Journal of Oleo Science
Copyright ©2008 by Japan Oil Chemists’ Society
J. Oleo Sci. 57, (2) 133-137 (2008)

NOTE

Effects of Dietary Supplementation of Ferulic
Acid and g -Oryzanol on Integument Color andSuppression of Oxidative Stress in Cultured Red
Sea Bream, Pagrus major
Takashi Maoka1＊, Fumio Tanimoto1, Mitsuhiko Sano2, Kanji Tsurukawa2,
Takuo Tsuno3, Satomi Tsujiwaki3, Katsuya Ishimaru4 and Kenji Takii5
1Research Institute for Production Development (15 Shimogamo-Morimoto-Cho, Sakyou-Ku, Kyoto 606-0805, JAPAN)
Dainichi Corporation (1385 Yorimatsu-Ko, Uwajima, Ehime 798-0084, JAPAN)
3
Tsuno Rice Fine Chemicals Co., Ltd. (94 Shinden, Katsuragi, Wakayama 649-7194, JAPAN)
4
Fisheries Laboratory, Kinki University (3153 Shirahama, Wakayama 649-5145, JAPAN)
5
Fisheries Laboratory, Kinki University (468-3 Uragami, Nachikatsuura, Wakayama 649-5145, JAPAN)
2

Abstract: The effects of ferulic acid (FA) and g-oryzanol (OZ) supplementation on cultured red sea bream
were examined. Commercial brown fish meal diets supplemented with FA (0.01-0.5%) or OZ (0.05-0.5%)
were given to zero-year, cultured red sea bream for 98 days. After the experiment, the brightness of theintegument color (“L” value) of FA- and OZ-administrated fish was higher than that of control fish.
Furthermore, 2-Thiobarbituric acid reactive substances (TBARS) in the liver of FA- and OZadministrated fish was lower than in control fish. These results indicate that FA and OZ suppressed not
only dark-color pigmentation but also oxidative stress in cultured red sea bream.
Key words: ferulic acid, g -oryzanol, red sea bream, integument color, TBARS

1 INTRODUCTION
Apart from body weight and flesh quality, the integument color is one of the important factors for dictating
commercial value of cultured red sea bream, Pagrus
major. The integument color of red sea bream is caused
not only by carotenoids (astaxanthin, lutein, and tunaxanthin) but also by melanin1, 2). Compared with wild red sea
bream, cultured fish are reared at a shallow depth (5-10 m)
in net pens and show a dark integument color due to exposure to sunlight2, 3) and several oxidative stresses4). Darkcolor pigmentation is caused by the excessive productionof melanin caused by sunburn 2, 3) . It is well-known thatdark-color pigmentation of the integument is prevented by
the inhibition of melanogenesis and/or presence of sunshade.
Also, astaxanthin given as a supplement to cultured red

sea bream led to red-color pigmentation1, 2). However, active
oxygen species and free radicals generated by several
oxidative stress decompose carotenoid and inhibit theiraccumulation in the fish integument. Therefore, it is necessary to administrate a melanogenesis inhibitor and/or
antioxidant to cultured red sea bream to achieve fine pigmentation similar to that in wild fish.
Ferulic acid (FA) and g -oryzanol (OZ), which is a sterol,
triterpene alcohol, and higher alcohol esters of ferulic acid,both have antioxidative and melanogenesis-inhibitory
activities5–7). Thus, we attempted to apply FA and OZ supplementation for the improvement of the integument colorand suppression of oxidative stress in cultured red sea
bream. This paper presents these results.

＊

Correspondence to: Takashi Maoka, Research Institute for Production Development, 15 Shimogamo-Morimoto-Cho, Sakyou-Ku, Kyoto
606-0805, JAPAN.
E-mail: maoka@mbox.kyoto-inet.or.jp
Accepted October 22, 2007 (received for review December 14, 2006)

Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
http://jos.jstage.jst.go.jp/en/133

T. Maoka, F. Tanimoto, M. Sano et al.

2 EXPERIMENTAL
2.1 Feeding experiment
Zero-year red sea bream obtained from the Fish Nursery
Center, Kinki University, Uragami, with an average bodyweight of about 250g, were randomly divided into eightgroups of 30 fish each in 2 × 2 × 2.5 m3 floating net penscovered with sunshade netting in Uragami Bay,
Nachikatuura, Wakayama Prefecture, Japan. The test fish
were fed test diets twice a day, six times a week, for 98
days. On the initial and final days of the feeding trial (allcases), fish were sampled and weighed individually foreach test group. On the final day of the feeding trial, hematology was assessed by the previously reported method8).
2.2 Test diets
The commercial diet for red sea bream containing 60
ppm astaxanthin (Madai EP45, Kirin Food Co., Ltd., Japan)
was used for the basal diet (diet 1). This diet was composedof brown fish meal (60%), grains (15%), plant oilcake (10%),rice bran (5%), fish oil (3%), and other components (7%)
such as brewery yeast, calcium carbonate, and calcium
phosphate. It contained 0.045% DL-a-tocopherol acetate.
Experimental diets (diets 2-8) composed 0.01-0.5 % of FA or
0.05-0.5 % of OZ added to the basal diet, as shown in Table
1. FA and OZ were provided from Tsuno Rice Fine Chemicals Co., Ltd., Japan. OZ consisted of 24-methylcycloartanal
ferulate (42%), cycloartenol ferulate (29%), campesterol ferulate (16%), b-sitosterol ferulate (9%), and some higher
alcohol esters of ferulic acid (4%). All experimental diets
were processed to dry pellets by the extruding method.

2.3 Estimates of integument color
On the final day of the feeding trail, integument color
was estimated by the “L” value (brightness), “a” value (redcolor), and “b” value (yellow color) obtained by the Chroma
Meter CR-300 (MINOLTA Co., Ltd., Japan)2, 3, 9). Three areas(head, lateral side, and ventral side) were assessed regarding integument color, as shown in Fig. 1.
2.4 Analysis of carotenoid content and compositions in the
integument
Carotenoid contents of integuments were measured
according to the methods described in previous literature10,11).
The whole integument of each fish except for the head, finsand tail were removed. Carotenoids were extracted withacetone from the integument. After transferring to hexane/ether (1 : 1) by adding water, the total carotenoid con1%
tent was calculated using an extinction coefficient of E cm=
2500 at l max. The individual carotenoid contents were
estimated by column chromatography on silica gel and
HPLC.

Fig. 1 Measuring Position of Integument Color in Red Sea
Bream.
A: head, B: lateral side, C: ventral side.

Table 1 Content of FA and OZ in Experimental Diet and Growth Performance and
Hematology of Red Sea Bream.
Group
1

2

3

4

5

6

7

8

0
0

0.01
0

0.05
0

0.1
0

0.5
0

0
0.05

0
0.1

0
0.5

Growth performance
Mean body weight (g)InitialFinal
Weight gain (kg)Feed intake (kg)Feed efficiency (%)
Survival

293
665
9.41
17.42
54.0
96.7

287
659
10.22
17.21
59.4
96.7

285
672
10.33
17.05
60.6
100

286
658
9.55
17.31
55.2
96.7

294
695
10.18
17.09
59.0
100

280
673
10.33
17.69
58.4
100

287
673
9.63
17.10
56.3
96.7

302
668
9.27
16.62
55.7
96.7

Hematology*
Hematocrit value (%)
Hemoglobin concentration (g/100 ml)

0.42
9.81

0.42
9.91

0.37
9.33

0.38
9.03

0.38
9.37

0.38
9.22

0.38
9.51

0.37
9.24

FA content (%)
OR content (%)* n=5.

134

J. Oleo Sci. 57, (2) 133-137 (2008)

Supplementation of Ferulic Acid and g-Oryzanol for Cultured Red Sea Bream

2.5 Analysis of 2-thiobarbituric acid reactive substances(TBARS) levels
2-Thiobarbituric acid reactive substances (TBARS) values in the liver were measured according to the method
described by Masugi and Nakamura 12) . Liver (1 g) was
homogenized with 5 mL of 0.05 M phosphate buffer (pH7.4). Then, 0.5 mL of homogenate was transfered to ascrew-capped tube containing 0.2 mL of 7% sodium dodecyl
sulfate and 2 mL of 1N HCl and mixed gently. After adding
0.3 mL of 10% tungsto phosphoric acid and 1 mL of 0.5% 2thiobarbituric acid, the solution was mixed and heated in awater bath at 95℃ for 45 min and then cooled quickly withrunning tap water. After adding 5 mL of n-butanol to thesolution, the mixture was shaken vigorously and centrifuged at 3000 rpm for 10 min. TBARS values wereexpressed as mg malondialdehyde (MDA) /g tissue. The
MDA content was calculated by absorbance at 532 nm of1%
the n-butanol layer using a coefficient of E cm
=156000.
2.6 Statistical analyses
The integumental color value and TBARS data areexpressed as the mean ±standard deviation for the mean.
Statistical comparisons were performed with Student’s ttest.

3 RESULTS AND DISCUSSION
3.1 Effects of ferulic acid and g-oryzanol supplementation
on growth performance
Table 1 shows the growth performance of fish fed thediets for 98 days. The survival rates of each group were
between 96.7 to 100%. No significant differences among the
dietary treatments were found in weight gain, feed efficiency, and survival, with a slightly higher trend in feed
efficiency in FA (diet 2-5 groups)- and OZ (diet 6-8 groups)administrated groups than the control group (diet 1 group).
There were no significant differences in hematology in thegroups (Table 1). Therefore, it was revealed that FA andOZ dietary supplementation caused no undesirable effecton growth, but rather enhanced the growth of red sea
bream.
3.2 Effects of ferulic acid and g-oryzanol supplementation
on integument color
Integument color in red sea bream was estimated by the“L”, “a”, and “b” values obtained by a chromatic meter2, 3).
The “L”, “a”, and “b” values indicated the index of brightness, red-color, and yellow-color values, respectively. It
was reported that the “L” value showed a negative correlation with the melanin content, whereas “a” and “b” values
showed a positive correlation with the astaxanthin and yellow xanthophyll (lutein and tunaxanthin) content, respec-tively2, 3). After 98 days of feeding, the integument color of
FA- and OZ-administrated fish (diet 2-8 groups) showed
higher-level brightness than that of the control group (diet1 group). Not only the “L” value but also the “a” and “b” values of FA- and OZ-administrated fish were higher thanthose of the control fish, as shown in Table 2. For the head
integument, the “L” values of FA- and OZ-administrated
groups were higher than that of the control group. Amongthem, the “L” value in diet groups 2, 3, 5, and 6 were significantly (p<0.05) higher than that of the control group (diet 1
group). In lateral and ventral areas, FA- and OZ-administrated groups also showed an increased “L” value compared to the control group. It is well-known that melanogenesis is induced by UV light and catalyzed by tyrosinase.
FA and OZ are not only effective UV absorbers6), but alsotyrosinase inhibitors5–7). Therefore, FA and OZ suppresseddark-color pigmentation in the integument of red sea
bream.
Concerning the red and yellow color, the “a” and “b” values of FA- and OZ-administrated groups (diets 2-8 groups)
were slightly higher than those of the control group (diet 1
group). This result showed that FA and OZ could intensify
the red and yellow coloration of the integument of red sea
bream. It was reported that the red color in the integument of marine fish is due to astaxanthin and the yellow
color is due to lutein and tunaxanthin13), and that astaxanthin was converted to tunaxanthin via lutein in marine
fish10–11, 13). The total carotenoid contents in the integuments in FA- and OZ-administrated groups were slightly
higher than those of the control group, as shown in Table
3. Therefore, supplementation with FA and OZ mightenhance the accumulation of carotenoid in the integumentof red sea bream. This effect might be due to their antioxidative activity. Details are discussed in the next section.
3.3 Effects of ferulic acid and g-oryzanol supplementationon oxidative stress
It was reported that oxidative stress caused some undesirable effects in marine animals14–16). TBARS comprise oneof the useful indicators to estimate the oxidative stress14–16).
TBARS values in the liver of FA- and OZ-administrated
groups (diet 2-8 groups) were lower than that of the control
group (diet 1 group), as shown in Fig. 2. Especially, TBARS
values in experimental groups 4, 5, and 8 were significantly(p<0.05) lower than the control group. These results clearly indicated that FA and OZ suppressed oxidative stress infish.
It was reported that astaxanthin itself was an excellent
antioxidant in fish, especially for singlet oxygen and lipid
peroxidation17, 18). These activities were higher than thoseof b-carotene and vitamin E17, 18). However, astaxanthin was
immediately decomposed when reacted with active oxygen
such as hydoxyl or superoxide radicals. On the other hand,
FA and OZ showed excellent quenching activity for

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J. Oleo Sci. 57, (2) 133-137 (2008)

T. Maoka, F. Tanimoto, M. Sano et al.

Table 2

Effect of Supplementation on FA and OZ for Integument Color of Red Sea Bream.
Group

Head
L valuea valueb value
Lateral side
L valuea valueb value
Ventral side
L value

1

2

3

4

5

6

7

8

32.7 ±0.8
8.0 ±0.3
10.7±0.7

37.1 ± 1.2**
7.9 ± 0.5
14.1 ± 0.8**

37.9 ± 1.7*
8.2 ± 0.4
13.6 ± 1.0*

34.2± 1.1
8.0 ±0.2
12.4 ±0.9

38.7± 1.0**
8.1 ±0.7
12.7 ±0.4*

40.9± 1.0**
8.5 ±0.2
10.5 ±0.6

37.1± 0.8
8.5 ±0.4
12.2 ±1.0

38.0±1.4
8.0±0.4
11.9± 0.7

62.5 ± 1.5
12.2 ± 0.6
20.8 ± 1.1

66.7 ± 1.8
14.3± 0.7*
27.7± 1.0*

66.7 ± 1.2*
13.7± 1.1
20.9± 0.6

68.3 ±1.3**
13.1 ±0.5
20.5 ±0.964.6 ±0.914.4 ±1.121.7 ±1.0

63.6 ±1.4
14.3 ±1.418.2 ±0.862.5 ±1.4 62.9± 1.2
15.7 ±0.6** 14.4± 0.9
21.9 ±0.7** 18.1± 0.8

82.2± 1.2

84.7 ± 0.5

86.1 ± 1.3*

85.3 ±0.7*

84.0 ±0.984.5 ±0.7

85.0 ±0.8

85.0± 0.8

Values are expressed as means± SE of 20 fish par group.
The asterisk indicates significant difference from the control group within the same row (* p < 0.05, ** P < 0.01).

Table 3

Total Carotenoid Content and Composition of Carotenoid in the Integument of Red Sea Bream.
Group
1

2

3

4

5

6

7

8

Total carotenoid content(mg/specimens)

0.30

0.30

0.34

0.31

0.31

0.30

0.32

0.38

Composition (%)
Astaxanthin
LuteinTunaxanthin
Others

65.0
10.0
17.0
8.0

65.0
11.0
16.0
8.0

66.0
11.0
17.0
6.0

65.0
10.0
17.0
8.0

65.0
10.0
17.0
8.0

65.0
10.0
18.0
7.0

66.0
11.0
17.0
6.0

68.0
12.0
15.0
5.0

Fig. 2 TBA Values in the Liver of Red Sea Bream.
TBA values are expressed as nmol of MDA /gtissue.
The same superscript in a row indicates nosignificant difference among the experimental group(p<0.05).

hydroxyl, superoxide, and lipid peroxide radicals6). Thus, it
was strongly suggested that FA and OZ could protect
astaxanthin from decomposition by active oxygen and free
radicals in fish. Therefore, supplementation with FA andOZ could enhance the accumulation of carotenoids in the
integument of red sea bream.
Some antioxidants such as glutathione9, 19), cysteine20),
kojic acid21), polyphenols from grape seeds, guava leaves
and soybeans 2 2 ) , and the leaf extract of Eucommia
ulmoides23) were administered for the improvement of pigmentation to cultured red sea bream. These compounds
suppressed melanogenesis. However, the carotenoid-accumulating effect of these compounds in red sea bream is
uncertain. Therefore, this is the first report to emphasize
the accumulation of carotenoids in the integument of red
sea bream by antioxidant presence.
FA and OR are widely used in food ingredients and cosmetics as an antioxidant and UV absorber. To our knowledge, this is the first report on the application of FA and
OR in aquaculture.

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J. Oleo Sci. 57, (2) 133-137 (2008)

Supplementation of Ferulic Acid and g-Oryzanol for Cultured Red Sea Bream

4 CONCLUSION
From the present experimental results described above,
FA and OZ might be valuable as integument color
improvers and oxidative stress suppressors in cultured
fish.

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