Antihyperglycemic Mechanisms of Allium sativum, Citrus sinensis and Persea americana Extracts: Effects on Inhibition of Digestive Enzymes, Glucose Adsorption and Absorption on Yeast Cells and Psoas Muscles

*Corresponding author: Boris Gabin Kingue Azantsa*, Guy Roussel Takuissu, Etienne Junior Tcheumeni, Martin Fonkoua, Edwige Ruth Kemadjou Dibacto, Judith Laure Ngondi and Julius Enyong Oben

Full-Text PDF:

original research

Abstract

Mechanisms by which some plants with antihyperglycemic effects reduce postprandial hyperglycemiaare not fully elucidated. This study was designed to investigate some action mechanisms of extracts from stem bark of Citrus sinensis, seeds of Persea americana and bulbs of Allium sativum including in vitro inhibition of α-amylase and invertase; glucophagic capacity, absorption capacity on yeast cells and psoas tissues.

Introduction

Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder of carbohydrates, fats and proteins resulting in an inability of insulinaction and/or secretion. T2DM is characterized by chronic hyperglycaemia with perturbations of glucose homeostasis due to absence of regulation of postprandial glycaemia. Postprandial glyceamia is at the onset of disturbances of glucose tolerance. Which appears earlier before high-levels of fasting blood glucose. Several factors influence postprandial glycaemia: foods rich in carbohydrates, glucose digestion and absorption, insulin secretion as a result of rise of glucose levels, incretins actions and glucose intake by cells. Postprandial glucose is also associated to protein glycosylation to the generation of reactive oxygen species which attack DNA and membrane lipids, to increase plasma lipid.4,5 It is also a risk factor for cardiovascular diseases.

Disturbances related to the glucose homeostasis as well as complications associated to dyslipidemia complexify management of diabetes, leading to alarming and increasing prevalence. In fact, 451 million individuals aged 18-99 years were reported to suffer from diabetes with more then 693 million of patients projected by 2045. Such increasing prevalence is a public health concern and constitutes economic burden for governments. It is why efforts are made everyday to improve health of diabetic patients. Several oral drugs developed are used to reduce blood sugar. These include inhibitors of digestive enzymes, which blocked alpha glucosidases (amylase and invertase), inhibitors of intestinal absorption of glucose which inhibit membrane transporters: inhibitors of Dipeptidyl peptidase-4 (DPP4) which promote insulin secretion and molecules involved in the capture of peripheral glucose.8,9 Thiazolidinediones, sylfonylureas, Metformin are oral antidiabetic drugs that play a prominent role in the treatment algorithm of T2DM. Unfortunately, despite the efficacy of those drugs side effects like hypoglycemia, nausea, vomitting, headache and constipation are still reported.12-14 Exploration of novel bioactive molecules from plants against diabetes has been intensified mainly with the availabilityof cell and tissue models like yeast cells and psoas mucles.

Cameroonian pharmacopea is very rich in plants with medicinal action including Citrus sinensis, Persea americana and Allium sativum. They have been reported to have antihyperglycemic effects
on rats in an oral glucose tolerance test. However, few studies on their action mechanisms exist to justify their efficacy. This study was designed to evaluate and compare effects of ethanolic and aqueous extracts of C. sinensis stem bark, P. americana seeds and A. sativum bulbson digestive enzymes (invertase and alpha amylase); to determine their adsorptive/glucophagic capacity; to evaluate their capacity to enhance glucose uptake by yeast cells and psoas musleas models.

methodology

Mechanisms by which some plants with antihyperglycemic effects reduce postprandial hyperglycemiaare not fully elucidated. This study was designed to investigate some action mechanisms of extracts from stem bark of Citrus sinensis, seeds of Persea americana and bulbs of Allium sativum including in vitro inhibition of α-amylase and invertase; glucophagic capacity, absorption capacity on yeast cells and psoas tissues.

Results and Discussion

Hyperglycemia is a major symptom of type 2 diabetes and a risk factor of cardiovascular diseases. However, many bioactive molecules derived from plants like polyphenols have proved their efficacy in the treatment of metabolic disorders. Polyphenols are highly soluble in polar sovents like water and ethanol, reason why those two extracts were prepared from stem bark of C sinensis, bulbs of Alium sativum and seeds of Persea americana. After consumption of foods, action of digestive enzymes α-amylase and invertase in the gastrointestinal tract produces smaller molecules.Monosaccharrides resulting from digestion are absorbed into enterocytes to reach the blood stream, where they enter cells to supply energy and perform other biological functions. Treatments administred in conditions of metabolic syndrome or diabetes target interaction with one or several of the abovementioned steps. In this study, the action of different plants have been reported on digestive enzymes. Starch and saccharose are the main polysaccharides found in meals.

An evaluation of invertase activity in the presence of both extract and sucrose revealed that all the extracts inhibited invertase (Figure 1) with IC50 varying from 1.92 (AE A. sativum) to 4.81 (EE A.sativum) (Table 1). Sucrase or invertase is a bifunctional enzyme also called sucrase-isomaltase which hydrolysis sucrose and isomaltose substrates.15 If aqueous extract of P. americana and ethanolic extracts of A. sativum showed no inhibition (null) of alpha amylase at the tested concentrations (Figure 2A), EE of Citrus sinensis inhibited α-amylase instead with an IC50=0.063 mg/ml (vs 2.73 with acarbose) (Figure 2B and Table 1). α-amylase hydrolyses the alpha bond linked polysaccharides of starch producing di- and mono-saccharides. Partial inhibition of the enzyme by C. sinensis extracts reduces its activity and prevents formation of monosaccharides.12-15 Inhibition of amylase and invertase by extracts delays absorption of consumed carbohydrates and reduces plasma glucose

All the extracts strongly inhibited invertase (Figure 1), but only the AE of P. americana and EE extract failed to inhibit the two enzymes. Inhibitors of those enzymes surely posssess in their structures functions similar to their substrates starch and sucrose although plant extracts do not possess such complex structures in their contents. Acarbose used as reference drug under the same experimental conditions provided an IC50 of 2.73 mg/mL, higher than C. sinensis extracts. Acarbose has a structure similar to oligosaccharides and therefore inhibits α-glucosidases in a dose dependent manner, preventing the formation of glucose.27 Flavonoids and alkaloids present in Allium sativum and americanaextracts can justify their efficiency as well.18,19 In fact, myricetin, quercetin, kaempferolare inhibitors of α-amylase and α-glucosidase while alkaloids like berberin are reported to be able to bind to α-glucosidases and prevent sucrose fixation.28-31 Their use to inhibit carbohydrates digestion is therefore beneficial in the management of diabetes because absorption of glucose is reduced and and postprandial hyperglycemia obviously.

The residual glucose resulting from digestion can be trapped in the intestine limiting its absorption by adsorption, reducing its availibility and diffusion into blood; reducing glucotoxicity as well. Adsorptive capacity through glucophagic activity of extracts revealed high percentages of glucose adsorption (Figure 3). Flavonoids and fibers content of extracts can justify such results. Insoluble fibers are capable of binding to glucose reducing its availibility and therefore its intestinal absorption.34 Also, they can reduce viscosity of secretions in intestial lumen slowing or preventing diffusion. Absorbed glucose present in the blood can condense with hydroxyl groups of flavonoids to form complex molecules like glycosyl-flavonoids.35 Decrease in glucose concentration contributes to reduce harmful effects of high plasma glucose on hemoglobin and other proteins, preventing glycation and complications such as retinopathy, neuropathy, and nephropathy observed in diabetic patients

Finally, the extracts were tested for their absorption capacity at the cellular levels. Glucose transport mechanism through cell membranes using yeast cells and psoas culture as models have been of great interest in the understanding of physiological processes of hypoglycemic activities of extracts.39,40 Results revealed that all the extracts stimulted glucose entry in yeast cells (Figure 4). In fact, extracts are capable of stimulating glucose membrane transporters, just like many oral antidiabetics drugs.39,40 On yeast membrane, glucose is transported via specialised stereospecific groups of tranporters called hexose transporters (HXT1 to HXT17) and specific molecules Snf3 and Rgt2, present on the membrane and involved
in its capture. Binding of glucose to Snf3/Rgt2 located on yeast cell membrane permits phosphorylation of two co-receptors Mthl and Std 1 of Rgt1 (transcriptional repressor in charge of down regulation of the HXT gene) exposing Rgt1 to phosphorylation by a protein kinase A, causing glucose to diffusion accross the membrane.

Effectiveness of glucose absorption on yeast membrane mediated by extracts was confirmed by muscle cells tested ex-vivo, on extracts capacity to boost insulin action (Figure 5). Glucose absorption by cells is very important in glusose homeostasis. Upto 80% of glucose fixation depend on insulin action.43 This study revealed that psoas in the presence of insulin produced 10.45% of glucose uptake. Insulin in the presence of each plant extract produced values higher than 10.45% in a dose dependent manner (p<0.05). Similar observation was done with A. aspera extract. Ability of extracts to boost insulin action can be attributed to polyhenols and alkaloids content of plants which act through stimultion of GLUT4 and/or AMPK activation or insulin secretory action. Polyhenols stimulate glucose capture by muscle via activation of AMPK by mechanisms similar to thiazolinediones. The enhanced action of extracts alongside with insulin on rat psoas muscles has been reported with maltitol.48 Alkaloids like berber in stimulates direct glucose absorption mediated by AMPK without involvement of GLUT4.49 Ability of extracts mainly ethanolic extracts to boost insulin action can be attributed to polyphenols known to stimulate binding of insulin to IRS receptors, to increase the number of insulin receptors or/and to increase insulin sensitivity.44-50 Enhanced uptake of glucose by cells via joint action of insulin and extracts may stimulate glucose clearance from blood, through improved insulin resistance. Enhanced uptake of glucose also promote post absorptive utilisation in glycolysis for energy supply.44 Absorption values above 10.45% and increments due to extracts varying from 4.21 to 28.11 % respectively for P. americana and A. sativum (Figure 5) cannot be intrinsically attributed to the stimulation of insulin action, because in vitro, extracts could adsorb part of glucose before insulin action, lowering its concentration in the medium. This study however demonstrated that, the cumulative actions of extracts on enzyme digestion, glucose adsorption and insulin action lead to reduced hyperglycemia, targeted in the first treatment outcome in type 2 diabetes. Reduced hyperglycemia also limits progression to complications.

Results

All extracts inhibited invertase with IC50 varying from 1.92 to 4.81 mg/ml for Allium sativum extracts. α-amylase was inhibited by EE C. sinensis (IC50=0.063 vs 2.73 mg/ml for arcabose) and not by EE of A. sativum and EE of P. americana. Glucophagic capacity of extracts varied signicanty from 47.55 % of AE P. americana (5 mg/ml) to 100% with C. sinensis (5 mg/ml). All extracts stimulated glucose uptake (p<0.05) from 2.62 % AE C. sinensis (2.5 mg/ml) to 54.74% for EE of Persea americana (10 mg/ml). All extracts enhanced glucose uptake by psoas tissues increasing absorption capacity to up to 38.56 % with A. sativum (10.45% insulin, p<0.05).

Conclusion

Cumulative actions of each plant extract on inhibition of carbohydrates’ digestive enzymes, adsorption of glucose in intestine and blood, stimulation of glucose uptake and insulin action on yeast cells and psoas tissues, contribute to lower hyperglycemia and diabetes related complications. Therefore, extracts from the plants could be good candidates for diabetes therapy