866 Asian Pac J Trop Biomed 2013; 3(11): 866-870

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Asian Pacific Journal of Tropical Biomedicine

journal homepage: www.elsevier.com/locate/apjtb

Document heading doi: 10.1016/S2221-1691(13)60170-7 襃 2013 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved.

In vitro hypoglycemic effects of Albizzia lebbeck and Mucuna pruriens

1 2
Mangesh Bhutkar *, Satish Bhise
Govt. College of Pharmacy, Karad (M.S), India
1

Sinhgad Institute of Pharmaceutical Sciences, Lonavala (M.S), India

2

PEER REVIEW ABSTRACT

Peer reviewer Objective: To verify the antidiabetic potential of stem bark of Albizzia lebbeck (A. lebbeck) and
Viroj Wiwanitkit seeds of Mucuna pruriens (M. pruriens) using various in vitro techniques.
Visiting Professor, Faculty of Medicine, Methods: The plant extracts were studied for their effects on glucose adsorption, diffusion
U niversity of N is, S erbia; V isiting amylolysis kinetics and glucose transport across yeast cells.
professor, Hainan Medical University, Results: Both the plant extracts adsorbed glucose and the adsorption of glucose increased
C hina; adjunct professor, J oseph remarkably with an increase in glucose concentration. No significant (P≤0.05) differences were
Ayobabalola University, Nigeria. observed between the adsorption capacities of A. lebbeck and M. pruriens. In amylolysis kinetic
experimental model the rate of glucose diffusion was found to increase with time from 30 to 180
Comments min, and both the plant extracts demonstrated significant inhibitory effects on movement of
This is a valuable research work in glucose into external solution across dialysis membrane as compared to control. The retardation
which authors have demonstrated of glucose diffusion by A. lebbeck extract was significantly higher (P≤0.05) than M. pruriens. These
the hypoglycemic activity of A. effects were reflected with higher glucose dialysis retardation index values for A. lebbeck than
lebbeck and M. pruriens using various M. pruriens. The plant extracts also promoted glucose uptake by yeast cells. The rate of uptake
in vitro methods. T he activity was of glucose into yeast cells was linear in all the 5 glucose concentrations used in the study. M.
assessed by studying the effects of pruriens extract exhibited significantly higher (P≤0.05) activity than the extract of A. lebbeck at all
plant extracts on glucose adsorption, concentrations.
diffusion amylolysis kinetics and Conclusions: The results verified the antidiabetic potential of A. lebbeck and M. pruriens. The
glucose transport across yeast cells. hypoglycemic effect exhibited by the extracts is mediated by increasing glucose adsorption,
It was found that the hypoglycemic decreasing glucose diffusion rate and at the cellular level by promoting glucose transport across
effect exhibited by the plant extracts the cell membrane as revealed by simple in vitro model of yeast cells.
is mediated by increasing glucose
adsorption, decreasing glucose
diffusion rate and at the cellular level
by promoting glucose transport across
the cell membrane as revealed by
simple in vitro model of yeast cells. KEYWORDS
Details on Page 870 Hypoglycemic, In vitro, Glucose diffusion

1. Introduction leading to various complications[2]. Currently available

pharmacotherapies for the treatment of diabetes mellitus
Diabetes mellitus is a common and very prevalent disease include oral hypoglycemic agents and insulin. However
affecting the citizens of both developed and developing these current drugs do not restore normal glucose
countries. It is estimated that 25% of the world population homeostasis and they are not free from side effects[3]. In
is affected by this disease[1]. It is characterized by group view of the adverse effects associated with the synthetic
of metabolic disorders. The deficiency or insensitivity drugs and as plants are safer, cheaper and much effective,
of insulin causes glucose to accumulate in the blood, conventional antidiabetic plants can be explored [4] .

*Corresponding author: Mangesh Bhutkar, Govt. College of Pharmacy, Karad. (M.S), Article history:
India. Received 18 Aug 2013
Tel: +91 94 2326 3907 Received in revised form 24 Aug, 2nd revised form 30 Aug, 3rd revised form 5 Sep 2013
E-mail: [email protected] Accepted 18 Oct 2013
Available online 28 Nov 2013
 Mangesh Bhutkar and Satish Bhise /Asian Pac J Trop Biomed 2013; 3(11): 866-870
867

Albizzia lebbeck Benth (Family: Leguminosae) (A. lebbeck) is determined by the method of Ou et al[8]. Briefly, the samples
a deciduous tree with compound leaves, flat oblong fruits, of plant extracts (1%) were added to 25 mL of glucose solution
round cream colored seeds, and grows wild. Barks are used of increasing concentration (5, 10, 20, 50 and 100 mmol/L).
in toothache and diseases of the gum. Decoction of the The mixture was stirred well, incubated in a shaker water
leaves and barks are protective against bronchial asthma bath at 37 °C for 6 h, centrifuged at 4 800 r/min for 20 min and
and other allergic disorders. Barks and seeds are astringent, the glucose content in the supernatant was determined. The
and are given in piles and diarrhea. Ethanolic extract of concentration of bound glucose was calculated using the
pods possesses antiprotozoal, hypoglycemic and anticancer following formula:
properties[5]. Mucuna pruriens Linn. (Family: Fabaceae) (M. G1- G6
Glucose bound= ×Volume of solution
pruriens) is one of the popular drug in Ayurvedic system Weight of the sample
of medicine[6]. Various preparations from the seeds of this
plant are used for the management of several free radical G1 is the glucose concentration of original solution.
mediated diseases such as ageing, rheumatoid arthritis, G6 is the glucose concentration after 6 h.
diabetes, atherosclerosis, male infertility and nervous
disorders[7]. The present study was undertaken to verify the 2.4.2. Effect of plant extracts on in-vitro glucose diffusion
antidiabetic potential of seeds of M. pruriens and bark of A. I t was performed according to the method stated by
lebbeck using various in vitro techniques as an attempt to Ahmed et al[9]. A total of 25 mL of glucose solution (20 mmol/
explore their mechanism of action. L) and the samples of plant extracts (1%) were dialyzed in
dialysis bags against 200 mL of distilled water at 37 °C in a
shaker water bath. The glucose content in the dialysate was
2. Materials and methods determined at 30, 60, 120 and 180 min using glucose oxidase
peroxidase diagnostic kit. A control test was carried out
2.1. Plant material without sample. Glucose dialysis retardation index (GDRI)
was calculated by using the following formula:
The plant material was collected from local areas of Glucose content with addition of sample (mg/dL)
GDRI%=100-
Karad and was further identified and authenticated by the Glucose content of the control (mg/dL)
×100
Department of Botany, Science College, Karad. The bark of
A. lebbeck and the seeds of M. pruriens were cleaned, dried 2.4.3. Effect of plant extracts on in-vitro amylolysis kinetics
in a hot air oven (50 °C), powdered, passed through 60 mesh A total of 40 g of potato starch was added to about 900 mL
sieve (BS) and stored in an airtight container at 4 °C till of 0.05 mol/L phosphate buffer (pH 6.5). The solution after
further use. stirring at 65 °C for 30 min was made up to a final volume
of 1 000 mL to give a 4% (w/v) starch solution. And 25 mL of
2.2. Chemicals the above starch solution, α-amylase (0.4%), and the plant
extracts (1%) were dialyzed in a dialysis bags against 200 mL
Glucose oxidase peroxidase kit was procured from Pathozyme of distilled water at 37 °C (pH 7.0) in a shaker water bath.
Diagnostics, Kagal, India. Dialysis bags (12 000 MW cutoff; The glucose content in the dialysate was determined at 30,
Himedia laboratories, India) were used. All the chemicals used 60, 120 and 180 min. A control test was carried out without
in the study were of extra pure analytical grade. sample[8].

2.3. Preparation of plant extracts 2.4.4. Glucose uptake by yeast cells

Yeast cells were prepared according to the method of
Aqueous extracts were prepared by extracting the powders of Cirillo[10]. Commercial baker’s yeast was washed by repeated
bark of A. lebbeck and the seeds of M. pruriens with hot water centrifugation (4 200 r/min, 5 min) in distilled water until the
(70 °C) in a mechanical shaker (24 h), filtered and freeze dried. supernatant fluids were clear and a 10% (v/v) suspension
was prepared in distilled water. Various concentrations of
2.4. Evaluation of antidiabetic activity of plant extracts using extracts (1-5 mg) were added to 1 mL of glucose solution
various in vitro methods (5-25 mmol/L) and incubated together for 10 min at 37 °C. The
reaction was started by adding 100 µL of yeast suspension,
2.4.1. Determination of glucose adsorption capacity vortexed and further incubated at 37 °C for 60 min. After
G lucose adsorption capacity of the samples was 60 min, the tubes were centrifuged (3 800 r/min, 5 min) and
868 Mangesh Bhutkar and Satish Bhise /Asian Pac J Trop Biomed 2013; 3(11): 866-870

glucose was estimated in the supernatant. The percent 3.2. Effect of A. lebbeck and M. pruriens extracts on in vitro
increase in glucose uptake by yeast cells was calculated glucose diffusion
using the following formula:
The effect of the plant extracts on retarding glucose
control–Abssample
Abs

Increase in glucose uptake (%)= ×100 diffusion across the dialysis membrane is shown in Table 1.
Abs
control
The rate of glucose diffusion was found to increase with time
W here, A bs control is the absorbance of the control from 30 to 180 min. In the present study, the movement of
reaction (containing all reagents except the test sample), and glucose across the dialysis membrane was monitored once in
Abs sample is the absorbance of the test sample. 30 min till 180 min and it was found that, both the samples of
plant extracts demonstrated significant inhibitory effects on
2.4.5. Statistical analysis movement of glucose into external solution across dialysis
All the determinations were carried out in triplicates and membrane compared to control. The retardation of glucose
data were analyzed by ANOVA followed by Tukey’s multiple diffusion by A. lebbeck extracts was significantly higher
comparisons test for significant differences. Values were (P≤0.05) than M. pruriens. These effects were reflected with
considered at P<0.05. Graphs were plotted using Graph Pad higher GDRI values for A. lebbeck than M. pruriens.
Prism 6 software.
Table 1
Effect of selected samples on glucose diffusion and GDRI.
Glucose content in dialysate (mmol/L)
Sample
3. Results 30 min 60 min 120 min 180 min
Control 0.90 依0.01 1.27 依0.01 1.77 依0.01 1.95 依0.01
c c c c

0.61 依0.01 (32.22) 1.06 依0.01 (18.11) 1.55 依0.01 (12.99) 1.70 依0.01 (12.82)
a a a a
A. lebbeck
3.1. Glucose adsorption capacity of A. lebbeck and M. M. pruriens 0.65
b
依0.01 (27.77) 1.08
b
依0.01 (14.96) 1.59
b
依0.01 (10.16) 1.79
b
依0.01 (8.20)

pruriens extracts Values in parenthesis indicate GDRI.

M ean values ( n= 3 ) with different superscript letters in columns differ
significantly from each other (P≤0.05)
Glucose adsorption capacity of the selected plant extracts
is depicted in Figure 1. The adsorption capacities of the 3.3. Effect of A. lebbeck and M. pruriens extracts on in vitro
samples were found to be directly proportional to the molar amylolysis kinetics
concentration of glucose and higher amounts of glucose was
bound with increased glucose concentration. No significant The effects of A. lebbeck and M. pruriens on the amylolysis
(P≤0.05) differences were observed between the adsorption kinetics are shown in the Table 2. The GDRI was found to be
capacities of A. lebbeck and M. pruriens. 42.85% and 33.33% in A. lebbeck and M. pruriens respectively
at 60 min which gradually got reduced to 20.68% and 13.79%
80 respectively at 120 min.
Table 2
Effect of selected samples on starch digestibility and GDRI.
60 Glucose content in dialysate (mmol/L)
Glucose adsorption (mmol/L)

Sample
30 min 60 min 120 min 180 min
Control 0.0 0.21 +0.01 0.29 +0.01 0.37 +0.01
c c c

A. lebbeck 0.0 (100) 0.12a+0.01 (42.85) 0.23a+0.01 (20.68) 0.33a+0.01 (10.81)

40 M. pruriens 0.0 (100) 0.14b+0.01 (33.33) 0.25b+0.01 (13.79) 0.35b+0.01 (5.40)
Values in parenthesis indicate GDRI.
Mean values (n=3) with different superscript letters in columns differ
significantly from each other (P≤0.05).
20 3.4. Effect of A. lebbeck and M. pruriens extracts on glucose
transport across yeast cells

0 The rate of glucose transport across cell membrane in

5 mmol/L 10 mmol/L 50 mmol/L 100 mmol/L yeast cells system is presented in Figure 2 and Figure 3.
Glucose concentration
A. lebbeck M. pruriens T he amount of glucose remaining in the medium after
Figure 1. Glucose binding capacity of A. lebbeck and M. pruriens at different a specific time interval serves as an indicator of the
concentrations of glucose. glucose uptake by the yeast cells. The rate of uptake of
Values are mean+SD of triplicate determinations.
glucose into the yeast cells was linear in all the 5 glucose
 Mangesh Bhutkar and Satish Bhise /Asian Pac J Trop Biomed 2013; 3(11): 866-870
869

concentrations. T he extract of M. pruriens exhibited Similar observations have been reported by Chau et al.
significantly higher (P≤0.05) activity than the A. lebbeck for insoluble fiber-rich fractions isolated from Averrhoa
extract at all concentrations. However, the percent increase carambola[11]. GDRI is a useful in vitro index to predict the
in the glucose uptake by the yeast cells was observed to be effect of a fiber on the delay in glucose absorption in the
inversely proportional to the glucose concentration and was gastrointestinal tract[12]. A higher GDRI indicates a higher
found to decrease with increase in the molar concentration retardation index of glucose by the sample. T he GDRI
of the glucose solution. was found to be 42.85% and 33.33% in A. lebbeck and M.
100
pruriens respectively at 60 min. The retardation of glucose
diffusion may also be due to the inhibition of α-amylase
by the plant extracts thereby limiting the release of glucose
80
from the starch. Ou et al. have mentioned several possible
Enhancement of glucose transport(%)

factors that may be responsible for α-amylase inhibition

60 such as fiber concentration, the presence of inhibitors on
fibers, encapsulation of starch and enzyme by the fibers
40 present in the sample, thereby reducing accessibility of
starch to the enzyme, and direct adsorption of the enzyme
on fibers, leading to decreased amylase activity [8]. The
20
mechanism of glucose transport across the yeast cell
membrane has been receiving attention as a lucrative
0
0 1 2 3 4 5
method for in vitro screening of hypoglycemic effect of
Concentration (mg) of extract of A. lebbeck various compounds/ medicinal plants. Both the extracts
promoted glucose transport across the yeast cells. The rate
5 mmol/L 10 mmol/L 15 mmol/L 20 mmol/L 25 mmol/L

Figure 2. Effect of A. lebbeck extract on the uptake of glucose by yeast cells.

Values are mean±SD of triplicate determinations. of uptake of glucose into the yeast cells was linear in all the
100 5 glucose concentrations used in the study. The studies on
the transport of non metabolizable sugars, metabolizable
80 glycosides have suggested that sugar transport across
Enhancement of glucose transport (%)

the yeast cell membrane is mediated by stereospecific

60 membrane carriers and takes place by facilitated diffusion
process[13,14].
I n conclusion, the results of this study verify the
40
antidiabetic properties as implied by the various in vitro
methods. The hypoglycemic effect exhibited by the extracts
20
of A. lebbeck and M. pruriens is mediated by increasing
glucose adsorption, decreasing glucose diffusion rate and
0 0 1 2 3 4 5 at the cellular level by promoting glucose transport across
Concentration (mg) of extract of M. pruriens
5 mmol/L 10 mmol/L 15 mmol/L 20 mmol/L 25 mmol/L the cell membrane as revealed by simple in vitro model of
Figure 3. Effect of M. pruriens extract on the uptake of glucose by yeast cells. yeast cells. However, these effects need to be confirmed by
Values are mean±SD of triplicate determinations.
employing different in vivo models and clinical trials for
their effective utilization as therapeutic agents.
4. Discussion

T he higher adsorption capacity of the extracts of Conflict of interest statement

A. lebbeck and M. pruriens may be attributed to their
constituents, as both insoluble and soluble constituents and We declare that we have no conflict of interest.
fibers from different sources are reported to adsorb glucose.
The results also revealed that the plant extracts under
study could bind glucose even at lower concentrations Acknowledgements
of glucose ( 5 mmol/ L ) thereby reducing the amount of
glucose available for transport across the intestinal lumen, The authors are thankful to Research and Development
consequently blunting the postprandial hyperglycemia. C ell of G ovt. C ollege of P harmacy, K arad for financial
870 Mangesh Bhutkar and Satish Bhise /Asian Pac J Trop Biomed 2013; 3(11): 866-870

support and other necessary facilities for successful glucose diffusion rate and at the cellular level by promoting
completion of work. glucose transport across the cell membrane as revealed by
simple in vitro model of yeast cells.

Comments
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