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Abstract
Galega ofcinalis L. (Papilionaceae) is widely used in folk medicine as antidiabetic or for increasing lactation. There is a little information
about its possible toxicity. In this study, acute and subchronic toxicity of aerial parts of Galega ofcinalis in Wistar rats have been evaluated. For
the acute toxicity study, the animals received orally four different single dose of plant suspension and were kept under observation for 14 days.
The results indicated that LD50 of Galega ofcinalis is higher than 5 g/kg. In the subchronic study, 48 rats were divided into four groups and were
fed a diet containing 0%, 0.15%, 1.5% and 3% (w/w) of Galega ofcinalis. After 90 days blood and tissue samples were taken for hematological,
biochemical and histopathological determinations. An increase in serum levels of cholesterol, creatine phosphokinase, lactate dehydrogenase and
total and conjugated bilirubin was observed. Some parameters such as calcium, albumin, albumin/globulin ratio, hematocrit, WBC and platelet
counts were decreased. In microscopic examination, sinusoidal congestion in liver and alveolar hemorrhage was observed. Other parameters
showed non-significant difference between treatment and control groups. Present data suggest that liver and lung could serve as target organs in
oral toxicity of this plant.
2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Galega ofcinalis; Acute toxicity; Subchronic toxicity
1. Introduction
Galega ofcinalis (Papilionaceae) is a native plant from
southeastern Europe and was used as a treatment for diabetes in
medieval times (Oubre et al., 1997). The discovery of the plants
active hypoglycemic agent led to the development of metformin,
a biguanide which has been used to treat type 2 diabetes mellitus (Oubre et al., 1997; Palit et al., 1999). In fact, the only
example of an approved antidiabetic drug that was developed
from an herbal source with a long history of use for diabetes
is metformin from Galega ofcinalis (Vuksan and Sievenpiper,
2005).
Also in traditional medicine, Galega ofcinalis (syn.: galega)
is well known as a galactagoguing plant to improve milk
secretion, both in human and in animals (Uncini Manganelli
et al., 2001; Leporatti and Ivancheva, 2003). Recent experimental investigations indicate that crude aqueous extracts and
gel-filtered fractions of the plant suppress platelet aggregation
(Atanasov, 1994; Atanasov and Spasov, 2000). Moreover, it was
0378-8741/$ see front matter 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2007.10.030
22
23
Hematological analyses were done using standard techniques. Erythrocyte (RBC), total and differential leukocyte
(WBC), hematocrit (Hct), hemoglobin (Hb), platelet count,
mean corpuscular volume (MCV), mean corpuscular
hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean platelet volume (MPV), platelet
distribution width (PDW), and red distribution width (RDW)
of blood samples were determined using Sysmex K-1000 fully
automated hematology analyzer.
2.3.5. Necropsy
Following blood collection, rats were sacrificed by decapitation and liver, heart, lung, and kidney of all animals and ovary in
female rats were removed. The macroscopic appearance of the
organs was noted and their weights were recorded. Livers and
lungs of the half of animals in each group (3 rats/(sex group))
were preserved in 10% neutral buffered formalin for microscopic
histopathological examinations. The tissues were embedded in
paraffin, and then sectioned, stained with haematoxylin and
eosin and were examined microscopically.
2.3.6. Statistical analysis
Analysis of one-way variance (ANOVA) and Dunnett test
was used to determine statistical significance between tests and
controls. Only the values with a p < 0.05 were considered statistically significant (Yamakoshi et al., 2002; Bidhe and Ghosh,
2004).
3. Results
3.1. Acute study
No mortalities had occurred during the study and clinical
observations and measurements did not indicate evidences of
substance-related toxicity. On gross histopathological observation, rare red spots indicating lung hemorrhage in some of
animals in the highest dose group of both sexes were found. The
oral acute toxicity of Galega ofcinalis was, therefore, considered as unclassified, since a dose of 5 g/kg did not induce deaths
or toxic symptoms.
3.2. Subchronic study
Fig. 1. Changes in female rats body weight with duration of treatment. Each
point represents mean S.D. of N = 6.
PDW levels for control rats were not significantly different from
those in treatment groups. However, WBC counts and MPV were
decreased significantly (p < 0.05) in 1.5% group of male rats.
A statistically significant decrease of platelet count in 0.15%
(p < 0.05) and 1.5% (p < 0.01) groups was also observed. In
females, a reduction in hematocrit levels in 1.5% group (p < 0.05)
had occurred.
3.2.3. Biochemical analysis
A slight, but significant reduction in urea was noted in
high dose male rats (Table 2). Cholesterol levels were elevated significantly among middle and high dose groups of
both sexes (p < 0.01). Calcium ion concentrations were significantly reduced in all treatment groups of females (p < 0.01). This
parameter in males had a reduction in the 0.15% (p < 0.05) and
elevation in the 3% (p < 0.01) groups. Phosphorous concentrations in the 0.15% (p < 0.01) and 3% (p < 0.05) groups of males,
albumin levels in the middle dose group (p < 0.05) and albumin/globulin ratio in middle and high dose groups (p < 0.01)
of females were decreased significantly. The mean LDH levels in the middle (p < 0.01) and high dose (p < 0.05) females
were significantly increased whereas, this parameter shows a
reduction in the 3% group (p < 0.01) in males. There was a statistically significant increase in CPK level in all female treatment
groups (p < 0.01). Total bilirubin in 1.5% females and conjugated bilirubin in 1.5% males and 3% females were increased
(p < 0.05).
Fig. 2. Changes in male rats body weight with duration of treatment. Each point
represents mean S.D. of N = 6.
24
Table 1
Hematological parameters at termination of treatment
Sex
Dose (%)
Male
Control
0.15
1.5
3
9.25
5.02
3.30
7.83
1.79
1.03
1.34*
0.80
8.50
7.94
6.99
8.15
0.09
0.17
1.18
0.22
15.47
14.38
12.12
15.15
0.16
0.58
2.86
0.26
47.17
43.52
38.00
44.60
0.60
1.03
6.70
0.84
55.67
54.83
54.00
55.00
0.56
0.70
0.71
1.73
Female
Control
0.15
1.5
3
5.37
4.87
4.55
5.73
0.65
0.83
0.69
0.63
7.72
7.54
6.54
7.35
0.14
0.17
0.88
0.14
14.30
13.95
11.97
13.80
0.22
0.38
1.49
0.65
43.40
40.15
34.7
40.35
0.60
0.95
3.96*
1.17
56.17
53.33
54.83
54.63
0.54
0.67
2.70
0.86
MCH (pg)
18.17
18.00
16.25
18.50
18.50
18.67
18.83
18.67
0.17
0.45
2.10
0.50
0.34
0.21
0.91
0.56
MCHC (%)
32.83
33.17
30.00
34.00
32.83
34.83
34.33
34.00
0.17
0.91
3.34
0.26
0.40
0.48
1.02
0.86
MPV (fl)
8.28
8.30
7.65
8.22
7.55
7.68
7.80
7.53
0.10
0.10
0.06*
0.22
0.08
0.09
0.12
0.11
Hb (g/dl)
HCT (%)
1.00
0.88*
1.08**
1.03
0.33
0.61
1.05
1.38
MCV (fl)
RDW (%)
16.62
15.47
16.52
16.37
15.48
15.50
14.62
14.73
0.21
0.53
0.26
0.26
0.41
0.07
0.35
0.34
PDW (%)
9.90
10.93
10.17
9.62
8.68
9.03
9.03
8.95
0.15
0.78
0.51
0.38
0.09
0.23
0.16
0.23
Data presented as mean S.E.M. for N = 6. Significantly different from control: *p < 0.05, **p < 0.01.
lished that, this plant has a weight reducing action in both normal
and genetically obese mice (Palit et al., 1999).
In an earlier toxicity study in sheep, no significant alterations in blood constituents were observed in treated animals
(Keeler et al., 1986). However, in present study a reduction in
Hct in females and WBC, platelet counts and MPV in males
were seen. The reduction of Hct is a result of slight and nonsignificant reduction in RBC, WBC, and platelets in females
and it does not appear to be toxicologically significant. It seems
that galega has toxic effects on bone marrow which leads to
reduce platelets count and volume and WBC count. Although,
it is not an established conclusion and surely needs more
investigations.
The results of the present study indicate that significant alterations in liver function have occurred. Urea reduction is a result
of liver damage. Hypercholesterolemia, another significant incident, could be an indicator of hepatic function failure. Although,
usually liver damage occurs along with fat accumulation in hepatocytes, this study did not show any remarkable pathological
changes including fatty changes in liver. Moreover, some other
parameters like bilirubin elevation and albumin reduction are
confirmations to varying liver function. A small elevation in
plasma bilirubin is an important indicator of liver damage in
laboratory animals or could be a sign of biliary duct obstruction. In liver microscopic examination, there is no obstruction
in intrahepatic bile duct, so it could be a result of problems
in extrahepatic bilirubin pathway. If, this problem continues,
hypercholesterolemia and ALP increase will be developed. It
should be noted that in this study any elevations in ALP or other
liver enzymes did not occur.
Determination of plasma proteins like albumin can act as a
criterion for assessing synthetic capacity of the liver, since nearly
all of them synthesized in hepatocytes. Decrease in plasma proteins therefore tend to reflect chronic damage. The common
pattern seen following significant hepatocellular damage is a
25
Table 2
Biochemical parameters at termination of treatment
Sex
Dose (%)
FBS (mg/dl)
Male
Control
0.15
1.5
3
124.2
179.2
177.2
163.7
12.7
9.1
31.7
14.7
0.72
0.63
0.73
0.72
0.04
0.02
0.03
0.02
47.17
42.17
40.75
39.00
1.92
1.97
1.65
1.79*
48.83
53.17
55.25
58.33
4.12
3.66
11.83
10.63
Female
Control
0.15
1.5
3
146.7
130.3
132.0
111.3
11.9
10.0
10.9
8.1
0.75
0.65
0.73
0.60
0.08
0.08
0.12
0.09
50.50
48.33
42.67
48.67
3.75
3.36
5.43
4.89
65.00
84.50
36.00
74.00
13.58
14.81
4.13
17.09
Cholesterol (mg/dl)
68.33
76.00
89.25
84.50
106.50
128.17
145.33
141.33
Na (mEq/L)
2.03
2.71
4.70**
3.95**
1.73
11.60
5.23**
4.69**
140.0
138.0
138.5
144.5
140.3
136.3
138.0
137.7
Creatinine (mg/dl)
K (mEq/L)
0.63
0.45
1.32
4.17
1.23
1.45
1.46
0.92
5.48
5.42
6.07
5.42
7.77
7.55
7.22
7.43
Urea (mg/dl)
Cl (mEq/L)
0.27
0.14
0.39
0.29
0.07
0.16
0.20
0.11
94.83
94.50
99.75
90.33
95.67
89.33
91.50
96.17
Ca (mg/dl)
4.33
3.84
5.76
3.61
4.22
2.72
3.81
2.89
8.53
6.88
8.90
10.43
10.57
5.35
3.35
3.25
0.31
0.11*
0.92
0.36**
0.31
1.17**
0.26**
0.15**
11.10
8.07
9.47
8.57
11.92
13.82
11.93
12.00
0.81
0.47**
0.52
0.53*
0.94
1.37
0.85
0.92
Male
Control
0.15
1.5
3
8.53
7.97
8.47
8.30
0.24
0.16
0.22
0.21
4.52
4.35
4.37
4.17
0.15
0.10
0.09
0.10
1.12
1.22
1.05
1.02
0.08
0.10
0.05
0.03
219.0
212.7
181.3
183.7
17.6
23.8
6.3
24.4
66.00
65.33
52.33
65.83
4.19
7.61
6.74
6.34
Female
Control
0.15
1.5
3
8.73
9.05
8.93
9.53
0.29
0.54
0.17
0.55
4.67
4.57
3.40
3.80
0.26
0.33
0.17*
0.38
1.17
1.05
0.62
0.65
0.11
0.10
0.06**
0.06**
204.5
220.2
224.3
221.0
1.4
28.0
10.1
23.2
65.50
63.83
42.67
62.67
12.65
9.50
1.98
10.52
2209
1965
2150
1033
1713
2702
3392
3013
109
107
72
324**
325
355
430**
210*
1212
1092
1261
864
954
2277
2771
2465
ALP (IU/L)
28
146
97
237
185
350**
103**
167**
289.3
250.8
317.7
267.2
153.5
250.4
114.0
252.7
AST (IU/L)
Dose (%)
Al/G ratio
Phosphorous (mg/dl)
Sex
LDH (U/I)
Albumin (gr/dl)
Triglycerides (mg/dl)
T. bilirubin (mg/dl)
28.0
44.1
45.7
46.3
65.9
49.9
35.8
37.7
0.35
0.33
0.45
0.48
0.58
0.72
1.13
0.93
ALT (IU/L)
0.15
0.03
0.17
0.08
0.08
0.13
0.12*
0.22
0.08
0.10
0.15
0.10
0.12
0.35
0.35
0.55
0.02
0.00
0.03*
0.00
0.02
0.09
0.05
0.22*
FBS = fasting blood sugar, Na = sodium ion, K = potassium ion, Cl = chloride ion, Ca = calcium ion. Al/G = albumin/globulin Ratio, T. = total, Conj. = conjugated.
Data presented as mean S.E.M. for N = 6. Significantly different from control: *p < 0.05, **p < 0.01.
reduction in albumin accompanied by a relative increase in globulins which leads to albumin/globulin ratio reduction, often with
little change in the level of total protein (Woodman, 1996). This
pattern was seen in the present study, although microscopic
Table 3
Relative organ weight at termination of treatment (g % body weight)
Sex
Dose (%)
Liver%
Male
Control
0.15
1.5
3
Control
0.15
1.5
3
2.69
2.79
3.09
3.13
3.13
3.01
3.13
3.77
Female
Kidney%
0.03
0.07
0.11**
0.07**
0.09
0.18
0.11
0.27*
0.29
0.32
0.35
0.35
0.35
0.36
0.35
0.36
0.01
0.01
0.04
0.01
0.01
0.01
0.01
0.02
Heart%
0.32
0.29
0.35
0.35
0.37
0.38
0.36
0.37
Data presented as mean S.E.M. for N = 6. Significantly different from control: *p < 0.05, **p < 0.01.
Lung%
0.01
0.01
0.02
0.01
0.01
0.01
0.01
0.02
0.50
0.64
0.50
0.52
0.70
0.66
0.72
0.70
Ovary%
0.02
0.08
0.03
0.02
0.04
0.02
0.03
0.02
0.07
0.05
0.06
0.06
0.00
0.00
0.01
0.01
26