Effect of collagen and collagen peptides from bluefin tuna abdominal skin on cancer cells

doi:10.4236/health.2011.33024

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Vol.3, No.3, 129-134 (2011) Health

doi:10.4236/health.2011.33024

Effect of collagen and collagen peptides from bluefin

tuna abdominal skin on cancer cells

Sung-Hee Han*, Yuki Uzawa, Tatsuya Moriyama, Yukio Kawamura

Department of Applied Biological Chemistry, Graduate School of Agriculture, Kinki University, Naka-Machi, Nara, Japan;

*Corresponding Author: ykawamur@nara.kindai.ac.jp

Received 27 December 2010; revised 17 February 17 2011; accepted 28 February 2011.

ABSTRACT

In the present study, we investigated the effect

of collagen and collagen peptides from bluefin

tuna abdominal skin on cancer cells. Collagens

were extracted from bluefin tuna (Thunnus ori-

entails) abdominal, mackerel, and carp skin. The

calf and salmon collagen were used reagent

grade as a standard samples. The main protein

band pattern produced by SDS-PAGE of all col-

lagen samples consisted of two chains and one

chain. For collagen peptides samples, bluefin

tuna abdominal skin collagen and salmon skin

collagen were hydrolyzed by trypsin. Among

samples, salmon, mackerel, carp collagen, and

their collagen peptides did not significantly re-

duce relative cell growth. However, the bluefin

tuna abdominal skin collagen dramatically re-

duced HepG2 and HeLa cell growth by over 50%

relative in a concentration-dependent manner

when added to cells seeded 96-well plates. This

suggests the collagen adding time was mightily

important for effect of the collagen.

Keywords: Bluefin Tuna Skin Collagen; Type I

Collagen; HepG2 Cell; HeLa Cell

1. INTRODUCTION

To date, many researchers have reported the preven-

tion of cancer both in vitro and in vivo by administration

of various functional foods or their extraction compo-

nents [1]. Collagen is a common protein and significant

part of the living bodies of mammals. As a structural

protein, collagen is essential to creating physical struc-

ture, and as an extracellular matrix protein, it acts as a

supporting framew ork over which cells are arrang ed [2].

Collagen has been utilized as a material in foods, cos-

metics, pharmaceuticals, and experimental reagents.

Commercial sources of collagen usually include mam-

mals such as cows or pigs; however, marine animals

have garnered increased attention as a backup collagen

resource ever since the onset of bovine spongiform en-

cephalopathy (BSE) in the cattle industry [3].

These days, consumption of fish worldwide has in-

creased, especially with the Japanese, who consume a

wide range of fish species [4]. Particularly, bluefin tuna

(Thunnus Orient ails) is one of the most popular types of

tuna in Japan, and its consumption is always increasing.

However, such large demand has resulted in large quan-

tities of fish wastes, including skin, bone, and fins, pro-

duced by many fish shops and fish-processing factories.

These wastes are usually dumped, resulting in pollution

and an offensive odor [5]. Therefore, recent studies have

focused on the extraction of collagen and from fish

wastes. Studies have reported the effect of animal colla-

gen on osteoporosis [6]. For example, there have been

numerous studies supporting th e hypothesis that collagen

from shark contains antiangiogenic and antitumor com-

pounds [7]. However, collagen from bluefin tuna as a

functional component as well as its effect on cancer cells

has not been studied.

In this study, we report the preparation of type I col-

lagen and collagen peptides from bluefin tuna as well as

their actions on HepG2 (Human hepa tocellular liver car-

cinoma cell line) and HeLa cells (Human cervical cell

line).

2. MATERIALS AND METHODS

2.1. Collagen Preparations

Bluefin tuna (Thunnus orientails) was obtained in an

unfrozen state at 4˚C within 24 hr after catching from

the tuna cultivation fields of Kinki University, Japan.

The skin of bluefin tuna was dissected from the body

and stored at –20˚C. Bluefin tuna abdominal skin type I

collagen and other collagens were isolated according to

previously reported procedures [1,8] with slight modifi-

S.-H. Han et al. / Health 3 (2011) 129-13 4

130

cation. The collagen extraction methods can be divided

into three steps as follows: Defatting, acid extraction

with protease, and salting out and dialysis. Our method

is based on previous research on the extraction of type I

collagen. The final protein recovery rate of type I bluefin

tuna abdominal skin collagen was 1.8 g/100 g and the

dry yield was 4.1%. This yield was similar to those of

previous reports featuring acetic acid soluble extraction.

Briefly, the abdominal skin of bluefin tuna without mus-

cle and scales was washed under running water and cut

into small pieces. The pieces were then defatted three

times with MeOH/CHCl3 (2:3) and then washed by

methanol and water. Second step was Extraction in acid

solution was carried out next at 4˚C. The defatted pieces

were homogenized in 10 vol (w/v) of 0.5 M acetic acid

for 5 min at 10,000 rpm using a homogenizer (AM-1,

NIHONSEIKI KAISHA Ltd., Japan). The homogenates

were kept at 4˚C for 24 hrs with continuous stirring. The

suspension was centrifuged (10,000 × g, 30 min) to re-

move any residue. Pepsin (3130 U/mg solid; Nacalai

tesque INC. Kyoto, Japan) was then added to the super-

natant (7 g/L), and the mixture was gently stirred for 2

days. Collagen was then precipitated by salting out with

25% (w/v) NaCl and centrifuged at 5,000 × g for 30 min.

The resultant precipitate was dissolved in 0.5 M acetic

acid and centrifuged (15,000 × g, 60 min). The super-

natant was dialyzed into distilled water for 5 days at 4˚C

and lyophilized. Calf and salmon skin type I collagens

were purchased from Wako Pure Chemical Industries,

Ltd., Japan and mackerel and carp collagen were ex-

tracted as mentioned above.

2.2. Protein Recovery Rate and Collagen

Yield

Protein recovery rate was estimated by the Bradford

method, and collagen yield (dry basis) of bluefin tuna

abdominal skin was calculated by the followin g:

(Weight of final collagen sample, g)/(weight of blue-

fin tuna sample, g) × 100

2.3. Sodium Dodecyl Sulphate

Polyacrylamide Gel Electrophoresis

(SDS-PAGE) and Western Blotting

SDS-PAGE was performed by the method of Laemmli

[9] using the Tris-HCl/glycine buffer system with a 7.5%

resolving gel and 4% stacking gel. The collagen sample

was dissolved in sample buffer (0.5 M Tris-HCl, pH 6.8,

containing 8% SDS, 30% glycerol, 0.2% bromophenol-

blue) containing 5% -ME and then boiled for 5 min.

The samples were loaded and electrophoresed. For de-

tection of bluefin tuna abdominal skin collagen, we pre-

pared anti-fish collagen antibodies as follows. Salmon

skin collagen type I (2 mg/mL) was immunized into

guinea pig five times. Then, the serum was drawn

four times on days 0, 24, 31, an d 45, and titers and reac-

tivities were checked. For Western blotting, the sample

was loaded onto SDS-PAGE gels, electrophoresed, and

then transferred onto a PVDF membrane, followed by

incubation for 1 hr with primary antibody (1:150) at

25˚C. Blots were washed with PBST buffer (3 × 5 min)

and incubated for 1 hr at 25˚C with secondary antibody

(anti-guinea pig IgG conjugat e d wi t h peroxi d a se (1: 1000)).

The protein band on the membrane was detected on

X-ray film using the standard enhanced chemilumines-

cent (ECL) method (GE health care, USA).

2.4. Preparation of Collagen Peptides

Preparation of collagen peptides was performed by the

method of Zhang et al. [10]. The extracted bluefin tuna

abdominal skin collagen and salmon skin collagen were

dissolved in 0.1 M sodium phosphate buffer (pH 7.8) at

a concentration of 6 mg/m L. Afte r adding 11,600 units/mg

of trypsin (EC.3.4.21.4., Sigma Chemical Co.) to colla-

gen solution (0.07 g/mL), the reaction mixture was

incubated at 37˚C for 5 min. To stop the reaction, the

mixture was heated immediately at 100˚C for 10 min,

followed by centrifugation at 10,000 × g for 10 min at

4˚C.

2.5. Determination of Degree of Hydrolysis

The degree of hydrolysis was determined by the

2,4,6,-trinitrobenzene sulfonic acid (TNBS) method

[11,12]. One milliliter of 0.1% TNBS solution was add-

ed to 1 mL of sample solution (0.15 mg/mL) containing

1% sodium dodecyl sulfate (SDS) and 4% NaHCO3

buffer (pH 9.5). The resulting solution was rapidly

mixed, reacted at 40˚C for 2 hrs in a water bath, and fi-

nally stopped by the addition of 0.5 mL of 1 N HCl and

10% SDS. The absorbance of the sample was read at 490

nm using a spectrophotometer against a blank control.

The total number of amino groups was determined in a

sample that was 100% hydrolyzed at 110˚C for 24 hrs in

6 N HCl [13 ]. The ab sorbance of the sampl e was read at

490 nm against a blank, and the readings were converted

to the number of free amino groups by comparison with

a standard curve prepared using glycine.

2.6. Cell Culture and MTT Assay

HepG2 cells were grown in Dulbecco’s-modified Ea-

gle’s medium (DMEM; Nissui, Tokyo, Japan) supple-

mented with 1% Non-Essential Amino acids, 7.5% Na-

HCO3, 100 units/mL of penicillin, 100 g/mL of strep-

tomycin, 200 mM glutamine, and 100 mM pyruvic acid

in a 5% CO2 incubator at 37˚C. HepG2 cells (1.0 × 104)

S.-H. Han et al. / Health 3 (2011) 129-13 4

131

were cultured in each well of a 96-well plate for 24 hrs

at 37˚C in a CO2 incubator. HeLa cells (4.0 × 103) were

grown in medium (DMEM; Sigma, MO, USA) supple-

mented with 1% Non-Essential Amino acids, 0.4% Na-

HCO3, 100 units/mL of penicillin, 100 g/mL of strepto-

mycin, and 100 mM pyruvic acid in a 5% CO2 incubator

at 37˚C. Sample solutions of various concentrations (5, 10,

15, and 20 g/100 L of medium) were then added to the

seeded HepG2 and HeLa cells, followed by 24 hrs of in-

cubation at 37˚C in a CO2 incu bator. Relativ e cell growth

was assayed by the MTT method, which is based on the

protocol described by Mossmann. Briefly, cells were in-

cubated for 4 hrs with 10 L of 3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyl-tetrazoliumbromide) (MTT) solution (5

g/L). After the detection of formazan under a micro-

scope, the medium in the 96-well plates was removed by

cen trifug ation (500 × g, 15 min). Isopropanol-0.04N HCl

was added to the 96-well plates, followed by shaking for

15 min. Then, 10 L of 3% SDS solution was added,

after which the absorbance was recorded using a dual

filter (595/655 nm).

2.7. Caspase Activity and Cytotoxicity

HepG2 (1.0 × 104) and HeLa cells (4.0 × 103) were

seeded in 96-well plates and incubated for 24 hrs at 37˚C

in a CO2 incubator. After incubation, samples (20 g/100

l of medium) were added, and the plate was incubated

for 24 hrs at 37˚C in a CO2 incubator. Caspase activity

and cytotoxicity were measured using a Caspase-Glo

assay kit (Promega Co. USA) and CytoTox96 non-ra-

dioactive cytotoxicity assay kit (Promega Co. USA).

Namely, cells in medium (100 L) were collected using

trypsin inhibitor and spinned down for 2 min. The me-

dium was then exchanged with 100 L of new medium,

followed by mixing of the samples. For measurement of

caspase activities, 20 L of sample medium was added

to the assay plate along with 20 L of substrate mixed

with buffer. The assay plate was then reacted for 30 min

at room temperature, and the luminescence of the plate

was measured. For the cytotoxicity assay, 30 L o f s a m-

ple medium was added to the assay plate, after which 30

L of substrate mixed with buffer was added, mixed, and

reacted for 30 min at room temperature while excluding

light using aluminum foil. Then, 30 L of 1 M acetic

acid was added to the plate, and the absorbance was

measured at 490 nm.

2.8. Statistical Analysis

Analysis of variance (ANOVA) was performed, and

differences among the samples were determined by

Duncan’ s Multiple Ran ge Test using the Statisti cal A na ly -

sis System. P values (p < 0.05) were considered signifi-

cant.

3. RESULTS AND DISCUSSION

3.1. Electrophoresis and Western Blotting

The collagen samples from bluefin tuna abdominal

skin and reagent grade salmon were analyzed by 7.5%

SDS-PAGE (Figure 1). The separation pattern shows

that bluefin tuna abdominal skin and salmon skin colla-

gen were composed of two  chains (1 and 2) and

one  chain. The purity of the collagen extracted from

bluefin tuna abdominal skin was 83.7%. The density of

the 1 chain was higher than that of the 2 chain for

both bluefin tuna abdominal skin collagen and salmon

skin collagen. This result is similar to previous reports

on other fish species and is typical of type I collagen

[14,15]. The estimated molecular weights for the and

2 chains compared to a standard were approximately

120 and 112 kDa, respectively [16]. From the results, the

1, 2, and  chains of bluefin tuna abdominal skin col-

lagen were lower purity than those of salmon skin colla-

gen. The  chain is a dimer with a molecular weight of

approximately 205 kDa. The SDS-PAGE patterns of the

 chain were similar regardless of fish species. The

topmost band was likely due to agglomeration of protein

particles by proteolysis during electrophoresis. The col-

lagen extracted from bluefin tuna abdominal skin showed

a clearer SDS-PAGE pattern at the low molecular weight

region compared to that of salmon. The prepared antisera

against salmon skin collagen were reacted with bluefin

tuna abdominal skin collagen and salmon skin collagen.

The reactivity of salmon collagen was high while the

reactivity of bluefin tuna abdominal skin collagen was

not relatively high. This result seems to suggest that

bluefin tuna abdominal skin collagen and salmon skin

collagen differed in sequence.

Figure 1. SDS-PAGE of fish skin collagen and Western blot

analysis by using antisera. SAC: Salmon skin collagen; TUC:

Bluefin tuna abdominal skin collagen.

S.-H. Han et al. / Health 3 (2011) 129-13 4

132

3.2. Degree of Hydrolysis

The peptides of bluefin tuna abdominal skin collagen

and salmon skin collagen were prepared by trypsin di-

gestion. The degree of hydrolysis of salmon skin colla-

gen peptides was 53 .1%, whereas the degree of hydroly-

sis of bluefin tuna abdominal skin collagen peptides was

96.2%. Therefore, the degree of hydrolysis of prepared

salmon skin collagen peptides was lower than that of

collagen peptides from bluefin tuna abdominal skin by

43%. The residue content of collagen (MW 300,00 0) upon

hydrolysis into only glycine residues produced 2.25 ×

1010 mol (1 mg of glycine = 1.33 × 10-5 mol). Th erefo re,

we assumed that the residue content of hydrolyzed

(96.2%) bluefin tuna abdominal skin collagen was 2.16

× 1010 mol while that of hydrolyzed (53.1%) salmon skin

collagen was 1.20 × 1010 mol. These results are perhaps

due to differences in amino acid and imino acid content

between bluefin abdominal skin collagen and salmon skin

collagen [15]. The high imino acid content of collagen

increased the thermal stability of collagen. Denaturation

of collagen occurs at above 25˚C during enzyme diges-

tion, and any lower temperature reduces enzyme activity

under the same conditions. Therefore, salmon skin colla-

gen contained higher imino acid content than that of

bluefin tuna abdominal skin collagen. Furthermore, amino

acid content had some effects on the thermal stability of

collagen. According to another report, differences in

amino acid composition affected the denaturation point

[17]. For instance, collagen sample with high leucine

and lysine content had a higher denaturation point.

3.3. Effect of Collagen and Collagen

Peptides on Growth Inhibition of Cells

Effects of collagen and collagen peptides on growth

inhibitor of cells are shown in Figure 2. We investigated

the adding timing effect of the collagen and collagen

peptides samples on HepG2 and HeLa cells. Samples at

the same concentration were added to HepG2 and HeLa

cells seeded in 96-well plates. HepG2 and HeLa cell

growth increased with the concentrations of salmon col-

lagen, salmon collagen peptides, and bluefin tuna ab-

dominal skin collagen peptides. However, cell growth

decreased upon the addition of bluefin tuna abdominal

skin collagen. Salmon skin collagen and salmon collagen

peptides induced increased HepG2 cell growth by 35%

while bluefin tuna abdominal skin collagen peptides

increased relative HepG2 cell growth by 30%. HeLa

cells also experienced similar increases in relative growth.

Salmon collagen and salmon collagen peptides increased

relative HeLa cell growth by over 50%. This result was

similar with other reported reports, which showed that

collagen is able to stimulate cell proliferation and

Concentration (g/well)

0510 15 20 25

Relative cell gr iw th (% )

120

150

180

210

HepG2 (A)

Concentrat ion (g/ well )

051015 2025

Relative cell griwth (%)

120

150

180

210

HepG2 (B)

Concentration (g/well)

0510 15 20 25

Relative cell griwt h ( %)

120

150

180

210

HeLa (A)

Concentration (g/well)

051015 2025

Relative cel l griwth (%)

120

150

180

210

HeLa (B)

SACSAP TUC TUP

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Figure 2. Effect of salmon skin collagen, bluefin tuna ab-

dominal skin collagen, and their peptides on HepG2 and HeLa

cell growth by MTT assay. The seeded cells were not incubated,

and the samples were added to cells seeded in a 96-well plate.

Then, the cells were incubated for 24 hrs in a 37˚C incubator

(HepG2 (A) and HeLa (A)). The seeded cells were incubated

for 24 hrs at 37˚C after which the samples were added. After

addition of the collagen samples, the cells were incubated

again for 24 hrs in a 37˚C incubator (HepG2 (B) and HeLa

(B)). Control: No addition sample; SAC: Salmon skin collagen;

SAP: Salmon skin collagen peptides; TUC: Bluefin tuna ab-

dominal skin collagen; TUP: Bluefin tuna abdominal skin col-

lagen peptides. *p < 0.05 compared with control.

adhesion when added during cell seeding. It may be that

the availability of collagen increases the stability of the

cytoskeleton as well as activation of signal transduction.

Moreover, the effect differs (increase or reduction of

relative cell growth) according to fish type and the mo-

lecular weight of collagen [18]. Bluefin tuna collagen

reduced cell growth when added to HepG2 and HeLa

cells seeded in 96-well plates (50% reduction of HepG2

cell growth and 38% reduction of HeLa cell growth).

More specifically, relative cell growth decreased with

the concentration of bluefin tuna abdominal skin colla-

gen with dependence on cell type. The reduction of cell

growth by collagen was affected by cell type. The both

prepared collagen peptides showed not relative cell

growth reduction, significantly. We also compared the

effects of other fish collagens on cells (Figure 3). Blue-

fin tuna abdominal skin collagen had a relatively large

effect on cell growth. These results show demonstrate

the effects of fish type, fish skin collagen, fish skin col-

lagen peptides, and the duration of collagen and collagen

peptide treatment. Particularly, addition of bluefin tuna

S.-H. Han et al. / Health 3 (2011) 129-13 4

133

(a) (b)

Figure 3. Effects of salmon skin collagen, bluefin tuna abdominal skin collagen, trout skin collagen, and carp skin collagen (20

g/well) on HepG2 and HeLa cell growth by MTT assay. The seeded cells were incubated for 24 hrs at 37˚C after which the samples

were added. After addition of the collagen samples, the cells were incubated again for 24 hrs in a 37˚C incubator (A). SDS-PAGE of

trout skin collagen and carp skin collagen.

abdominal skin collagen during cell attachment resulted

in decreased relative cell growth. This result sugg ests the

possibility that collagen may have a negative effect on

cancer cell growth and also that the duration of collagen

treatment is very important. It was reported that shark

cartilage modulates the immune response [19]. Specifi-

cally, shark cartilage inhibits the proliferation and mi-

gration of endothelial cells through the fibrin matrix,

which implies the modulation of adhesion molecules on

the surfaces of endothelial cells. Namely, when collagen

was added to attach cells, cell-cell adhesion was inter-

rupted. However, there was a positive effect observed

when collagen was added during cell seeding. These

results suggest the possibility that other fish collagens

have effects on cells. For example, collagen is a sort of

optically-active protein. However, the hydrolysis of col-

lagen induces collapse of the triple helix conformation

[20]. This implies that the different structures of collagen

and collagen peptides influenced adhesion molecules on

the surfaces of HepG2 and Hela cells. Furthermore, the

shape and density of the collagen strands had an effect

on cell growth. According to our data (Ta bl e 1 ), the in

hibition of relative cell growth was most likely not due

to cell apoptosis or necros is. However, understanding the

reduction in cell growth is difficult since the related

mechanisms are very complex. Therefore, further inves-

tigation of the purified  and  bands of collagen in the

cells as well as the mechanisms of apoptosis, necrosis,

and growth inhibition is needed.

4. CONCLUSION

In this paper, we describe the preparation and effects

of collagen and collagen peptides from bluefin tuna ab-

dominal skin on HepG2 and HeLa cells. The main pro-

tein band of bluefin tuna abdominal skin collagen and

reagent grade salmon skin collagen consisted of two 

chains and one  chain. The degree of hydrolysis (DH)

of peptides from bluefin tuna abdominal skin collagen

was higher than that of salmon skin collagen by 43.1%.

Bluefin tuna abdominal skin collagen had a large inhibi-

tory effect on HepG2 and HeLa cell growth. The dura-

tion of collagen treatment was also important. These

results suggest a role for collagen from bluefin tuna ab-

dominal skin waste as a functional component in the

treatment of attached human cancer cells.

Table 1. Caspase activity and cytotoxicity.

Caspase 3/7 Caspase 9 Cytotoxicity

HepG2 cell

Control 100.0 ± 2.6a1 100.0 ± 1.2a 100.2 ± 0.9a

Calf 94.7 ± 3.7a 94.1 ± 7.1a 98.6 ± 4.8a

Salmon collagen 99.6 ± 3.9a 103.6 ± 5.6a 98.1 ± 1.9a

Tuna collagen 96.8 ± 5.7a 97.7 ± 6.3a 98.3 ± 2.0a

Mackerel collagen91.5 ± 5.0a 95.7 ± 6.0a 100.2 ± 1.1a

Carp collagen 97.3 ± 7.5a 101.6 ± 2.2a 100.9 ± 0.9a

HeLa cell

Control 100.2 ± 1.2a 100.2 ± 3.0a 100.0 ± 3.0a

Calf 99.7 ± 5.6a 92.1 ± 8.0a 96.6 ± 2.5a

Salmon collagen 101.0 ± 2.1a 94.6 ± 2.1a 97.2 ± 2.8a

Tuna collagen 98.5 ± 5.9a 103.2 ± 3.4a 96.2 ± 5.1a

Mackerel collagen100.9 ± 3.8a 100.9 ± 4.4a 101.4 ± 3.8a

Carp collagen 98.10 ± 3.2a 100.6 ± 1.7a 102.3 ± 0.4a

1Superscripts in an each low of HepG2 and HeLa cell indicated signifi-

cantly different difference at p < 0.05 by Ducan’s multiple com parisons.

S.-H. Han et al. / Health 3 (2011) 129-13 4

134

5. ACKNOWLEDGEMENTS

This work was supported by GCOE project Grant of Kinki Univer-

sity 2009.

REFERENCES

[1] Knekt, P., Jarvinen, R., Seppanen, R., Heliovaara, M.,

Teppo, L., Pukkala, E. and Aromaa, A. (1997) Dietary

flavoniods and the risk of lung cancer other malignant

neoplasmas. American Journal of Epidemology, 146,

223-230.

[2] Jongjareonrak, A., Benjakul, S., Visessanguan, W., Nagai,

T. and Tanaka, M. (2005) Isolation and charaterisation of

acid and pepsin-solubilised collagens from skin of

Brownstripe red snapper (Lutjanus vitta). Food Chemis-

try, 93, 475-484. doi:10.1016/j.foodchem.2004.10.026

[3] Mizuta, S., Fujisawa, S., Nishimoto, M. and Yoshinaka,

R. (2005) Biochemical and immunochemical detection of

type I and V collagens in tiger puffer Takifugu ruripes.

Food Chemistry, 89, 373-377.

doi:10.1016/j.foodchem.2004.02.043

[4] Nagai, T. and Suzuki, N. (2002) Preparation and partial

characterization of collagen from paper nautilus (Argo-

nauta argo, Linnaeus) outer skin. Food Chemistry, 76,

149-153. doi:10.1016/S0308-8146(01)00255-2

[5] Hwang, J.H., Mizuta, S., Yokoyama, Y. and Yoshinaka, R.

(2005) Purification and characterization of molecular

species of collagen in the skin of skate (Raja Kenojei).

Food Chemistry, 100, 921-925.

doi:10.1016/j.foodchem.2005.10.046

[6] Tsuruoka, N., Yamato, R., Sakai, Y., Yoshitake, Y. and

Yonekura, H. (2007) Promotion by collagen tripeptide of

type I collagen gene expression in human osteoblastic

cells and fracture healing of rat femur. Bioscience Bio-

technology Biochemistry, 71, 2680-2687.

doi:10.1271/bbb.70287

[7] Gozalea, R.P., Leyva, A. and Moraes, O. (2001) Shark

cartilage as source of antiangiogenic compound: from

basic to clinic research. Biological & Pharmaceutical

Bulletin, 24, 1097-1101. doi:10.1248/bpb.24.1097

[8] Omenn, G.S., Goodmay, G.E. , Thornquist, M.D., Balmes,

J., Cullen, M,R., Glass, A., Keogh, J.P., Meysken, F.L.,

Valanis, B., Williams, J., Barnhart, S. and Hammar, S.

(1996) Effects of a combination of beta carotene and vi-

tamin A on lung cancer and cardiovascular disease. The

New England Journal of Medicine, 334, 1150-1155.

doi:10.1056/NEJM199605023341802

[9] Laemmli, U.K. (1970) Cleavage of structural proteins

during assembly of head bacterriophage T4. Nature, 277,

680-685. doi:10.1038/227680a0

[10] Zhang, Y., Liu W., Li, G., Shi, B., Miao, Y. and Wu, X.,

Physicochemical properties of collagen, gelatin and col-

lagen hydroysate derived from bovine limed split waste.

(2005) Food Chemistry, 103, 906-912.

doi:10.1016/j.foodchem.2006.09.053

[11] Achouri, A., Zhang, W. and Shiying, X. (1998) Isolation

and partial characterization of pepsin-soluble collagen

from the skin of grass carp (Ctenopharyngodon idella).

Food Research International, 31, 617-623.

doi:10.1016/S0963-9969(98)00104-5

[12] Adler-Nissen, J. (1979) Enzymatic hydrolysis of soy

protein isolate and effect of succinylation on the func-

tional properties of resulting protein hydrolysates. Jour-

nal of Agriculture Food Chemistry, 27, 1256-1262.

doi:10.1021/jf60226a042

[13] Shoji, Y., Isemura, M., Muto, H. and Isemura, S. (2000)

Isolation of 41-kDa protein with cell adhesion activity

for animal tumor cells from the mushroom Hypsizigus

marmoreus by affinity chromatogra phy with type IV col-

lagen immobilized on agaros. Bioscience Biotechnology

Biochemistry, 64, 775-780. doi:10.1271/bbb.64.775

[14] Muyonga, J.H., Cole, C.G.B. and Doudu, K.G. (2004)

Charaterisation of acid soluble collagen from skins of

young and adult nile perch (Lates niloticus). Food Ch em is-

try, 85, 81-89. doi:10.1016/j.foodchem.2003.06.006

[15] Yata, M., Yoshida, C., Fujisawa, S. and Yoshinaka, Y.,

(2001) Identification and characterization of molecular

species of collagen in fish skin. Journal of Food Science,

66, 247-251. d oi:10.1111/j.1365-2621.2001.tb11325.x

[16] Nalinanon, S., Benjakul, S., Visessanguan, W. and Ki-

shimura, H. (2007) Use of pepsine for collagen extrac-

tion from the skin of bigeye smapper (Priaca nthus tay-

enus). Food Chemistry, 104, 591-601.

doi:10.1016/j.foodchem.2006.12.035

[17] Nomura, Y., Sakai, H. and Shirai, K. (1996) Preparation

and some properties of type I collagen from fish scales.

Bioscience Biotechnology Biochemistry, 60, 2092-2094.

doi:10.1271/bbb.60.2092

[18] Nagai N., Mori K., Satoh Y., Takahashi N., Yunoki S.,

Tajima K. and Munekata M., (2007) In vitro growth and

differentiated activities of human periodontal ligment fi-

broblasts cultured salmon collagen gel. Journal of Bio-

medical Materials Research Part A, 395-402.

doi:10.1002/jbm.a.31110

[19] Close, M.J., Howlett, A.R., Roskelley, C.D., Desprez,

P.Y., Bailey, N., Rowning, B., Teng, C.T., Stampfer, M.R.

and Yaswen, P. (1997) Lactoferrin expression in mam-

mary epithelial cell is mediated by changes in cell shape

and action cytoskeleton. Journal of Cell Science, 110,

2861-2871.

[20] Hassan, Z.M., Feyz i, R., Sheikhian, A. , Bargahi, A., Mo-

stafaie, A., Mansouri, K., Shahrokhi, S., Ghazanfari, T.

and Shahabi, S. (2005) Molecular weight fraction of

shark cartilage can modulated immine responses and

abolish angiogenesis. International Immunophamacology,

5, 961-970. doi:10.1016/j.intimp.2005.01.006