2.1. Phytochemical Characterization
In our investigation the TLC analysis showed a predominance of phenolic compounds and flavonoids in the crude extract (CE) and the
n-butanol fraction (BF) of
B. articulata.This was verified using the Natural Reagent A/UV356 that showed yellow spots corresponding to phenolic compounds (data no shown) as previously described [
6]. In addition, it can be observed (
Table 1) that the BF showed significantly higher total flavonoids content (44.9 ± 0.71 mg of RE/g of DW,
p < 0.05) when compared to the CE (38.9 ± 0.66 mg of RE/g of DW) and ARF (25.3 ± 0.42 mg of RE/g of DW). On the other hand the total phenolic content of CE and their related fractions did not shown differences.
Some of the different biological activities of
Baccharis species are related to the presence of phenolic compounds and flavonoids [
7,
8]. A large number of studies have demonstrated that phenolic compounds as flavonoids and phenolic acids derivatives have different biological activities, such as antioxidant, anticancer, anti-inflammation and cardioprotective properties, and they can also prevent lipoperoxidation, induce favorable changes in the lipid profile, improve endothelial function, and disclose antithrombotic properties [
9]. In addition, the hypoglycemic and/or antihyperglycemic activity of flavonoids has been previously reported [
10].
Table 1.
Total phenolic a and total flavonoids b content in crude extract (CE), n-butanol fraction (BF) and aqueous residual fraction (ARF) of B. articulata.
Table 1.
Total phenolic a and total flavonoids b content in crude extract (CE), n-butanol fraction (BF) and aqueous residual fraction (ARF) of B. articulata.
Extract/fraction | Total phenolic | Total flavonoids |
---|
CE | 151.8 ± 0.93 a | 38.9 ± 0.66 a |
BF | 154.1 ± 1.08 a | 44.9 ± 0.71 b |
ARF | 135.6 ± 0.95 b | 25.3 ± 0.42 c |
2.2. Effect of Crude Extract and n-Butanol and Aqueous Residual Fractions of B. Articulata on Oral Glucose Tolerance Curve
As expected, in the oral glucose tolerance test, after 15 min of glucose loading the glycemia was significantly increased when compared with zero time. Glipizide (100 mg/kg) an oral hypoglycemic agent of the sulfonylurea class was used as a positive control and produced a typical serum glucose lowering at all periods analyzed (15 to 180 min) compared to the hyperglycemic group (
Table 2). At all doses tested (50, 100 and 200 mg/kg) the CE of
B. articulata leaves was effective in reducing the glycemia at different times after oral treatment compared with the respective hyperglycemic control group (
Table 2). The dose of 100 mg/kg of the CE produced the best antihyperglycemic profile at 15 to 60 min and the maximum reduction observed was 26% at 30 min. Administration of 50 mg/kg of the BF of
B. articulata decreased serum glucose levels significantly at 15, 30 and 60 min, and the glycemic reduction was around 23, 23 and 18%, respectively, whereas the dose of 100 mg/kg was not effective in reducing the serum glucose levels during the times studied (
Table 2). In addition, oral administration of the ARF of
B. articulata also reduced the serum glucose levels in hyperglycemic rats at both doses tested (50 and 100 mg/kg) and the antihyperglycemic effect was better with the lower dose. The reduction was around 15 and 20% at 15 and 30 min after treatment, respectively. At 180 min, glycemic levels were similar to the respective results for the hyperglycemic control groups.
On the other hand, the CE, BF and ARF (100 mg/kg) of B. articulata were studied in rats with induced diabetes and no significant changes in the serum glucose levels in an acute treatment were observed (data not shown). These results point a potential insulin secretagogue effect for B. articulata compounds.
Another
Baccharis species,
Baccharis trimera reportedly has potential antidiabetic activity. Oliveira
et al. [
11] investigated the effect of its extracts and fractions on glycemia in non-diabetic mice and mice with streptozotocin-induced diabetes. After 7 days of treatment, the aqueous fraction (2,000 mg/kg, twice daily) reduced the glycemia of diabetic mice. However, in contrast to our results for
B. articulata, none of the extracts or fractions (200 or 2,000 mg/kg) of
B. trimera induced any effect on glycemia after acute administration on hyperglycemic mice. Thus, to the best of our knowledge, our results represent the first report of the potential antihyperglycemic effect of
B. articulata.Diterpenoids, flavonoids and other phenolic compounds have been reported as the major phyto-constituents of the
Baccharis species and this diverse chemical composition is related to a variety of biological activities described for these species [
8]. The results reported herein demonstrate that
B. articulata has a significant content of flavonoids and other phenolic compounds (
Table 1) and the presence of these constituents may be associated with the antihyperglycemic effect observed, since the hypoglycemic activity of phenolics compounds has been previously reported [
10].
2.3. Effect of Crude Extract and n-Butanol and Aqueous Residual Fractions of B. Articulata on Insulin Secretion and Glycogen Content
In order to evaluate the possible mechanism of action of the extract (CE) and fractions (BF and ARF) of
B. articulata, their effects on glycogen content and on insulin secretion were investigated. Serum insulin levels were determinated in fasted rats after an oral glucose loading (4 g/kg) as shown in
Table 3. As expected a sulfonylurea agent, glipizide, stimulated the insulin secretion by 295, 149 and 191% at 15, 30 and 60 min, respectively, compared to the hyperglycemic control group. The CE potentiated insulin secretion induced by glucose at 15 (167%), 30 (141%) and 60 min (268%), after oral treatment. In addition, the BF increased significantly the insulin secretion by 162, 189 and 244% at 15, 30 and 60 min, respectively. However, the ARF was not able to increase serum insulin levels. The treatments with the CE and BF resulted in around a 2.3-fold increase in the insulinogenic index (II) compared with the hyperglycemic control group (hyperglycemic control 0.44 ng/mg; CE 0.96 ng/mg and BF 1.03 ng/mg), achieving values similar to that calculated for glipizide (1.09 ng/mL). These results indicate, for the first time, the powerful effect of
B. articulata on insulin secretion.
The glycogen content in the soleus muscle and liver samples of hyperglycemic rats and those which received acute treatments with the CE (100 mg/kg), BF (50 mg/kg) and ARF (50 mg/kg) was determinate as shown in
Figure 1A and B. After 3 h of oral treatment with the BF and ARF the glycogen content in the soleus muscle increased significantly, by around 443 and 212%, respectively, compared to the hyperglycemic control group (
Figure 1A).
Table 2.
Acute effect of crude extract (CE), n-butanol fraction (BF) and aqueous residual fraction (ARF) of Baccharis articulata on serum glucose levels (mg/dL) in oral glucose tolerance curve a.
Table 2.
Acute effect of crude extract (CE), n-butanol fraction (BF) and aqueous residual fraction (ARF) of Baccharis articulata on serum glucose levels (mg/dL) in oral glucose tolerance curve a.
Time (min) | Group I Hyper Glucose (4 g/kg) | Group II Hyper + glipizide (10 mg/kg) | Group III Hyper + CE | Group IV Hyper + BF | Group V Hyper + ARF |
---|
50 mg/kg | 100 mg/kg | 200 mg/kg | 50 mg/kg | 100 mg/kg | 50 mg/kg | 100 mg/kg |
---|
0 | 112 ± 4 # | 104 ± 3 | 110 ± 6 | 116 ± 5 | 103 ± 3 | 116 ± 2 | 113 ± 3 | 116 ± 3 | 103 ± 2 |
15 | 162 ± 8 | 121 ± 4 *** | 128 ± 5 ** | 123 ± 2 *** | 134 ± 6 ** | 124 ± 6 *** | 187 ± 4 | 138 ± 4 ** | 136 ± 10 * |
30 | 185 ± 6 | 148 ± 7 *** | 144 ± 4 *** | 137 ± 1 *** | 146 ± 3 *** | 143 ± 2 *** | 178 ± 6 | 148 ± 3 *** | 159 ± 6 * |
60 | 164 ± 4 | 122 ± 4 *** | 145 ± 5 | 130 ± 8 ** | 140 ± 6 * | 134 ± 9 ** | 186 ± 8 | 154 ± 4 | 161 ± 10 |
180 | 135 ± 4 | 116 ± 5 ** | 130 ± 3 | 129 ± 3 | 136 ± 4 | 145 ± 5 | 126 ± 4 | 142 ± 5 | 133 ± 5 |
Table 3.
Acute effect of crude extract (CE), n-butanol fraction (BF), aqueous residual fraction (ARF) of Baccharis articulata on serum insulin levels (ng/mL) and insulinogenic index (II; ng/mg) a.
Table 3.
Acute effect of crude extract (CE), n-butanol fraction (BF), aqueous residual fraction (ARF) of Baccharis articulata on serum insulin levels (ng/mL) and insulinogenic index (II; ng/mg) a.
Time (min) | Hyper Glucose (4 g/kg) | Hyper + glipizide (10 mg/kg) | Hyper +
B. articulata |
---|
CE 100 mg/kg | BF 50 mg/kg | ARF 50 mg/kg |
---|
0 | 0.57 ± 0.03 | - | - | - | - |
15 | 0.77 ± 0.06 # | 2.27 ± 0.20 *** | 1.29 ± 0.27 ** | 1.25 ± 0.10 * | 0.82 ± 0.04 |
30 | 0.90 ± 0.10 | 1.37 ± 0.02 * | 1.30 ± 0.14 * | 1.74 ± 0.06 *** | 0.79 ± 0.09 |
60 | 0.54 ± 0.04 | 1.03 ± 0.15 * | 1.45 ± 0.24 *** | 1.32 ± 0.20 *** | 0.63 ± 0.10 |
II | 0.44 | 1.09 | 0.96 | 1.03 | 0.49 |
In addition, only the BF treatment was able to significantly increase the glycogen content in the liver when compared with the hyperglycemic control group 3 h after treatment. In percentage terms, this change was 137% (
Figure 1B). Taking it in account, it seems that the serum glucose lowering could be related with the effect on increased glycogen content similar to that known to insulin, points to an insulin-mimetic effect.
Figure 1.
Effect of crude extract (CE), n-butanol fraction (BF) and aqueous residual fraction (ARF) of B. articulata on glycogen content in comparison to hyperglycemic rats (control). (A) soleus muscle and (B) liver 3 h after treatment by oral gavage. Values are expressed as mean ± S.E.M; n = 6 in duplicate for each group. Significantly different to the corresponding hyperglycemic group; * p< 0.05; ** p< 0.001; *** p< 0.0001.
Figure 1.
Effect of crude extract (CE), n-butanol fraction (BF) and aqueous residual fraction (ARF) of B. articulata on glycogen content in comparison to hyperglycemic rats (control). (A) soleus muscle and (B) liver 3 h after treatment by oral gavage. Values are expressed as mean ± S.E.M; n = 6 in duplicate for each group. Significantly different to the corresponding hyperglycemic group; * p< 0.05; ** p< 0.001; *** p< 0.0001.
Insulin is secreted into the bloodstream by β-cells of the endocrine pancreas and glucose is the main insulin secretagogue. Insulin is the most important hormone that regulates energy metabolism and has hypoglycemic effect. An absolute or relative lack of insulin, as in the case of diabetes, leads to severe dysfunction in the major insulin target organs such as muscle, liver and adipose tissue [
12].
The stimulation of β-cells and subsequent release of insulin and activation of the insulin receptors is a possible mechanism of natural products with potential antidiabetic activity. Folador
et al. [
13] showed that the crude extract, the
n-butanol fraction and two isolated
C-glycosylflavones, isovitexin and swertisin, of
Wilbrandia ebracteata can have an antihyperglycemic action, which was related to the stimulation of
in vivo insulin secretion.
Glucose homeostasis is maintained by the balance of liver glucose production and glucose utilization by peripheral tissues. In mammals, glucose is stored as glycogen in the liver and muscle, which are the major sites for glycogen synthesis and storage. It is well known that glycogen deposition from glucose is regulated by insulin. However, it is well reported that flavonoids and plant extracts with proven antihyperglycemic activity can also influence glycogen deposition in different tissues as well as interact with key enzymes of the glycolytic route in rats [
14,
15].
In addition, it is worth noting that plants and phytochemicals that have a hypoglycemic effect may act by different mechanisms of action to regulate glucose homeostasis, including an increase in insulin secretion from the pancreatic islets (insulin-secretagogue) and/or enhancing or reproducing the effect of insulin (insulin-mimetic) as we demonstrated for B. articulata.
2.4. Effect of Crude Extract and n-Butanol and Aqueous Residual Fractions of B. Articulata on the Disaccharidases
The intestine plays an important role in glucose homeostasis. A therapeutic approach to decreasing postprandial hyperglycemia is to retard the absorption of glucose via inhibition of carbohydrate-hydrolyzing enzymes, such as α-glucosidase, in the intestine. These disaccharidase enzymes are located in the brush border of the small intestine and are required for the breakdown of carbohydrates before monosaccharide absorption. The α-glucosidase inhibitors delay the absorption of ingested carbohydrates, reducing the postprandial glycemia and insulin peaks [
16]. Some plants which exhibit properties similar to those of known classes of anti-diabetic drugs, for instance, inhibitors of α-glucosidase such as acarbose, have been identified [
17].
The effect of the extract and fractions of
B. articulata in disaccharidase assays were determined (
Figure 2). A significant effect on maltase inhibition was observed after 5 min of incubation of the intestine homogenate in the presence of a maltose substrate. The CE, BF and ARF were effective at inhibiting the enzyme maltase at both doses tested (500 and 1,500 μg/mL) after 5 min of incubation.
Figure 2.
In vitro effect of crude extract (CE), n-butanol fraction (BF) and aqueous residual fraction (ARF) of B. articulata on specific activity of maltase, in the duodenal portion of rat intestine. Incubation = 5 min. Values are expressed as mean ± S.E.M.; n = 6 for each group. Significant at ** p< 0.001; *** p< 0.0001 compared to control group.
Figure 2.
In vitro effect of crude extract (CE), n-butanol fraction (BF) and aqueous residual fraction (ARF) of B. articulata on specific activity of maltase, in the duodenal portion of rat intestine. Incubation = 5 min. Values are expressed as mean ± S.E.M.; n = 6 for each group. Significant at ** p< 0.001; *** p< 0.0001 compared to control group.
The inhibitory effect observed ranged from 15 and 32% and the maximum inhibitory effect on maltase activity, above 30%, was observed for the CE and ARF at the higher dose studied, compared with the respective controls. On the other hand, none of the treatments affected the sucrase and lactase activity at any concentration tested (data not shown).
A number of plants are known to exert antihyperglycemic activity through the inhibition of disaccharidase enzymes in the small intestine, impeding the absorption of carbohydrates. Recently, De Souza
et al. [
18] reported that aqueous and methanolic extracts of
B. trimera efficiently inhibited β and α-glycosidase activity.
2.5. Effect of Crude Extract and n-Butanol and Aqueous Residual Fractions of B. Articulata on in vitro Albumin Glycation
Chronic hyperglycemia and increased oxidative stress during diabetes results in the irreversible formation of advanced glycation endproducts (AGEs), which are a heterogeneous group of molecules formed from non-enzymatic glycation of reducing sugars with free amino groups of proteins, lipids, and nucleic acids. The Schiff’s bases formed by glycation rearrange further through stable reactions to form Amadori products which later, by isomerization, condensation, and rearrangement reactions, form AGEs. The AGEs are known to have a wide range of chemical, cellular, and tissue effects implicated in the development and progression of diabetic complications, like nephropathy, neuropathy, retinopathy, and cardiovascular diseases [
19].
In the method adopted in this study, BSA was chosen as the model protein and glucose or fructose was used as the glycated agent. This BSA-reducing sugar system is an
in vitro model widely used in non-enzymatic glycation studies. Proteins can be modified when exposed to reducing sugars through the spontaneous glycation process. The sugar-mediated fluorescence intensity, which is a characteristic of AGEs, increases during incubation at 37 °C for a long period.
Figure 3 shows the fluorescence intensity of the products (AGEs) formed in the BSA-glycation model. After incubation at all periods analyzed (7, 14 and 28 days) it was clearly observed that the formation of AGEs was significantly increased in the BSA/glucose (
Figure 3A–C) and BSA/fructose (
Figure 3D–F) systems when compared with the basal control.
Figure 3B and C show the efficiency of the CE, BF and ARF in the inhibition of albumin glycation with glucose after 14 and 28 days. After 28 days of
in vitro incubation all treatments caused a glycation reduction of over 30% when compared with positive glycation group (albumin plus glucose). However, a slight increase in glycation was observed for the CE, BF and ARF treatments after 7 days in the BSA/glucose system (
Figure 3A). These results show the significant capacity of the extract and fractions of
B. articulata to reduce the AGE formation after a long period of incubation, the fluorescence intensity being stronger.
The capacity of the CE, BF and ARF to inhibit albumin glycation with fructose for different periods is shown in
Figure 3D–F. The reduction in the glycation of albumin by glucose in the presence of the extract and fractions of
B. articulata increased from 7 to 28 days of treatment. With 7 days of incubation only the CE and BF, at both doses tested, were able to inhibit significantly the albumin glycation. At the maximum period evaluated, more that 75% of glycation reduction was observed when compared with the positive glycation group (albumin plus fructose).
Figure 3.
Inhibitory effect of crude extract (CE), n-butanol fraction (BF) and aqueous residual fraction (ARF) of B. articulata on the formation of fluorescent AGEs in a BSA/glucose or BSA/fructose system. (A, B and C) 7, 14 and 28 days BSA/glucose; (D, E and F) 7, 14 and 28 days BSA/fructose. Values are expressed as mean ± S.E.M; n = 6 in duplicate for each group. Significantly different to the corresponding control group (BSA/glucose or BSA/fructose); * p< 0.05; ** p< 0.001; *** p< 0.0001.
Figure 3.
Inhibitory effect of crude extract (CE), n-butanol fraction (BF) and aqueous residual fraction (ARF) of B. articulata on the formation of fluorescent AGEs in a BSA/glucose or BSA/fructose system. (A, B and C) 7, 14 and 28 days BSA/glucose; (D, E and F) 7, 14 and 28 days BSA/fructose. Values are expressed as mean ± S.E.M; n = 6 in duplicate for each group. Significantly different to the corresponding control group (BSA/glucose or BSA/fructose); * p< 0.05; ** p< 0.001; *** p< 0.0001.
Several natural compounds have been proposed and tested as inhibitors of glycation and AGE formation, providing additional therapeutic options for the treatment of the various complications associated with diabetes [
20]. As the incidence of diabetes continues to rise worldwide, the study of natural products for the treatment and prevention of diabetes, and its associated complications, offers an important opportunity for the development of complementary interventions that may be more acceptable to high-risk populations in the search for non-pharmaceutical alternatives. Our results demonstrate that the extract and fractions of
B. articulata are potent inhibitors of
in vitro AGE formation in chronic treatments and this mechanism may help to provide a protective effect against hyperglycemia-mediated protein damage. Recent reports have shown that flavonoids inhibit the formation of AGEs, which is related to their well-known antioxidative effects [
21]. In this regard, we verified the high total flavonoid content of
B. articulata, which may explain the significant AGE inhibition observed for this species.