via anti-inflammatory activity and inhibition of carbohydrate metabolizing enzymes
T. Olabisi Obafemia,b , M. Tolulope Olaleyea , Afolabi C. Akinmoladuna
Ethnopharmacological relevance: Miracle fruit plant (Synsepalum dulcificum) has many applications in folk medicine. The leaves are used traditionally to treat diabetes and other diseases. The antidiabetic potential of Synsepalum dulcificum leaves in rats has been studied but the mechanisms involved are yet to be elucidated.
Aim: The present study aimed to provide insight into the antidiabetic mechanisms of methanol and flavonoidrich leaf extracts of Synsepalum dulcificum (MSD and FSD, respectively).
Methods: In vivo effects of administering 30 mg/kg or 60 mg/kg MSD and FSD for 21 consecutive days to rats after type II diabetes was induced through 14 days of fructose feeding and injection of one dose of streptozotocin, were assessed. Glibenclamide (5 mg/kg) served as the reference drug. In addition, in vitro inhibitory activity of MSD and FSD on the carbohydrate metabolizing enzymes, α-amylase and glucokinase, were evaluated, with acarbose as the reference drug. Moreover, in silico analyses to elucidate the contribution of key polyphenolics to the antidiabetic activity of the extracts through docking with glucokinase were performed.
Results: MSD and FSD significantly reduced HbA1c and serum levels of interleukin-6 and TNF-α (p < 0.05) in diabetic animals. Conversely, serum level of insulin and hepatic hexokinase activity were increased (p < 0.05) in extract treated groups. Both extracts showed α-amylase and α-glucosidase inhibitory activities. Quercetin, caffeic acid and chlorogenic acid in extracts showed strong binding affinities with glucokinase in the molecular docking analyses.
Conclusion: Results from this study indicate that increased insulin synthesis, reduction of inflammation and inhibition of carbohydrate metabolizing enzymes are likely mechanisms by which MSD and FSD exert antidiabetic action in type II diabetic rats.
Diabetes mellitus is characterized by hyperglycemia resulting from either the inability of the pancreas to produce adequate insulin or the ineffective use of the insulin produced by the pancreas (World Health Organization, 2016). Hyperglycemia, which can result from persistent diabetes may lead to heart, blood vessels, eyes, kidneys and nerves damage. Recent studies show that about 451 million people have diabetes worldwide in 2017, a figure that is expected to increase to 693 million by 2045. It was further revealed that about half of the people living with diabetes are undiagnosed.
Multiple studies support the concept that inflammation strongly associates with insulin resistance (IR), which, in addition to loss of islet function, defines type II diabetes (T2D) (Rehman and Akash, 2016). Many approaches which target inflammation as a means of managing type II diabetes have been investigated, however none has been used in clinical practice (Garber et al., 2015). Moreover, chronic subclinical inflammation has been described as the specific mechanism by which insulin resistance leads to type II diabetes mellitus (Odegaard et al., 2016). T the chief acute phase reactant has been shown to be a powerful independent risk predictor of type II diabetes (Bao et al., 2015). In vivo production of TNF-α increases under chronic hyperglycemia and may exacerbate insulin resistance and eventually contribute to the development of diabetic complications (Akash et al., 2018). The importance of glycated hemoglobin in the diagnosis of diabetes has been advocated, largely on the basis of its established association with microvascular diseases. Estimated risk of cardiovascular disease (CVD) was shown to increase by 18% for every 1% increase in absolute HbA1c value in diabetics (Memon and Soomro, 2016). Also, hepatic hexokinase (glucokinase) expression is insulin-controlled and is an essential molecular target for the development of antidiabetic drugs (Choi et al., 2013). Glucokinase activators are emerging as a new and appealing class of drugs for managing type II diabetes through mechanisms that differ from other oral antidiabetic agents, including insulin. It was reported that an agent that activates the enzyme lowered blood glucose in a dose-dependent manner without medically significant side effects (Matschinsky et al., 2011). Molecular docking is a widely used computer analytical procedure to predict the conformation of receptor-ligand complex and this process has become a vital means of identifying novel drugs. The molecular docking method determines interaction between ligand and target molecule and predicts binding affinity of ligand to form a stable complex with protein by finding preferred orientation of minimum free binding energy (Ferreira et al., 2015).
Synsepalum dulcificum (Scumach. & Thonn.) Daniell otherwise known as miracle plant belongs to the Sapotacae family. It is native to West Africa and known as Agbayun by the Yorubas and Uni by the Ibos of Nigeria. S. dulcificum also has many applications in folk medicine and is used either raw or processed (e.g. powder, infusion, or decoction). A clear understanding of the mechanisms of action of antidiabetic drugs is essential in order to optimize treatment of diabetic patients and attain the target for glycemic control as set by various clinical bodies (Bösenberg and Zyl, 2008). Antidiabetic medicinal plants owe their activity to the presence of phytoconstituents such as alkaloids, flavonoids, phenolic acids, saponins and terpenoids and plants exert their antidiabetic effects via several mechanisms including insulin secretion, inhibition of carbohydrate metabolizing enzymes and regeneration of beta-cells (Kumar and Veeranjaneyulu, 2018). The present study therefore aimed to establish the antidiabetic action of Synsepalum dulcificum leaf extracts in a type II model of diabetes and elucidate the probable mechanisms involved along the lines of inhibition of carbohydrate metabolizing enzymes, inflammation, activation of hexokinaseIV activity and insulin secretion.
Materials and Methods
2.1. Chemicals Glibenclamide, sodium citrate, citric acid, acarbose, streptozotocin, quercetin, quercitrin, isoquercitrin, rutin, catechin, epicatechin and kaempferol were purchased from Sigma-Aldrich (St-Louis, MO, USA). Acetonitrile, formic acid, gallic acid, chlorogenic acid, ellagic acid and caffeic acid purchased from Merck (Darmstadt, Germany). All other reagents used were analytical grade.
2.2. Plant material and extraction Synsepalum dulcificum (Schumach. & Thonn.) Daniell Sapotaceae leaves were obtained from a farmland in Olode Village, Osun State, Nigeria in June 2015 and authenticated at the Botany Department, University of Ibadan, Ibadan, Nigeria (voucher number: UIH-22457). The plant name was checked at http://www.theplantlist.org and found to be approved. The leaves were air-dried for three weeks, pulverized, and ground sample (700 g) extracted in 80% methanol by maceration for 72 h. The methanol extract was concentrated in a rotary evaporator, lyophilized and preserved for further use. The yield of extract was 121.46 g (17.35%). Flavonoid-rich fraction (FSD) was obtained from the methanol extract (MSD) according to the method of Chu et al. (2002). Identification of phenolic compounds was performed by comparing their retention time and UV absorption spectrum with those of commercial standards. All chromatography operations were carried out at ambient temperature and in triplicate.
2.4. Experimental animals Male Wistar rats weighing 200 ± 20 g were obtained from the Animal Care Facility, Afe Babalola University, Ado-Ekiti, Nigeria. The animals were allowed to acclimatize for seven days before commencement of the study. They had unfettered access to standard chow (Ladokun farms Ibadan, Nigeria) and water. The study adhered to the Principles of Laboratory Animal Care (NIH publication 85−23, revised in 1985). Ethical clearance (No: ABUAD/ET/2017/091) was obtained for the study from the university's ethical committee.
2.5. Experimental design Diabetes was induced in rats through intraperitoneal injection of 40 mg/kg body weight streptozotocin dissolved in ice-cold 0.1 M citrate buffer (pH 4.5), after administration of 10% fructose in their drinking Abbreviations CVD Cardiovascular disease HBA1c Hemoglobin A1c FSD flavonoid-rich leaf extract of Synsepalum dulcificum LOD limit of detection LOQ limit of quantification MSD methanol leaf extract of Synsepalum dulcificum TNF-α Tumor necrosis factor alpha T.O. Obafemi, et al. Journal of Ethnopharmacology 244 (2019) 112124 2 water for 14 days (Wilson and Islam, 2012). Type II diabetes is characterized by impaired insulin secretion due to loss of beta cells and insulin resistance (Diabetes Canada Clinical Practice Guidelines Expert Committee et al., 2018). Diabetes was confirmed after 72 h and animals with blood glucose ≥250 mg/dl were considered diabetic and used for the study. Distilled water was used as a vehicle and administered to both control groups. Treatment was carried out orally for 21 days after confirmation of diabetes.
2.5.1. Preparation of blood sample and liver homogenate Twenty-four hours after the last treatment, rats were sacrificed by cervical dislocation and blood collected through cardiac puncture into EDTA bottles and plain bottles. Blood in a plain bottle was centrifuged at 4000 rpm for 10 min to separate the serum. Both serum and whole blood were stored at −20 °C until used. Livers were rapidly excised and placed in saline solution on ice. The organs were weighed after being dried with a paper towel and homogenized in a cold Tris-HCl buffer (1:10 w/v). The homogenate was centrifuged at 3000×g for 10 min to yield the clear supernatant used for biochemical estimations.
2.5.2. Biochemical analyses Serum glucose level was estimated using a Randox assay kit. Alphaamylase inhibitory activity was estimated as previously reported (Adisakwattana et al., 2009). Alpha-glucosidase inhibitory activity was estimated according to an earlier reported method (Kim et al., 2005). Serum insulin, IL-6 and TNF-α were estimated using enzyme-linked immunosorbent assay (ELISA) with kits from Elabscience Biotechnology (Houston, Texas, USA). HbA1c was evaluated using a previously described method (Nayak and Pattabiraman, 1981). Hepatic hexokinase activity was measured following as previously reported (Shalaby et al., 2014).
2.6. Homeostasis model assessment (HOMA) Homeostasis model assessment (HOMA-IR) was calculated using the formula: HOMA-IR = (fasting glucose (mg/dl) x fasting insulin (mmol/l)/ 405 (Matthews et al., 1985). Homeostasis model assessment of β-cell function (HOMA-B) was calculated using the formula: HOMA-B = (360 x fasting insulin (mmol/ l))/(fasting glucose (mg/dl) – 63) (Matthews et al., 1985).
2.7. Molecular docking analyses The three dimensional structure of Hexokinase IV/Glucokinase (1V4T) was downloaded from Protein Databank (http://www.rcsb.org). The structure of quercetin (5280343), rutin (5280805), chlorogenic acid (1794427) and caffeic acid (689043) were downloaded from https://pubchem.ncbi.nlm.nih.gov/compound and were then converted to PDB format using Open Babel 2.2.1 for docking studies. Molecular Docking was carried out using Autodock vina. The Root Mean Square Deviation (RMSD) and Affinity Energy (Trott and Olson, 2010) were used in selecting the best interaction prose.
2.8. Statistical analysis Results were expressed as mean ± standard error of mean (SEM). Data analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey-test using GraphPad Prism 5. p < 0.05 was considered significant.
The extracts showed appreciable and concentration-dependent alpha amylase inhibitory potential with FSD showing a comparatively higher activity than MSD (p < 0.05) especially at the higher concentrations tested. However, both MSD and FSD displayed moderate alpha glucosidase inhibitory activities which were not significantly different (p > 0.05) at all concentrations tested. The HPLC fingerprints of MSD and FSD revealed some of the constituent bioactive polyphenols. In in vivo experiments, extracts at both doses effectively lowered serum glucose level of diabetic after 21 days of treatment. In addition, hexokinase activity was increased in extract treated groups when compared with diabetic controls.
Traditional methods of drug design could be costly, slow, and inefficient necessitating the introduction of better rational methods involving virtual screening. In the present study, docking analysis of the most abundant polyphenols present in MSD and FSD (quercetin, rutin, chlorogenic acid and caffeic acid) showed that they could bind with relatively strong binding affinities with glucokinase, with rutin having the highest bond affinity while caffeic acid had the lowest. However, it was observed that only quercetin, caffeic acid and chlorogenic acid met Lipinski's rule of 5 which stipulates the criteria for an orally administered drug to be bioavailable and drug-like (Lipinski et al., 2001). This suggests that quercetin, caffeic acid and chlorogenic acid might significantly contribute to the increased glucokinase activity observed in the extracts and glibenclamide treated rats in this study.
MSD and FSD showed marked improvements in most parameters investigated and outperformed glibenclamide, the reference drug, in some instances. This study indicated that the mechanisms of action of Synsepalum dulcificum leaves against TIIDM might include increased insulin synthesis, reduction of inflammation and modulation of carbohydrate metabolizing enzymes. The formulation of the extracts into drugs is suggested after further studies including elucidation of the molecular mechanisms of the observed effects of the extracts and clinical evaluation. Author declaration We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Author contributions TOO, MTO and ACA contributed to the design of the work. TOO carried out most of the bench work and drafted the manuscript. MTO and ACA provided laboratory space, supervised the work and corrected the manuscript draft. All authors agreed to the final form of the manuscript.
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