Anticarcinogenic Effect of Red Ginseng on the Development of Liver Cancer Induced by Diethylnitrosamine in Rats

J Korean Med Sci 2001; 16(Suppl): S61-5
Copyright ? The Korean Academy
ISSN 1011-8934
of Medical Sciences
Anticarcinogenic Effect of Red Ginseng on the Development of Liver
Cancer Induced by Diethylnitrosamine in Rats
Anticarcinogenic effect of red ginseng (Panax ginseng C.A. Meyer cultivated in
Xiu-gan Wu, Da-he Zhu, Xun Li
JiLin, China) on the development of liver cancer induced by diethylnitrosamine
Department of Pathology, Tongji Medical
(DEN) in rats was studied, especially in preventive and curative groups. In the
University, Wuhan, China
preventive group, the rats were given with DEN concomitantly with red ginseng
fluid, and in the curative group, the rats were administered with red ginseng fluid
after they developed liver cancer nodules induced by DEN. The result of the
preventive group revealed that the developmental rate of liver cancer in the
experimental group was 14.3%, while 100% in the control group, with the differ-
ence being statistically significant. DNA, RNA, glycogen, -GT, SDH, and 5?
-NT
were maintained at relatively normal level in experimental group, and decreased
or increased in the control group. The result of curative group showed that hep-
atoma nodules of the DEN-red ginseng group I were smaller than those of con-
trol group I, the structure of hepatic tissue was well preserved, the area with -
GT positive was smaller, and the ultrastructure of hepatocytes was normal. The
average life span the DEN-red ginseng group II and the DEN control group II
Address for correspondence
Xiu-gan Wu, M.D.
were 72.8 and 42.3 days, respectively. To sum up, all findings on preventive
Department of Pathology, Tongji Medical Universi-
and curative groups had clearly proved that the red ginseng had the anticarcino-
ty, Wuhan Apt. 407, Dong-cui-nan Street 7 Hai-lian
genic effect on the development of liver cancer induced by DEN in rats.
Road Guangzhou 510230 P.R. China
Tel : 86(20)-3429 4545
Key Words : Ginseng; Diethylnitrosamine; Liver Neoplasms; Anticarcinogenic Agents
E-mail : denda020@publicguangzhou.gd.cn
INTRODUCTION
in the experimental group were continuously given red gin-
seng fluid. The total amounts of DEN and red ginseng given
Panax ginseng has the noticeable effects to protect liver and
to each rat were 180-200 mg and 17-25 g, respectively. In
to elevate immune function. In recent years, it was proven
the 49th day and 103rd day, 5 rats each from the experimen-
that ginseng had anticarcinogenic effects on transferred
tal and control groups were sacrificed. In the 161st day, 7 rats
tumors, cultured hepatoma cells, animal and human cancer
in the experimental group and 6 rats in the control group
(1-6). This paper describes anticarcinogenic effect of red gin-
were sacrificed. The liver cancer morphology and its histo-
seng on the development of liver cancer induced by diethyl-
chemical changes of the rats in the two groups were studied
nitrosamine (DEN) in rats particularly in the preventive and
by paraffin section, HE stain, Feulgen reaction, methyl green
curative aspects.
pyronine and periodic acid-Schiff (PAS) stain. Some fresh
liver tissues were taken and processed by freezing section for
SDH, 5?
-NT and -GT. DNA, RNA, glycogen, -GT and
MATERIALS AND METHODS
SDH were detected, and measured with Univar microspec-
trometer, and analyzed statistically.
Experiment of preventive group
The experiment of curative group
33 Wistar white rats, weighing 130-180 g, were random-
ly divided into experimental and control groups. In first 15
From 60 Wistar male rats, weighing 140-200 g, 40 rats
weeks, the rats in the experimental group were given 10%
were randomly taken and given 10% DEN water solution
DEN water solution (70 mg/kg per week) concomitantly
(70 mg/kg per week) via gastric tube for 15 weeks. Another
with red ginseng fluid (Panax ginseng C.A. Meyer cultivated
20 rats served as the control group, and were randomly divid-
in JiLin, China, 3.78 g/kg per week) via gastric tube; the rats
ed into null control group and red ginseng control group.
in the control group were given only 10% DEN water solu-
In the 20th week, all 60 rats were operated with laparoto-
tion (70 mg/kg per week). In the following 8 weeks, the rats
my. It was found that 20 of the 40 rats treated with DEN
S61

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S62
X.-G. Wu, D.-H. Zhu, X. Li
had been found with liver cancer nodules smaller than 3 mm
with 17% of red ginseng water solution (5.6 g/kg per week)
in diameter, and the other 19 rats with liver cancer nodules
via gastric tube and continued for 8 weeks. At the same time,
over 3 mm diameter (one rat died during the experiment).
the rats in the DEN-control group II were administered
The former 20 rats were randomly divided into DEN red
with tap water, left for natural death, and their life spans
ginseng group I and DEN control group I, 10 rats in each
were compared (7).
group. Starting from the 21st week, the rats in the DEN-
red ginseng group I and the red ginseng control group were
administrated with 17% red ginseng water solution (5.6 g/
RESULT
kg per week) via gastric tube for 8 weeks. At the same time,
the rats in the DEN control group I and the null control
The result of preventive group
group were administered with tap water. In the 29th week,
the rats in the above four groups were all sacrificed, and the
The developmental rate of hepatoma
body and liver were weighed. The fresh liver tissues about 1
mm3 and 5×5×2 mm were taken from the low edge of
All the rats sacrificed on the 49th day and the 103rd day
middle or left lobes and fixed with 3% amyldialdehyde buffer
were found not to have liver cancer developed. But on the
fluid and 1:1 alcohol-acetone. The rest of liver was fixed with
161st day, all 6 rats in the control group which were admin-
10% formalin, serially cut in 2 mm thick, simulated the sec-
istered with the total average amount of DEN 200 mg per
tion surface of liver and cancer nodules with transparent paper
rat developed heptoma, and developmental rate was 100%.
was measured with Leitz Asm 68K Image Analysis, and the
At the meantime, only 1 of 7 rats in the experimental group
total size of the cancer nodules was calculated according to
which were administered with the total average amount of
the formula of size=area×thick. The liver tissues fixed with
DEN 196 mg and ginseng 19.6 g per rat developed hep-
alcohol-acetone were sectioned and stained for -GT, scanned
atoma, and developmental rate was 14.3%. The difference
in 4 continuous field per section with Univar-mspm Micro-
in the developmental rates of hepatoma is highly significant
spectrophotometer, and the average -GT positive area per
between the two groups (p<0.001).
unit area was calculated. The liver tissues fixed with amyl-
dialdehyde buffer fluid were embedded in Epon812 for elec-
Morphologic change
tromicroscope analysis. The latter 19 rats with liver cancer
nodules over 3 mm diameter were randomly divided into
The liver surface of all the rats sacrificed on the 49th day
DEN-red ginseng group II with 10 rats and DEN control
was smooth, mild in degeneration, necrosis and prolifera-
group II with 9 rats. At the beginning of the 21st week, the
tion of hepatocytes. In the 103rd day, the liver surface of the
rats in the DEN-red ginseng group II were administered
rats in the experimental group remained smooth, but the
Fig. 1. There was only one liver cancer nodule (?4) at the later
Fig. 2. Hepatic glycogen of the liver cancer cells, revealed with
stage in the Experimental Group.
PAS reaction, disappeared completely (×400).

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Anticarcinogenic Effects of Ginseng on Liver Cancer
S63
Fig. 3. Hepatic glycogen of the hepatic cells with PAS reaction
Fig. 4. -GT stain is strongly positive in the liver cancer nodule
is apparent (×400).
of the control group (×100).
control group showed severe degeneration, necrosis, prolif-
eter. But only one cancer nodule with about 0.4 cm in diam-
eration of hepatocytes and connective tissue in portal areas,
eter was found in the experimental group (Fig. 1).
and granular cirrhosis. On the 161st day, the liver surface of
all rats in the control group was found with granular cirrho-
Histochemical change
sis and liver cancer nodules in total quantity of up to 45,
and the size of the cancer nodules were 0.5-1.5 cm in diam-
The details of histochemical changes are shown in Table 1
Table 1. Hitochemical changes in the liver in rats (qualitative evaluation)
Degree of histochemical changes in rats
Groups (cases)
DNA
RNA
PAS
-GT
SDH
5?
-NT
+
++ +++ +
++ +++
-
+
++ +++
+
++ +++
-
+
++ +++ ±
+
++ +++
Early stage: nomal group
1
1
1
1
1
1
exp. group
5
5
4
1
5
3
2
4 1
control group
5
5
1
3
1
1
4
3
2
2
3
middle stage: normal group 1
1
1
1
1
1
exp. group
5
5
4
1
3
2
1
4
3 2
control group
5
5
2
2
2
3
2
3
1
4
Later stage: normal group
3
3
3
3
3
3
exp. group
5
1
1
4
2
1
1
1
5
5
1
1
1
6
1
5 1
control group
6
6
6
6
3
1
4
Note:
“-”negative, “±”weak positive, “+”positive, “++”middle, “+++”strong positive early, middle and later stages were the lesion in the 49th,
103rd and 161st day after administration with DEN and red ginseng.
Table 2. Quantitative histochemical changes in rat liver on the 161st day
DNA
PAS
-GT
SDH
Groups
Amount in average
Nuclear area in average
Amount in average
Amount in average
Amount in average
Nomal group
1.4
30.5
1.1
5.3
2.5
Exp. group
2.40±0.4641
3.37±6.3021
3.2±0.7742
6.8±0.8981
3.3±0.8361
Control group
6.6±0.5308
72.3±5.9641
0.1±0.0391
13.0±1.0132
0.7±0.1384
Control group was compared with normal and experimental groups, both of them with p<0.01.

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S64
X.-G. Wu, D.-H. Zhu, X. Li
Fig. 5. -GT stain was focally weak positive in the hepatic tissue
Fig. 6. Mitochondria increased and were large in size and
of the experimental group (×100).
rough endoplasmic reticula and Golgi apparatus well devel-
oped, under electron microscopy (×10,000).
and Table 2. Feulgen reaction and methyl green pyronine
stain revealed that the stain of liver cancer tissues was stronger
longed than the DEN- control group II (42.37±24.42 days,
than that of hepatic tissues. The average amounts of DNA
p<0.05).
and area of nuclei of liver tissue in the experimental group
were apparently lower than those in the control group (p<
Microscopic findings
0.01). PAS stain was negative in the cancer tissues, and pos-
itive in hepatic and proliferative hepatic cells (Fig. 2, 3).
Liver tissue surrounding cancer in the DEN- control group
Cancer nodules were strongly positive in -GT stain, but
I was severely changed, and showed fat degeneration and
the liver tissues of the experimental group were essentially
focal hydropic degeneration, but they were maintained essen-
normal (Fig. 4, 5). The activity of SDH and 5?
-NT in can-
tially normal and seldom saw fat degeneration and hydropic
cer tissues were apparently decreased or disappeared.
degeneration in the DEN-ginseng group I. In addition, the
hepatic cells around cancer in both two groups showed sim-
The result of curative group
ple or atypical proliferation, and the proliferations were dif-
ferent in morphology, but both were positive in -GT stain.
Statistics
The ultrastructural changes
Comparing the ratio of liver weight to body weight, there
was no significant difference between ginseng control group
On the 20th week, the nuclear membrane of hepatocytes
and null control group and between DEN-ginseng group I
was not smooth, nucleoli were larger and increased in quan-
and null control group. But the obvious increase between
tity, mitochondria increased and were different in size and
DEN-control group I and null control group (p<0.01), and
form, fat drop increased, and the contact surface of hepato-
apparent decrease between DEN-ginseng group I and DEN-
cytes was widened. On the 29th week, the hepatocytes of the
control group I (p<0.01). Morphometry revealed that the
DEN-control group I revealed severe atypia, large nucleus
total size of liver cancer nodules in each rat of DEN-ginseng
and dental nuclear membrane, chromatin increased and coag-
group I was 5.76±2.81 L, apparently smaller than that of
ulated pieces, mitochondria degeneration, rough reticuloen-
DEN control group I (43.81±42.46 L,
p< 0.05). The result
doplasma decreased, microvilli in sinus surface of hepatocytes
of microspectrophotometer showed that -GT positive areas
decreased and endothelium was defective. But on the 29th
per unit area of the DEN-ginseng group I was 0.10±0.12
week, the form of hepatic nuclei of the DEN-ginseng group
mm2, significantly smaller than the DEN control group I
I was maintained regular and intact, chromatin increased but
(0.27±0.15 mm2, p<0.05). The life spans of the DEN- gin-
not prominent, mitochondria increased and became larger
seng group II were 72.80±31.40 days, significantly pro-
with the intact structure, rough reticuloendoplasmic reticu-

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Anticarcinogenic Effects of Ginseng on Liver Cancer
S65
lum developed, and Golgi apparatus was normal (Fig. 6).
the life spans of the rats with cancer.
To sum up, all findings on preventive and curative groups
had proved clearly that the red ginseng has anticarcinogenic
DISCUSSION
effect on the development of liver cancer induced by DEN
in rats.
The results of the preventive group revealed that red gin-
seng had significantly inhibited the development of liver
cancer induced by DEN in rats. In the early stage, degener-
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