Background
Diabetes mellitus is a disease characterised by chronic hyperglycaemia due to defects in insulin secretion, insulin action or both [
1]. Of the different forms of diabetes, based on pathophysiology, type II diabetes is the predominant form of the disease accounting for 90 % of all cases globally in both developed and developing countries. The pathogenesis of type II diabetes is complex and involves an interaction between genetic susceptibility and environmental factors, especially the consumption of diets that promote obesity coupled with physical inactivity. With an increase in sedentary lifestyles globally, type II diabetes mellitus is considered one of the most rapidly growing non-communicable diseases and is currently the fourth to fifth leading cause of death [
2]. The World Health Organisation (WHO) has estimated that 171 million people were diagnosed with diabetes mellitus in 2000 and predicts an increased prevalence of 366 million cases by 2030 if no action is taken [
3].
While many therapies such as lifestyle intervention with moderate exercise and weight loss with pharmacologic agents can control many aspects of type II diabetes, none has so far convincingly demonstrated an ability to decrease the progressive loss of pancreatic insulin secretory function that eventually requires exogenous insulin supplementation [
4,
5], or the development of other pathological complications. Other concerns associated with the use of conventional treatments are their inherent side effects such as abdominal discomfort, anorexia, diarrhoea, hepatic and renal impairment [
4,
5]. As a result, newer and more effective drugs need to be developed. One proposed method to find a safe and effective therapeutic agent would be to use a medicinal plant which has a history of being safe, effective, low cost and having a lower incidence of adverse effects, although the latter may just be an unproven perception [
6].
Numerous phytochemicals have been recognised for their potential health benefits. Recent studies have indicated that the consumption of polyphenol-rich remedies are associated with reduced risks for a variety of non-communicable diseases including diabetes [
7]. In a previous study, we could show that a
Ficus lutea acetone extract contains polyphenol compounds that influenced the inhibition of α-amylase and α-glucosidase activities [
8], as well as glucose uptake of cells and insulin secretion [
9]. For this study we evaluated
F. lutea acetone extracts by fractionation to determine if potentizing is possible both
in vitro assays and
in vivo in an obese murine model.
Discussion
Our results revealed that the in vitro hypoglycaemic activities of F. lutea extract were in the ethyl acetate and n-butanol fractions (intermediate polarity) with the ethyl acetate fraction having a superior antidiabetic activity. The ethyl acetate fraction containing many different compounds at a concentration of 250 μg/ml had a similar level of activity as the positive control glibenclamide, a pure compound at a concentration of c. 50 μg/ml. This may indicate that the ethyl acetate fractions could contain compounds as effective as or better than glibenclamide.
The ethyl acetate fraction was a polyphenol-rich extract containing compounds including epiafzelechin [
9], and catechins and epicatechins (unpublished data), that potently inhibited α-glucosidase activity and influenced glucose uptake of C2C12 muscle cells and H-4-II-E liver cells as well as enhanced insulin release of RIN-m5F pancreatic cell. In some
Ficus species the polyphenol rich extracts were identified in the intermediate polar fractions either in the ethyl acetate fraction or the butanol fraction [
17,
18], supporting our findings. Polyphenol-rich extracts are known to influence carbohydrate metabolism and glucose homeostasis through various mechanisms of which the most common are delaying of glucose absorption through inhibition of the activities of α-amylase and α-glucosidase thereby blunting post-prandial hyperglycaemia, stimulating of insulin release and increasing the numbers of glucose transporters [
9,
19,
20].
Based on its superior in vitro antidiabetic activity, the ethyl acetate fraction was investigated further for in vivo activity. In this study obesity and its concomitant pre-diabetic condition, characterised by mild pancreatic changes and associated changes in GTT were induced in normal male CD1 mice placed on HCD ad libitum for a total period of 13 weeks. Hereafter they were placed in one of the four treatment plans. Treatment plan 1 was to simulate the practice of failing to reduce caloric intake in a weight loss programme while treatment plan 2 was essentially to simulate the standard practice of decreasing caloric intake in a weight loss programme.
One of the problems with induction diabetic studies is proving that the animals are pre-diabetic for proper evaluation of the efficacy results. For this study we believe that the animals were pre-diabetic for the following reasons. The mice on the high caloric induction diet showed an increase in body mass and obesity, with elevated blood glucose concentrations. The trend towards diabetic nephropathy which develops as a result of chronic hyperglycaemia was also present in the study animals as seen with the increase in plasma creatinine and urea concentrations [
21,
22]. The histopathology report also indicated that the animals were pre-diabetic as the animals had moderate to excessive fat deposit in the abdominal cavity, mild to moderate cell swelling with vacuolated changes within the cytoplasm of the hepatocytes with accumulation of fatty acids suggesting metabolic-induced fatty acid changes of the liver and enlargement of the pancreatic islets of β-cell [
23,
24].
With insulin resistance being fundamental in the pathogenesis of type II diabetes, intervention is initially aimed towards improvement in tissue sensitivity/responsiveness with modulation of weight being suggested as a better treatment modality. Weight loss is known to improve insulin sensitivity and overall glycaemic control, and to decrease mortality rates [
25]. While a change in diet alone can result in moderate weight loss, this is difficult to achieve in the long term as weight lost is slowly regained due to the physiological abnormalities induced by obesity [
25]. As a result it has been suggested that weight loss may be enhanced through combination with pharmacological agents that can reduce or control weight by altering appetite, metabolism, decreased fat absorption or consumption of calories [
26,
27]. Of these, much emphasis has been place on the inhibition of the glucosidase and amylase enzymes. Inhibition of these enzymes, particularly of α-glucosidase in the small intestine, which catabolises non-absorbable complex carbohydrates into absorbable monosaccharides will modulate blood glucose concentration and weight gain [
28]. The α-glucosidase and α-amylase enzyme inhibitors regulate blood glucose, especially postprandial blood glucose by limiting the rate of starch and sucrose metabolism, delaying the absorption of glucose and fructose in the gastrointestinal tract and the gastric emptying rate, which further alters the secretion of insulin [
28]. In addition, the limiting the rate of starch metabolism may promote weight loss by reducing the digestive availability of carbohydrate derived calories [
29].
The search for newer weight controlling agents from plant polyphenols may be an alternate source or adjunct means of controlling diabetes [
30]. For this reason the obese mice were exposed to the ethyl acetate fraction of
F. lutea, in an attempt to establish the effect of the extract in the pre-diabetic condition in the presence or absence of dietary modification. Since the animals were allowed
ad lib. access to food, the test extract was included within the food. The reason for this was that acarbose, a known α-glucosidase inhibitor, is recommended for use on consumption of the main meals [
31]. For this study, the mice placed on treatment without a change in their caloric intake maintained a steady weight throughout the study in comparison to further increases in the control animals which received the HCD alone. This suggests that in cases of high caloric intake, the ethyl acetate fraction of
F. lutea could potentially mitigate further weight gain. When the mice were switched from high caloric to a normo-caloric intake, it did not lead to statistically significant gain or loss of weight, in the presence or absence of the
F. lutea ethyl acetate fraction. This indicates that the extract fraction in itself was not a weight loss agent. The failure of the mice to lose weight when switched from the high caloric to the normo-caloric diet was also not unexpected as other authors found that switching to a low caloric diet is required for a substantial weight loss [
30,
32]. This was similar to findings of Veerapur et al. [
33], who observed that
Ficus racemosa extract did not affect body weight and food intake in high fat diet (HFD) fed Albino Wistar male rats. Also supplementation of feed with cyanidin 3-glucoside did not affect the body weight in either HFD and
db/
db male mice or significantly change food intake during the experimental period [
34]. Hou et al. [
35] found no significance body weight change between the group receiving a normal standard diet and high carbohydrate – high fat diet (HC-HF) as well as between the groups of the HC-HF diet and metformin administration.
Further evidence supporting the inability of the ethyl acetate fraction of
F. lutea to stimulate weight loss can be seen with the failure of the extract to induce a change in either faecal weight or the extent of nutrient absorption in the mice per treatment group in comparison to their control group. This differed from other studies, where the faecal weights of animals fed polyphenol-rich diet were significantly higher than those on a control diet [
36,
37]. These authors speculated that polyphenols adsorb cholesterol, bile acids and dietary lipid thereby increasing faecal excretion. The reason for the failure of the ethyl acetate fraction to induce similar changes is unclear, but it could be speculated that in contrast to other studies, the ethyl acetate fraction of
F. lutea does not inhibit the activity of pancreatic lipase, a key enzyme in the digestion and absorption of fat [
36,
37].
An important result of this study was the decrease in the AUC of the glucose concentration versus time profiles in those animals that were treated with both a change in diet and the plant extract. With all the treatment groups showing the same time to peak in blood glucose concentrations (Tmax), conventional pharmacological principles indicate that the induced changes were not due to a change in the rate of glucose absorption, but rather to a change in the total extent of absorption or an increased depletion rate of plasma concentrations subsequent to absorption. Based on the in vitro effect of the plant extract, both these effects are possible. However considering that the plant extract failed to induce a change in faecal weights, the more likely mechanism is an increase in the depletion of glucose within the plasma. Since the latter is linked to increased uptake within the cells, the most likely mechanism is either an increased insulin response or increased insulin secretion. From the in vitro work, we believe that the effect is due to the stimulation of insulin secretion and perhaps not inhibition of activities of the α-amylase and α-glucosidase.
Competing interests
The authors declare that they have no competing interest.
Authors’ contributions
OOO carried out the study and wrote the manuscript; IJ assisted with the animal study; VN and JNE contributed to conception, design, analysis and interpretation of data; LJM assisted with cell culture assays, and LJM, JNE and VN assisted with and supervised the manuscript writing. All authors have read and approved the final manuscript.