Journal of Complementary and

Integrative Medicine
Volume 6, Issue 1 2009 Article 6

Exploring Antidiabetic Mechanisms of Action

of Galactomannan: A Carbohydrate Isolated
from Fenugreek Seeds

Sirajudheen Anwar, Principal K. M. Kundnani College of

Pharmacy
Sandhya Desai, Principal K. M. Kundnani College of
Pharmacy
Rahul Mandlik, Principal K. M. Kundnani College of
Pharmacy

Recommended Citation:
Anwar, Sirajudheen; Desai, Sandhya; and Mandlik, Rahul (2009) "Exploring Antidiabetic
Mechanisms of Action of Galactomannan: A Carbohydrate Isolated from Fenugreek Seeds,"
Journal of Complementary and Integrative Medicine: Vol. 6: Iss. 1, Article 6.
DOI: 10.2202/1553-3840.1218
Available at: http://www.bepress.com/jcim/vol6/iss1/6
©2009 Berkeley Electronic Press. All rights reserved.
Exploring Antidiabetic Mechanisms of Action
of Galactomannan: A Carbohydrate Isolated
from Fenugreek Seeds
Sirajudheen Anwar, Sandhya Desai, and Rahul Mandlik

Abstract
The previously studied hypoglycemic effect of fenugreek galactomannan was confirmed in
normal rats. Additional investigations were carried out to study the effect of galactomannan on
utilization of glucose by hemidiaphragm and its antioxidant activity in diabetic rats. As compared
to diabetic control rats, the galactomannan enhanced the uptake of glucose by hemidiaphragm but
it was not comparable to the standard drug glibenclamide. Furthermore, galactomannan lowered
lipid peroxidation and elevated the levels of antioxidant enzymes. The present study demonstrates
that fenugreek galactomannan exhibits little antioxidant activity and little effect on peripheral
glucose uptake. Further scope is there to study galactomannan's different antidiabetic
mechanism(s) of actions.

KEYWORDS: fenugreek galactomannan, glucose uptake, glucose utilization, antioxidant

Author Notes: Author Sirajudheen Anwar Research Fellow, is grateful to AICTE, New Delhi,
India and Hyderabad(Sind) National Collegiate Board for this work.
 Anwar et al.: Fenugreek Galactomannan - Antidiabetic Activity

Introduction

Diabetes is possibly the world’s fastest growing metabolic disease and as

knowledge of the heterogeneity of this disorder increases, there is need for more
appropriate therapies (Baily and Flatt 1986). Traditional plant medicines are used
throughout the world for a range of diabetic presentations. The study of such
medicines might offer a natural key to unlock a diabetologist’s pharmacy for the
future. Combination of conventional drugs along with herbal drug is the key to
control hyperglycemia and to avoid further complications. Several reports are
available on the hypoglycemic effects of the seeds of fenugreek
(Trigonella foenum-graecum) extract (Shani and Golschmied 1974; Clifford and
Caroline 1989; Ajabnoor and Tilmisamy 1988). Various constituents of
fenugreek (Trigonella foenum-graecum) seed are responsible for hypoglycemic
activity one among them being fenugreek galactomannan. Fenugreek
galactomannan has unique property of increasing viscosity when dissolved in
water; it swells instantly in the presence of moisture as soon as it reaches the
stomach. As a result, the absorption of glucose is delayed, which leads to decrease
in blood sugar level. For combating diabetes, antidiabetic drugs reduce
hyperglycemia and its complication by different modes of action, two among
them being improved peripheral utilization of glucose and antioxidant activity-
which scavenges free radicals formed due to hyperglycemia (Ashok K T 2004;
Melander et al 1996).
As fenugreek galactomannan lowers blood glucose levels significantly, it
was essential to correlate its activity with other probable modes of action. Hence
in our laboratory an attempt was made to explore fenugreek galactomannan’s
possible modes of action. For this purpose, in addition to its glucose lowering
activity, its effect on glucose utilization by hemidiaphragm and its antioxidant
activity was studied.

Material and methods

Fenugreek seeds. Fenugreek seeds purchased from local market Colaba Mumbai
was identified and taxonomically authenticated at Department of Pharmacognosy,
Prin.K.M.Kundanani College of Pharmacy Mumbai.

Isolation and characterization of fenugreek galactomannan. The seeds were

subjected to dry, milling in a mixer and the seed coats were separated from
endosperm of the seeds. The endosperm of seeds were defatted with petroleum
ether and soaked with distilled water and shaken frequently for 4-5 hrs. The
viscous solution obtained was passed through the muslin.

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 Journal of Complementary and Integrative Medicine, Vol. 6 [2009], Iss. 1, Art. 6

The mucilage was precipitated out by addition of 95% ethanol in the ratio
1:1 by continuous stirring. The coagulated mucilage, which formed as a white
mass was transferred to an evaporating dish and treated successively with ethanol.
The coagulated mass was dried in oven at 40 – 45º C powdered and stored in
airtight container. The percent yield obtained was 48 %. (Mulimani and Prashant
2002).
Isolated fenugreek galactomannan was subjected to phytochemical
analysis. Various test for carbohydrate [Molish test and Fehling’s test] and protein
[Biuret test] identification was carried out (Smith U 1959). Specific test for
fenugreek galactomannan was carried out (Khandelwal 2001). Fenugreek
galactomannan was characterized by chemical tests and standardized by paper
chromatography (Mukherjee and Srivastava 1952). Sample is stored for reference.

Animals. Albino mice of Haffkine strain (weighing 15-25g) and albino rats of
Wistar strain (weighing 100-160 g) of either sex, obtained from Bharat Serum and
Vaccines, Thane, India were housed under standard conditions of temperature
(24+1 0C), relative humidity (65+10 %), 10-h light and 14-h dark cycle and fed
with standard pellet diet (Chakan Mill Ltd, Pune, India) and water ad libitum.
Experimental protocols were approved by Institutional Ethics Committee.

Acute toxicity studies. The homogenous suspension of fenugreek galactomannan

was prepared freshly, using 0.5% (w/v) Na-Carboxyl Methyl Cellulose (CMC)
using a mortar and pestle.
The different groups of mice were administered various doses (1-8 g/kg
p.o.) of fenugreek galactomannan suspension. The mice were then critically
observed for clinical symptoms, behavioral changes and mortality up to 72 h
period (Luster et al 1982; Muralidhara etal 1999).

Normal fasted rats. Overnight fasted normal albino rats were randomly divided
into five group of six animals each and treated orally as follows (Skim et al 1999;
Sabu and Kutan 2002)
Group 1. Control: given only vehicle i.e. 0.5%w/v (1 ml/kg)
Group 2. Treated with fenugreek galactomannan (150 mg/kg)
Group 3. Treated with fenugreek galactomannan (300 mg/kg)
Group 4. Treated with fenugreek galactomannan (600 mg/kg)
Group 5. Treated with fenugreek glibenclamide (4 mg/kg)
Glibenclamide (4 mg/kg) was used as standard antidiabetic drug
(Fernandes N P, et al. 2007; Adebajo A C, et al. 2007). Blood samples were
collected from tail vein prior and 1, 2, 4 and 6 h after treatment. Fasting blood
glucose (FBG) was determined by the glucose oxidase method using Hypogaurd’s
advanced Microdraw test strips.

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 Anwar et al.: Fenugreek Galactomannan - Antidiabetic Activity

Glucose loaded normal rats. Vehicle, fenugreek galactomannan and

glibenclamide were orally administered to groups of six rats each (as mentioned
above). Subsequently (30 min later) all the animals were loaded with glucose
solution (1 g/kg) orally. Blood samples were taken from tail vein just before drug
administration and at 0.5, 1 and 2h (after glucose loading) for the determination of
glucose (Williams et al 1996; Rastogi A.K. 1997).

Induction of experimental diabetes. In overnight fasted albino rats, diabetes was

induced by a single intraperitoneal injection of alloxan monohydrate in ice cold
saline at a dose of 150 mg/kg body weight (Antia et al 2005). The diabetic state
was confirmed 72 h after alloxan injection by measuring FBG. Rats with FBG
above 250 mg/dl were considered to be diabetic and were used in this study
(Chattopadhyay et al 1997; Vogel 2002).

Sub-chronic treatment in experimentally induced diabetic rats. The diabetic rats

were divided into five groups of six animals each and treated orally as follows:
Group 1. Normal control: normal rats given only vehicle i.e. 0.5%w/v (1
ml/kg)
Group 2. Diabetic control: diabetic rats given only vehicle i.e. 0.5%w/v (1
ml/kg)
Group 3. Diabetic rats treated with fenugreek galactomannan (300 mg/kg)
Group 4. Diabetic rats treated with fenugreek galactomannan (600 mg/kg)
Group 5. Diabetic rats treated with glibenclamide (4 mg/kg)
All the animals were continued with the same dose of vehicle, fenugreek
galactomannan and glibenclamide once daily for 15 days. After 15 days
treatment, overnight fasted rats were sacrificed and diaphragm and pancreas were
dissected out and washed with ice cold saline immediately.

Study of glucose uptake by hemidiaphragm (in vitro).Isolated diaphragm was

divided into approximately 2 equal halves. The hemidiaphragms were rinsed with
cold Tyrode solution, to remove blood clots. The hemidiaphragms were placed in
2 small tubes containing 2 ml of Tyrode solution with 2% glucose and incubated
for 30 min at 37 ± 0.2 0C with appropriate aeration to enable stirring and also to
provide oxygen. Following 30 min of incubation, the hemidiaphragms were taken
out, dried at 600C till constant weight was obtained. The glucose content of the
incubated medium was measured (Trinder 1969). Glucose uptake by the
hemidiaphragm was calculated as the difference between the initial and final
glucose content in the incubation medium (Chattopadhyay et al 1992].

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 Journal of Complementary and Integrative Medicine, Vol. 6 [2009], Iss. 1, Art. 6

Estimation of antioxidant activity. Isolated pancreatic tissue was homogenized

and the extract was used for the estimation of enzymatic antioxidants [catalase,
CAT (Claiborne 1991) and glutathione peroxidase, GPx (Rotruck et al 1973;
Wendel 1985; Mannervik 1985)] activities including lipid peroxidation (Ohkawa
et al 1979) process to see the effect of 15 days treatment with fenugreek
galactomannan.

Statistical analysis. All the values are expressed as means ± S.E.M. The results
were analyzed statistically by Analysis of Variance (ANOVA) followed by
Dunnett’s test. P values <0.05 were considered significant.

Results and discussion

In the preliminary acute toxicity study, fenugreek galactomannan seems to be safe

up to 8 g/kg because even at this high dose no toxic or deleterious effects were
observed immediately or during 3 days of observation period.
In the present experiment, fenugreek galactomannan like glibenclamide,
showed significant (P<0.05 and P<0.01) hypoglycemic effect at 150, 300 and 600
mg/kg in dose related manner both in normal (Table 1) as well as glucose loaded
rats (Table 2). The results were more pronounced in glucose loaded rats at all the
three doses tried i.e. 150 (25 %), 300 (28.20 %) and 600 mg/kg (43.78 %) which
were comparable to glibenclamide - dose 4mg/kg (50 %), the standard
antidiabetic drug.
Table 1
Effect of fenugreek galactomannan of blood glucose level in normal fasted rats

Treatment Blood glucose levels (mg/dL)

and Time Interval
Dose 0(h) 1(h) 2(h) 4(h) 6(h)

Normal (CMC 1ml/kg) Group I 61.33 ± 1.17 59.66 ± 0.71 58 ± 1.21 54.66 ± 1.11 58.5 ± 1.08
GL (150 mg/kg) Group II 61.66 ± 1.45 60.5 ± 1.38 55.16 ± 1.66 50.33 ± 1.74 51.83 ± 1.40 *
GL (300 mg/kg) Group III 59.33 ± 2.17 58.66 ± 2.34 50.33 ± 1.23** 44.83 ± 2.51** 48.66 ± 2.55**
GL (600 mg/kg) Group IV 59.33 ± 1.12 57.66 ± 1.15 49.16 ± 1.20** 42.83 ± 1.28 ** 49.0 ± 1.155**
Glibenclamide(4mg/kg) Group V 60.88 ± 1.42 56.5 ± 1.18 48.83 ± 1.20 ** 42.5 ± 1.06 ** 46.83 ± 0.98**

GL –Fenugreek galactomannan
N = 6 in each group. Values are mean ± S.E.M.
* P< 0.05 compared to normal group (ANOVA followed by Dunnett’s);
** P< 0.01 compared to normal group (ANOVA followed by Dunnett’s).

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 Anwar et al.: Fenugreek Galactomannan - Antidiabetic Activity

Table 2
Effect of fenugreek galactomannan on blood glucose level in normal glucose (1 g/kg) loaded rats

Treatment and dose Blood glucose level (mg/dL)

Time Interval
-0.5 (h) + 0.5 (h) +1 (h) +2(h)

Normal (CMC 1ml/kg+Glucose) Group I 62.33 ± 0.95 85.5 ± 1.50 80.66 ± 1.94 75 ± 1.61
GL (150 mg/kg+Glucose) Group II 62.16 ± 1.30 71.83 ± 1.11** 67.66 ± 1.20** 60 ± 1.27**
GL (300 mg/kg+Glucose) Group III 63.33 ± 1.52 69.33 ± 0.49** 66.16 ± 2.27** 58.5 ± 1.84**
GL (600 mg/kg+Glucose) Group IV 62 ± 1.29 62.66 ± 1.15** 60.33 ± 1.26** 52.16 ± 0.95 **
Glibenclamide (4mg/kg+Glucose) Group V 61.83 ± 1.82 60 ± 1.48** 52.33 ± 1.09** 50 ± 1.13 **
% effect was calculated by % decrease in BGL at +2(h)
GL –Fenugreek galactomannan
N = 6 in each group. Values are mean ± S.E.M.
** P< 0.01 compared to normal group (ANOVA followed by Dunnett’s).

It is well known that insulin and antidiabetic drugs promotes glucose

uptake by peripheral cells and tissues (Melander et al 1996). So it is one of the
possible modes of action for lowering blood glucose level. The hemidiaphragms
taken from rats treated with the fenugreek galactomannan and glibenclamide
showed a significant (P<0.01) enhancement in the glucose uptake process as
compared to diabetic untreated rats (26.31 %) (Table 3). But glucose uptake in
fenugreek galactomannan (GL 300 – 38.49 % and GL 600 – 51.86 %) treated
hemidiaphragms was much less than that of glibenclamide (82.48 %).
Table 3
Effect of fenugreek galactomannan on glucose uptake by hemidiaphragm in Alloxan
(150 mg/kgx1 i.p.) induced diabetic rats

Treatment and dose Glucose uptake (mg/g) % effect #

Normal (CMC 1ml/kg); Group I 11.825 ± 0.66 ---

a
Normal (CMC 1ml/kg) + Alloxan; Group II 3.11 ± 0.41 26.31 %
GL (300 mg/kg) + Alloxan; Group III 4.55 ± 0.18 * 38.49 %
GL (600 mg/kg) + Alloxan; Group IV 6.13 ± 0.13 * 51.86 %
Glibenclamide (4mg/kg) + Alloxan; Group V 9.75 ± 0.32 * 82.48 %
# - % glucose uptake by hemidiaphragm is calculated
N = 6 in each group. Values are mean ± S.E.M.
a
P< 0.01 compared to normal group (ANOVA followed by Dunnett’s);
* P< 0.01 compared to diabetic group (ANOVA followed by Dunnett’s)

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 Journal of Complementary and Integrative Medicine, Vol. 6 [2009], Iss. 1, Art. 6

Diabetics and experimental animal models inhibit high oxidative stress

due to persistent and chronic hyperglycemia, which depletes the activity of the
anti oxidative defense system and thus promotes de novo generations of free
radicals (Albers and Beal 2000). The activity of antioxidant enzymes such as
superoxide dismutase, catalase, and glutathione peroxidase, which is low in islet
cells when compared to other tissues, becomes further worsened under diabetic
conditions (Kawamura M and Heinecke JW 1994). Further, the presence of higher
glucose or glycated protein concentration enhances lipid peroxidation (Hicks M
and Delbridge L 1989) and furthermore lipid peroxides may increase the extent of
advanced glycation end products (Tiedge M and Lortz S 1997).
Lipid peroxidation may bring about protein damage and inactivation of
membrane bound enzymes either through direct attack by free radicals or through
chemical modification by its end products, MDA and 4-hydroxy nonenal
(Halliwell and Gutteridge 1999). Increased lipid peroxidation under diabetic
conditions may be attributed to increased oxidative stress in the cells as a result of
the depletion of antioxidant systems. Diminished levels of both enzymatic
antioxidants (CAT and GPx) and increased levels of MDA were observed in
diabetic rats. It is observed that in fenugreek galactomannan treated diabetic rats;
there was slight decrease in lipid peroxidation (GL 300 – 9.3 %; GL 600 –
18.09%; Glibenclamide – 43.67 %) and increase in the levels of antioxidant
enzymes (CAT : GL 300 -70 %; GL 600 – 76.3 % ; Glibenclamide – 88.2 % and
GPx : GL 300 – 79 %; GL 600 – 83.4 %; Glibenclamide – 94 %) (Table 4).
Antioxidant activity of fenugreek galactomannan was significant as compared to
diabetic untreated group but it was much less when compared with glibenclamide
treated group. This shows that fenugreek galactomannan does not possess very
effective antioxidant activity when compared against standard drug.

Table 4a
Antioxidant effects of fenugreek galactomannan (Catalase activity)

Treatment and dose CAT (U/mg Prot) % Effect #

Normal (CMC 1ml/kg); Group I 3.23 ± 0.09 ---
Normal (CMC 1ml/kg) + Alloxan; Group II 1.981 ± 0.08 a 61 %
GL (300 mg/kg) + Alloxan; Group III 2.261 ± 0.09* 70 %
GL (600 mg/kg) + Alloxan; Group IV 2.465 ± 0.05 ** 76.3 %
Glibenclamide (4mg/kg) + Alloxan; Group V 2.851 ± 0.07** 88.2 %
# - % increase in CAT is calculated; GL- Fenugreek galactomannan, CAT- Catalase
N = 6 in each group. Values are mean ± S.E.M.
One way ANOVA is applied for statistical analysis.
a
P< 0.01 compared to normal group; * P < 0.05 compared to diabetic group;
** P < 0.01 compared to diabetic group.

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 Anwar et al.: Fenugreek Galactomannan - Antidiabetic Activity

Table 4b
Antioxidant effects of fenugreek galactomannan (Glutathione peroxidase activity)

Treatment and dose GPx (U/mg Prot) % Effect #

Normal (CMC 1ml/kg); Group I 2.68 ± 0.13 ---
Normal (CMC 1ml/kg) + Alloxan; Group II 1.721 ±0.08 a 64.2 %
GL (300 mg/kg) + Alloxan; Group III 2.121 ± 0.16 ** 79 %
GL (600 mg/kg) + Alloxan; Group IV 2.235 ± 0.06 ** 83.4 %
Glibenclamide (4mg/kg) + Alloxan; Group V 2.53 ± 0.022** 94 %

# - % increase in GPx is calculated

GL- Fenugreek galactomannan, GPx- Glutathione peroxidase
N = 6 in each group. Values are mean ± S.E.M.
One way ANOVA is applied for statistical analysis.
a
P< 0.01 compared to normal group; * P < 0.05 compared to diabetic group; ** P < 0.01 compared to
diabetic group.

Table 4c
Antioxidant effects of fenugreek galactomannan (Lipid peroxidation)

Treatment and dose % LP % Effect #

Normal (CMC 1ml/kg); Group I ----------
Normal (CMC 1ml/kg) + Alloxan; Group II 100 0%
GL (300 mg/kg) + Alloxan; Group III 91.44 ± 1.27 * 9.3 %
GL (600 mg/kg) + Alloxan; Group IV 84.68 ± 1.66 ** 18.09 %
Glibenclamide (4mg/kg) + Alloxan; Group V 69.60 ± 3.31 ** 43.67 %

# - % decrease in Lipid peroxidation is calculated

GL- Fenugreek galactomannan, LP- Lipid peroxidation
N = 6 in each group. Values are mean ± S.E.M.
One way ANOVA is applied for statistical analysis.
a
P< 0.01 compared to normal group; * P < 0.05 compared to diabetic group;
** P < 0.01 compared to diabetic group.

Conclusion

On the basis of results obtained, it can be concluded that fenugreek

galactomannan is a potential blood glucose reducing agent; but its antidiabetic
activity is not fully supported by different mechanisms like peripheral glucose
uptake and antioxidant effect. Hence our study demands to investigate different
pancreatic and extra-pancreatic mechanism(s) of action of fenugreek
galactomannan and also its long term toxicity studies in different animal species.

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