INTRODUCTION
Diabetes mellitus is responsible for about 5% of global deaths (WHO,
2005). The underlying symptom of this condition is chronic hyperglycemia
which eventually culminates into abnormal fat and protein metabolism due to
defects in insulin secretion or action. Type 1 diabetes mellitus result from
failure of pancreatic β-cells to produce insulin while the type 2 diabetes
mellitus (which is more common) is caused by a decreased sensitivity of target
cells to insulin (Burcelin et al., 2010).
The available conventional therapies for diabetes include stimulation of endogenous
insulin secretion, enhancement of the action of insulin at the target tissues,
administration of oral hypoglycemic agents such as biguanides, thiazolidinediones,
sulfonylureas and alpha-glucosidase inhibitors (Zimmet,
2009). One of the methods employed to treat diabetes mellitus is the inhibition
of carbohydrate-digesting enzymes such as α-amylase and α-glucosidase
in the gastro-intestinal tract, thereby slowing down intestinal glucose absorption
and decreasing postprandial blood glucose levels (Rhabasa-Lhoret
and Chiasson, 2004).
Since, time immemorial, medicinal plants have been employed in the management
of many diseases such as malaria, inflammations and diabetes mellitus (Babu
et al., 2006). These plants include Abrus precatorius, Ageratum
conyzoides, Allium sativum, Alstonia boonei, Bridelia micrantha and Ficus
exasperata (Gbolade, 2009). Ficus exasperata
is a plant found in the tropics and sub-tropics region and it has been found
to improve glucose in tolerance as assessed by glucose index (Taiwo
et al., 2010). It possesses anti-oxidant, anti-convulsant, anti-arthritic,
anti-bacterial, anti-inflammatory, anti-pyretic, antinociceptic, anti-candidal,
anti-diabetic and hypotensive properties (Woode et al.,
2009). Its leaves has also been reported to have anti-diabetic, lipid-lowering
and anti-fungal potentials (Sonibare et al., 2006).
Though, several studies have reported the antidiabetic potential of Ficus
exasperata (Harati et al., 2003; Taiwo
et al., 2010; Adewole et al., 2011)
but none has been able to clarify the mechanism by which this plant elicits
its hypoglycemic potential. Therefore, the aim of this study was to investigate
the α-amylase and α-glucosidase inhibitory potentials of Ficus
exasperata, as a possible mechanism behind the hypoglycemic action of the
plant and its usage in the management of diabetes mellitus.
MATERIALS AND METHODS
Plant material: The leaf of Ficus exasperata was obtained from
Badagry Area of Lagos in Nigeria in May 2012. It was identified and authenticated
by Dr. A. B. Kadiri of the Department of Botany, University of Lagos, Nigeria
and voucher specimen (LUH 4720) was deposited in the University herbarium.
Chemicals and reagents: Alpha-amylase from Aspergillus oryzae, α-glucosidase from Saccharomyces cerevisiae and paranitrophenyl-glucopyranoside were products of Sigma-Adrich Co., St Louis, USA while starch soluble (extra pure) was obtained from J.T. Baker Inc., Phillipsburg, USA. Other chemicals and reagents were of analytical grade and water used was glass-distilled.
Preparation of plant extracts: Fresh leaves of Ficus exasperata were cut and washed with water to remove all contaminants; they were dried under room temperature and grounded to powder. The powdered leaves were divided into three portions and each portion was extracted with acetone, ethanol or water. They were all left to steep in covered containers for 24 h; the resulting infusions were decanted, filtered and evaporated in a rotary evaporator (Cole Parmer SB 1100, Shangai, China). The extracts were freeze-dried using Virtis Bench Top (SP Scientific Series, USA) freeze dryer. Dried extracts were weighed and dissolved in 10% dimethylsulphoxide to yield a stock solution from which lower concentrations were prepared.
Phytochemical screening: Phytochemical compositions of the leaves were
determined using the methods variously described by Trease
and Evans (1996) and Sofowara (2006).
Test for tannins: In the test for tannins, 0.5 g of dried powdered sample was boiled in 20 mL of water in a test tube and filtered. Few drops of 0.1% ferric chloride was added and observed for brownish green or a blue black colouration as indication of tannins.
Test for saponin: Approximately 2 g of powdered material was boiled in 20 mL of distilled water in a water bath and filtered. Next, 10 mL of the filtrate was mixed with 5 mL of distilled water and shaken vigorously and observed for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously again and then observed for the formation of emulsion as indication of saponin.
Test for flavonoids: A portion of the powdered material was heated with 10 mL of ethyl acetate over a steam bath for 3 min. The mixture was filtered and 4 mL of the filtrate was shaken with 1 mL of dilute ammonia solution. Development of yellow colouration is an indication of the presence of flavonoids.
Test for steroids: In this test, 2 mL of acetic anhydride was added to 0.5 g of extract with 2 mL concentrated H2SO4. The colour change from violet to blue or green is indication of steroids.
Test for terpenoids: In brief, 5 mL of extract was mixed with 2 mL chloroform and 3 mL H2SO4 was carefully added to form a layer. A reddish brown colouration of the interface was indication of terpenoids.
Test for anthraquinones: Briefly, 5 mL of chloroform was added to 0.5 g of the powdered plant materials of each specimen. The resulting mixture was shaken for 5 min after which it was filtered. The filtrate was then shaken with equal volume of 10% ammonia solution. The presence of a bright pink colour in the aqueous layer indicated the presence of anthraquinones.
Test for reducing sugar: To about 1 g of each sample in the test tube was added 10 mL distilled water and the mixture boiled for 5 min. The mixture was filtered while hot and the cooled filtrate made alkaline to litmus paper with 20% sodium hydroxide solution. The resulting solution was boiled with an equal volume of Benedict qualitative solution on a water bath. The formation of a brick red precipitate depicted the presence of reducing compound.
Alpha-Amylase inhibitory assay: This assay was carried using a modified
procedure of McCue and Shetty (2004). A total of 250
μL of extract was placed in a tube and 250 μL of 0.02 M sodium phosphate
buffer (pH 6.9) containing α-amylase solution was added. This solution
was pre-incubated at 25°C for 10 min, after which 250 μL of 1% starch
solution in 0.02 M sodium phosphate buffer (pH 6.9) was added at timed intervals
and then further incubated at 25°C for 10 min. The reaction was terminated
by adding 500 μL of dinitrosalicylic acid (DNS) reagent. The tubes were
then incubated in boiling water for 5 min and cooled to room temperature. The
reaction mixture was diluted with 5 mL distilled water and the absorbance was
measured at 540 nm using spectrophotometer. A control was prepared using the
same procedure replacing the extract with distilled water. The α-amylase
inhibitory activity was calculated as percentage inhibition:
Concentrations of extracts resulting in 50% inhibition of enzyme activity (IC50) were determined graphically.
Mode of α-amylase inhibition: The mode of inhibition of the leaf
extract was conducted using the extract with the lowest IC50 according
to the modified method described by Ali et al. (2006)
Briefly, 250 μL of the (5 mg mL-1) extract was pre-incubated
with 250 μL of α-amylase solution for 10 mins at 25°C in one set
of tubes. In another set of tubes α-amylase was pre-incubated with 250
μL of phosphate buffer (pH 6.9). Also, 250 μL of starch solution at
increasing concentrations (0.30-5.0 mg mL-1) was added to both sets
of reaction mixtures to start the reaction. The mixture was then incubated for
10 min at 25°C and then boiled for 5 min after addition of 500 μL of
DNS to stop the reaction. The amount of reducing sugars released was determined
spectrophotometrically using a maltose standard curve and converted to reaction
velocities. A double reciprocal plot (1/v versus 1/[S]) where v is reaction
velocity and [S] is substrate concentration was plotted. The type (mode)
of inhibition of the crude extract on α-amylase activity was determined
by analysis of the double reciprocal (Lineweaver-Burk) plot using Michaelis-Menten
kinetics.
Alpha-Glucosidase inhibitory assay: The effect of the plant extracts
on α-glucosidase activity was determined according to the method described
by Kim et al. (2005), using α-glucosidase
from Saccharomyces cerevisiae. The substrate solution p-nitropheynyl
glucopyranoside (pNPG) was prepared in 20 mM phosphate buffer, pH 6.9. 100 μL
of α-glucosidase (E.C. 3.2.1.20) was pre-incubated with 50 μL of the
different concentrations of the extracts (acetone, ethanol and water) for 10
mins. Then 50 μL of 3.0 mM (pNPG) as a substrate dissolved in 20 mM phosphate
buffer (pH 6.9) was then added to start the reaction. The reaction mixture was
incubated at 37°C for 20 min and stopped by adding 2 mL of 0.1 M Na2CO3.
The α-glucosidase activity was determined by measuring the yellow colored
para-nitrophenol released from pNPG at 405 nm. The results were expressed as
percentage of the blank control.
Percentage inhibition calculated as:
Concentrations of extracts resulting in 50% inhibition of enzyme activity (IC50) were determined graphically.
Mode of α-glucosidase inhibition: The mode of inhibition of the
extracts was determined using the extract with the lowest IC50 according
to the modified method described by Ali et al. (2006).
Briefly, 50 μL of the (5 mg mL-1) extract was pre-incubated
with 100 μL of α-glucosidase solution for 10 min at 25 °C in one
set of tubes. In another set of tubes α-glucosidase was pre-incubated with
50 μL of phosphate buffer (pH 6.9). 50 μL of PNPG at increasing concentrations
(0.63-2.0 mg mL) was added to both sets of reaction mixtures to start the reaction.
The mixture was then incubated for 10 min at 25°C, and 500 μL of Na2CO3
was added to stop the reaction. The amount of reducing sugars released was determined
spectrophotometrically using a paranitrophenol standard curve and converted
to reaction velocities. A double reciprocal plot (1/v versus 1/[S]) where v
is reaction velocity and [S] is substrate concentration was plotted. The type
(mode) of inhibition of the crude extract on α-glucosidase activity was
determined by analysis of the double reciprocal (Lineweaver-Burk) plot using
Michaelis-Menten kinetics.
Statistical analysis: Statistical analysis was performed using GraphPad Prism 5 statistical package (GraphPad Software, USA). The data were analysed by one way analysis of variance (ANOVA) followed by Bonferroni test. All the results were expressed as mean ± SE for triplicate determinations.
RESULTS
From Table 1 which shows the yield of various leaf extracts, ethanolic extract of Ficus exasperata has the highest yield of 9.47% compared to the other extracts. The phytochemical constituent of each extract of Ficus exasperata was tested for the presence of anthraquinones, flavonoids, reducing sugar, saponins, steroids, tannins, and terpenoids (Table 2). However, it is only reducing sugar was present in all of the three extracts whereas anthraquinone was not detected in any these extracts. Ethanolic extract of Ficus exasperata has more phytochemical constituent when compared with acetone and aqueous extracts of Ficus exasperata. However, saponins were only detected in the aqueous extract of the plant.
Figure 1 shows the percentage inhibition of α-amylase activity by acetone, ethanol and aqueous extracts of Ficus exasperata. At concentrations of 0.63 and 2.50 mg mL-1, the values obtained from the three extracts are not significantly different from one another (p > 0.05). At all the concentrations tested except at 5 mg mL-1, acetone extract exhibited the highest percentage inhibition of the enzyme while aqueous displayed the highest value at 5 mg mL-1. The IC50 values generated from the percentage inhibition reveals that out of all the extracts, aqueous extract of Ficus exasperata has the lowest IC50 value (3.70 mg mL-1) (Table 3). Kinetic analysis of the α-amylase inhibition by aqueous extract of Ficus exasperata using Lineweaver-burke plot shows that it displayed a non-competitive mode of inhibition (Fig. 2).
Figure 3 shows the percentage inhibition of α-amylase
activity by acetone, ethanol and aqueous extracts of Ficus exasperata.
At all concentrations tested(1.25-10.00 mg mL-1), aqueous extract
of Ficus exasperata displayed significantly higher inhibition (p<0.05)
from acetone and ethanolic extracts. This was corroborated by the lowest IC50
value (1.70 mg mL-1) generated by the aqueous extract compared to
the other extracts (Table 3). This is because the lower the
IC50, the more potent the extract will be for treatment of the disease.
| Table 1: |
The percentage yield of different extracts of Ficus exasperata
leaf |
 |
|
| Table 2: |
The phytochemical constituent of different extracts of Ficus
exasperata leaf |
 |
| +: Present, -: Not detected |
| Table 3: |
IC50 values of various extracts of F. exasperata
against α-amylase and α-glucosidase |
 |
|
|
| Fig. 1: |
Percentage inhibition of α-amylase by different extracts
of Ficus exasperata Bars carrying different letters at the same concentration
are significantly different |
|
| Fig. 2: |
Mode of inhibition of α-amylase by aqueous extract of
Ficus exasperata |
|
| Fig. 3: |
Percentage inhibition of α-glucosidase by different extracts
of Ficus exasperata Bars carrying different letters at the same concentration
are significantly different |
|
| Fig. 4: |
Mode of inhibition of α-glucosidase by acetone extract
of Ficus exasperata |
The kinetics of inhibition by the aqueous extract of Ficus exasperata
against α-glucosidase using Lineweaver-Burke plot reveals that the mode
of inhibition of the enzyme is near competitive (Fig. 4).
DISCUSSION
Hyperglycemia is a state characterized by an abnormal postprandial increase
of blood glucose level and it has been linked to the onset of type 2 and associated
vascular complications (Dicarli et al., 2003).
Recent studies have indicated that hyperglycemia induced vascular complications
are likely from oxidative dysfunction from reactive oxygen species (ROS) produced
by the mitochondrial electron transport chain (Kwon et
al., 2007).
In order to manage diabetes, various inhibitors of disaccharide hydrolysing
enzymes (α-amylase and α-glucosidase) had been used as oral hypoglycemic
agents especially in patients with type 2 diabetes mellitus (Oboh
et al., 2012). Inhibition of these enzymes delay carbohydrate digestion
and prolong overall carbohydrate digestion which causes reduction in the rate
of glucose absorption and consequently reducing the postprandial blood glucose
rise (Kwon et al., 2007).
From this study, the results of the enzymes (α-amylase and α-glucosidase)
inhibitory assay showed that the aqueous extract of Ficus exasperata is
a mild inhibitor of α-amylase and strong inhibitor of α-glucosidase.
This however, is in agreement with earlier reports that plant phytochemicals
are mild inhibitors of α-amylase and strong inhibitors of α-glucosidase
activity with minimal side effect (Kwon et al.,
2007). This has an advantage over synthetic drugs such as acarbose; used
by diabetics in the management of postprandial blood glucose which strongly
inhibit α-amylase (Oboh et al., 2012).
Strong inhibition of both α-amylase and α-glucosidase by acarbose
causes indigested starch-linked complications such as abdominal distention,
flatulence, meteorism and possibly diarrhoea (Pinto et
al., 2009) which results to abnormal bacteria fermentation of undigested
carbohydrate in the colon (Kwon et al., 2007).
The characteristic non-competitive inhibition displayed by the aqueous extract
of this plant towards α-amylase indicates that aqueous extract of Ficus
exasperata binds to a site other than the active site of the enzyme and
combines with either free enzyme or the enzyme-substrate complex, possibly interfering
with the action of both (Mayur et al., 2010).
Lineweaver- Burke plot also showed that aqueous extract of this plant inhibited
α-glucosidase competitively. This suggests that the active components in
the extract compete with the substrate for binding to the active site of the
enzyme thereby preventing the breaking down of oligosaccharides to disaccharides
(Matsuda et al., 2002; Shai
et al., 2010).
The presence of phytochemicals such as flavonoids, saponins and tannins may
be responsible for the inhibitory potential of this plant (aqueous) extract
on the activities of the enzymes studied. These phenolic compounds can protect
the human from free radicals, chelate metal catalysts, activate antioxidant
enzymes and inhibit the action of oxidases (Oboh et
al., 2012). Flavonoids is one of the most diverse and widespread group
of natural compounds, having hydroxyl group present in it which confers scavenging
ability and also plays an important role in preventing lipid peroxidation (Mayur
et al., 2010). They may preserve beta cell function by reducing oxidative
stress-induced tissue damage and therefore protect against the progression of
insulin resistance to type 2 diabetes (Song et al.,
2005). On the other hand, tannin, which is composed of a central glucose
molecule derivatized at its hydroxyl groups with one or more galloyl residue
and in the presence of copper ions, act as an antioxidant suppressing hydroxyl
radical formation (Andrade et al., 2005).
CONCLUSION
It can be concluded from this study that only aqueous extract of Ficus exasperata exhibited the most effective inhibition of α-amylase and α-glucosidase and that these enzymes were inhibited non-competitively and competitively respectively. The study also suggests that the inhibitory potential of the aqueous extract of Ficus exasperate on the two enzymes may not be unconnected to the presence of phytochemicals like flavonoids, tannins and saponins. Therefore, this study suggests that one of the mechanisms of antidiabetic potential of Ficus exasperate is through the inhibition of α-amylase and α-glucosidase.
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