/v/
v]) (HEAT) solvent and extracted using a mortar and
pestle. The sample was then extracted two or three more
times until the extraction solvent was colorless. The
extracts were combined and the volume was brought up
to +** mL in ethanol in a volumetric flask. The extract
(+* mL) was mixed with *., g pyrogallol, 0*ῌ (w/v)
methanolic KOH (+ mL) and *./ g solid KOH and sapo-
nified for -* minutes at .*ῌC. The pigments were extra-
and evaporated to dryness. Subsequently, the residue
248
was dissolved in +* mL of a HEAT : ethanol (.* : 0* [v/v])
solution and filtered through a *../-mm filter for the HPLC
analysis.
The carotenoid levels were determined using HPLC as
previously described (Hart et al., +33/ ; Yeum et al., +332).
The HPLC system consisted of a LC-+*AD vp pump
(Shimadzu, Kyoto, Japan), a SIL-+*AD vp autosampler
(Shimadzu, Kyoto, Japan), a C-* carotenoid column (S/
mm, ,/*ῌ..0 nm, YMC, Wilmington, NC), a CTO-+*AC vp
column oven (at .*῎C, Shimadzu, Kyoto, Japan) and a SCL-
+*A vp system controller (Shimadzu, Kyoto, Japan). The
SPD-M+* vp diode array detector (Shimadzu, Kyoto,
Japan) was set at .0* nm for carotenoid. The HPLC
mobile phase was methanol : methyl-tert-butyl ether : water
(2- : +/ : ,, [v/v/v], with *.*/ῌ triethylamine and *.+ῌ
butylhydroxyltoluene ; solvent A) and methanol : methyl-
tert-butyl ether : water (2 : 3* : ,, [v/v/v], with *.*/ῌ tri-
ethylamine and *.+ῌ butylhydroxyltoluene ; solvent B).
The gradient procedure at a flow rate of + mL/min was
+**ῌ solvent A for ,/ minutes to +**ῌ solvent B, +**ῌ
solvent A for , minutes (linear steps). Using this method,
capsanthin, lutein, zeaxanthin, b-cryptoxanthin, a-caro-
tene, b-carotene, and lycopene were adequately separated.
The peaks were identified by comparing their specific
retention times and absorption spectra with the authentic
standard. Carotenoids were quantified by determining
peak areas in the HPLC chromatograms, calibrated against
known amounts of standards. The carotenoid concen-
tration was expressed as milligrams per +** g fresh weight.
Result
Table + shows the carotenoid composition of 1* vegeta-
bles commonly consumed in Japan. a-Carotene was
detected in // of 1* vegetable samples. High a-carotene
levels were only found in carrots, orange type (,.-+ mg/
+** g fresh wt.), but other samples did not show high
levels (῍+ mg/+** g fresh wt.). b-Carotene was detected
in all 1* vegetable samples. High b-carotene levels were
found in tossa jute (+,.*. mg/+** g fresh wt.), perilla (+*.+2
mg/+** g fresh wt.) and mugwort (3.+3 mg/+** g fresh wt.).
Also, many dark green leafy vegetables (such as parsley,
komatsuna, water dropwort, and garland chrysanthe-
mum) and orange carrots showed high b-carotene levels.
Lycopene was detected in only two kinds of vegetables :
four types of tomatoes (Red type ; 3./+ mg/+** g fresh wt.,
Cherry, red type ; 0.+3 mg/+** g fresh wt., pink type ; -.3.
mg/+** g fresh wt., orange type ; ,.*3 mg/+** g fresh wt.)
and Kintoki carrot (+,.+3 mg/+** g fresh wt.). Similarly,
capsanthin was detected in only two types of red sweet
peppers (Tomapi red type ; +1./0 mg/+** f fresh wt., red
type ; 2.02 mg/+** g fresh wt.). Lutein was the second
most frequently detected carotenoid and occurred in 02 of
1* samples, with the highest lutein levels found in perilla
(+..,/ mg/+** g fresh wt.), tossa jute (+-.0- mg/+** g fresh
wt.) and mugwort (++.,0 mg/+** g fresh wt.). Along with
high levels of b-carotene, many dark green leafy vegeta-
bles also showed high lutein levels. Two types of red
sweet peppers were the only vegetable in which lutein
was not detected. Zeaxanthin was detected in -* of 1*
K. AIZAWA et al.
samples, but no samples showed high levels (῍+ mg/+** g
fresh wt.). Similarly, although b-cryptoxanthin was detect-
ed in +, of 1* samples, no samples showed a high level of
b-cryptoxanthin.
The content of carotenoids may be a#ected by cul-
tivars, culture conditions, and seasons of the year. Thus,
several samples were analyzed to determine the di#er-
ence in carotenoid profiles with varieties of color and
varying seasons of purchase.
The variation in carotenoid contents between color
varieties in tomatoes and sweet peppers are shown in
Table +. There were relatively large di#erences in carote-
noid contents in these vegetables. Lycopene content in
tomatoes ranged from * mg (not detected) to 3./+ mg/+** g
fresh wt. The highest lycopene levels were found in red
tomatoes, but lycopene was not detected in yellow toma-
toes. Also, capsanthin content in sweet peppers ranged
from * mg (not detected) to +1./0 mg/+** g fresh wt.
The variation in carotenoid content of vegetables pro-
duced in the same prefecture but purchased in di#erent
seasons was also determined (Table ,). There were rela-
tively large di#erences in the carotenoid content of 0 out
of +, vegetable samples : watercress, Chinese chives, spin-
ach, tomatoes, Japanese radishes, and tossa jute. In these
vegetables, an approximate ,* to -*ῌ di#erence in
carotenoid content was confirmed due to the seasons of
purchase.
Discussion
The aim of this study was to obtain qualitative and
quantitative carotenoid data for vegetable samples that
could be used to estimate individual carotenoid intake
from vegetables. Since the Japanese consume many vege-
tables that are specific to Japan and uncommon in other
countries, we wanted to determine the carotenoid levels
of the vegetables most frequently consumed in Japan.
We studied 1 carotenoids in 1* vegetables : -. leafy
vegetables (with or without stems), +0 fruits, . heads, -
beans, - pods, - inflorescences, , roots (with skin), , pears,
+ shoot, + bulb, and + scape.
All leafy vegetables contained relatively high levels of
b-carotene and lutein. In addition, minor carotenoids,
such as a-carotene and zeaxanthin, were detected in almost
all of these vegetables. It was suggested that dark green
leafy vegetables widely contributed to the consumption
of carotenoids, especially b-carotene and lutein. Leafy
vegetables have a strikingly constant carotenoid pattern,
the main carotenoids being lutein (about ./ῌ) and b-
carotene (usually ,/ῌ-*ῌ) (Britton, +33+). The composi-
tion of carotenoids in leafy vegetables obtained in the
present study are approximately the same as reported in
that study.
In contrast to leafy vegetables, fruits showed a variable
carotenoid composition. A high content of lycopene was
quantified in tomatoes (3./+ mg/+** g fresh wt.) and
capsanthin was found exclusively in red sweet peppers
(+1./0 mg/+** g fresh wt.) in this study. The Japanese
pumpkin did not contain relatively high levels of b-
carotene (*.1. mg/+** g fresh wt.) in spite of its orange
Quantitation of Carotenoid in Vegetables
Table +. Carotenoid content of raw vegetablesa.
color. Other fruits, such as zucchini, eggplant, bitter
gourd, and cucumber, did not show a high level of any
carotenoids. The composition and content of the carote-
noids found in sweet pepper has been investigated for a
long time, and it is widely known that sweet peppers are
very rich sources of carotenoids, particularly capsanthin.
Deli et al. (,**+) reported that the capsanthin content of
the red sweet pepper was 0.02 mg/+** g fresh wt., calculat-
ed by dry weight. Also, Sugita (,***) reported in a book
of Japanese agricultural products that red sweet peppers
contained 0.,ῌ1.. mg/+** g fresh wt. of capsanthin. In the
USDA-NCC Carotenoid Database, the b-carotene level in
pumpkins (canned, without salt) was reportedly higher
(0.3. mg/+** g) than the level of the Japanese pumpkin
researched in this report. The di#erence can probably
be attributed to variations in the cultivars investigated.
In the Standard Tables of Food Composition in Japan
(Ministry of Education, Culture, Sports, Science and Tech-
nology, Japan, ,**/), the most useful resource concerning
food composition in Japan, large di#erences in b-carotene
contents were observed between Japanese pumpkins
249
(Cucurbita moschata) and Western pumpkins (Cucurbita
maxima) (*.1* mg and -.3* mg/+** g fresh wt. respectively).
In root crops, the carotenoid levels of two types of
carrots were found. Orange carrot, available worldwide,
contained high levels of a-carotene and b-carotene.
Carrots are well known to be carotenoid rich roots
(Heinonen, +33*). However, in this study, a high level of
lycopene was found in the Kintoki carrot, which is mainly
cultivated in Japan and is uncommon in other countries.
Reports show that lycopene is contained in red vegetables
and fruits such as tomatoes, apricots, watermelons,
papaya, guava, and red or pink grapefruits, with tomatoes
providing over 2/ῌ of total lycopene consumed (Gerster,
+331). Although it was thought that lycopene was almost
exclusively provided by tomatoes and its products, the
Kintoki carrot might also contribute somewhat to the
consumption of lycopene in Japan.
In other crops, such as heads, beans, pods, inflore-
scences, pears, shoots, bulbs, and scapes, the carotenoid
content was not found to be high in this study. These
results suggest that these vegetables do not significantly
250
Table +.
a
: All vegetable samples were purchased at a local market.
b
: , or . vegetables of di#erent production area and seasons were selected.
c
: Carotenoid quantitation was performed in duplicate. Results presented as meansῌSE.
ND, not detected.
contribute to carotenoid consumption.
The carotenoid composition of vegetables is likely to be
a#ected by factors such as cultivar or variety, stage of
maturity, climate of production, geographic or geologic
conditions, harvesting and postharvest handling, as well
as the part of the plant that is consumed. In this study,
there were relatively large di#erences in carotenoid con-
tent by color or variety in tomatoes and sweet pepper. It
was generally confirmed that lycopene increased in con-
centration throughout the ripening process in tomatoes.
Also, carotenoid content in ripe fruit of di#erent tomato
varieties showed quite a wide variation ; the highest con-
tent being approximately ,* times that of the lowest
(Davis, +32+). In sweet peppers, it has been reported that
the ripening stage and some cultural conditions a#ect the
K. AIZAWA et al.
(Continued)
carotenoid content and quality of sweet peppers (Markus,
+333). In addition, we showed that there were relatively
large di#erences in carotenoid content based on the season
of purchase (Table ,). Although the data is not shown in
this paper, it is natural for carotenoid content in vegeta-
bles to vary between di#erent parts of the vegetables.
For example, the peel of the Japanese pumpkin contained
high levels of b-carotene and lutein (/.+* mg and +0.,* mg/
+** g fresh wt., respectively), whereas the fruit did not
(*.1. mg and +./* mg/+** g fresh wt., respectively). As
shown above, it is likely that the carotenoid content
changes according to a vegetable’s variety, cultivation
environment, seasons, and parts. Our research suggests
that vegetables with light color contain less carotenoid.
We should be aware of these results in order to increase
Quantitation of Carotenoid in Vegetables
Table ,. Content of b-carotene, lycopene and lutein in vegetables purchased in di#erent seasons.
a
: All vegetable samples were purchased at a local market.
b
: Carotenoid quantitation was performed in duplicate. Results presented as means῍SE.
ND, not detected.
Fig. +. Correlation between b-carotene contents of the
Standard Tables of Food Composition in Japan and the
data retrieved from this study.
the intake of carotenoids from vegetables.
The Standard Tables of Food Composition in Japan
show nutritional contents such as proteins, lipids, carbo-
hydrates, minerals, and vitamins in commonly consumed
food in Japan, including +0, kinds of vegetables. The
carotenoid contents reported therein are limited to provi-
tamin A carotenoids, a-carotene, b-carotene and b-crypto-
xanthin. Therefore, the correlation between the b-caro-
tene content reported therein and that reported in this
study was investigated. As shown in Fig. +, our study
confirms a strong correlation (correlation factor ; r῎*.221.),
which suggests that the data analyzed in this study is
comparatively reliable.
In conclusion, the approach described herein has en-
251
abled the investigation of 1 carotenoid contents in 1* raw
vegetables commonly consumed in Japan. Our data will
be helpful for estimating the intake of carotenoids from
raw vegetables and provide a base for epidemiological
studies investigating the relation between carotenoid
intake and the risk of cancers or other chronic diseases in
Japan.
Acknowledgements This study was partially supported by Spe-
cial Coordination Funds from the Ministry of Education, Culture,
Sports, Science and Technology, Japan, and New Food Creation
Technology Research Association, a project of the Ministry of
Agriculture, Forestry and Fisheries of Japan.
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