Studies on Leukogenic Effect of Leucozepin®
Abstract:
The
subjects of the experiment are normal mice and S180-bearing
mice. Mice leukopenia
models were prepared by iP cyclophosphamide
to observe the effects of Leucozepin® on the peripheral
leukocyte count, bone marrow karyocyte count and
tumor weight of mice models. The results
show that 8g/kg and 4g/kg of Leucozepin® can obviously
increase the counts of white blood cell and bone marrow karyocyte
of the leukopenia model and does not affect the
inhibitive effect of cyclophosphamide on tumors.
Materials:
1.
Mice, Kunming-breed mice, half of whom are male, with the weight of 18-22g,
were provided by the Animal Experiment center of
2. Tumor cells, sarcoma 180 (S180) of mice, was provided
by the Tumor Research Section of the Pharmacy college of
3. Leucozepin®，provided by LIFEnhance, Inc., is pyknotic
extract. 1g crude drug is equal to 0.11g extract. It was mixed with distilled water into the
needed concentration.
4. Cyclophosphamide (CTX), 200mg per bottle, is made by Jiang
Su Heng Rui Medicine
Limited Company and the batch number is 02022821.
5. Leucogen (a
medicine that increases white blood cells) is made by Shanghai Jinshan Medicine-making Limited Company and the batch
number is 000502.
6. Tabellae Batiloli is produced by
Jiang Su PengYao Pharmacy Limited Company and the
batch number is 000316-2.
Methods:
1. Effect
of Leucozepin® on cyclophosphamide
induced leukopenia in mice
84 mice, according to sex and weight, were divided into 6
groups at random: normal control group, model control group, large dosage
group(8g/kg), medium dosage group(4g/kg), small dosage group(2g/kg), and Tabellae Batiloli positive group
(40mg/kg). Before the model was
prepared, the mice were given the extract (ig) for 6
days (the normal control group and the model control group were given the same
amount of distilled water). From the
seventh day iP cyclophosphamide
was given to the five groups - excluding the normal control group - once daily
for two days. From the day when the
model was prepared, they were given Leucozepin® for six consecutive
days. On the first and the fourth day
after the model was prepared, the tails of the mice were cut to take a blood
sample to check the peripheral leukocyte count. After the last blood sample was
taken, the mice died due to dislocation of cervical vertebra. The right femur was removed according to the literature[1] to count the bone marrow
karyocyte and t-check was carried out among groups.
2. Effect
of Leucozepin® on cyclophosphamide
induced leukopenia in S180-bearing mice
According to the literature [2], S180
cells were inoculated under the skin of the right armpit of 72 mice, half of
whom were male (0.2ml each). The next
day, the mice were divided into 6 groups at random, S180-bearing
control group, (20ml/kg distilled water), CTX model group (20ml/kg distilled
water), large dosage group, medium dosage group, small dosage group (i.e. 8, 4,
2g/kg) and Leucogen positive group (20mg/kg). The next day after the inoculation they were
given the medicine (ig) or distilled water, once
daily for 12 consecutive days. From the
7th day, ip cyclophosphamide
100mg/kg was given to the five groups – excluding the S180-bearing
control group – once daily for two days.
On the first and the fourth day after the model was prepared, the tails
of the mice were cut to take a blood sample to check the peripheral leukocyte
count. After the last blood sample was
taken, the mice died due to dislocation of cervical vertebra. The mice were dissected and the tumors were
removed and weighed. According to the
literature [2], the inhibitive rate of the tumors was
calculated. Simultaneously, the right
femur was removed to count the bone marrow karyocyte
and t-check was carried out among the groups.
Results:
- The effect of Leucozepin®
on cyclophosphamide induced leukopenia
and the decrease of bone marrow karyocyte count
in normal mice is shown in table 1.
The results reveal that ip cyclophosphosphamide could induce leukopenia
and decrease bone marrow karyocyte count, which
is obviously different from that of the control group. 8g/kg and 4g/kg of Leucozepin®
and Tabellae Batiloli
can obviously increase the counts of white blood cell and bone marrow karyocyte as compared to the results of the model
control group.
Table1. The effect of Leucozepin® on cyclophosphamide induced leukopenia
and the decreased bone marrow karyocyte count in
normal mice （
）
|
Groups |
Dosage （/kg） |
WBC（ |
Bone marrow karyocyte
count
|
|
|
1st
day after model |
4th
day after model |
|||
|
Normal control |
— |
12.09±3.28 |
12.92±2.59 |
29.07±4.69 |
|
Model control |
— |
3.65±0.81*** |
4.92±1.50*** |
16.88±3.33*** |
|
Large dosage |
8g |
4.37±0.78* |
7.03±2.20** |
20.12±2.42* |
|
Medium dosage |
4g |
4.68±0.99* |
7.36±1.84*** |
20.52±2.67** |
|
Small dosage |
2g |
4.50±2.01 |
5.77±1.76 |
20.01±4.74 |
|
Tabellae Boatiloli |
40mg |
4.52±0.79* |
5.96±0.98* |
21.74±3.60** |
n=14 compared with
normal control group：***P<0.001
compared with model control group： *P<0.05，**P<0.01，***P<0.001
2.The effect of Leucozepin®
on cyclophosphamide induced leukopenia
and the decreased bone marrow karyocyte in S180-bearing
mice is shown in table 2. The results
have shown that ip cyclophosphamide
could induce leukopenia and decrease bone marrow karyocyte count in S180-bearing mice, which is
obviously different from that of the S180-bearing control group.
8g/kg and 4g/kg of Leucozepin® and Leucogen
can obviously increase the counts of white blood cells and bone marrow karyocyte as compared to that of the model control group.
Table2. The effect of Leucozepin®
on cyclophosphamide induced leukopenia
and the decrease of bone marrow karyocyte count in S180-bearing
mice（）
|
Groups |
Dosage （/kg） |
WBC（ |
Bone
marrow karyocyte count
|
|
|
1st
day after model |
4th
day after model |
|||
|
S180-bearing control |
— |
10.78±2.46 |
11.19±3.32 |
12.16±4.23 |
|
Model control |
— |
2.62±0.88*** |
4.52±1.28*** |
7.39±1.02** |
|
Large dosage |
8g |
3.79±1.49* |
6.27±1.86* |
9.37±2.35* |
|
Medium dosage |
4g |
4.64±1.61** |
8.52±2.27*** |
10.33±3.92* |
|
Small dosage |
2g |
3.42±1.16 |
5.62±2.89 |
7.53±1.97 |
|
Leucogen |
20mg |
4.36±1.14** |
8.41±2.68*** |
11.67±3.28*** |
n=12 compared with S180-bearing control
group：**P<0.01，***P<0.001compared with model
control group：*P<0.05，**P<0.01，***P<0.001
3. The effect of
Leucozepin® on cyclophosphamide
inhibiting the growth of S180 cells in mice is shown in table
3. The results have shown that CTX can
treat S180 in mice and the inhibitive rate of tumors is
47.2%. When Leucozepin® was given to the mice,
there were no obvious effects on tumor cells.
This shows that while Leucozepin® increases the count
of white blood cells it did not affect the anti-tumor effect of CTX, nor did it
have any obvious effect on the decrease of the mice’s weight induced by cyclophosphamide.
Table3. The effect of Leucozepin® on CTX
inhibiting the growth of S180 cells in mice （）
|
Groups |
Dosage （/kg） |
Mice weight (g) |
Tumor weight （g） |
Inhibitive rate of tumor （%） |
|
|
Beginning |
End |
||||
|
S180-bearing
control |
— |
18.91±1.22 |
30.45±3.33 |
2.90±0.83 |
— |
|
CTX |
100mg |
19.25±1.60 |
24.50±2.54 |
1.53±0.66*** |
47.24 |
|
CTX+Large dosage |
100mg+8g |
18.92±1.08 |
25.25±2.63 |
1.59±0.51 |
45.17 |
|
CTX+Medium dosage |
100mg+4g |
19.28±1.27 |
25.14±2.63 |
1.39±0.28 |
52.07 |
|
CTX+Small dosage |
100mg+2g |
19.00±1.35 |
24.75±1.91 |
1.48±0.50 |
48.96 |
|
CTX+Leucogen |
200mg+20mg |
19.42±1.24 |
24.50±2.47 |
1.48±0.34 |
48.96 |
n=12 compared
with S180-bearing control group：***P<0.001
References:
[1]
Li Yikui. Experimental Methodology
of Pharmacology of Traditional Chinese Medicine [M]. Shanghai: Shanghai
Science and Technology Press, 1991: 253.
[2]
Xu Shuyun, Bian Rulian, Chen Xiu. Experimental Methodology of
Pharmacology [M].