239 Asia Pac J Clin Nutr 2007;16 (Suppl 1):239-

243                                                                                                                                

Original Article

Study onf the radio-protective effect of cuttlefish ink on

hemopoietic injury

Min Lei PhD, Jingfeng Wang PhD, Yuming Wang PhD, Long Pang, Yi Wang, Wei Xu
PhD and Changhu Xue PhD

Ocean University of China, Qingdao 266003, PR China

Irradiation leads to immunosuppression, hemopoiesis injury as well as sub-health of human being. The
protective and therapeutic effects of cuttlefish ink on hemopoiesis in 60Co γ radiated model mice were
investigated. One hundred and twenty female ICR mice aged 6 weeks (20-24g) were randomly divided into five
groups: the control group, the model group, and the low, medium, high dosage groups. The mice in different
groups were orally administered normal solution (N.S.) or cuttlefish ink of different dosage daily for 40 days.
Hemopoiesis impaired model was induced by 60Co γ irradiating with lethal dose of 8.0 Gy. The number of bone
marrow nucleated cells (BMNC), colony-forming unit in spleen (CFU-S), colony-forming unit of granulocyte
and monocyte (CFU-GM), peripheral blood pictures and superoxide dismutase (SOD) activity in serum have
been measured. Compared with model group, the decrease of BMNC, CFU-S, CFU-GM, peripheral leukocytes
and SOD activity in serum in 60Co γ irradiated mice of cuttlefish ink feeding groups were resisted
significantly (p<0.05 or p<0.01). Moreover, the restoration of those indices was promoted significantly (p<0.05
or p<0.01). The cuttlefish ink showed no significant effect on peripheral erythrocytes, thrombocytes and
hemoglobin. The results showed that cuttlefish ink had significant effects on granulopoiesis. The mechanism
underlining these effects may be that the increase of antioxidant level in mice, the improvement of bone marrow
haematopoietic microenvironment and the inducement of cellular factors promoted the proliferation and
differentiation of CFU-S and CFU-GM and thus enhance the defensive system of organism.

Key wWords: cuttlefish ink, irradiation injury, hemopoiesis, immune, superoxide dismutase.

traditional Chinese medicine. The physical activities of

cuttlefish ink,
Introduction
Irradiation is one of the extensive natural phenomena in the
universe and our ambient environment. With the rapid
development and the application of nuclear technology in Chinese medicine. The physical activities of cuttlefish ink,
agriculture, industry, medical and life science, people are such as anti-tumour, immunity promotion, and induction of
increasingly exposed to artificial irradiation hazards. There many cytokines, have been widely researched in recent
are numerous health risks associated with irradiation such years. 9. Between two major components, melanin is an
as immune organ atrophy and immunosuppression1, irregular polymer consisting of indole structure, which can
hemopoiesis injury and decrease of hematocytes2, as well as resist free radicals chain reaction as the acceptor of free
sub-health of human being. In addition, as an important radical. 10. Previous study illustrated that cuttlefish ink can
therapeutic method for cancer3, radiotherapy has to be given prevent acute irradiation syndrome11 but the mechanism is
up by many patients due to its strong side effects. One of not clear. However, little information on the hemopoietic
the most important mechanisms of irradiation damage is function of cuttlefish ink was available. In this study, the
peroxidation.4. Reactive oxygen species such as super oxide effect of cuttlefish ink on hemopoietic function in
anion radical and hydroxyl radical lead to cell death via hemapoiesis impaired model mice induced by 60Co γ
inducing DNA oxidation, DNA strand breakage or base pair irradiation was investigated. The mechanism underlining
mismatch. 5-6. Although some effective chemical irradiation- such effect was proposed as well. The objective of this
protective agents have been found, the heavy adverse study is to deduce the effect of cuttlefish ink on humans via
reactions prevented them from clinical application.7-8. animal experiments, and develop a theoretical foundation
Therefore, it is one of our major concerns to find effective for the clinical application of cuttlefish ink in health care
nutrition measurements, e.g., nontoxic anti-radiation and medicine.
functional foods and medicines from natural products.
Cuttlefish ink is a natural substance discharged by
cuttlefish confronting enemies. It is mainly composed of
melanin and protein-polysaccharide compound. Due to its
hemostasia effects, cuttlefish ink is widely used in
 239 Asia Pac J Clin Nutr 2007;16 (Suppl 1):239-
243                                                                                                                                

while, the dynamic change of peripheral blood picture in

Table 1. The effect of cuttlefish ink on the spleen and thymus index, the number of BMNC, CFU­S and CFU­GM in
model mice after 60Co γ irradiation (n=8,  x s )

Dose Spleen index Thymus index BMNC CFU-S CFU-GM

groups
(mg.kg–1.d–1) (mg.g-1) (mg.g-1) (106.femur-1) (1105 cells) -1 (1105cells) -1
Control 0 4.040.47 3.760.26 7.451.11 41.84.79 86.36.65
Preventive effect (D3 after irradiation)
Model 0 1.170.13 a 0.560.14 a 1.180.06 a 19.8±3.77 a 47.7±4.11 a
Low dose 100 1.500.16 b
0.690.15 1.890.42 b 31.5±4.39 b 65.3±3.29 c
Medium dose 300 1.540.08 c
0.920.20 b
1.93017 c 29.2±4.45 b 73.7±2.05 c
High dose 900 1.580.08 c
1.000.14 c
2.020.24 c 31.2±5.15 b 75.3±5.73 c
Therapeutic effect (D10 after irradiation)
Model 0 0.950.16 0.740.18 0.820.21 16.8±1.79 46.0±6.97
Low dose 100 1.080.11 1.090.31 1.030.13 21.3±1.89 b 74.2±5.20 c
Medium dose 300 1.040.08 1.110.17 b 1.150.21 b 27.3±5.31 b 77.0±6.98 c
High dose 900 1.050.08 1.290.31 b 1.310.21 b 29.5±4.61 b 81.7±2.36 c
Therapeutic effect (D30 after irradiation)
Model 0 3.910.27 1.380.35 1.890.05 28.2±2.74 54.7±3.68
Low dose 100 3.860.32 1.830.77 2.110.11 37.6±2.15 c 79.3±3.77 c
Medium dose 300 4.840.74 2.000.58 b 2.250.04 c 38.2±4.11 c 81.3±1.69 c
High dose 900 4.410.31 1.980.51 b 2.450.09 c 40.3±5.41 c 85.7±0.47 c
BMNC=bone marrow nucleated cells, CFU-S=colony-forming units in spleen, CFU-GM=colony-forming units of granulocyte and
monocyte. a Pp<0.01, Compared with control; bpP<0.05, cpP<0.01, Compared with model

Corresponding Author: Professor Changhu Xue, College of mice was observed on Days 0, 1, 3, 6, 8, 10, 14, 16, 21,
Food Science and Technology, Ocean University of China, 5 25 and 30 after irradiation, respectively.
Yushan Road, Qingdao, Shandong, PR China, 266003
Tel: +86 532 8203 2468, Fax: +86 532 8203 2468
The ratios of spleen/body weight and thymus/body
Email: xuech@ouc.edu.cn
weight
Materials and methods
The spleen and thymus of every mouse were taken out
Cuttlefish ink product
and weighed immediately after sacrifice. The ratios of
Fresh cuttlefish ink was hydrolyzed with 0.5% pawpaw
spleen/body weight and thymus/body weight were
proteinase for 6 hours, then boiled for 10 minutes to
calculated at milligram per gram (mg/g).
denature the enzyme and washed with distilled water. The
cuttlefish ink powder was obtained by vacuum drying at
Bone marrow nucleated cells (BMNC) counting
60°C, stored at 4°C and suspended in distilled water for
Bone marrow cells were drawn from femoral bones with
usage.
3% acetic acid. Single-cell suspension was counted with
haemacytometer.
Animals and design of the experiment
Healthy female ICR mice (weighing 222g, SPF,
Spleen Colony Forming Unit (CFU-S) Assay12
purchased from the Centre of Laboratory Animal of
Bone marrow cells from each donor were injected into tail
Qingdao) were divided into 5 groups randomly: control
veins of irradiated (8Gy) recipients (1105 cells per
(neither irradiation nor cuttlefish ink), model (irradiation,
mouse). After 9 days, recipient spleens were removed and
no cuttlefish ink), low dosage (irradiation plus cuttlefish
fixed in Bouin’s, and numbers of macroscopic colonies
ink of 100mg/kg bw), medium dosage (irradiation plus
were counted.
cuttlefish ink of 300mg/kg bw) and high dosage group
(irradiation plus cuttlefish ink of 900mg/kg bw), 24 mice
Lung conditioned culture medium of mice 13
in each group. Mice in control and model groups were
Two lung leaves of adult ICR mice were cut into pieces
administered N.S. as placebo. On Day 10, all mice except
under the asepsis condition and cultured in 2ml RPMI-
those in control group were placed in ventilated
1640 with 10% horse serum in incubator (2711, QUEUE
containers and were exposed to concentric revolving 60Co
Systerms, USA) at 37°C, 5% CO 2 and saturated humidity
treatment machine (FCC-7000, XINHUA iatrical
for 7 days. Then the supernatant was separated and stored
instrument factory, China) at a rate of 50.8 cGy/min. The
at -20°C.
total dose of 8 Gy was given to the whole body. On Days
3, 10 and 30 after radiation, 8 mice in each group were
Granulocyte-monocyte colony forming units (CFU-GM)
sacrificed respectively and the number of bone marrow
Assay
nucleated cells (BMNC), hemopoietic colony forming
CFU-GM was quantified in semisolid culture: 1105 /ml
units in spleen (CFU-S), granulocyte-monocyte colony
bone marrow cells were cultured in RPMI-1640
forming units (CFU-GM), the ratios of spleen/body
containing 0.3% agar, 24% horse serum (Haoyang
weight and thymus/body weight were examined. Mean
Biology Scientific Production Inc, Tianjin), 10% lung
 Homocysteine in an Aboriginal community 4

conditioned culture medium of mice and incubated at The hemopoietic function in mice was destroyed
37°C with 5% CO2 for 7 days. Usually, cluster containing obviously by 60Co γ irradiation (Table 1). Compared with
50 or more cells was counted as a colony the control, the numbers of BMNC, CFU-S, CFU-GM in
mice of model group declined by 84.2%, 52.2% and
Peripheral blood picture 53.3%, respectively. However, when the mice were given
Blood was collected from tail veins of mice on different different dosages of cuttlefish ink for 9 days pre-
days following irradiation and the fluctuation of irradiation, the numbers of BMNC, CFU-S, CFU-GM of
peripheral blood cells, including leukocytes, erythrocytes 10 10
Model control
thrombocytes and hemoglobin levels were measured with

Erythrocytes(x1012cells/ L)
Leukocyte(x109cells/ L)
Low dose 9
8
an automatic hematocyte counter (SYSMAX F-820, Mid dose
High dose
8
6 7
Japan). 6
4
5
4
The SOD activity in serum 2
3
One hour after the last administration, blood was 0 2
0 1 3 6 8 101316212530 (d) 0 1 3 6 8 101316212530 (d)
collected from the eyes and serum was separated. The
SOD activity in serum was determined with the SOD
reagent kit (catalogue No: 20020329, Nanjing Jiancheng
1200 180
Bioengineering Institute).

Thrombocyte(x109cells/ L)
1000 160
140
800
Statistical analyses

Hb(g/L)
120
600
The results were expressed as the meansstandard 100
400
80
deviation. The statistical significance of the differences 200 60
was evaluated with software Quattro Pro 9 and p<0.05 0 40
was considered as significant. 0 1 3 6 8 101316212530 (d) 0 1 3 6 8 101316212530 (d)

Figure 1. The effects of cuttlefish ink on dynamic change of

Results peripheral blood picture in mice induced by 60Co γ irradiation. Hb=
The ratio of spleen/body and thymus/body hemoglobin
The functions of spleen and thymus in mice cuttlefish ink feeding groups were 10.3%, 22.1% and
were depressed by 60Co γ irradiation (Table 1). On Day 3 34.4% higher than those of model group, respectively. Up
after irradiation, the spleen and thymus indices of model to Day 30 after irradiation, the indices mentioned above
group decreased by 71.0% and 85.1%, respectively. When of cuttlefish ink feeding groups recovered to 30.5%,
mice were administered different dosage of cuttlefish ink 96.6% and 99.2% of normal level, respectively, compared
for 9 days pre-irradiation, the spleen and thymus indices with model group did merely 25.4%, 67.6% and 59.9%. It
increased obviously in comparison with those of the illustrated that cuttlefish ink resisted the impairment and
model group. When mice were given cuttlefish ink after enhanced the recovery of hematopoisis effectively.
irradiation for 30 days, thymus index of the medium and
high dosage groups recovered to 52.9% on average of the The fluctuation of peripheral blood picture
control, while the model group did merely to 36.7%. The The numbers of leukocyte, erythrocyte, thrombocyte and
results showed that cuttlefish ink had significant (p<0.05 hemoglobin changed markedly due to the hemopoietic
or p<0.01) protective and therapeutic effects on immune damage in all irradiated groups. Among them, leukocytes
organs in 60Co γ injured mice. were most sensitive to 60Co γ irradiation (Figure 1). The
leukocyte number declined rapidly at the beginning and
Numbers of BMNC, CFU-S and CFU-GM then elevated gradually from Day 3 after irradiation.

10 10
Model control
/L)
/ L)

9
x10cells
Leukocyte(x10

Low dose
cells

8 Mid dose 8
Erythrocytes(
12

High dose
9

6 7
6
4
5
2 4
3
0 2
0 1 3 6 8 1013 16 2125 30 (d) 0 1 3 6 8 101316212530 (d)

1200 180
/L)
(x10cells

1000 160
Hb(g/L)
Thrombocyte

140
9

800
120
600
100
400 80
200 60
0 40
0 1 3 6 8 101316212530 (d) 0 1 3 6 8 1013 16 212530 (d)

Figure 1. The effects of cuttlefish ink on dynamic change of peripheral blood picture in mice induced by 60Co γ irradiation. Hb= hemoglobin.
239 Asia Pac J Clin Nutr 2007;16 (Suppl 1):239-
243                                                                                                                                

During post-irradiation period, the recovery of leukocytes

of cuttlefish ink feeding groups was significantly rapider
than those of model group (p<0.01). On Day 30 after
irradiation, the number of leukocyte of cuttlefish ink
feeding groups almost returned to the normal level, as
comparison with 65.7% of the model group. The numbers
of erythrocytes, thrombocytes and hemoglobin decreased
slowly and reached to minimum values on Days 16, 10
and 13 after irradiation, respectively, then elevated
gradually. But there was no statistical significance
between dosage groups and model group.

The peroxidatic level

The SOD activity in serum was reduced by 60Co γ
irradiation (p<0.01). However, the SOD activity of
cuttlefish ink feeding groups was markedly higher than
that of model group during the post-irradiation period
(p<0.05 or p<0.01) (Fig 2). On Day 30 after irradiation,
the average SOD activity of cuttlefish ink feeding groups
was 12.6% higher than that of the model group. The
results suggested that cuttlefish ink had protective and
therapeutic effects on the peroxidation induced by
irradiation.
 M Lei, J Wang, Y Wang, L Pang, Y Wang, W Xu and C Xue 242
Effects of cuttlefish ink on hemopoiesis

stromal cells to secrete GM-CSF in hemopoietic

microenvironment thereby promoting the proliferation,
Discussion differentiation and maturity of CFU-GM in bone marrow.
Hemopoiesis relates closely to the immunity of organism. It has been proved that cuttlefish ink can activate T cells
BMNC denotes the hemopoietic function of marrow. and B cells which in turn secrete large number of GM-
CSF and other cytokines, which finally promote the
proliferation and differentiation of myelocyte progenitor
A cell19. Our findings evidenced further that the cuttlefish
SOD Act i vi t i es ( U/ ml )

500
B ink can promote the granulopoiesis in bone marrow.
400 C Leukocyte plays a key role in the defensive system of
D organism. Both heterophile granulocytes (neutrophile in
300
c man) and monocytes have a marked capacity for ingesting
b c
bb
c c c small, discrete particles. The phagocytosis and digestion
a b
200 a a by the heterophile granulocytes is one of the means by
which the host destroys bacteria, and the issue of some
100
infections may depend upon the extent of phagocytosis.
Moreover, activated monocyte and macrophage can
0
synthesize and release various cytokines such as CSF, IL-
E F G H 1, IL-3, IL-6, tumour necrosis factor α (TNF-α),
Figure 2. The effects of cuttlefish ink on the level of peroxidation in
interferon-α and interferon-β (INF-α, INF-β), regulating
mice induced by 60Co γ irradiation. SOD=superoxide dismutase, cell growth and acting as a key factor in the inducement
A=Model, B=Low dose, C= Medium dose, D=High dose, E= D3 and regulation of immune responses. Cuttlefish ink could
after irradiation, F= D10 after irradiation, G= D30 after irradiation, enhance non-specific immunity and specific immunity via
H=Control. a p <0.01, Compared with control; b p <0.05, c p <0.01,
promoting granulocytopoiesis and monopoiesis in bone
Compared with model.
marrow.
Discussion The mechanism of hemopoietic injuries by irradiation
Hemopoiesis relates closely to the immunity of organism. may be the lipid peroxidation injury of stromal cells in
BMNC denotes the hemopoietic function of marrow. bone marrow microenvironment20. Our results showed
Relative stability of peripheral blood cell number depends that cuttlefish ink could effectively resist the decrease of
on the differentiation and proliferation of hemopoietic SOD activity in serum and promote its recovery. Melanin,
stem cells and progenitor cells. The interaction between as the main component of cuttlefish ink, has the ability of
hematopoietic cells and cell growth factors regulates the scavenging free radicals and antioxidation21 thereby
proliferation and differentiation of hematopoietic cells. It protecting hemotopoitic system from irradiation injury.
has been approved that cuttlefish ink can induce many
kinds of cytokines such as IL-1 14 and colony stimulating Conclusion
factor (CSF) 15. CSF stimulates the proliferation and Cuttlefish ink can promote the proliferation and the
differentiation of hemopoietic stem cell and many kinds differentiation of granulocyte-monocyte progenitor cells,
of progenitor cells, increase the numbers of granulocyte, enhance non-specific immunity and specific immunity
monocyte in blood and macrophage in tissue 16-17. Recent significantly. The mechanism may be that cuttlefish ink
research showed that IL-1 can promote the proliferation, weakens the irradiation injury on hemopoietic
differentiation and maturity of granulocytes while inhibit microenvironment and hemopoietic cells via regulating
these processes of erythrocytes 18. Our results showed that immunological function, inducing GM-CSF and other
different dosages of cuttlefish ink could effectively resist cytokines and elevating SOD activity in mice. As a safe
the decrease of BMNC CFU-S, CFU-GM and peripheral natural product, cuttlefish ink has potential clinical
leukocytes (p<0.05 or p<0.01) in 60Co γ irradiated model application in health care and medicine.
mice. Moreover, the restoration of those indices
mentioned above was promoted significantly (p<0.05 or Acknowledgements
p<0.01). However, there is no significant effect on This study was supported by the National High-tech Research
and Development Project of China (2006AA09Z444) and the
peripheral erythrocytes, thrombocytes and hemoglobin. It
New Century Excellent Talents in University (NCET-04-0642).
suggested that cuttlefish ink could promote the
proliferation of CFU-S and CFU-GM and induce them References
differentiating into granulocytes and monocytes, but had .
no effect on erythropoiesis. 1. Sapin MR, Grigoriev AI, Erofeeva LM, Grigorenko DE
Myelocytes and monocytes or macrophages derive and Fedorenko BS. .Immune organs and haemopoietic
from CFU-GM. There is GM-CSF receptor on the surface system under modelling of the mission factors. Acta
of CFU-GM membrane. Granulopoiesis and monopoiesis Astronautica.1997; 41: 57–-62.
in organism are regulated mainly by GM-CSF during 2. Wu DC, Chen JP, Mao BZ. Radiation Medicine. Beijing:
such derivation. CFU-GM semisolid cultured in vitro Military Medicine Science Press, 2001; 16: 70–76.
assay showed that the increase of CFU-GM was dose- 3. Shen Y. Development in the Study on Radiotherapy. Chin
dependent when exogenous GM-CSF presents. Our J Radiat Oncol. 2005; 14: 17.
4. Ganasoundari A, Devi U, Rao N. Protection against
results illustrated that cuttlefish ink could stimulate
radiation-induced chromosome damage in mouse bone
 marrow by Ocimum sanctum. Mutat Res. 1997; 373: 6. Ning H. Studing on the Protection of Macromolecule
271–6. Against Light by Melanin from Engineering Bacteria
5. Xia SX, Wei K, Zhang YP. Molecular irradiation biology. (E.Coli/p WSY). Journal of Central China Normal
Beijing: Atomic Energy Press. 1992. University (Nat Sci Ed). 2001; 35: 85–-88 (Ch)
7.
8. Liang X, Hai CX, Zhao KT, Qin XJ. Protective effect of
Vit.C, Vit.E on peripheral blood composition and dose-
effect relationship in rabbit injured by irradiation of 60Co
game rays. J Fourth Mil Med Univ. 2002; 23: 187–190.
9. Guo CH, Kong PY, Zou ZM. The preventive and 16. Xie GL, He S. Study about CSF activity induced by
treatment affect of several radioprotectants on irradiation cuttlefish ink in mice. Chin Ocean Med. 2001,; 20: 25–-
injured mice. Chongqing Medicine. 2002; 3: 186–188. 27.
10. Lu CL,Yan JZ, Hu PL, He S. Activation of Squid Ink 17. Ganser A, Linelemannn A, Seipelt G, Ottmann OG,
Extracts on Macrophages in Vivo. Chin Med Univ 1999; Herrmann F, Eder M, Frisch J. Clinical effects of
6: 410–-411. recombinant human interleukin-3. Am J Clin Oncol.
11. Wan SY, Zhao YJ, Li L. Food antioxidants. Beijing: 1991; 14: S51–-63.
Chinese light industry Press, 1998; 153–-162. 18. Guba SC, Sartor CI,Gottschalk LR, Ye-Hu J, Mulligan T
12. Health services of Chinese navy logistic. Chinese and Emerson SG. Bone marrow stromal fibroblasts
Medical marine biology. Shanghai: Shanghai Demos secrete interleukin- 6 and granulocyte-macrophage
Press. 1977 . colony-stimulating factor in the absence of inflammatory
13. Sharma Y, Astle CM, and Harrison DE. Heterozygous Kit stimulation: Demonstration by serum-free bioassay,
mutants with little or no apparent anemia exhibit large enzyme-linked immunosorbent assay, and reverse
defects in overall hematopoietic stem cell function. transcriptase polymerase chain reaction. Blood. 1992; 80:
Experimental Hematology. 2007; 35: 214–-220. 1190-1198.
14. Chen DH, Luo X, Yu MY, Zhao YQ, Cheng YF, Yang ZR. 19. Rao JJ, Wu SG, Yu CL, Xu W. Positive and negative
Effect of Spatholobus suberectus on the bone marrow effects of interleukin-1 on myeloid cells and erythroid
cells and related cytokines of mice. Chin J of Chin Mater cells. Chinese Pharmacological Bulletin. 1996; 16: 465-
Med. 2004; 29: 352–-355. 468.
15. He S, Meng SN, Xie GL. Study on secretion of 20. Rao JJ, Wu SG, Yu CL, Xu W. Positive and negative
interleukin-1 induced by cuttlefish ink in mice. Chin effects of interleukin-1 on myeloid cells and erythroid
Ocean Med. 2003; 22: 17–-19. cells. Chinese Pharmacological Bulletin. 1996; 16, 465–
468.
21. Liu XZ. Hand book for hemopoietic progenitor cell
cultural technology. Beijing: Beijing Press. 1993.
22. Van Den Heuvel R, Leppens H, Nemethova G,
Verschaeve L. Haemopoietic cell proliferation in murine
bone marrow cells exposed to extreme low frequency
(ELF) electromagnetic fields. Toxicol in Vitro. 2001; 15:
351–-355.
23. Sarna T,Pilas B, Land, EJ, Truscott TG. Interaction of
radicals from water radiolysis with melanin. Biochem.
Biophys. Acta. 1986; 883:162–-167.