Hypoglycaemic activity of ethanolic extract of Garcinia mangostana Linn. in normoglycaemic and streptozotocin-induced diabetic rats

The number of diabetic mellitus (DM) cases is rapidly increasing in recent years. According to the National Diabetes Statistics Report [1], approximately 29.1 million Americans, or 9.3 % of the population, had diabetes in 2012 with around 8.1 million of them were undiagnosed. Moreover, there were about 3.3 million cases of diabetes reported in Malaysia in 2015, according to the International Diabetes Federation [2]. Diabetes mellitus is a chronic disease characterised by the high blood glucose levels as a result of impaired glucose metabolism. Impaired glucose metabolism occurs when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces [3]. Hyperglycaemia, or raised blood sugar, is a general effect of uncontrolled diabetes that eventually leads to severe damage to many of the body’s systems, especially the nerves and blood vessels. Elevated blood glucose triggers oxidative damage of cell membranes through the production of superoxide anions, which generate hydroxyl radicals via Haber-Weiss reaction, inducing peroxidation of membrane lipids and protein glycation. These radicals further cause derangement of major biomolecules including carbohydrate, protein, lipid, and DNA [3]. Antioxidants play an important role in the prevention of cell damage against reactive oxygen species (ROS). Thus, herbal preparations containing both antioxidants and hypoglycaemic properties would be useful in diabetic management.

Garcinia mangostana Linn., generally known as mangosteen and belongs to the family Clusiaceae, is a tree cultivated for centuries in the tropical rainforest. Its fruit hull is used as phytomedicine in the Southeast Asia for the treatment of skin and wound infection [4], inflammation, diarrhoea [5], cholera, and dysentery [6]. Phytochemical studies of G. mangostana extracts (GME) report that this plant contains a variety of secondary metabolites such as oxygenated and prenylated xanthones [7]. Moreover, 50 xanthones have been identified in G. mangostana [8]. The ethnopharmacological views of the fruit rinds suggest remarkable properties such as antioxidants [9], antitumour [10], anti-inflammatory [11], analgesic [12], antiviral activities [13], cardioprotective effects [14], antifungal [15], antiallergy [16], antibacterial [17], antituberculosis [18], and immunomodulation [19]. Xanthone backbones of mangosteen have been reported as a potent molecular basis for in vitro α-glucosidase inhibition and proven to reduce postprandial hyperglycaemic condition by suppressing the glucose absorption [20]. The extract of G. mangostana has also been reported to retard glucose absorption through inhibition of carbohydrate-hydrolysing enzymes such as α-glucosidase and α-amylase [21]. The major component of the extract, α-mangostin, demonstrates a protective role in sexual dysfunction and exerts an antidiabetic effect in streptozotocin-induced diabetic male rats possibly via the reduction of blood glucose levels [22]. Preliminary studies in our laboratory indicated potent antioxidant and hypoglycaemic effects of crude ethanolic extracts on normoglycaemic models in rats. Hence, the present study was performed to evaluate the hypoglycaemic efficacy of crude ethanolic extracts of GME on the normal and streptozotocin-induced diabetic rats.

Diabetes mellitus is a metabolic disorder characterised by derangement in carbohydrate, protein, and fat metabolisms resulting from impairment in glucose homeostasis, lack of insulin secretion, and development of macro- and microvascular dysfunctions [23]. Modern antidiabetic agents like sulphonylureas, thiazolidinediones, and biguanides are commercially prescribed but failed to provide a prolonged glycaemic control effect. Medicinal plants have been widely used to treat diabetes due to their effectiveness, safety, affordability, and acceptability. The present study highlighted the potential hypoglycaemic action of GME in normal and diabetic-induced rats. In this study, diabetes was induced in the rats using 50 mg/kg STZ, a glucosamine derivative of nitrosurea, which selectively destroys pancreatic islets of β-cells and causes development of hyperglycaemia and glycosuria, as seen in type 1 diabetic patients. Glibenclamide (glyburide), a member of the second generation sulphonylureas, provides an effective treatment for patients with moderate diabetes. Other than its glucose-lowering properties, glibenclamide seems to have antioxidant properties and is capable of restoring liver antioxidant enzymes, superoxide dismutase (SOD), and catalase (CAT) in STZ-induced diabetic rats [24]. The increase in blood glucose level might be due to shortage of insulin as a result of destruction of pancreas interceded by STZ action, which boosts ATP dephosphorylation, which in turn generates superoxide anions, hydrogen peroxide, and hydroxyl radicals. Elevated intracellular peroxides in pancreatic islets further mediate a damage induced by reactive oxygen species (ROS) [25]. Under hyperglycaemic conditions, antioxidants are supposed to regenerate damaged extracellular matrix proteins and cell growth as a result of ROS elevation through nonenzymatic glycation of proteins, as well as through auto-oxidation [26]. This indication marks the increased levels of indicators of oxidative stress in diabetic patients. Because oxidative stress is associated with pathogenesis of diabetes, therefore, it can be postulated that the antioxidants may exert a key role in the management of DM.

Mangosteen’s pericarps, leaves, and barks have been used traditionally to treat various ailments such as arthritis, diarrhoea, dysentery, inflammation, and skin disorders, besides being applied in the healing of wound [27]. However, no study has addressed its hypoglycaemic effect although it has been commercially claimed as an antidiabetic agent. To date, numerous articles have reported the use of in vitro assays to evaluate the pharmacological effect of G. mangostana’s purified compounds mainly α-mangostin, which is the major bioactive secondary metabolite of xanthone derivatives [28] and the first substance to be isolated [29]. There are limited animal models testing on α-mangostin, which could be attributed to its low bioavailability via oral treatment when compared to the i.v. administration [28]. Although there is an issue of low bioavailability when α-mangostin is given orally, GME is still given orally and tested in diabetic animal models as its beneficial bioactive components consist of not only pure conjugated xanthones, but also a combination of free and unconjugated compounds [30]. This combination, as far as the pharmacokinetic is concerned, can improve the bioavailability of GME. This fact could be one of the best evidences of better health-promoting properties of the extract over the administration of pure compounds like xanthones. Furthermore, the extract has been reported to exhibit α-glucosidase inhibitory activity, which has been proven to reduce postprandial hyperglycaemia by inhibiting glucose absorption [31]. Moreover, the ethanol extract of G. mangostana also shows a significant hypoglycaemic effect following the oral maltose administration [20], and the effect is similar to the reference drug, acarbose [32].

In this study, for the normoglycaemic rats, significant blood glucose-lowering actions were observed in GME- and glibenclamide-treated groups when compared to the normal control group. The effect of GME on normoglycaemic animals suggests that the pericarp of G. mangostana has a mild lowering effect on normal glucose levels. This effect was comparable to that of glibenclamide, an insulin secretagogue, which also lowers blood glucose in normal animals. Provided the β-cells are fully functional, sulphonylureas such as glibenclamide can cause hypoglycaemia because insulin release is initiated even when glucose concentrations are below the normal threshold for glucose-stimulated insulin release (approximately 5 mmol/L or 90 mg/dL) [33]. In the same study on STZ-induced rats, we reported the increase of hypoglycaemic action of glibenclamide and GME in a dose-dependent manner. This was indicative of the potentiation of GME on insulin secretion and the enhanced peripheral utilisation of insulin in diabetic rats as supported by the histopathological findings. Glibenclamide was used as a reference hypoglycaemic agent to compare the efficacy of a variety of glucose-reducing compounds via enhanced activity of β-cells of the pancreas resulting in the secretion of larger amounts of insulin. In the multiple-dosage study, the elevated blood glucose improved after 28 days of GME treatment, suggesting that the extract’s antioxidant activity mediated the protective effect of β-cells in the diabetic rats. It is widely accepted that the enhancement of antioxidant activity could be one of the mechanisms responsible in the prevention of diabetic complications [34].

The STZ-induced rats initially showed a reduction in their body weight, suggesting abnormal water and food intake. However, the administration of GME in diabetic rats improved their weight commencing from Day 14 onwards in comparison to the diabetic control group. The increase in body weight could be due to amelioration of glycaemic control and structural proteins synthesis [35]. This finding was actually contradicting the finding made by Chivapat et al. [36], who reported that GME at the dose of 1,000 mg/kg or above caused reduction in the body weight of male rats. This observation was related to the effect of condensed tannins in the extract of G. mangostana pericarp [37] that are believed to be present in a high quantity due to the high dose (1,000 mg/kg) of GME used [36]. In other studies, the mice fed with a diet containing tannic acid underwent a growth retardation [38], while those fed with a high dose of tannins suffered from a slower growth rate when compared to the mice that consumed a low dose of tannins [39]. It is plausible to suggest that the amount of condensed tannins present in the GME at the dose of 50–200 mg/kg as used in the present study was not enough to trigger body weight reduction. In addition, weight loss in the diabetic rats could be the result of high catabolic rate of protein to amino acid for gluconeogenesis during insulin deficiency, which could be enhanced in the presence of enough concentration of tannins [39]. In the present study, the lack of protein catabolism could be related to the insufficient amount of condensed tannins present in the GME.

The high cholesterol and lipid levels mark an increased risk of atherosclerosis and coronary heart diseases development, which are considered secondary complications of diabetes [40, 41]. In the present study, GME was found to reduce the total cholesterol and triglycerides in STZ-treated rats, suggesting the extract’s capability to reduce atherogenic-related complications. Liver plays a crucial function in the metabolism, storage, detoxification, and excretion of xenobiotics and their metabolites. Liver function tests provide vital indicators of liver activities, which include SGOT, SGPT, and ALP [42]. High levels of these enzymes in the blood indicated leakage from the liver cytosol into the bloodstream, indicating a liver injury [42]. In the present study, GME reduced the levels of important liver enzymes, particularly SGOT and SGPT, in all the treated groups. Because of the promising reduction of liver transaminases, long-term studies should be conducted to observe the improvement of liver function markers following the consumption of GME. It is important to highlight that the effect of GME on the level of liver enzymes was observed in a dose-independent manner with the maximum reduction observed at the dose of 100 mg/kg. This observation could be associated with the phenomenon known as “therapeutic windows” [43], which could possibly be related to the optimal hepatoprotective effect of GME or reduction in the enzyme activity. Other than that, GME was also observed to cause an increase in the urea and creatinine levels, which were reduced in the diabetic groups. The ability of diabetic rats to decrease both parameters seems to suggest that diabetes could lead to renal dysfunction. The significant improvement in the level of renal markers indicated the potential of GME to ameliorate the kidney dysfunction. Additionally, the total protein content was also found to improve in the diabetic rats following a treatment with GME, suggesting the remedial role of the extract towards the kidney function in the diabetic rats. Histopathological examination revealed an improved preservation of normal pancreatic islets and diminished necrotic changes as compared to the STZ-induced diabetic rats. The effect of GME might be due to the pancreatic rejuvenation via enhanced protein synthesis, accelerated detoxification, potentiation of antioxidant defence, and neutralisation of free radicals, which confirmed the insulinotropic effect of GME through the regeneration of insulin-producing β-cells.

The hypoglycaemic activity of GME could also be attributable to the extract’s phytoconstituents. Polyphenolics like tannins have been reported to be present in the pericarp of G. mangostana [44]. In addition, several polyphenols have been identified from the pericarp namely 4-aryl-2-flavanylbenzopyran derivative, 3,4,3′,5’-tetrahydroxy-5-methoxybenzophenone, 2,3-dihydrochromone derivative, epicatechin, and procyanidin B2 [45]. Previous studies have also reported the presence of various types of xanthones in different parts of G. mangostana, including the pericarp. Some of the identified xanthones derived from pericarp are mangostinone, α-mangostin, β-mangostin, γ-mangostin, gartanin, garcinone E, 1,5-dihydroxy-2-(3-methylbut-2-enyl)-3- methoxyxanthone, 1,7-dihydroxy-2-(3-methylbut-2-enyl)-3-methoxyxanthone [46], 8-hydroxycudraxanthone G, mangostingone, cudraxanthone G, 8-deoxygartanin, garcimangosone B, garcinone D, garcinone E, gartanin, 1-isomangostin, mangostinone, smeathxanthone A, tovophyllin A, mangostanaxanthones I, mangostanaxanthones II, 9-hydroxycalabaxanthone, parvifolixanthone C, rubraxanthone [47], 1,3,7-trihydroxy-2-(3-methyl-2-butenyl)-8-(3-hydroxy-3-methylbutyl)-xanthone, 1,3,8-trihydroxy-2-(3-methyl-2-butenyl)-4-(3-hydroxy-3-methylbutanoyl)-xanthone, garcinones C, garcinones D, gartanin, xanthone I [48], garcimangosxanthone F (1), garcimangosxanthone G [49], and mangostanate [50].

The presence of xanthones and polyphenols like tannins in the pericarp of G. mangostana could also be used to explain the hypoglycaemic activity of GME observed. Tannins, one of the major groups of antioxidant polyphenols, can be classified into two broad groups namely hydrolysable tannins and condensed tannins. Hydrolysable tannins are molecules with a polyol (D-glucose) as a central core. The hydroxyl groups of these carbohydrates are partially or totally esterified with phenolic groups (i.e., gallic acid or ellagic acid). Hydrolysable tannins are usually present in low amounts in plants and are easily hydrolysed by mild acids and bases to yield carbohydrate and phenolic acids. Condensed tannins are a group of naturally occurring polyphenolic bioflavonoids, specifically taking the form of oligomers or polymers of polyhydroxy flavan-3-ol units such as (+)-catechin and (−)-epicatechin. Condensed tannins are more common in the plant kingdom and have been reported to have a wide range of biological and pharmacological activities including antioxidative activity without inducing significant toxicological effects [51]. These protective effects are related to their capacity (a) to act as free radical scavengers and (b) to stimulate antioxidant enzymes. Moreover, tannins have been observed to improve the glucose uptake through mediators of the insulin-signalling pathways (i.e., phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (p38 MAPK) activations and GLUT-4 translocation. The reduction in glycaemia (blood glucose levels) caused by tannins and other phenolic compounds has been attributed to actions such as (a) a decrease in the absorption of nutrients (b) a decrease in food intake (c) initiation of β-cell regeneration, (d) direct action on adipose cells that enhances insulin activity, and (e) sharing insulin-signalling pathways in hepatocytes possibly by modulation of the redox state of cell [51]. In other words, tannins work to establish an organised network of metabolic sensors that incorporates glucose homeostasis, lipid metabolism, inflammation, drug metabolism, bile acid synthesis, and some other processes. One of the restorative approaches to decrease postprandial hyperglycaemia is by preventing or delaying the absorption of glucose via inhibition of carbohydrate hydrolysing enzymes, α-amylase, and α-glucosidase in the digestive organs. Interestingly, tannins are natural inhibitors of α-amylase and α-glucosidase with a potent inhibitory effect on the latter, but a mild inhibitory effect on the former. Therefore, phenolic antioxidant-mediated inhibition of these enzymes can significantly decrease the postprandial hyperglycaemia after ingestion of a mixed carbohydrate diet and could be an effective strategy in the control of diabetes [51]. Epicatechin, one of the natural condensed tannins present in G. mangostana pericarp [45], prevents hyperglycaemia by inducing β-cell regeneration [52]. This effect of epicatechin is supported by the histopathological examination of the rat’s liver tissue treated with GME in comparison to the tissue of diabetic rats. Taking other reports into consideration, several other possible mechanisms of hypoglycaemic condition could also be suggested to be triggered by tannins in order to induce hypoglycaemia, and these mechanisms include (a) reduction in food intake, (b) inhibition of intestinal glucose absorption, and (c) direct action on adipose cells by enhancing insulin activity in rat epididymal adipocytes. Catechins are powerful antioxidants that inhibit oxidation of LDL-cholesterol, reduce cholesterol levels, and reduce body fat, resulting in a decreased risk of heart disease.

Other than that, several xanthones have also been reported to exert antihyperglycaemic activity. According to Jariyapongskul et al. [53], the damage in the retinal microvasculature in type 2 diabetic rat model such as reduction of ocular blood flow (OBF) and leakage of the blood-retinal barrier (BRB) permeability is associated with hyperglycaemia and the accumulations of free radicals, advanced glycation end products (AGEs), receptor of advanced glycation end products (RAGE), tumour necrosis factor alpha (TNF-α), and vascular endothelial growth factor (VEGF) levels in the retinal tissues. α-Mangostin has also been reported to prevent retinal microvascular complications in diabetic rats possibly via its antihyperglycaemic, antioxidant, anti-inflammatory, and antiglycation properties [53]. Other xanthone, mangiferin reduces the level of blood glucose by increasing the level of insulin in STZ-induce diabetic rats. Moreover, mangiferin also increases the level of hexokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, glycogen synthase, and glycogen content to near normal in diabetic rats. The levels of these enzymes are reduced following a treatment with STZ. On the other hand, the activities of lactate dehydrogenase, glucose-6-phosphatase, fructose-1,6-diphosphatase, and glycogen phosphorylase in the liver tissue of diabetic rats are decreased after a treatment with mangiferin [54]. Besides, mangiferin has also been shown to exert an antidiabetic activity in KK-Ay mice (an animal model of type-2 diabetes) by decreasing insulin resistance [55]. Moreover, several xanthones isolated from the fruit case of G. mangostana have also been reported to exert α-glucosidase inhibitory activity [56].