Biotechnology and Bioprocess Engineering 15: 407-413 (2010)
DOI 10.1007/s12257-009-3037-9
RESEARCH PAPER
Inhibitory Effect of Fenugreek Galactomannan on Digestive
Enzymes Related to Diabetes, Hyperlipidemia, and Liver-kidney
Dysfunctions
Khaled Hamden, Bassem Jaouadi, Serge Carreau, Samir Bejar, and Abdelfattah Elfeki
Received: 17 September 2009 / Revised: 5 November 2009 / Accepted: 6 November 2009
© The Korean Society for Biotechnology and Bioengineering and Springer 2010
Abstract The present study was undertaken to investi- study indicate that fenugreek galactomannan displays a
gate the effect of fenugreek galactomannan on intestinal
glucose uptake in surviving diabetic rats. It explored their
potential action with respect to lowering maltase, lactase,
and sucrase activities in the small intestine of galacto-
mannan-treated diabetic group compared to the diabetic
control group. The findings indicate that the increase of
blood glucose levels was significantly suppressed in the
galactomannan-treated group than those in the diabetic
rats. Moreover, the galactomannan isolated from fenugreek
exhibited a prominent selective inhibitory effect against
intestinal lipase activity. It was found to significantly delay
the absorption of LDL-cholesterol and triglycerides and the
increase in HDL-cholesterol. In addition, fenugreek galac-
tomannan efficiently protect the hepatic function observed
by the considerable decrease of aspartate and alanine
transaminases (AST and ALT) and lactate deshydrogenase
(LDH) contents in the serum of diabetic rats. The bene-
ficial effects of fenugreek galactomannan were also evi-
denc-ed by their capacity to inhibit diabetes-induced
kidney injury through lowering the urea and creatinine
content in plasma. Overall, the conclusion of the present
Khaled Hamden*†, Abdelfattah Elfeki
Animal Ecophysiology Laboratory, Faculty of Sciences of Sfax, P.O. Box
95, Sfax 3052, Tunisia
Tel: +216-97-469-111; Fax: +216-74-274-437
E-mail: khaled.hamden@yahoo.fr
Bassem Jaouadi†, Samir Bejar
Laboratory of Prokaryotic Enzymes and Metabolites, Centre of
Biotechnology of Sfax, P.O. Box 1177, Sfax 3018, Tunisia
Serge Carreau
Department of Biochemistry, University of Caen-Basse Normandie, USC
INRA, EA2608, CHU-Caen, France
†
Both authors have equally contributed to this work.
number of promising properties and attributes for future
applications as therapeutic agents in biotechnological and
bioprocess-based technologies, particularly those interested
in the development of anti-diabetic and hypolipidemic
drugs.
Keywords: fenugreek galactomannan, diabetes, lipase,
disaccharidases, drugs
1. Introduction
Diabetes is a serious health problem growing at alarming
rates around the world. It is reported to constitute the 16th
leading cause of global mortality [1]. Current estimates
indicate that worldwide about 150 million people suffer
from diabetes and that by 2025 this number is expected to
reach 300 million or more [2]. It is generally recognized
that patients with diabetes are at risk for numerous severe
complications, including diabetic nephropathy and hepato-
logy [3-6]. One of the therapeutic approaches for de-
creasing postprandial hyperglycaemia and hyperlipidemia
is to retard absorption of glucose and lipid by the inhibition
of lipid and carbohydrate-hydrolyzing enzymes, such as
disaccharidases and lipase, in the digestive organs [7,8].
In this respect, many synthetic disaccharidase and lipase
inhibitors have been reported to reduce plasma glucose and
lipid levels via delaying glucose and lipid absorption and
retarding the liberation of glucose from oligosaccharides
and disaccharides from dietary complex carbohydrates [9].
Nevertheless, the use of these inhibitors often induced
disturbances in the gastrointestinal tract, including flatu-
lence, diarrhea, and abdominal pain [9].
408
Accordingly, recent research seems to have granted
special interest for the search of effective natural gluco-
sidase and lipase inhibitors. In the same vein, polysac-
charides, isolated from plant sources, have attracted a great
deal of attention in the biomedical arena particularly for
their broad spectrum of therapeutic properties and relative-
ly low toxicity [8,10,11]. Dietary polysaccharides are well
established as beneficial food components that help to
alleviate a wide range of complications pertaining to
various diseases, including cancer and obesity [12,13].
Of particular interest to the aims of the present study,
fenugreek (Trigonella foenum-graceum), which is a member
of the leguminosae family, is often appreciated for its
nutritional and medicinal benefits [14]. Its well-document-
ed nutritional, functional, and multiple health effects have
recently been reviewed in the literature [15]. This product
is high in polysaccharide (92%), which is a galactomannan,
a highly branched water soluble polysaccharide similar to
that of guar and locust bean gums [14]. Galactomannans
are polysaccharides consisting of a mannose backbone
with galactose side chains attached at a C6 position. They
consist of linear chains of (1-4)-diequatorially linked -D-
mannose residues, some of which carry single-sugar side
chains of -D-galactose attached by (1-6) glycosidic bonds
[16].
The current study attempts to contribute to this line of
research by investigating the therapeutic action of fenu-
greek polysaccharides on diabetic status as well as the
activities of disaccharidases and lipase with respect to
small intestinal and liver-kidney toxicity in streptozotocin-
induced diabetic rats.
2. Materials and Methods
2.1. Fenugreek
The galactomannan used in the present study was extracted
from fenugreek seeds at a laboratory setting. The seed coat
of fenugreek seed (0.5 mm mesh) was separated and di-
spersed in reverse osmosis distilled water for 4 hours at a
ratio of 1:40 (w/v). After centrifugation (16,000 × g, 30
min), the supernatant was mixed with absolute ethanol at a
ratio of 1:1 (v/v) to precipitate gum, which contained
91.4% (w/w) galactomannan on a dry weight basis [14].
2.2. Induction of diabetes
The assays of the present study were conducted on adult
male wistar rats, weighing 179 ± 10 g, obtained from the
local Central Pharmacy, Tunisia. All rats were kept in an
environmentally controlled breeding room (temperature:
20 ± 2oC, humidity: 60 ± 5%, 12-h dark/light cycle) where
they had free access to tap water. The animals fasted
Biotechnology and Bioprocess Engineering 15: 407-413 (2010)
overnight before blood and tissue collection. Diabetes was
induced in the rats by intraperitoneally administering a
nicotinamide solution (1,000 mg/kg) that was followed 90
min later by a streptozotocin solution (150 mg/kg) [17].
Non-diabetic rats were intraperitoneally administered phy-
siological saline and were kept for the next 24 h on 5%
glucose solution bottles, in their cages, to prevent hypo-
glycemia. Two weeks later, the rats with moderate diabetes
having glycosuria and hyperglycemia (i.e., with blood
glucose levels of 2 g/L) were chosen for the subsequent
experimental assays. The handling of the animals was
approved by the Tunisian Ethical Committee for the care
and use of laboratory animals.
2.3. Experimental procedure
A total of 40 rats (30 surviving diabetic rats and 10 control
animals) were subdivided into different groups. Group 1,
surviving diabetic rats obtained two weeks after strepto-
zotocin injection, were scarified and referred as diabetic
rats at the start of treatment and named as “diabetic heath
rats (DHC) (beginning of treatment) (glycemia ≥ 2 g/L)”.
Group 2, surviving diabetic rats were scarified at the end of
the experimentation (two months after “DHC (beginning
of treatment) group” and named as “diabetic heath rats
(DHC) (end of treatment)”. Group 3 referred to galacto-
mannan-treated diabetic health rats named DHC + F (Gm)
(8% in food). Ten normal rats were used as controls which
were named as normal heath rats (NHC).
After two months of fenugreek galactomannan admini-
stration to diabetic rats, the NHC, DHC (end of treatment)
and DHC + F (Gm) animals were sacrificed by decapita-
tion and the trunk blood was collected. The serum was
prepared by centrifugation (1,500 × g, 15 min at 4°C), the
small intestine was removed, and all samples were stored
at –80°C until further use.
2.4. Analytical methods
The mucosal small intestine of each rat was excised and
the lumen was flushed out several times with 0.9% NaCl.
The mucosal washing and the scraped mucosa were
pooled, homogenized, and centrifuged (5,000 × g, 15 min).
The supernatant was frozen and stored for use in sub-
sequent enzymatic assays. The activities of intestinal di-
saccharidases were determined by measuring the amount
of glucose released from various substrates [18]. Lipase
activity was assayed, with olive oil as substrate, using the
method described by Tietz and Fiereck [19]. The activities
of aspartate and alanine transaminases (AST and ALT) and
lactate deshydrogenase (LDH) as well as the levels of total-
cholesterol, triglyceride, and HDL-cholesterol in the serum
were measured using commercial kits from Biomagreb
(Tunis, Tunisia) and Biomerieux (Lyon, France).
Inhibitory Effect of Fenugreek Galactomannan on Digestive Enzymes Related to Diabetes, Hyperlipidemia… 409

2.5. Statistical analysis

Data are presented as means ± SD. Determinations were
performed from ten animals per group. The differences
were examined using one-way analysis of variance (ANOVA)
and Fisher test (Stat View) and the significance value was
accepted at p < 0.05.

3. Results

3.1. Intestinal maltase, lactase, and sucrase activities

and plasma glucose level
Figs. 1 and 2 show that, when compared to control, the
intestinal maltase, lactase, and sucrase activities were signi-
ficantly amplified in diabetic rats; they increased by 39, 47,
and 48%, respectively. An increase in glucose concent-
ration in plasma by 384% could also be observed. How-
ever, the supplementation of fenugreek galactomannan to
surviving diabetic rats was found to significantly decrease
the activities of intestine maltase (Fig. 1A), lactase
(Fig. 1B), and sucrase (Fig. 1C) by 39, 47, and 48%,
respectively. In addition, the administration of fenugreek
galactomannan to diabetic rats reduced glucose concent-
ration in plasma by 56% as compared to diabetic untreated
rat (Fig. 2).
3.2. Intestinal lipase activity and plasma lipid concent-
ration
Figs. 3 and 4 indicate that, when compared to the non-
diabetic rats, the intestinal lipase activity in the diabetic rats
underwent a potent increase of 164% (Fig. 3). The total-

Fig. 1.Effect of fenugreek galactomannan, denoted F (Gm), supple-

ments on maltase (A), lactase (B), and sucrase (C) activities in the
small intestine of diabetic heath rats (DHC) as compared to normal
heath control (NHC). Values are given as mean ± SD for group of
10 animals each. Values are statistically presented as follows:
*p < 0.05 significant differences compared to controls.
#
p < 0.05 significant differences compared to diabetic rats before Fig. 2. Effect of fenugreek galactomannan supplements on blood
treated groups (BT). glucose level of diabetic heath rats (DHC) as compared to normal
@
p < 0.05 significant differences compared to diabetic rats after heath control (NHC). Statistical analyses are as given in Fig. 1
treated groups (AT). legend.
410 Biotechnology and Bioprocess Engineering 15: 407-413 (2010)

Fig. Intestinal lipase activity of diabetic heath rats (DHC)

3.

treated with fenugreek galactomannan as compared to normal

heath control (NHC). Statistical analyses are as given in Fig. 1
legend.

cholesterol and triglycerides concentrations in plasma were

also noted to significantly increase in the diabetic rats. The
administration of galactomannan to surviving diabetic rats,
however, was observed to have reverted the activity of
lipase in intestine back by 46%. This supplement also led
to a considerable decrease in cholesterol and triglycerides
in plasma. It was also observed that diabetes induced a
decrease in the HDL-cholesterol concentration and that
galactomannan prevented this decrease (Fig. 4).
3.3. Liver dysfunction indices

Fig. 5 illustrates that the activities of AST (Fig. 5A), ALT

(Fig. 5B), and LDH (Fig. 5C) in the plasma of the diabetic
rats witnessed a significant increase of 38, 109, and 129%,
respectively, when compared to non-diabetic rats. In fact,
the supplement of galactomannan was found to bring about
a potent decrease in terms of the three indices of liver
toxicity.
3.4. Kidney dysfunction indices

Fig. 6 demonstrates that, compared to non-diabetic rats, the

diabetic rats undertook an increase in terms of the urea
(Fig. 6A) and creatinine (Fig. 6B) rates in plasma by 56
and 50%, respectively. More interestingly, the admini-
stration of galactomannan to diabetic rats seems to have
reverted this increase back.

Fig. 4. Effect of fenugreek galactomannan supplements on total

4. Discussion
and HDL-cholesterol and triglyceride levels in blood, as compared
to diabetic heath rats (DHC) and normal heath control (NHC).
The literature presents a variety of therapeutic proposals to Statistical analyses are as given in Fig. 1 legend.
Inhibitory Effect of Fenugreek Galactomannan on Digestive Enzymes Related to Diabetes, Hyperlipidemia…
Fig. 5.AST, ALT, and LDH activities before and after fenugreek
galactomannan administration to diabetic heath rats (DHC) as
compared to normal heath control (NHC). Statistical analyses are
as given in Fig. 1 legend.
411
Fig. 6.Effect of fenugreek galactomannan supplements on indices
of kidney dysfunction in (urea and creatinine contents) diabetic
heath rats (DHC) as compared to normal heath control (NHC).
alleviate and prevent risks, disorders and dysfunctions
pertaining to the concerns presented above. One of the
therapeutic approaches used for the decrease of post-
prandial hyperglycemia and hyperlipidemia is the retarda-
tion of glucose and lipid absorption through the inhibition
of lipid and carbohydrate-hydrolyzing enzymes, such as
maltase, lactase, sucrase, and lipase, in the digestive organs
[20]. In an attempt to contribute to the current body of
knowledge, the present study was undertaken to investigate
the inhibitory effect of fenugreek galactomannan on the
disaccharidase and lipase activities in the intestine of
diabetic rats. Interestingly, the findings indicate that the
administration of galactomannan to surviving diabetic rats
significantly decreased the maltase, lactase, and sucrase
activities present as key intestinal enzymes and involved in
the conversion of oligosaccharides into monosaccharides.
412
The inhibitory effects exhibited by intestinal disacchari-
dases seem to have limited the process of carbohydrate
hydrolysis and absorption in the intestine. In fact, the
results of the present study are in accordance with the data
recently reported by Srichamroen et al., stipulating that
fenugreek galactomannan in vitro affects intestinal glucose
uptake in genetically determined lean and obese rats [14].
Similarly, the administration of fenugreek at dose 100 g to
diabetic subjects during 10 days was observed to improve
glucose level [21]. In humans, fenugreek seeds exert hypo-
glycemic effects by inhibiting the activities of -amylase
and sucrase [22,23], two intestinal enzymes involved in
carbohydrate metabolism. The inhibition of intestine di-
saccharidases by food containing polysaccharides has also
been reported to retard this digestive process through their
inhibition of intestinal digestive enzymes and this was
observed to bring about better control of hyperglycemia
[24,25]. Therefore, the findings of the present study pro-
vide sheer evidence that fenugreek galactomannan has a
promisingly efficient potency as a therapeutic agent and
can, therefore, be considered as potential strong candidate
for future biotechnological applications interested in hypo-
glycemic drug development.
The findings of the present study also demonstrated that
diabetes increased lipase activity in the intestine and that
the increased lipid absorption from the intestine con-
sequently amplified the hypercholesterolemia and hyper-
lipidemia effects [26,27]. The administration of fenugreek
galactomannan to surviving diabetic rats, on the other
hand, clearly reverted the activity of intestinal lipase nearly
back to that of the non-diabetic rats. The inhibitory action
of lipase in the intestine decreased the hydrolysis of dietary
triglycerides to monoglycerides and free fatty acids as it
lowered the lipid level of the blood [26,27]. Furthermore,
the hypocholesterolemic effect of this polysaccharide
resulted in empowering these compounds with the capacity
to elevate the viscosity in the lumen of the small intestine,
which, in turn, resulted in the decrease of intraluminal
mixing. The positive effect of fenugreek galactomannan
was confirmed in humans specified that several studies
have found hypocholesterolemic effects associated with
oral fenugreek. Sharma et al. investigated human subjects
who ingested 100 g of defatted fenugreek powder per day
for three weeks and found that their triglyceride (TG) and
LDL-C levels were lower than baseline values [28]. In
another study, Sharma et al also reported a decrease in total
cholesterol levels in five diabetic patients treated with
fenugreek seed powder (25 g orally per day) for 21 days
[29] with no change in HDL-C level. Accordingly, the
findings indicate that galactomannan may be considered as
a potentially natural and safe approach for the control of
obesity and cardiovascular diseases in humans [30]. In
Biotechnology and Bioprocess Engineering 15: 407-413 (2010)
addition, hyperglycemia and hyperlipidemia in diabetic rats
are involved in a variety of metabolic disorders and other
serious diseases such as liver-kidney dysfunctions [26,31].
The authors of the present work have shown in previous
studies [4-6] that the acceleration and amplification of the
increase of glucose and lipid levels caused the formation of
free radicals of oxygen (ROS). The oxidative atmosphere
in cells caused the damage of cells and tissues in liver-
kidney [31]. Moreover, the oxidative atmosphere that
attacked the hepatic and kidney function appeared by an
increase in the level of AST, ALT, and LDH, an indice of
liver dysfunction, and of urea and creatinine, which are
considered as significant markers of renal dysfunction.
However, both the hypoglycemic and hypolipidemic action
of fenugreek galactomannan prevented glucose and lipid
toxicity as ROS formation. In fact, this study showed that
this drug exhibit a strong promising potential as a pro-
tective therapeutic agent against liver and kidney toxicity
in diabetic rats. They proved remarkably efficient in the
decrease of liver and kidney dysfunction indices in surviv-
ing diabetic rats, namely the AST, ALT, and LDH activities
and the urea and creatinine levels. The strong therapeutic
effect and potential of fenugreek galactomannan is pre-
sumably associated with its potent hypoglycemic and
hypolipidemic properties.
5. Conclusion
The findings of the current study demonstrate that fenu-
greek galactomannan exhibited promising therapeutic effects
on postprandial hyperglycemia and hyperlipidemia and
significantly prevented diabetic toxicity in the liver and
kidney. They also suggest that fenugreek galactomannan
can be safely and fruitfully used in future therapeutic and
medicinal applications as a therapeutic agent for the treat-
ment of diabetes, cardiovascular diseases, liver, and kidney
dysfunctions. Accordingly, further studies are currently
under way in our laboratories to further explore this anti-
diabetic agent and to make its application suitable for the
therapeutic and medicinal industries interested in the
development of anti-diabetic and hypocholesterolemic
drugs.
Acknowledgments
This research was supported by the Tunisian Ministry of
Higher Education and Scientific Research and Technology
and the Tunisian Ministry of public heath. The authors
would like to express their gratitude to Prof. Anouar
Smaoui, from the English department at the Sfax Faculty
Inhibitory Effect of Fenugreek Galactomannan on Digestive Enzymes Related to Diabetes, Hyperlipidemia…
of Science for carefully proofreading and editing the
manuscript of the present work.
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