Abrus Precatorius Leaf Extract Reverses Alloxan/NicotinamideInduced Diabetes Mellitus in Rats Through Hormonal (Insulin, GLP-1, And Glucagon) And Enzymatic (α-Amylase/α- Glucosidase) Modulation Part 2

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3. Results

3.1.Phytochemical Screening and Quantification of Total Phenols and Flavonoids in APLE. An average yield of 9.6%APLE was obtained from the initial powdered(120g)leaves of Abrus precatorius. Standard phytochemical screening showed the presence of phenols, flavonoids, tannins, alkaloids, and saponins in APLE. From the calibration curve of rutin (standard), flavonoid content in APLE was estimated to be 220.29ug/mL of rutin equivalent (RE)(Figure 2(a)). Also, from the calibration curve of gallic acid (standard), phenolic content in APLE was estimated to be 85.51 ug/mL of gallic acid equivalent (GAE)(Figure 2(b)).

3.2.APLE Restored Loss in Bodyweight Associated with Alloxan/Nicotinamide-Induced Diabetic Rats. Compared to control rats, model rats significantly(P≤0.05)lost body weight. However, treatment of Alloxan/nicotinamide-induced diabetic rats with APLE, particularly APLE (100mg/kg), significantly restored bodyweight loss relative to model rats (Table 1). Although there were differences in the organ weight/body weight ratios between control and model and also between model and APLE, these differences were statistically insignificant (P>0.05).

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3.3.APLE Decreased Elevated Blood Glucose Levels of Alloxan/Nicotinamide-Induced Diabetic Rats. Sequential exposure of rats to Alloxan monohydrate(120mg/kg; ip)and nicotinamide(48 mg/kg; ip) resulted in elevated blood glucose levels in model rats (diabetic rats) compared to control rats. Treatment of Alloxan/nicotinamide-induced diabetic rats with APLE(100,200, and 400mg/kg)for 18 days resulted in a significant(P<0.05) decrease in average blood glucose levels of diabetic rats. Over the 18-day treatment/observation period, control rats had a percentage decrease in mean blood glucose levels from the initial blood glucose level by 11.96% as against 4.3% by model rats(diabetic rats). Compared to model rats, APLE treatment, particularly APLE(100mg/kg; PO) produced a 68.67% decrease in mean blood glucose levels from the initial blood glucose level of Alloxan/nicotinamide-induced diabetic rats over 18 days of treatment (Table 2).

3.4. APPLE Treatment Increased the Number and Median Cross-sectional Area of Pancreatic Islets of Langerhans of Alloxan/Nicotinamide-Induced Diabetic Rats. The number of pancreatic islets of Langerhans was not different between control and model rats; however, the median cross-sectional area of pancreatic islets of model rats was decreased compared to that of control rats. Treatment of Alloxan/nicotinamide-induced diabetic rats with APLE significantly increased both the number and cross-sectional area of pancreatic islets of Langerhans compared to model rats(Figure 3 and Table 3).

3.5.APLE Increased Serum Insulin and GLP-1Levels Inversely with Glucagon in Alloxan/Nicotinamide-Induced Diabetic Rats. Sequential exposure of rats to Alloxan monohydrate (120mg/kg; IP) and nicotinamide(48 mg/kg; IP) led to a decrease in serum insulin in model rats(diabetic rats) compared to control rots. However, treatment of alloxan/nicotinamide-induced diabetic rats with APLE(100,200, and 400 mg/kg) over a period of 18 days restored serum insulin levels even more than that of control rats. Surprisingly, APLE

(200mg/kg)had no effect on Alloxan/nicotinamide-induced decrease in serum insulin(Figure 4(a)).Serum glucagon levels decreased in Alloxan/nicotinamide-induced diabetic rats compared to that of control rats. Relative to Alloxan/nicotina-mide-induced diabetic rats (model rats), APLE treatment,par-ticularly APLE(400mg/kg), significantly(P<0.05) decreased serum glucagon levels(Figure 4(b)). Serum GLP-1 signif-cantly (P<0.05) decreased in Alloxan/nicotinamide-induced diabetic rats(model rats) compared to control rats. However, treatment of Alloxan/nicotinamide-induced diabetic rats (model rats) with APLE(100, 200, and 400mg/kg) restored serum GLP-1 levels in a dose-related manner(Figure 4(c)).

3.6. APLE Concentration Dependently Decreased Enzymatic Activity of α-Amylase. At equivalent concentrations, both APLE and acarbose concentration-dependently inhibited the enzymatic activity of a-amylase; however, the concentration-% inhibition curve for APLE was shifted to the left of that of acarbose (Figure 5(a)). From the Lineweaver-Burk and Michaeles-Menten plots (Figures 5(b) and 5(c)), APLE decreased maximum velocity(Vmax) of α-amylase/substrate reaction relative to control but increased the Michaelis constant (Km)relative to control (Table 4). Respectively, the ICsoestimates for APLE and acarbose were 259ug/mL and 297 ug/mL (Table 5).

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3.7.APLE Decreased Enzymatic Activity of a-Glucosidase.At equimolar concentrations, both APLE and acarbose demonstrated concentration-dependent inhibitory effects on α-Glu-oxidase enzymatic activity; however, the concentration-%inhibition curve for APLE was shifted to the left of that of acarbose(Figure 6(a)). From the Lineweaver-Burk and Michaeles-Menten plots (Figures 6(b) and 6(c)), APLE decreased maximum velocity(Vmax)of α-glucosidase/substrate reaction relative to control but increased Michaelis constant(Km)relative to control(Table 4). Respectively, the ICsestimates for APLE and acarbose were 176ug/mL and 1090 ug/mL (Table 5).

3.8. APLE Increased DPPH and NO Radical Scavenging Activity as well as Demonstrating Ferric Reducing Antioxidant Capacity (FRAC). In vitro, APLE demonstrated concentration-dependent DPPH radical scavenging activity, but it was lower than that of ascorbic acid (Figure 7(a)). Compared to ascorbic acid and gallic acid, APLE demonstrated concentration-dependent scavenging activity on nitric oxide (NO) radicals. While APLE and gallic acid

FIGURE 3: Effect of APLE and metformin on Alloxan/nicotinamide-induced pancreatic β-cell damage and necro-apoptosis in diabetic rats. (a)A photomicrograph of representative H&E-stained pancreatic islets of Langerhans showing(A)control,(B)model,(C) APLE (100 mg/kg PO),(D)APLE(200mg/kg; PO),(E)APLE(400mg/kg; PO),and (F)metformin(300mg/kg; PO).(b)A bar graph showing the median area of pancreatic islets of Langerhans. Each bar is the mean±SD median area of pancreatic islets of Langerhans."P≤0.05 (model vs. Control); PP≤0.05(APLE and metformin vs. model); APLE: Abrus precatorius leaf extract.

morphologically demonstrated a flat concentration-response (% NO radical scavenging activity)curve, ascorbic acid showed a steep concentration-response(% NO radical scav-enging activity)curve(Figure 7(b)).From the ICestimates, APLE had the lowest IC5 value relative to ascorbic acid and gallic acid (Table 5). At equimolar concentrations, quercetin demonstrated significant concentration-dependent ferric reducing antioxidant capacity relative to APLE(Figure7(c)).

4. Discussion

Herbs have been used in many capacities by mankind to improve human health and also serve as a source of natural templates for the pharmaceutical synthesis of novel drugs. Many local communities across Afro-Asian regions of the world rely heavily on their ethnobotanical heritage to meet most of their primary healthcare needs. This study demonstrated that the antidiabetic effect of APLE in experimental diabetes mellitus in rats is mediated through multiple mechanisms including inverse modulation of insulin and GLP-1 with glucagon, inhibition of α-amylase, and α-glucose-date enzymatic activity, free radical scavenging, antioxidant, and recovery of necro-apoptosis pancreatic β-cells. As the present results corroborate earlier reports on Abrus pre-categories [1,35], it further confirms folk claims on Abrus precatorius especially those made by the local communities in western Ghana, where the leaves are used to treat diabetes mellitus.

Alloxan was used as a diabetogenic agent in this study, in view of its specific pancreatic β-cell toxicity to establish experimental diabetes mellitus in rats. Upon exposure of Alloxan to rats, it undergoes phase 1 reaction; specifically,

figure 4:Effect of APLE on serum levels of insulin, glucagon, and GLP-1 of Alloxan/nicotinamide-induced diabetic rats. Each bar is the mean±SD, n=3.(a)Effect of APLE on serum insulin,(b)effect of APLE on serum glucagon, and(c)effect of APLE on serum GLP-1."P ≤0.05(model vs. control);βP≤0.05(APLE and metformin vs. model); ns: not significant; APLE: Abrus precatorius leaf extract;metformin (300 mg/kg; po).

it is biotransformed by hepatic metabolic enzymes via reduction into dialuric acid. The dialuric acid is reoxidized back to Alloxan establishing a redox cycle leading to the production of superoxide radicals(O,), which dismutate to form hydrogen peroxide(H, O,). From the H, O, reactive hydroxyl radicals(OH)are formed through the Fenton reaction.

Resultant ROS induces an increase in cytosolic calcium concentrations which in turn induces rapid destruction and necro-apoptosis of pancreatic β-cells. Extensive destruction of pancreatic β-cells occasions insulin insufficiency and hyperglycemic episode leads to glucose toxicity(glucose-toxicity). Expectedly, rats in the model group(Alloxan/nicotinamide-induced diabetic rats) developed sustained hyperglycemia due to insulin insufficiency culminating from extensive destruction of pancreatic β-cells. However, treatment of diabetic rats with APLE over 18 days reversed the chronic hyperglycemia in diabetic rats relative to model rats (Table 2). To ascertain the mechanism by which APLE produced glucose lowering over the 18 days, serum concentrations of insulin, glucagon, and GLP-1 were measured across all groups by using a rat-specific ELISA kit. Physio-logically, at any given time point, blood glucose concentration reflects a balance between glucose production (dietary sources of glucose, glycogenolysis, and gluconeogenesis)and utilization (uptake of glucose by insulin-responsive

FiGURE 5:Effect of APLE on α-amylase enzymatic activity. Each plotted point is the mean±SD, n=3.(a)% inhibitory effect of APLE on α-amylase enzymatic activity,(b)Lineweaver-Burk plot showing the mode of inhibition of a-amylase enzymatic activity by APLE. (c)Michaels-Menten plot showing the effect of APLE on α-amylase kinetics (Vmax and Km)."P<0.05(APLE ys, acarbose);APLE: Abrus precatorius leaf extract; Vmax: maximum velocity; Km: Michaelis constant.

tissues and conversion of excess glucose to storage carbohydrate)primarily in response to secretion pattern and the actions of insulin and glucagon. While insulin decreases peripheral glucose concentration by increasing glucose utilization by insulin-responsive tissues such as the brain, muscles, liver, and other body cells as well as inhibition of glucagon secretion, glucagon on the other hand increases peripheral glucose levels by promoting break down of glycogen(glycogenolysis)and biosynthesis of glucose from non-carbohydrate sources (fatty acids, pyruvate, and amino acids, i.e., gluconeogenesis). Interestingly, APLE treatment reversed decreased insulin concentration inversely with glucagon in diabetic rats compared with model rats, indicating

FIGURE 6: Effect of APLE on a-glucosidase enzymatic activity. Each plotted point is the mean± SD, n=3.(a)% inhibitory effect of APLE on a-glucosidase enzymatic activity,(b)Lineweaver-Burk plot showing the mode of inhibition of α-glucosidase enzymatic activity by APLE.(c)Michaelis-Menten plot showing the effect of APLE on α-glucosidase kinetics(Vmax and Km)."P≤0.05(APLE vs.acarbose);APLE: Abrus precatorius leaf extract; Vmax: maximum velocity; Km: Michaeles constant.

that APLE treatment improved utilization of peripheral glucose in an insulin-dependent manner. APLE-dependent increase in insulin inversely with glucagon confirms the already established observation that insulin inhibits glucagon secretion and action. To assess how APLE increased insulin but decreased glucagon in diabetic rats, the pancreatic islets of Langerhans were histologically examined; specifically, the number of islets and the median area of the islets were studied across groups. Of note, APLE treatment did not only recover partially damaged pancreatic β-cells but also increased the number and median cross-sectional area of islets relative to that of model rats(Figure 3 and Table 3). Since insulin concentration in blood is directly related to pancreatic β-cell population and mass, it is possible that APLE-dependent increase in insulin inversely with glucagon was through the recovery of damaged pancreatic ß-cells as well as an increase in the number of islets and mass of pancreatic β-cells, which enhanced insulin secretion and utilization of peripheral glucose by insulin-responsive tissues. Also, APLE produced an increase in GLP-1 inversely with glucagon. GLP-1 is one of the incretins (INtestine seCRETion Insulin), and just like glucose-dependent insulinotropic polypeptide(GIP), they exert an insulinotropic effect in response to the presence of glucose in the duodenum. GLP-1 and GIP are, respectively, produced by enteroendocrine L and K cells. These two hormones exert their insulinotropic effect by binding to and activating G-protein-coupled receptors(GIP receptor(GIPR) and GLP-1 receptor(GLP-1R)) in the plasma membrane of pancreatic β-cells. Binding and activation of the G-protein receptor lead to a decoupling of the α-subunit of the G-protein and its transactivation of adenylate cyclase, which dephosphorylates ATP to cyclic AMP. An increase in cyclic AMP activates protein kinase A which mediates the closure of K ion-gated channels. Subsequently, the influx of Ca2 via voltage-gated Ca2+ channels causes depolarization of the β-cell membrane which eventually leads to the secretion of insulin by pancreatic β-cells [45]. GLP-1 and GIP promote pancreatic β-cell proliferation, inhibit necro-apoptosis of pancreatic β-cells, thereby expanding pancreatic β-cell mass [46]. While GIP enhances postprandial glucagon response, GLP-1 suppresses postprandial glucagon response. Pancreato-protective effects of APLE could be due to enhanced release of GLP-1 since the decrease in GLP-1 in diabetic rats corresponded with decreased

FIGuRE 7:Freeradical scavenging and antioxidant effects of APLE.(a) DPPH radical scavenging activity of APLE. (b) NO radical scavenging activity of APLE.(c) Ferric reducing the antioxidant activity of APLE."P<0.05(APLE vs.ascorbic acid and quercetin);APLE: Abrus precatorius leaf extract; DPPH:2,2,-diphenyl-1-picrylhydrazyl; NO: nitric oxide.

a number of islets as well as the cross-sectional area of pancreatic islets. Also, APLE-dependent increase in insulin could be related to GLP-1 mediation.

Enzymatically, carbohydrate digestion in humans begins in the mouth by rapid hydrolysis of both amylopectin and amylose in cooked starch by a-amylase, which is secreted by both salivary glands and the pancreas.Alpha(a)-amylase is an endoglycosidase that specifically hydrolyses internal α-1,4 linkages yielding maltose, maltotriose, and α-dextrin. Unlike α-amylase,a-glucosidase is a brush border enzyme of duodenal enterocytes. Functionally,α-glucosidase hydro-lyzes the terminal nonreducing (1-4)α-glucose residues of maltose to release a single a-glucose. These two enzymes serve as key targets for pharmacological modulation of carbohydrate digestion in people suffering from diseases related to errors in carbohydrate metabolism such as DM. Inhibition of these two enzymes results in a significant delay in the release of glucose from disaccharides thereby reducing glucose availability and absorption. Indeed, among the available conventional oral hypoglycemic agents,α-glucosidase inhibitors (e.g, acarbose)enjoy therapeutic preference for the treatment of type 2 DM. Interestingly, APLE concentration-dependently inhibited these two enzymes, which reveals yet another mechanism by which APLE lowers postprandial blood glucose levels, and this observation corroborates an earlier study [47], which demonstrated that a triterpene ketone(lupenone)isolated from the leaves of Abrus precatorius exerted potent α-amylase inhibitory effect. The inhibitory effects of APLE against α-amylase and α-Glu-oxidase mirror that of other medicinal plants known for their antidiabetic properties including Chrysobalanus orbicu-laris[11], Spondias mombin and Mangifera indica[12], Sesa-mum indicum [13], and Bryophyllum pinnatum [14].

Chronic hyperglycemia induces nonenzymatic glycosylation of various macromolecules leading to the generation of unstable chemical species including ROS. ROS inhibits glyceraldehyde-3-phosphate dehydrogenase (GADPH) in the glycolytic pathway, thereby increasing upstream intermediates of GADPH. These glycolytic intermediates(glucose, fructose-6-phosphate, and glyceraldehyde-3-phosphate) are shunted into other biochemical pathways which are implicated in DM. Also, ROS is implicated in lipid peroxidation and oxidative stress. Oxidative stress induced by ROS as a result of chronic hyperglycemia plays a key role in the onset of various diabetic complications including insulin resistance and pancreatic β-cell dysfunction. In this study, the free radical scavenging activity of APLE was assessed by using DPPH and NO assays, while the antioxidant capacity of APLE was assessed using FRAC.DPPHis a stable free radical as a result of the delocalization of electrons all over the molecule.Delocalization of electrons in DPPHresults in deep violet color, and upon reduction by any hydrogen or electron donor, the violet color of DPPH fades and leads to the formation of pale-yellow hydrazine. The color change reflects the shifting of wavelength in the visible spectra from 517nm to 330nm. As a result, free radical scavenging activity corresponds to a reduction of DPPH, which can be quantified by measuring absorbance at 517nm[48]. Similarly, NO partakes in a series of reactions leading to a decrease in mitochondrial ATP and aconitase, which in turn induce an increase in xanthine oxidase. Nitric oxide(NO) donors such as STZ promote the reaction between superoxide(O2)and hydrogen peroxide (H, O), which yields reactive hydroxyl(OH) and nitro radicals that cause DNA damage of pancreatic β-cells. Conversion of ferric（Fe+） to ferrous（Fe²2）by donation of an electron by an electron donor (antioxidant agent) forms the basis of FRAC[49]. Therefore, FRACassay provides a direct measure of the reducing or electron-donating ability of an antioxidant. In this study, APLE demonstrated concentration-dependent scavenging activity against DPPH and NO and also demonstrated reducing capacity in the FRAC assay. These observations point to the ability of APLE to mop-up unstable chemical intermediates generated by Alloxan exposure to rats, thereby preventing ROS-mediated cell damage and necro-apoptosis, which accounted for extensive damage of pancreatic β-cells and attendant hyperglycemia in model rats. The antioxidant and free radical scavenging effects of APLE are attributable to the bioactive secondary plant metabolites identified in APLE, particularly the phenolic compounds(Figure 2). Phenolic compounds derived from plants exhibit many biological properties which account for their health benefits and a justification for their use in food and drug discovery industries. Phenolic compounds exert their biological effects by interacting with diverse cellular components including membrane transporters, protein kinases, catechol-O-methyltransferases, membrane-bound NADPH oxidases, xanthine oxidase, cyclo-oxygenases, lipoxygenases, and some transition metals [50-52]. Phenolic com-pounds exert antioxidant and free radical scavenging activities either directly or indirectly. Mostly, antioxidant effects of phenolic compounds are exerted indirectly by the ability of phenolic compounds to induce cellular events that lead to the production of ROS-scavenging enzyme systems in vivo, while direct antioxidant effects of phenolic compounds are related to their ability to suppress the initiation step needed for the generation of oxidant species or direct inter-action with these unstable chemical species. Phenolic compounds derived from many plants have demonstrated inhibitory effects on the activity of α-amylase and α-glucosidase [53-55], which supports the assertion that the inhibitory effects of APLE against the activity of α-amylase and α-glucose-dose observed in the present study could be due to the phenolic compounds detected in APLE. Also, tannins, saponins, and alkaloids were identified in APLE confirming a previous report [1]. Further, it is suspected that inhibitory effects of APLE on a-amylase and α-glucosidase enzymatic activity could be due to the combined effects of its phytoconstituents including tannins and saponins whose inhibitory effects against α-amylase and α-glucosidase have already been established [56-58].

Putting together this study has demonstrated that glucose-lowering and pancreatic-protective effects of APLE are mediated through multiple mechanisms including hormonal modulation, enzyme inhibition, free radical scavenging, antioxidant activity, and repair of damaged pancreatic β-cells. This study could have benefited from investigating the effect of APLE on the counterregulatory hormonal systems particularly, the catecholamines, and the stress hormone(cortisol)in gluconeogenesis (a major contributor to peripheral glucose) as well as the effect of APLE on specific glucose transporters; nonetheless, the present results provide a compelling basis for further mechanistic elucidation of antidiabetic effects of APLE.

5. Conclusion

Increase in insulin and GLP-1 inversely with glucagon, inhibition of α-amylase/α-glucosidase enzymatic activity, free radical scavenging, antioxidant, and pancreatic β-cell recovery underpin antidiabetic effects of Abrus precatorius leaf extract(APLE), and these pharmacological effects are attributable to phenolic and flavonoid contents of APLE. As this finding confirms folk use of APLE as an antidiabetic herbal medicine by local communities, it also lays a foundation for possible translational studies on APLE.

This article is extracted from Hindawi BioMed Research International Volume 2021, Article ID 9920826, 17 pages https://doi.org/10.1155/2021/9920826