Total phenolic contents of the selected coffee extracts
The total phenolic contents (TPC) of selected coffee extracts were analyzed using the Folin-Ciocalteu method. Fig. 1 shows the TPC of the tested water and ethanol coffee extracts. The highest average TPC value from water extract samples was observed in Sumatra coffee (67.65 ± 1.59 mg GAE/100 g) followed by Guatemala (71.58 ± 1.55) and Ethiopia (73.00 ± 0.57). In the case of coffee extracted by ethanol the higher value of TPC was recorded for Guatemala coffee with Sumatra and the lowest value was recorded from Ethiopia.
An excess of free radicals in the body is one of the causes of lifestyle diseases such as cancer or diseases of the circulatory system [14]. Therefore, it is essential that the human diet contains, among other nutrients, phenolic compounds. phenolic compounds have a number of beneficial health properties related to their potent antioxidant activity as well as hepatoprotective, hypoglycemic, and antiviral activities [23]. It has been reported that coffee is one of the dietary sources of phenolic compounds [24]. Also, it is reported that plant variety, species and growing/harvesting conditions can affect phenolic content in plants [31].
α-Glucosidase Inhibitory Activity
The α-glucosidases inhibitors, which interfere with enzymatic action in the brush-border of the small intestine, could slow the liberation of D-glucose from oligosaccharides and disaccharides resulting in reduced postprandial plasma glucose levels [28].
α-Glucosidase inhibitory activities of water and ethanol extracts of coffee samples are listed in Figs. 2 and 3 respectively.
In water extracts, the higher α-glucosidase inhibitory activity was obtained from SWE (4.39 mg/mL of IC50) followed by EWE (4.97 mg/mL of IC50) and GWE (5.19 mg/mL of IC50) (Table 1).
Table 1. The half-maximal inhibitory concentration (IC50) values for rat intestinal α-glucosidase by water extracted coffee types
|
IC50 (mg/mL)
|
Extracts
|
SWE
|
GWE
|
EWE
|
α-glucosidase
|
4.39
|
5.19
|
4.97
|
The dose-dependent α-glucosidase inhibitory activity water extracted coffee samples were observed in Fig. 2. At 5 mg/mL, SWE showed the higher α-glucosidase inhibitory activity (4.39 mg/mL of IC50); followed by EWE and GWE; however, there was no significant difference between SWE and EWE.
In similar pattern ethanol extracted coffee showed α-glucosidase inhibitory activity in a dose-dependent manner. SEE showed higher α-glucosidase inhibitory activity at higher concentrations followed by GEE and EEE (Fig. 3). Table 2 showed that the IC50 (mg/mL) values of ethyl alcohol extracted coffee.
Table 2. The half-maximal inhibitory concentration (IC50) values for rat intestinal α-glucosidase by ethyl alcohol extracted coffee types
|
IC50 (mg/mL)
|
Extracts
|
SEE
|
GEE
|
EEE
|
α-glucosidase
|
2.97
|
3.19
|
3.27
|
α-Amylase Inhibition assay
The α-amylase inhibitors, which interfere with enzymatic action in the small intestine, could slow the liberation of maltose from starch, resulting in delaying maltose conversion to glucose and decreasing postprandial plasma glucose levels [30]. In our case, little or no inhibition of α-amylase was observed by coffee extracts (data not shown) linked to the side-effect due to increase non-digested starch in large intestine. reported that chlorogenic acid and phenolic acid from coffee are very weak inhibitors of human salivary α-amylase [32].
Sucrase, Maltase, and Glucoamylase Inhibition Assay
It has been reported that most yeast α-glucosidase inhibitors did not show significant activities against mammalian α-glucosidase, due to the difference in molecular recognition in the target binding site of these enzymes. Therefore, rat small intestinal sucrase, maltase, and glucoamylase, the key α-glucosidases that catalyze the hydrolysis of disaccharides to glucose were used for estimating the inhibitory activities of coffee extracts [33]. To determine the specificity of the observed inhibitory activity, we examined the effect of coffee extracts of all three coffee types on rat small intestinal sucrase, maltase, and glucoamylase. Both water and ethanol coffee extracts showed intestinal sucrase inhibitory activity in a dose-dependent manner. Water extracted coffee EWE showed higher inhibitory activity in all concentrations followed by SWE and GWE (Fig. 4a). In ethanol extracts, SEE showed higher inhibitory activity than EEE and GEE (Fig. 4b). At 3 mg/mL concentrations GWE showed higher maltase inhibitory activity in water extracts followed by EWE and EWE whereas in ethanol extracts SEE showed maltase inhibitory activity in a similar percentage with GEE but significantly higher than EEE (Figs. 4 c and d). For glucoamylase, a higher inhibitory percentage was obtained by GWE in water extracts and GEE in ethanol extracts and showed in both water and ethanol extracts (Figs. 4e and 4f).
The IC50 value of sucrase in both extracts and all coffee types was lower than maltase and glucoamylase implying that sucrase inhibitory activity of all coffee types was higher than that of maltase and glucoamylase (Table 3). With the exception of glucoamylase, ethanol extracts of all coffee types showed higher inhibitory activity than DW extracts. The overall result revealed that in a dose-dependent manner the coffee extracts showed similar but significant inhibitory effects on sucrase, maltase, and glucoamylase. This suggested that the coffee extracts may be used as potential inhibitors of α-glucosidases with particular inhibitory effect on sucrase than maltase and glucoamylase.
Table 3. Half maximal inhibitory concentration (IC50) of coffee water (DW) extract and ethyl alcohol extract on rat small intestinal sucrase, maltase, and glucoamylase activity
|
|
|
IC50 (mg/mL)
|
|
|
|
Sumatra
|
Guatemala
|
Ethiopia
|
Sucrase
|
DW
|
0.52
|
0.50
|
0.47
|
|
Ethanol
|
0.43
|
0.39
|
0.41
|
Maltase
|
DW
|
2.03
|
1.93
|
1.94
|
|
Ethanol
|
1.60
|
1.67
|
1.83
|
Glucoamylase
|
DW
|
1.11
|
1.01
|
1.08
|
|
Ethanol
|
1.38
|
1.20
|
1.27
|
Inhibition of sucrase, maltase, and glucoamylase plays an important role in controlling the rapid rise in blood glucose levels after excessive mixed carbohydrate meals. Therefore, limiting the amount of glucose/calories that can be absorbed by inhibiting the activity of α-glucosidases in the small intestine plays an important role in improving postprandial hyperglycemia.
Blood glucose-lowering effect of coffee extracts in-vivo
To confirm the in vitro inhibition of sucrase activity, the time courses of plasma glycemic response were measured at 0, 30, 60, and 120 min after sucrose-loading (2.0 g/kg body weight (bw)) in SD rats. Since all the three coffee types showed similar inhibitory potential in vitro we assessed the blood-lowering effect of the three coffee types in in-vivo. Figs. 5 and 6 show the comparison of the plasma glucose-lowering effect of water and ethanol extracts of Sumatra, Guatemala, and Ethiopia coffee types at the concentration of 0.5 g/Kg bw with the control group (sucrose or starch only) and a known pharmacological α-glucosidase inhibitor, acarbose (5 mg/kg bw) as a positive control.
At half an hour after sucrose loading, SWE and EWE significantly reduced plasma glucose level when compared to the control in SD rats (Fig. 5). However, no significant plasma glucose-lowering effect was observed from GWE. On the other hand, EEE and GEE significantly decreased plasma glucose levels as compared to the control in SD rats at 30 minutes following sucrose loading (Fig. 6).
When male SD rats were administered with starch solution EWE and SWE significantly decreased blood glucose levels (Fig. 7). Meanwhile, SD rats treated with SEE, EEE, and GEE showed significant decreases in plasma glucose levels when compared to the control (starch). Acarbose, the well-known α-glucosidase inhibitor pharmacology therapy drug, showed significantly higher blood lowering than all the coffee types and the controls.
Both EWE and EEE showed significant blood lowering effect in sucrose and starch loading tests showing its potential as an α-glucosidase inhibitor (Fig. 8). Overall, these results revealed the potential of both water and ethanol coffee extracts to reduce plasma glucose levels after a meal.
Pharmacodynamics Parameters
Pharmacodynamics (PD) parameters of the sucrose and starch loading tests with GWE, EWE, SWE and acarbose are shown in Table 4. Changes in PD parameters of control and after administration of GWE, EWE, SWE, and acarbose with sucrose or starch ingestions4. EWE-treated groups resulted significantly reduced Cmax (Both sucrose and starch were p < 0.01) and AUCt (sucrose was p < 0.05), however this reduction was less effective than the acarbose-treated group.
Table 4. Changes in pharmacodynamics (PD) parameters of control and after administration of GWE, EWE, SWE, and acarbose with sucrose or starch ingestions
Groups
|
PD Parameters
|
Cmax (mg/dL)
|
Tmax (hr)
|
AUCt (hr·mg/dL)
|
Sucrose 2.0 g/kg
|
222.3 ± 16.0
|
0.7 ± 0.3
|
516.0 ± 29.0
|
Acarbose 5.0 mg/kg
|
137.4 ± 8.3***
|
0.8 ± 0.3
|
376.2 ± 17.2***
|
GWE 0.5 g/kg
|
208.8 ± 14.4
|
0.8 ± 0.3
|
496.3 ± 23.9
|
EWE 0.5 g/kg
|
182.5 ± 15.4**
|
1.4 ± 0.9
|
434.8 ± 50.8*
|
SWE 0.5 g/lg
|
191.0 ± 9.1**
|
1.0 ± 0.0*
|
482.3 ± 12.7
|
Starch 2.0 g/kg
|
236.2 ± 25.1
|
0.5 ± 0.0
|
529.1 ± 13.4
|
Acarbose 5.0 mg/kg
|
144.1 ± 18.8***
|
1.3 ± 0.5**
|
389.0 ± 36.8***
|
GWE 0.5 g/kg
|
218.3 ± 20.6
|
1.0 ± 0.6
|
536.3 ± 49.4
|
EWE 0.5 g/kg
|
191.3 ± 13.2**
|
0.8 ± 0.3*
|
490.5 ± 34.6
|
SWE 0.5 g/lg
|
222.9 ± 30.1
|
0.5 ± 0.0
|
548.9 ± 47.2
|
The results were expressed as mean ± S.D. All parameters were compared between control and treatment group (GWE, EWE, SWE, and Acarbose) by unpaired Student’s t-test (*p < 0.05; **p < 0.01; and ***p < 0.001).
On the other hand, in Table 5. Changes in PD parameters of control and after administration of GEE, EEE, SEE and acarbose with sucrose or starch ingestions. All the ethanol extract coffee was shown significantly reduced Cmax (GEE: p < 0.01, p < 0.05; EEE: p < 0.05, p < 0.01; SEE p < 0.01, p < 0.05). However, in terms of AUCt there was no significant difference among sucrose and all of ethanol extract coffee. Starch loading tests are shown GEE, EEE, and SEE significantly reduced AUCt (GEE: p < 0.01; EEE: p < 0.05; SEE p < 0.01) of blood glucose in rats ingested with starch compared to control.
Table 5. Changes in pharmacodynamics (PD) parameters of control and after administration of GEE, EEE, SEE, and acarbose with sucrose or starch ingestions
Groups
|
PD Parameters
|
Cmax (mg/dL)
|
Tmax (hr)
|
AUCt (hr·mg/dL)
|
Sucrose 2.0 g/kg
|
222.3 ± 16.0
|
0.7 ± 0.3
|
516.0 ± 29.0
|
Acarbose 5.0 mg/kg
|
137.4 ± 8.3***
|
0.8 ± 0.3
|
376.2 ± 17.2***
|
GEE 0.5 g/kg
|
188.7 ± 13.5**
|
0.8 ± 0.3
|
480.7 ± 34.1
|
EEE 0.5 g/kg
|
194.1 ± 18.3*
|
0.7 ± 0.3
|
483.6 ± 27.6
|
SEE 0.5 g/lg
|
185.9 ± 8.9**
|
0.9 ± 0.2
|
481.4 ± 11.8
|
Starch 2.0 g/kg
|
236.2 ± 25.1
|
0.5 ± 0.0
|
529.1 ± 13.4
|
Acarbose 5.0 mg/kg
|
144.1 ± 18.8***
|
1.3 ± 0.5**
|
389.0 ± 36.8***
|
GEE 0.5 g/kg
|
188.0 ± 25.7*
|
0.6 ± 0.2
|
454.9 ± 46.7*
|
EEE 0.5 g/kg
|
195.0 ± 13.9**
|
0.7 ± 0.3
|
477.8 ± 34.5*
|
SEE 0.5 g/lg
|
203.4 ± 14.5*
|
0.8 ± 0.3*
|
468.2 ± 37.0**
|
The results were expressed as mean ± S.D. All parameters were compared between control and treatment group (GEE, EEE, SEE, and Acarbose) by unpaired Student’s t-test (*p < 0.05; **p < 0.01; and ***p < 0.001)