Background
Diabetes mellitus is one of the major metabolic conditions with leading morbidity and mortality rate, which results in macrovascular and microvascular complications caused due to persistent glucose toxicity [
1]. It is now said to have reached epidemic status and the figure of people living with diabetes is projected to increase to 522 million globally by the year 2030 [
2]. Despite having standard drugs for the management of diabetes mellitus, it continues to be a challenge because so far, there are no reports of drugs that are capable of curing the condition. Standard drugs available have been associated with undesirable effects, in addition to their inaccessibility [
3,
4]; meaning that financially challenged people, especially from developing and underdeveloped countries such as most African countries, including South Africa, cannot access these medications [
2,
5]. Because of historical recognition [
6] and economic pressure, people have resorted to the use of medicinal plants as a conventional way to treat diabetes and its complications. Although there are no reports that one completely recovered from diabetes mellitus because of using medicinal plants, there has been a surge in trying to isolate compounds of therapeutic interest from plants. Reduction of postprandial glucose levels is very important in the management of diabetes mellitus, and plants provide such effects which are similar to available therapies with less to no side effects [
7,
8]. Nonetheless, their modes of action are poorly explored.
Acacia karroo, commonly known as sweet thorn (English), belongs to the family Fabaceae. The plant is common to the southern Africa region and is widely distributed throughout South Africa, Zambia, Zimbabwe and Angola [
9]. The plant is very easy to identify due to its characteristic white paired thorns, brilliant yellow flowers and red like coffee colored bark [
10]. The name of the plant has recently been changed to
Vachellia karroo [
11]. The gum produced by
A. karroo is used against oral thrush and can also be harvested for food during hard times. The Shona people of Zimbabwe use the roots against conditions such as convulsions, gonorrhea and dizziness while the Ndebele people use them for general body pains. Herbivores enjoy a great deal of nutritious forage from
A. karroo, especially during dry seasons when the grass is dried out.
Acacia karroo is also effective against fever, malaria, cholera, diarrhoea, dysentery and high blood pressure [
12‐
14].
Acacia karroo, just like
Acacia nilotica contains compounds with hypoglycaemic effects; however, there are no reports that indicate that
A. karroo induces hypoglycaemic effects. The ability of this plant to exhibit hypotensive effects and interact with biomolecules resulting in inhibitory effects against digestive enzymes [
14] prompted us to examine its potential to promote hypoglycemic effects. The aim of this study was to determine the phytochemical, antioxidant, and possible hypoglycemic effects (alpha amylase enzyme inhibition and increased glucose uptake) of the defatted and non-defatted extracts of
A. karroo and possible modes of action (GLUT4 translocation and phosphorylation of MAPKs) of the most active extract on cells with high glucose uptake.
Discussion
Modulation of postprandial glucose levels through decreased glucose liberation from diet and glucose disposal by peripheral tissues such as muscle and adipose tissues are widely acceptable primary modalities for testing hypoglycaemic effects. In this study, we examined the phytochemical, phenolic and antioxidant constituent differences of the defatted and non-defatted extracts of A. karroo. Furthermore, biological activities that are determinants of antidiabetic effects were evaluated.
Medicinal plants contain diversity of phytochemicals that serve to protect them against pathogens. These phytochemicals have a proven history in the treatment of various diseases in humans and have served as a source for proven templates for drug development [
26]. A review of available literature on antidiabetic effects of plants report that phytochemicals such as polyphenols, flavonoids, tannins, saponins, coumarins, anthraglycosides and terpenoids have the potential to induce antidiabetic effects [
5,
26]. Our qualitative tests for the presence of phytochemicals showed the presence of phenols, tannins, saponins, flavonoids and cardiac glycosides in both defatted and non-defatted extracts and absence of terpenoids, carbohydrates and steroids. Presence of steroids [
27] and carbohydrates in the extract were evaluated in the study because they can negatively affect the associated hypoglycaemic effects. Furthermore, defatting of the plant material did not influence the presence of inherent compounds.
Phenols are a diverse group of polar compounds attributed to the treatment and management of various conditions including; haemorrhagic shock, ageing, ischemia, Alzheimer, Parkinson’s disease, arthritis, gastrointestinal disorders, carcinogenesis, atherosclerosis and most importantly diabetes mellitus [
28‐
30]. Following the determination of the qualitative presence of phenols in both extracts of
A. karroo, the amounts of phenols contained in each extract were quantified. Defatting of the plant material influenced the outcome of the amounts of phenols, although the difference was not significant, the non-defatted extract had slightly higher amounts of phenols compared to the defatted extract. A previous study reported that the acetone leaf extract of
Vachellia karroo (
Acacia karroo) showed low amounts of phenols [
31]. This is contrary to our finding in this study, where the acetone extract (non-defatted) is shown to have high amounts of phenols possibly due to geographical location, environmental factors, parasitic infestation or season of plant collection. On the other hand, we could not correlate the findings of the defatted extract with any previous study on the plant. In trying to understand the reasons behind the observed differences, we correlated our findings to findings on other plants other than
A. karroo. A previous study [
32] compared the phytochemical, antioxidant and bioactivity differences between the defatted and non-defatted extracts of various plants. Defatting of plant materials have been associated with yields containing high amounts of highly polarisable medium sized compounds, mostly phenols; which could be the exact reason why defatted extracts had high amounts of phenols. Hence we could attribute our findings to the nature of compounds present in the plant extract.
Phenols are considered as natural hosts of antioxidants. Antioxidants are molecules with the ability to prevent auto oxidation of bio-molecules and have also been greatly implicated in the prevention and progression of diabetes. The current findings in this study indicated that defatting of the plant material influenced the outcome of its antioxidant potential. The DPPH free radical scavenging potential was shown to decrease with defatting (0.40 ± 0.012), although the difference was not significant. Both the defatted and non-defatted extracts were shown to have very high electron donating abilities to quench DPPH free radicals when compared to ascorbic acid (0.59 ± 0.003) at
P < 0.001. Contrary to the findings of DPPH free radical scavenging activity, the ferric reducing power was significantly (
P < 0.001) improved with defatting (1.13 ± 0.017). Ascorbic acid had the high ability to reduce ferric ions Fe
3+ to ferrous ions Fe
2+ (1.06 ± 0.006) when compared to both extracts, with the non-defatted extract showing significantly low (
P < 0.001) ferric reducing power (1.65 ± 0.023). This finding is consistent with a previous study [
32] where the same trend was observed with other plants and was attributed to the removal of fats and oils (defatting). Adding to that, the nature of compounds present in the plant might have a direct bearing on the outcome, since phytochemicals produced in plants are a function of environment influences. However, looking at other phenolic compounds such as flavonoids and tannins might also give a clear picture to the outcome. Furthermore, the reducing power potential could be as a result of the number of phenol groups and position/number of hydroxyl groups and the scavenging activity could be attributed to the number of hydroxyl groups with redox properties [
33].
Partial inhibition of carbohydrate digesting enzymes is one of the primary targets in the discovery of drugs with antidiabetic effects. These enzymes liberate glucose entering the blood circulation, thereby contributing to the levels of postprandial glucose [
34,
35]. Hence, partial inhibition of these enzymes would result in the reduction of postprandial glucose levels. The extracts of
A. karroo had high inhibitory effects on pancreatic α-amylase compared to acarbose, by more than 3 fold for the defatted and 2 fold for the non-defatted extracts. The observed inhibitory effects against α-amylase could be as a result of polyphenols [
36,
37], in which case, antioxidants in this study could have also influenced the outcome of the inhibitory effects. Also, that defatting of the plant material yielded more compounds with inhibitory effects or unmasks their inhibitory potential against alpha amylase is a possibility. Furthermore, the interactive ability of proteins with phenols, perhaps due to the degree of hydroxylation and conjugation, could be the reason for the observed outcome. To the best of our knowledge, this is the first time this plant is reported to have α-amylase inhibitory effects.
Cytotoxic evaluation of the extracts showed the defatted and non-defatted extracts to be non-toxic against 3T3-L1 and C2C12 cells, with CC50 values >300 μg/ml. However, defatting of the plant material resulted in low toxicity effects against both cell lines. Furthermore, the toxic effects of the extracts were less on the C2C12 cells compared to the 3T3-L1 cells, possibly due to distinctive roles of the cell lines.
Apart from reducing enzyme activity, uptake of glucose from the circulation into peripheral tissues such as the liver, muscle and adipose tissue is the major process regulating blood glucose homeostasis [
38]. It is widely accepted that adipose and muscle tissues are the major site of insulin-mediated glucose disposal due to their large contribution to body mass and serve as great modalities in diabetes studies. A majority of diabetics suffer a great loss of insulin resistance; hence compounds that mimic or increase insulin sensitivity are of major importance in the treatment of diabetes mellitus [
7]. We examined the ability of the extracts of
A. karroo to induce glucose disposal into the muscle and adipose cells. Results obtained revealed that both extracts have the potential of being a source of drug leads for treating diabetes mellitus; this is because both extracts increased glucose uptake at 25 and 50 μg/ml in both cell lines. Defatting of the plant material seemed to have improved the biological efficacy, probably as a consequence of the removal of interfering compounds. Also glucose uptake may differ from cell to cell as seen in our findings, where the C2C12 muscle cells improved glucose uptake as compared to the 3T3-L1 cells, which could be attributed to the distinct roles of different cells, as the compounds present in each extract may have distinctive effects on different cell lines. As to whether the observed glucose uptake is due to insulin mimicking/sensitising effects or other mechanisms is yet to be conclusively elucidated. Furthermore, we could as well not correlate our findings to any hypoglycaemic study on
A. karroo because there are no reports on its hypoglycaemic potential, at least to the best of our knowledge. However, the capacity to which glucose was taken up in a period of 3 h is comparable to available studies on other plants with antidiabetic effects [
8,
39].
Glucose disposal in peripheral tissues is mediated by insulin through special glucose transporters. Any defect in the translocation of these glucose transporters (most importantly GLUT4) from the intracellular vesicles to the surface membrane is associated with insulin resistance, thereby leading to hyperglycaemia. Glucose disposal in the muscle and adipose tissues is dependent upon GLUT4 translocation [
40,
41]. In our study, we determined the major cause of the observed glucose uptake in the C2C12 muscle cells. The reason for this choice was the potential to induce high glucose uptake and low toxicity when treated with the extracts. Furthermore, the extract with overall best enzyme inhibition activity and glucose uptake was chosen; in this case, the defatted extract of
A. karroo at 25 μg/ml. In this assay, a combination of insulin and the extract was included in order to ascertain whether the extract has additive or antagonistic effects when combined with insulin. The results obtained correlate with that of glucose uptake, suggesting that the glucose uptake observed was due to GLUT4 translocation. Furthermore, a combination of insulin and the extract showed antagonistic effects, suggesting the possibility of the extract’s ability to interact with one or more factors associated with insulin-mediated glucose transport signalling pathway, thereby blocking signal transduction caused by insulin. The possible role of chemicals, physical or biological alterations of factors involved in GLUT4 translocation can also not be ruled out. Despite the antagonistic effects observed with the treatment combination, this finding is beneficial since an extract that exerts such effects and equally promote GLUT4 translocation can serve as an alternative to insulin (insulin mimetic) treatment; and could be highly beneficial in the case of absolute absence of insulin and severe insulin resistance.
For GLUT4 molecules to translocate to the surface membrane, activation of the cascade involving various mitogen activated protein kinases and serine/threonine kinases augment to bring about desired effects. Having been certain that the observed glucose uptake was through GLUT4 translocation, we further assessed the modes of action by which the extract induce GLUT4 translocation. The phosphorylation profile of MAPKs [Akt1, Akt pan (Akt1, 2 and 3), p38α, CREB, ERK2, GSK3α/β and GSK3β] which are involved in glucose transport either directly or indirectly were examined using dot blot analysis.
The results obtained indicated that GLUT4 translocation indicated by insulin was through Akt1. Treatment combination of the defatted extract of
A. karroo and insulin was shown to decrease the expression of Akt1, thus buttressing the notion that the plant extract in combination with insulin exert antagonistic effects, which could also be seen in the translocation of GLUT4 to the surface membrane. Contrary to insulin, treatment, the plant extract alone is shown to have completely suppressed the expression of Akt, which may suggest that the observed glucose uptake and GLUT4 translocation might not have been through Akt activation but rather through other pathways such as aPKCλ/ζ or p38α or proteins downstream Akt, although further studies need to be conducted in order to elucidate the exact mechanisms responsible for GLUT4 translocation. It was also evident that the defatted extract of
A. karroo supresses all isoforms of Akt, as seen by the total suppression of Akt pan which is composed of all the three isoforms of Akt. The expression of Akt pan with insulin and the treatment combination could have been entirely through phosphorylation of Akt1 unlike Akt2 and Akt3. These findings are consistent with previous report [
42], where compounds present within plants such as apigenin, kaemferol, quercetin and luteolin among a few, inhibit Akt-mediated glucose uptake, which might explain why Akt was completely suppressed and phosphorylation levels of Akt1 following treatment combination was reduced; this is also suggestive that the plant contains such compounds with the capabilities of inhibiting Akt. A study undertaken [
43] demonstrated that some compounds such as garlic acid within plants inhibit GLUT4 translocation through suppressing Akt phosphorylation but stimulates GLUT4 translocation through aPKC ζ/λ phosphorylation.
Besides insulin as a stimulus, mitogenic or other stress stimulus activates p38α, resulting to glucose uptake via GLUT1 and GLUT4, protein synthesis and monitor cell survival [
44,
45]. This study supports the claims that insulin activates p38α and could also account for the observed GLUT4 translocation among other functions; the phosphorylation levels of p38α due to insulin treatment was significantly higher than the control (
P < 0.01). Surprisingly, the defatted extract of
A. karroo was shown to slightly induce the phosphorylation of p38α than that of insulin and was also significantly different from the control (
P < 0.01). This could suggest that the observed GLUT4 translocation induced by the defatted extract of
A. karroo might be due to activation of proteins downstream of Akt (more especially the p38α) or other unknown pathways. Similarly, a treatment combination of insulin and extract also resulted in the phosphorylation of p38α slightly above that of insulin. Relating GLUT4 translocation induced by the extract alone and the treatment combination to phosphorylation levels of p38α may suggest that high phosphorylation levels of these treatments above that of insulin could be due to other stimuli such as compounds present in the extract. Furthermore, the possibility that p38α serves as the major regulator of GLUT4 translocation for the treatments, particularly the defatted extract of
A. karroo cannot be ruled out.
Akt has also been associated with downstream phosphorylation of glycogen synthase kinases at positions Ser21 for alpha and Ser9 for beta respectively, thereby deactivating them. This enables glycogen synthase (GS) to remain active and promote glucose uptake and glycogen synthesis. Although both isoforms of GSK-3 have no direct bearing on GLUT4 translocation, they help in maintaining glucose regulation and cellular proliferative processes [
46‐
48]. In this study, all the treatment groups exhibited significantly high phosphorylation levels of GSK-3β than the control. This suggests that there was increased glycogen synthesis from all treatment groups and that utilisation of energy for various metabolic processes was rather through a compensatory mechanism than the entire glucose taken up by the cells [
49]. The results of GSK-3α/β phosphorylation were enhanced compared to GSK-3β with an exception of insulin, which suggests that insulin was more involved with phosphorylation of GSK-3β than GSK-3α. These results confirm that the plant extract has the ability to phosphorylate proteins downstream of Akt. Dysregulation of GSK-3 results in insulin resistance as observed in diabetic patients; which could possibly make the extracts beneficial in this aspect.
The cAMP response element binding protein (CREB) is one of the major important proteins that mediate nuclear transcription of various factors depending on the stimuli. The CREB is commonly known to be responsible for adaptation, cell survival, differentiation and proliferation [
50,
51]. CREB can be activated by upstream proteins such as Akt, PKA, PKC, MSK-1, p90RSK and CAMK, each with a desired transcriptional response [
50,
52,
53]. It was not surprising in this study that all treatment groups showed phosphorylation of CREB, even though only the extract and treatment combination of extract and insulin were significantly higher than the control at
P < 0.01 and
P < 0.05, respectively. These results indicate that there was transcription of various proteins responsible for executing desired functions based on the stimuli. Elevated levels in the extract and treatment combination could suggest increased protein transcription probably due to the fact that plants contain an array of compounds which may act as stimulus for transcription of different proteins and as such, the observed differences. Similar to the function of CREB, another protein called ERK-2 has also been associated with transcription of proteins and is involved in differentiation, cell proliferation, migration, adhesion and survival [
54,
55]. Phosphorylation of ERK-2 due to the defatted extract of
A. karroo and insulin was significantly lower than the control (
P < 0.001) and higher in the treatment combination than the control (
P < 0.001). It is not clear as to why the control exhibited very high phosphorylation levels of ERK-2, although it could be suggested that a recovery from starvation might have played a role in various processes which could be compensatory to the cells. Also, slow glucose uptake seems to have played a role in the untreated control relative to insulin and the extract alone. Very high phosphorylation levels of the treatment combination might suggest that there was no interference between the extract and insulin resulting to an additive response. As to whether this might have played a role in the reduction of glucose uptake requires further investigation.