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The effects of Trigonella foenum-graecum on induced

diabetes in rats

Roioru Corina Luminia1, Bolog Daniela Maria2


1

- School of Biology and Geology, Babes-Bolyai University, Clinicilor 5-7,

Cluj-Napoca, Romania
2

- School of Biology and Geology, Clinicilor 5-7, Babes-Bolyai University,

Cluj-Napoca, Romania;
Corresponding author.: Bolog Daniela, 0040754581612, danabolog@yahoo.com, Clinicilor 5-7, Cluj-Napoca, Romania

Abstract: Trigonella foenum-graecum seeds are used as spices in the


oriental cuisine or as a traditional medicine for different affections. Certain
compunds found in the seeds have hypoglicemic and hypocolesterolemiant
properties. Its use in treating diabetes has been recently studied. This paper
aims to investigate the effects of Trigonella foenum-graecum extracts on
diabetes. The necessary studies were made using made using an experimental
model in vivo. The animals were fasted for a period of 24 hours. Each rat
found under light anesthesia with diethyl ether, received in the tail vein at a

dose of streptozotocin calculated according to body weight (60 mg / kg).


Streptozotocin was dissolved in a solution of 10 mM Na-citrate pH 7.5. After
30 minutes, the rats were intraperitoneally administrated 1 ml 33% glucose
saline solution for preventing hypoglycemic shock. To ensure that animals
injected with STZ became diabetic, we monitorised blood glucose concetration.
The animals with a glycemia bigger than 200 mg/dl were included in the study.
The rats were divided into four groups: first control group, second control
group, Trigonella 5% group and Trigonella 10% group. At the end of 28 days
of treatment the animals were sacrificed under anesthesia. Blood was colected
from jugular vein for further morphological and biochemical examinations.
10% Trigonella dose restored the track parameters to normal value.
KEY WORDS: fenugreek, diabetes, rats, glucose, cholesterol

1. Introduction
Trigonella foenum-graecum is originar from the mediteranean region
where it is cultivated on large areas. In the acient Egipt the plant has been used
for parfumes or for the emmbalment of mummies. In Rome it has been used to
aid labour. In chinese traditional medicine it is used as tonic for asthenia or as a
treatment for renal diseases

(Pribac et all, 2006; Amr et all, 2005).


It is an annual species about 70 cm tall. The stem is cylindrical and with
no hair. The leaves are compound pinnate and trifoliate. The flowers are
axillary with a white to yellow colour or slightly violet. The fruit is a long thin
pointed beaked pod which contains oblong yellowish seeds. These seeds are
aromatic, bitter and carminative. It contains about 45-60% carbohydrates, 7.5%
lipids, 6.3% tryglicerides and 450mg/phospholipids. Almost 50% dry weight of
the seeds is eatable dietary fibre which delays gastric emptying, induce satiety,
supress apetite and reduce risk of constipation. Fenugreek seeds also contain 48% saponins and 1% alkaloids. Saponins stimulate uterin contractions in
females, testosterone release in males and increase secretory functions.
Trigonelline decreases glycosuria in diabetes. 4-hydroxi-isoleucine has
hypoglicemic and hypocholesterolemiant properties and regulates insulin
secretion(Srinivasan, 2006; Pribac et all, 2008). The seeds also contain micro-,
and macroelements: Ca, Fe, Na, K, Cu, Cr,Zn.
Polyphenolic flavonoids like quercetin have protective effects against
EtOH-induced liver cell damage by adjusting the activity of alchool
metabolizing enzymes and stress protein expression (Kaviarasan et all, 2006).

Recent studies revealed the anticarcinogenic potential of certain


compounds found in fenugreek seeds. The intra-peritoneal administration of the
alcohol extract of the seed in mice with EAC(Ehrlich ascites carcinoma) cells
inhibited tumor cell growth, activated macrophage cells and induced
antiinflammatory effects (Sur et all, 2001).
The anti-cancer activity of Trigonella foenum graecum in rats against
7,12-dimethylbenz(a)anthracene(DMBA)-induced breast cancer has been
recently investigated. Tumor progression was inhibited and stimulatory effects
on macrophages have been observed (Amr et all, 2005).
Many complications in diabetes appear because of an increase
production of oxygen free radicals due to hyperglicemia. By administrating
insuline, sodium orthovandate and fenugreek for 21 days to a group of diabetic
mice, peroxide concentration was restaured to control values. This treatment
may protect the organism against oxidative damage and degenerative changes
that occur in many tissues (Genet et all, 2002).
The administration of trigonella seed powder for 3 weeks on another
diabetic mice group restored the levels of piruvate kinase and
phosphoenolpyruvate carboxykinase in liver. The alterations occurred in

translocation of GLUT-4 to plasma membrane in the skeletal muscle were also


corrected (Mohammad et all, 2011).
4-hydroxyisoleucine was administrated orally to type II diabetic mice. A
decrease in plasma glucose tryglicerides and LDL-cholesterol was noticed.
HDL -cholesterol levels were increased (Singh et all, 2010).
The administration of trigonella seed powder in combined treatment
with sodium orthovanadate in alloxan diabetic rats for 21 days showed an
amelioration of altered lipid metabolism in plasma and tissues and prevented
the development of hyperglicemia (Yadav et all, 2004).
The present study aimes to investigate the effects of Trigonella seed
powder on streptozotocin induced diabetes in rats.
2. Materials and methods
Trigonella seed powder was purchased from SC.ADSERV SRL.,
Brasov, Romania and added to animal fodder 5% and 10 % respectively.
We used white female Wistar rats with a weight of 17020 g. The animals were
housed in the zoobase of Experimental Biology Chair, School of Biology and
Geology, in hygienic conditions, at a constant temperature, under 12/12 h
light/dark cycle, water ad libitum and a suitable diet with or without fenugreek
seed powder.

The animals were starved for 24 hours. Every rat received a dose of
streptozotocin(STZ) (60mg/kg body weight, in sodium citrate 10 mM) in the
codal vein. To prevent the hypoglicemic shock, 30 minutes after the
administration of the diabetogenic agent animals received i.p. 1 ml of a 33%
glucose saline solution. Blood glucose concentration was measured before STZ
administration and in the third and seventh day after. The animals that had a
glycemia >200 mg/dL were included in the experimental groups. 30-40 % of
the animals that receive STZ dont develop diabetes in the next days after
injection. These animals received another dose of streptozotocin (50 mg/ kg
body weight, in sodium citrate 10 mm). For the experiment have only been
recruted the animals that had developed hyperglycemia.
Animals were organized in four groups:
-first control group(C) with healthy rats and standard diet;
-second control group(D) with diabetic rats and standard diet;
- Trigonella 5% group(D+T5%) with diabetic rats which received 5% trigonella
seed podder in the fodder;
- Trigonella 10% group(D+T10%) also with diabetic rats which received 10%
trigonella seed powder in the diet.

The treatment period was 28 days. Animals were treated gently. At the
end of this period animals were sacrificed under anesthesia.
Blood was colected from jugular vein for further morphological and
biochemical examinations.
For the white and red cell count whole blood was used. The packed cell
volume has been measured using the Windrobe method. The haemoglobin
concentration was determined by photocolorimetric method with Drabkin
reagent. Blood concentration of glucose was determined colorimetrically by
Somogy-Nelson method. Protein determination was made using serum and
Bradford reagent (Gornall et al.,1949). We also used serum for measuring
cholesterol concentration and the activities of lactate dehydrogenase (LDH)
(Bergmeyer and Bernt, 1974) and catalase (Vives-Bauza et all., 2007).
The results are given as meansSE. The data were examined for statistical
significance using Students t test. Variations which have values of 0 p were
considered to be significant, statistically, as follows: P < 0.05 significantly ( * );
P <0.01 distinctly significantly ( ** ); P <0.001 very significant ( *** ).

3. Results
3.1 Red cell count
Parameter
x ES
n
Red cell
count/mm3

C
7.350.17
10

p1(C)

D
5.790.25
4
p<0,001
***

p2(D)

D+T5%
5.680.78
3

D+T10%
6.050.60
4

p< 0,01 **

p<0,05 *

p>0,05 NS

p>NS

xES: mean valuestandard error, n: sample number used for statistical analysis of data, p1(C): statistical significance
against the control group, p2(D): statistical significance of fenugreek-treated groups against the diabetic group

The toxic effects of the excess of glucose on erythrocytes are: low


plasticity, changes occuring in the membrane structure, cytoskeleton structure
and in the composition of the lipid bilayer. Red cell count from the D+T10%
group are near the normal values.
3.2 White cell count
Parameter
White cell
count/mm3

x ES
n
p1(C)
p2(D)

C
11.060.89
10

D
8.470.44
4
p>0,05 NS

D+T5%
8.900.58
3
p>0,05 NS
p>0,05 NS

D+T10%
9.470.31
4
p>0,05 NS
p>0,05 NS

xES: mean valuestandard error, n: sample number used for statistical analysis of data, p1(C): statistical significance
against the control group, p2(D): statistical significance of fenugreek-treated groups against the diabetic group

In diabetes the imune system is affected so the people who have this
affection are incled to inffection and have a diminished capacity of defense.
WBC count at the animals from the diabetic group is low compared to the
control group. On the other hand, in the D+T10% group we observ a slight
increase of the WBC count.
3.3 Packed cell volume
Parameter

Packed cell
volume %

x ES
n
p1(C)
p2(D)

C
42.080.62
10

D
241.34
4
p<0,001***

D+T5%
17.973.21
6
p<0,001 ***
p>0,05 NS

D+T10%
23.412.56
8
p<0,001 ***
p>0,05 NS

xES: mean valuestandard error, n: sample number used for statistical analysis of data, p1(C): statistical significance
against the control group, p2(D): statistical significance of fenugreek-treated groups against the diabetic group

Semnificative changes are observed in all three diabetic groups. The


decreases recorded in this parameter are corelated with the low red cell count.
3.4 Hemoglobin
Parameter
Hemoglobin
g/dL

x ES
n
p1(C)
p2(D)

C
17.940.47
10

D
10.540.93
8
p<0,001***

D+T5%
6.711.53
6
p<0,001***
p<0,05 *

D+T10%
7.560.42
8
p<0,001***
p<0,05 *

xES: mean valuestandard error, n: sample number used for statistical analysis of data, p1(C): statistical significance
against the control group, p2(D): statistical significance of fenugreek-treated groups against the diabetic group

The hemoglobin concentration in the 3 groups of diabetic rats is


significantly low raported to the control group. Its value at the fenugreek
treated groups is lower than the value from the diabetic group, although the iron
content of the seeds should have been able to stimulate hemoglobin synthesis.
Low hemoglobin concentration correlates with low packed cell volume.
3.5 Serum glucose
Parameter

D+T5%

D+T10%

x ES

108.927.44

424.1651.46 373.4783.01

172.5728.09

Serum

10

glucose

p1(C)

p<0,001 ***

p<0,001 ***

p<0,05 *

mg/dL

p2(D)

p>0,05 NS

p<0,001 ***

xES: mean valuestandard error, n: sample number used for statistical analysis of data, p1(C): statistical significance
against the control group, p2(D): statistical significance of fenugreek-treated groups against the diabetic group

The diabetic group with a standard diet presents increased glucose


values compared to the control group. We can observe a sligh decrease at the
D+T5% group. At the D+T10 % group we notice a semnifcative decrease, the
values approach the values obtained at the control group. According to
Mohamad et all. (2011), 4 hydroxyisoleucine acts directly on the beta cells

adjusting insulin secretion. Other compunds with hypoglicemic properties are


arginine and tryptofan.
3.6 Serum cholesterol
Parameter

D+T5%

D+T10%

x ES

156.125.55

109.842.17

119.835.53

137.804.06

Serum

cholesterol

p1(C)

mg/dL

p2(D)

p<0,001 ***

p<0,01 **

p<0,05 *

p>0,05 NS

p<0,001 ***

xES: mean valuestandard error, n: sample number used for statistical analysis of data, p1(C): statistical significance
against the control group, p2(D): statistical significance of fenugreek-treated groups against the diabetic group

Compared to the control group the values obtained at fenugreek-treated


groups confirm the hypercholesterolemic potential of the fenugreek seeds. We
also observe a decrease of the cholesterol value at the diabetic group in relation
to the control group.

3.7 Serum protein concetration

Parameter

Protein
concentration
g/dL

D+T5%

D+T10%

x ES

6.000.20

3.100.36

2.930.66

3.350.43

n
p1(C)
p2(D)

10

6
7
p<0,001 *** p<0,001***
p>0,05 NS
p>0,05 NS

p<0,001***

xES: mean valuestandard error, n: sample number used for statistical analysis of data, p1(C): statistical significance
against the control group, p2(D): statistical significance of fenugreek-treated groups against the diabetic group

Insulin enahces protein synthesis and diminishes protein degradation. In


diabetes protein metabolism is affected and free aminoacid concentration
increases. We didnt notice any semnifcative change at the fenugreek-treated
groups.

3.8 Lactat dehydrogenase-LDH

Parameter

LDH
moli
piruvat/dL
ser/min

D+T5%

D+T10%

x ES

0.910.12

1.040.17

0.850.15

0.580.05

n
P1(C)

10

8
p>0,05 NS

6
p>0,05 NS

6
p>0,05 NS

p>0,05 NS

p<0,05 *

P2(D)

xES: mean valuestandard error, n: sample number used for statistical analysis of data, p1(C): statistical significance
against the control group, p2(D): statistical significance of fenugreek-treated groups against the diabetic group

Comparative to the healthy animals, LDH activity at the diabetic


animals is elevated. The values registered at the D+T5% group approach the
values from the control group.

3.9 Superoxid dismutase and seric catalase- SOD and CAT

Parameter
x ES
n
P1(C)

C
0.110.01
10

SOD
USOD/min/mg
P2(D)
prot

CAT
k/s/g prot

x ES
n
P1(C)
P2(D)

45.635.03
8

D
0.210.03
4
p<0,05 *

26.215.79
7
p<0,05 *

D+T5%
0.290.13
3
p<0,05 *
p>0,05
NS

D+T10%
0.150.008
3
p>0,05 NS

22.085.43
5
p<0,05 *
p>0,05 NS

30.695.90
3
p>0,05 NS
p>0,05 NS

p>0,05 NS

xES: mean valuestandard error, n: sample number used for statistical analysis of data, p1(C): statistical significance
against the control group, p2(D): statistical significance of fenugreek-treated groups against the diabetic group

The activity of these antioxidant enzymes is disturbed in diabetes. SOD


activity is elevated at the diabetic group with a standard diet. 5% fenugreek
dose added in the animal diet lead to increased activity of this enzyme. Instead
at the D+T10% group the activity decreased. Catalase activity is decreased at
the diabetic groups. We also observe a decrease at the D+T5% group. There is a
statistically insignificant increase at the D+T10% group, that correlates with the
antioxidant potential of the fenugreek powder.

4. Discussion
After the administration of fenugreek powder in the animal diet for 28
days semnificative decreases of blood glucose were observed in the diabetic
groups Trigonella 5%, respectively 10%. Maintaing normal blood glucose
values certifies the hypoglycaemic potential of fenugreek seed powder. Hannan
et all (2007) affirm that the diets rich in Trigonella fiber slow intestinal
absorbtion of sugars by reducing the activity of -amylase and sucrase, the key
enzymes in the metabolism of carbohydrates. Also Trigonelline reduces
glycosuria in diabetic people.
The results also validated the hypercholesterolemic potential of
fenugreek. Its seeds contain 4-6 % saponins, which are transformed into
sapogenins in the gastrointestinal tract. Biliary cholesterol secretion is
enhanced so the plasmatic concentration decreases.
According to Bin-Hafeez et all. (2003) fenugreek has stimulatory effects
upon immunitary functions increasing macrophages phagocytizing function.
Most complications that accompany diabetes occur due to the presence
of reactive oxygen species. The activity of antioxidant enzymes SOD and CAT
is disturbed. The treatment with 10% fenugreek seed powder restaured their
activity to control values. There by we can affirm that one of the benefic effects
of Trigonella powder is the protection of the membrane system against the
destructive action of free radicals.Genet et all (2002) assert that a treatment
with insulin, sodium orthovanadate and fenugreek powder restored elevated
peroxide levels to normal values. Polyphenolic compounds found in fenugreek
seeds have antioxidant properties and protected the erythrocytes against
oxidative stress.

10% Trigonella dose administered in the feed for 4 weeks, had


promising effects on restoring the track parameters to control values.

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