Chinese Journal of Oceanology and Limnology

Vol. 26 No. 3, P. 307-312, 2008

DOI: 10.1007/s00343-008-0307-x

Antiviral active peptide from oyster*

ZENG Mingyong ()**, CUI Wenxuan (), ZHAO Yuanhui (),
LIU Zunying (), DONG Shiyuan (), GUO Yao ()
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China

Received Dec. 5, 2006; revision accepted Jan. 29, 2007

Abstract
An active peptide against herpes virus was isolated from the enzymic hydrolysate of oyster
(Crassostrea gigas) and purified with the definite direction hydrolysis technique in the order of alcalase
and bromelin. The hydrolysate was fractioned into four ranges of molecular weight (>10 kDa, 105 kDa,
51 kDa and <1 kDa) using ultrafiltration membranes and dialysis. The fraction of 105 kDa was purified
using consecutive chromatographic methods including DEAE Sephadex A-25 column, Sephadex G-25
column, and high performance liquid chromatogram (HPLC) by activity-guided isolation. The antiviral
effect of the obtained peptide on herpetic virus was investigated in Vero cells by observing cytopathic
effect (CPE). The result shows that the peptide has high inhibitory activity on herpetic virus.
KeywordCrassostrea gigas; antiviral activity; herpetic-virus; active peptide

1 INTRODUCTION
Previous researches indicate that oyster extract
has many biological activities, such as anti-tumor
(Wang et al., 1997), anti-cancer (Li et al., 2002;
Huang et al., 2002), anti-oxidization (Yoshikawa et
al., 1997), anti-bacteria (Zeng and Zeng, 2002) and
reducing the blood pressure (Yu et al., 2004; Achour
et al., 1997) because the oyster extract can scavenge
free radicals and create middle leucocyte in
immunocytes.
Recently, research on antivirus activity of oyster
extract has made big progress. Achour et al. (1997)
found that oyster extract could enhance proliferation
of immunocytes in human immunodeficiency virus
(HIV). Lee and Susumu (1998) isolated two
peptides (Leu-Leu-Glu-Tyr-Ser-Ile and Leu-LeuGlu-Tyr-Ser-Leu) inhibiting HIV-1 protease from
hydrolysate of oyster (Crassostrea gigas) proteins
prepared with thermolysin. However, no
information about active peptide on herpetic virus
from the enzymic hydrolysate of oyster has yet been
available.
In this study, an active peptide on herpetic virus
was isolated and purified from enzymic hydrolysis
of oyster with definite direction hadrolysis
technique, providing forerunner compounds for the
development of antivirus medication, and
developing the utilization of the low value shellfish.

2 MATERIALS AND METHODS

2.1 Material and reagent
Live Pacific oysters (Crassostrea gigas) were
purchased from a local seafood market in Qingdao,
China, alcalase was purchased from Novo Nordisk
and bromelin from Sandon Biological Engineering
Co. Ltd, Shanghai. The two enzymes are of food
grade. All other chemicals were of the highest grade
available.
Vero cell (African green monkey kidney cell,
ATCCCCL 81), pseudorabies virus (PrV) were
obtained from Graduate School of Wuhan Institute
of Virology, Chinese Academy of Sciences.
2.2 Preparation of oyster hydrolysate
Fifty grams of fresh raw oysters were minced and
homogenized in 150 ml of Tris-HCl buffer (50
mmol/L, pH8.5, containing 10 mmol/L CaCl2).
Samples were first digested by alcalase at 600 U/g
for 3 h at 50C. pH of the solution was adjusted to
5.5 and the solution was digested by bromelin at 700
U/g for another 3 h. After two-step digestion, the
* Supported by the Science and Technology Project of Qingdao
(04-2-HH-70).
** Corresponding author: mingyz@mail.ouc.edu.cn

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CHIN. J. OCEANOL. LIMNOL., 26(3), 2008

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reaction was terminated by boiling for 10 min at

100C and the hydrolysate was centrifuged (9 000
r/min, 15 min) (Ace Homogenizer, Jouan MR23i,
France). The supernatant was collected.

incubated at 37C in 5% CO2 for 72 h. Cells from

six wells without sample treatment served as basic
controls.

2.3 Isolation of active peptide

Antivirus activity was evaluated by cytopathic

effect inhibitory assays, which was a rapid assay for
evaluation of antiviral activity on herpes virus
(Young et al., 2004; Fang and Hu, 2005). The
cytopathic effect inhibitory assay involved the lysis
of Vero cells by PrV, and the agents acting at any
stage of the virus reproductive cycle were detected.
The antiviral evaluation was often carried out by
scoring the cytopathic effect microscopically;
therefore, it is subjective.
Like the cytotoxic assay, the cytopathic effect
(CPE) inhibitory assay was carried out in 2-day-old
confluent monolayers of Vero cells (PrV) in 96-well
flat-bottomed microtitre plates (Falcon 3075). After
the aspiration of the cell growth medium, 50 L of
sample diluted in test medium (the same
concentrations as used in the cytotoxicity assay)
were added to the cell monolayers. Six wells of
noninfected and six wells of infected cells without
the test fraction served as the cell and virus control,
respectively, on each plate. To calibrate the assay,
50% and 100% plaque inhibitory concentrations of
reference compounds (each three wells) were
included as positive controls in each microtiter plate.
Plates were incubated at 37C in a humidified
atmosphere with 5% CO2. The development of
cytopathic effect was monitored by optical
microscopy.

The supernatant was fractionated using UF

membranes of three different molecular weight
cutoffs (MWCO) at 10 kDa, 5 kDa and 1 kDa. Four
portions (molecular weight ranges were <1 kDa, 15
kDa, 510 kDa and >10 kDa, respectively) were
obtained. Antivirus activity assay showed that the
portion of 510 kDa has inhibitory effect on
pseudorabies virus. Therefore, 1 ml of the portion of
510 kDa was loaded onto a DEAE Sephadex A-25
column (16 mm 300 mm), which was equilibrated
with 0.05 mol/L Tris-HCl buffer (pH8.0) and eluted
with a linear gradient of NaCl concentration (00.5
mol/L) at a flow rate of 30 ml/h. The active fraction
was collected and loaded onto a Sephadex G-25
column (16 mm 500 mm), then eluted with
distilled water at a flow rate of 30 ml/h. The active
fraction was further purified by HPLC on a
reverse-phase C18 column (Agilent Inc., 4.6
mm300 mm), which was at first eluted with a
linear gradient of acetonitrile (0 to 30%) at a flow
rate of 0.8 ml/min, and then was eluted with a linear
gradient of acetonitrile (0 to 10%). Each
chromatography was monitored by the absorbance
at 220 nm. The active fraction was collected and
lyophilized.
2.4 Cell and virus

2.6 Antivirus activity assay

Vero cells were cultured with DMEM

supplemented with 10% (v:v) fetal bovine serum
(FBS). All cells were proved free of mycoplasma
contamination before using.
Virus stock of the herpes virus strain (PrV;
provided by Graduate School of Wuhan Institute of
Virology, Chinese Academy of Sciences) was
prepared in Vero cells. About 50 L aliquots of the
virus stocks were stored at 70C until use. The titer
of virus stocks was determined by plaque assay on
the respective cell monolayers.

2.7 Amino acid analysis of AVP

2.5 Cytotoxicity

Four portions in molecular weight ranges <1 kDa,

15 kDa, 510 kDa and >10 kDa were obtained
after ultrafiltration and concentration. Among them
the portion of <1 kDa was obtained by dialysis,
which composed of water, minerals and a small
amount of free amino acids. The portion of >10 kDa
was very turbid because it contained a great amount

For cytotoxicity assay, Vero cells were seeded in

96 well plates (Falcon, 3075). After the cells had
been incubated for 24 h at 37C, the growth medium
was removed, several ten-fold dilutions of the
samples in 100 L test medium were added and

AVP was hydrolyzed for 24 h in 6 mol/L HCl

containing 0.1% thioglycolic acid at 110C in
vacuum. Amino acids were identified and quantified
using an automatic amino acid analyzer (Hitatch
835-50, Japan).
3 RESULTS AND DISCUSSION
3.1 Purification of active peptide and its antivirus
activity

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ZENG et al.: Antiviral active peptide from oyster

of cell wall and other impurities. As reported in the

literature (Young et al., 2004), most active peptides
had low molecular weight. Therefore, the portions
of 15 kDa and 510 kDa were applied to further
experiment.
As shown in Fig.1, all cells underwent
pathological changes after infected with the virus
for 72 h, thus the portion of 15 kDa could not
inhibit herpetic virus.
When the portion of 510 kDa was diluted
twenty-fold or forty-fold, it showed distinct inhibitory
effect (Fig.2D, E). It was cytotoxic to cells when the
concentration of sample was as high as 1:10 (Fig.2C).

309

When the concentration was as low as 1:80, it showed

a weak inhibitory effect (Fig.2F).

Fig.1 Inhibitory effects of portion 1-5 kDa on herpetic-virus

A: Cell control; B: Virus control; C: Sample (1:10); D: Sample (1:20)

Fig.2 Inhibitory effects of portion of 510 kDa on herpetic-virus

A: Cell control; B: Virus control; C: Sample (1:10); D: Sample (1:20); E: Sample (1:40); F: Sample (1:80)

inhibitory activity (Fig.4). When D3 was

diluted to 40 or 80 multiples, it exhibited
obvious inhibitory effect to herpetic virus
(Fig. 4-D, 4-E). However, it was cytotoxic to
cells when its density was high.

Fig.3

Elution profile
chromatography

of

DEAE

Sephadex

A-25

According to above result, sample of 510

kDa was chosen for purification. Fig. 3 shows
the elution profile of the DEAE Sephadex
A-25 chromatogram. There were three
fractions, noted as D1, D2 and D3
respectively, which were collected and
concentrated. Fraction D3 has the maximum

Fraction D3 was loaded onto the Sephadex G-25

column for further purification (Fig.5). Four
fractions (noted as D3S1, D3S2, D3S3, D3S4,
respectively) were obtained. Antivirus activity assay
indicated that D3S3 had inhibitory activity on
herpetic virus (Fig.6). When D3S3 was diluted
fifty-fold, it showed an obvious inhibitory effect on
herpetic virus (Fig.6D). However, it was cytotoxic
to cells when the concentration was greater than
1:20.
D3S3 was further purified by HPLC for two
times on a reverse-phase C18 column. At first time,
the peak with arrowhead on the RP-HPLC
chromatogram (Fig.7), noted as AVPI, showed
better inhibitory activity. The AVPI was collected
and then purified by RP-HPLC again (Fig.8); a high
antiviral active peptide (AVP) with a mono-peak on
RP-HPLC chromatography was obtained.

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310

3.2 Amino acid analysis of AVP

As summarized in Table 1, among the amino acid
composition of AVP, the content of Cys was the
highest, and that of leu, Glu, Asp, Phe, Tyr, Ile and

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Gly was also very high, occupying 75.39% of all the

amino acids. It is suggested that AVP is primarily
composed of the eight types of amino acids.
Frank and Curtis (1999) isolated an antiviral

Fig.4. Inhibitory effects of fraction D3 on herpetic-virus

A: Cell control; B: Sample (1:10); C: Sample (1:20); D: Sample (1:40); E: Sample (1:80); F: Sample (1:160)

Fig.5 Elution profile of Sephadex G-25 chromatogram

Fig.6 Inhibitory effects of fraction D3S3 on herpetic-virus

A: Cell control; B: Virus control; C: Sample (1:20); D: Sample (1:40); E: Sample (1:80); F: Sample (1:160)

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ZENG et al.: Antiviral active peptide from oyster

311

present, and the amino acid categories of antivirus

peptides from different sources diversify greatly
from each other. Thereby, studying the relation
between amino acid composition of antiviral
peptides and their activities is greatly demanded in
the future.

4 CONCLUSION
Fig.7 Elution profile of RP-HPLC of D3S3

Fig.8 Elution profile of RP-HPLC of AVPI

Table 1 Amino acid composition of AVP
Amino acid

Content (mg/100ml)

Relative content (%)

Asp

2.5348

8.21

Thr

1.2744

4.13

Ser

1.6148

5.22

Glu

2.6856

8.70

Gly

1.7208

5.57

Ala

1.2348

4.00

Cys

7.2808

23.58

Val

1.0816

3.50

Ile

2.0216

6.55

Leu

2.7428

8.88

Tyr

2.0408

6.61

Phe

2.2508

7.29

Lys

0.8576

2.78

His

0.4456

1.44

Arg

1.0924

3.54

peptide from edible mushroom (Rozites caperata)

which inhibited herpes simplex virus types 1 and 2
replication, varicella zoster virus, and influenza A
virus. Sequence analysis revealed two amino
terminal sequences and they consisted of Ala, Asn,
Val, Thr, Tyr, Pro, His, Ser, etc. Artificial antiviral
peptide can be used to reduce or inhibit herpes
simplex virus infection; its amino acids sequence
was Arg-Arg-Trp-Trp-Cys-Arg-X, where X is an
amino acid or an amino acid analog. However,
reports on sequence of antiviral peptides are few at

An active peptide was isolated and purified from

the enzymic hydrolysate of oyster with definite
direction bienzyme hydrolysis, and with consecutive
chromatographic
methods
including
DEAE
Sephadex A-25 column, Sephadex G-25 column,
and high performance liquid chromatogram (HPLC)
by activity-guided isolation. In cytopathic effect
inhibitory assays, at certain concentration (1:40) of
peptide (105 kDa) and its purified products, they
had significant inhibitory effects on herpetic virus.
However, if the concentrations were greater than
1:20 or smaller than 1:80, they were cytotoxic to
cells or had no inhibitory effects on herpetic virus.
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