https://orcid.org/0000-0001-9331-396X
Figures
Abstract
Background and objective
Annona muricata L. peel has been recognized for many ethnobotanical uses, including diabetes management. However, limited detailed scientific information about its mechanism of antidiabetic activity exists. The objective of this study was to evaluate the anti-diabetic properties of an aqueous extract of A. muricata peel (AEAMP) and its mechanism of action on alloxan-induced diabetic rats.
Methods
In vitro antidiabetic assays, such as α-amylase and α-glucosidase were analyzed on AEAMP. Alloxan monohydrate (150 mg/kg b.w) was used to induce diabetes in the rats. 150 mg/kg b.w positive control group doses of 6.67, 13.53, and 27.06 mg/kg were administered to 3 groups for twenty-one days. The positive control group was administered 30 mg/kg of metformin. The negative and normal control groups were administered distilled water. The fasting blood glucose, serum insulin, lipid profile, inflammatory cytokines, antioxidant markers, carbohydrate metabolizing enzymes, and liver glycogen were analyzed as well as PI3K/AKT and apoptotic markers PCNA and Bcl2 by RT-PCR.
Results
AEAMP inhibited α-amylase and α-glucosidase enzymes more effectively than acarbose. AEAMP reduced FBG levels, HOMA-IR, G6P, F-1,6-BP, MDA, TG, TC, AI, CRI, IL-6, TNF-α, and NF-κB in diabetic rats. Furthermore, in diabetic rats, AEAMP improved serum insulin levels, HOMA-β, hexokinase, CAT, GST, and HDL-c. Liver PI3K, liver PCNA and pancreas PCNA were not significantly different in untreated diabetic rats when compared to normal rats suggesting alloxan induction of diabetes did not downregulate the mRNA expression of these genes. AEAMP significantly up-regulated expression of AKT and Bcl2 in the liver and pancreatic tissue. It is interesting that luteolin and resorcinol were among the constituents of AEAMP.
Conclusions
AEAMP can improve β-cell dysfunction by upregulating liver AKT and pancreatic PI3K and AKT genes, inhibiting carbohydrate metabolizing enzymes and preventing apoptosis by upregulating liver and pancreatic Bcl2. However, the potential limitation of this study is the unavailability of equipment and techniques for collecting more data for the study.
Citation: Ojo OA, Grant S, Amanze JC, Oni AI, Ojo AB, Elebiyo TC, et al. (2022) Annona muricata L. peel extract inhibits carbohydrate metabolizing enzymes and reduces pancreatic β-cells, inflammation, and apoptosis via upregulation of PI3K/AKT genes. PLoS ONE 17(10): e0276984. https://doi.org/10.1371/journal.pone.0276984
Editor: Abhishek Kumar Singh, Amity University, INDIA
Received: June 24, 2022; Accepted: October 18, 2022; Published: October 27, 2022
Copyright: © 2022 Ojo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Diabetes is a long-term health condition and has been ranked as the 9th leading cause of adult mortality globally [1]. In 2014, it was estimated that five to ten percent of the world’s 386 million diabetics had type 1 diabetes (T1DM) [2–4]. T1DM is characterized by insulin deficiency due to pancreatic β-cell loss, leading to hyperglycaemia. The hallmark of T1DM is the destruction of the pancreas, which leads to the non-secretion of insulin required to maintain normal blood glucose levels [5].
One of the recognized pathologies of T1DM is an autoimmune disease that leads to the destruction of insulin-producing pancreatic beta cells over months or years [6]. Damage to beta-cells can result in high blood glucose, a prevalent metabolic condition involving carbohydrates, lipids, and proteins that is rapidly becoming an epidemic [7]. Insulin deficiency leads to hyperglycemia and ketosis. Chronic hyperglycemia is associated with long-term damage, leading to dysfunction of the kidney, eyes, nerves, heart, and blood vessels [8]. Based on statistics, 2.8% of the world’s population currently has diabetes, with that proportion anticipated to rise to around 5.4% by 2045 [7]. The high prevalence, varied pathogenesis, and complications of the disease are pointers to the urgent need for effective treatment options [9]. In recent times, different treatments, like insulin, drug administration, and food therapy, have recently been effective in T1DM management [10].
Many oral medications exert anti-diabetic effects via various mechanisms [11, 12]. Metformin, imatinib, gliptines, α-glucosidase inhibitors, and gliflozines are some of the drugs used to treat T1DM. However, further studies are necessary for conclusive validation of their efficacy and safety. Recent evidence-based investigations did not necessarily confirm the efficacy and safety of these oral medications as adjuvants for T1DM patients. Thus, they still signify off-label medication for its management [11, 12]. Many treatment regimens with the exploitation of medicinal plants are recommended for diabetes. Allium sativum, Azadirachta indica, Blighia sapida, and Caesalpinia bonducella are a few of the hundreds of plants documented to have produced positive anti-diabetic effects [13–18].
Annona muricata L. (A. muricata) is a medicinal plant in the Annonaceae family and is recognized for its numerous traditional benefits [19, 20]. Annona is utilized in traditional medicine in its entirety. It is believed to be a rich source of antioxidants for several ailments [19–21]. The peels are traditionally used among Abia villagers in Nigeria to treat diabetes, headaches, and liver complications. The efficacy of most medicinal plants has been linked to their constituents [22, 23]. There is, though, limited detailed information linking the antidiabetic activity of the A. muricata peel to a mechanism of action. Thus, the study examined the antidiabetic activity of an aqueous extract of A. muricata peel (AEAMP) in an alloxan-induced diabetic rat model and also determined the mechanisms of action of this extract. The objective of this study is to examine the role of A. muricata peel (AEAMP) in abrogating alloxan-induced diabetes via suppressing oxidative stress, inflammation, and β-cell apoptotic death, and by upregulating glucose uptake by stimulating the PI3K/AKT genes. Furthermore, to identify potential bioactive compounds present in the plant that might be responsible for the antidiabetic activity observed and to understand the mechanisms by which potential mRNA genes are upregulated or downregulated in the management of diabetes.
Materials and methods
Plant material
The peels of A. muricata were obtained from a local farm in Abia village, Ohafia Local Government Area, Abia State. The plant was identified at the Forestry Research Institute of Nigeria (FRIN), Nigeria, voucher number FHI 113160.
Chemicals, apparatus, and general procedures
All of the chemicals were of analytical grade. Methanol, phosphoric acid, vanillin, cyanidin, and benzoic acid were purchased from Merck (Darmstadt, Germany). Quercetin, rescorcinol, and luteolin were acquired from Sigma Chemical Co. (St. Louis, MO, USA). High performance liquid chromatography (HPLC-DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector, and LC solution 1.22 SP1 software.
Preparation of aqueous extract of A. muricata peel (AEAMP)
Peels of A. muricata were sliced into little pieces and shed-dried at room temperature for 8 weeks. We obtained the aqueous extract by freeze-drying the filtrate obtained from the extraction of 50 g of the powdered material with distilled water for 48 hours.
High-performance liquid chromatography (HPLC-DAD)
We carried out HPLC analysis of AEAMP, following the standard procedure described by Ojo et al. [24]. Reverse phase chromatographic analyses were carried out under gradient conditions using a C18 column (4.6 mm x 150 mm) packed with 5μm diameter particles; the mobile phase was water containing 1% phosphoric acid (A) and acetonitrile (B), and the composition gradient was 17% of B until 10 min and changed to obtain 20%, 30%, 50%, 60%, 70%, 20%, and 10% B at 20, 30, 40, 50, 60, 70, and 80 min, respectively. The samples and mobile phase were filtered through a 0.45 μm membrane filter (Millipore) and then degassed in an ultrasonic bath prior to use. The Annona muricata peel extract was analyzed at a concentration of 10 mg/mL. The flow rate was 0.7 mL/min, the injection volume was 50 μl and the wavelength was 327 nm for benzoic acid, cyaniding, and vanillin, and 366 nm for quercetin, resorcinol, and luteolin. Stock solutions of standards references were prepared in the HPLC mobile phase at a concentration range of 0.030–0.500 mg/mL. Chromatography peaks were confirmed by comparing their retention times with those of reference standards and by DAD spectra (200 to 600 nm). All chromatographic operations were carried out at ambient temperature and in triplicate.
Evaluation of α-amylase activity
We used the method described by Shai et al. [25] to assess AEAMP’s inhibitory activity against the -amylase enzyme. The samples (15–240 μg/ml) were incubated for 20 minutes at 37°C with 500 μl of porcine pancreatic amylase (2 U m/L) in 100 mmol/L of phosphate buffer (pH 6.8) in a known volume (250 μl). A known volume (250 μl) of 1% starch dissolved in 100 mmol/L phosphate buffer (pH 6.8) was added to the reaction mixture and incubated at 37°C for 1 hour. A milliliter of dinitrosalicylic acid color reagent (1%, 3,5-dinitrosalicylic acid, 0.2% phenol, 0.05% Na2SO3, and 1% sodium hydroxide) was added and heated for 10 minutes at 100°C. After cooling to 25°C in a cold water bath, the absorbance of the resulting mixture was read at 540 nm. Acarbose was used as a standard. The result was calculated and expressed as a percentage.
Evaluation of α-glucosidase activity
We used the method described by Nguelefack et al. [26] to assess AEAMP’s inhibitory activity against the α-glucosidase enzyme. One mg of α-glucosidase (Saccharomyces cerevisiae, Sigma-Aldrich, USA) was dissolved in 100 ml of phosphate buffer (pH 6.8) containing 200 mg of bovine serum albumin. The reaction mixture, consisting of 10 μl of sample at varying concentrations (15–240 μg/ml) was premixed with 490 μl of phosphate buffer (pH 6.8) and 250 μl of 5 mM p-nitrophenyl α-D-glucopyranoside. After pre-incubating at 37°C for 15 minutes, the reaction was terminated by the addition of 2000 μl of 200 mM Na2CO3. α-Glucosidase activity was determined spectrophotometrically at 400 nm by measuring the quantity of p-nitrophenol released from p-NPG. Acarbose was used as a positive control of the α-glucosidase inhibitor.
Dosage determination and experimental animals
Male Wistar rats (36) (240.20 ± 10.52 g) were purchased from the Department of Biochemistry, University of Ilorin. The rats were then housed in cages under standard conditions (room temperature with a 12 h light-dark cycle). The rats were acclimatized for two weeks before the experiment began. From the ethnobotanical survey conducted in an Abia village in Nigeria during the study, the mentioned human therapeutic doses of A. muricata peels are between 1 and 9 g. The dose for rats was calculated considering the human to albino rat conversion factor (conversion factor = 0.162) according to body surface area. This was done by dividing it by the adult human weight of 60 kg and multiplying it by a factor to accommodate the body surface area of the animal [27]. The calculated dose obtained falls within the range of approximately 100 mg/kg b.w rat. Therefore, dose levels of 6.76, 13.53, and 27.06 mg/kg b.w. were used in this research work.
Induction of DM
Rats that were deprived of food pellets overnight were each treated with a single intraperitoneal injection of alloxan at a dose of 150 mg/kg [24]. Alloxan was freshly prepared by dissolving it in 0.9% normal saline. The Wistar rats were given a 150 mg/kg body weight single injection of alloxan dissolved in normal saline (NaCl) and a 5% glucose solution for 24 hours to prevent hypoglycaemic shock. The alloxan-diabetic rats’ fasting glucose levels were measured on the third day after injection, resulting in a blood glucose level of ≥ 250 mg/dL.
Animal groups and extract treatment
Thirty-six male Wistar rats were chosen and put into a group of six containing six rats each as follows:
Group 1: Distilled water + normal rats
Group 2: Untreated diabetic rats
Group 3: Diabetic rats were given 30 mg/kg body weight of metformin.
Group 4: Diabetic rats were given 6.76 mg/kg body weight of AEAMP.
Group 5: Diabetic rats were given 13.53 mg/kg body weight of AEAMP.
Group 6: Diabetic rats were given 27.06 mg/kg body weight of AEAMP.
Following feeding, both the drug (metformin) and the extract were administered. By reconstituting the extract in water, it was supplied via oral gavage.
Ethical approval
The Landmark University animal ethics committee accepted this work with the permission number LUAC/2021/007A.
Sample collection and analysis of organs
This study lasted three weeks, after which the rats were euthanized via halothane anesthesia; the liver and pancreas were harvested and homogenized in cold phosphate buffer (0.01M, pH 7.4, 1:5 w/v) following the procedure described by [17]. Blood was drawn from the heart and placed in a clean, dry centrifuge tube, which was then allowed to clot at 25°C before being centrifuged at 5,000 rpm for 20 minutes to preserve the sera for further analysis. The tissues harvested were centrifuged for 15 min at 10,000 rpm, and the supernatant was separated with Pasteur pipettes, transferred into specimen bottles, and frozen at -80°C for further biochemical analysis.
Biochemistry parameters in serum and liver
We used the technique described by [28] to assess serum insulin concentration, which used an ELISA kit from England in a multiple plate ELISA reader (Winooski, Vermont, USA). We evaluated liver glycogen following the method of Murat and Serfaty [29]. Serum total cholesterol was evaluated following the procedure described by Fredrickson et al. [30]. We also assessed triglyceride via the procedure illustrated by Tietz [31]. The Jacob et al. method was employed to determine the HDL-c [32]. We estimated LDL and VLDL cholesterol by employing the technique illustrated by Friedewald et al. [33]. The atherogenic index (AI) was calculated by using the expression in Eq 1 [34]. We calculated the coronary artery index (CRI) by using the described expression in Eq 2 [35]. The Homeostasis model assessment of insulin resistance (HOMA-IR) and β-cell score (HOMA-β) were determined via the expression described by Wilson and Islam [36] in Eqs 3 and 4.
Determination of antioxidant biomarkers
In this study, antioxidant biomarkers including reduced glutathione (GSH) levels, catalase, superoxide dismutase activities, and MDA levels were analyzed [37–40].
Determination of the activities of glycolytic enzymes
We analyzed the activities of hexokinase, glucose-6-phosphatase (G6P), and fructose 1,6-bisphosphate (F-1,6-BP) on the liver supernatant [41–43].
Evaluation of inflammatory biomarkers in rat serum
We analyzed for TNF-α, IL-6, and NF-κB, via the procedure illustrated in the ELISA kits (Biosource, USA).
RT (reverse transcriptase) PCR (RT-PCR) analysis
Isolation of RNA.
We isolated total RNA from the liver and pancreas following the procedure illustrated by Ojo et al. [24, 44]. TRIzol reagent (Zymo Research, USA, Cat: R2050-1-50, Lot: ZRC186885) was used to homogenize the liver and pancreas at 4°C. Absolute RNA was apportioned in chloroform solvent (BDH Analytical Chemicals, Poole, England, Cat: 10076-6B) and centrifuged at 15,000 rpm/15 min (Abbott Laboratories, Model: 3531, Lake Bluff, Illinois, United States). The RNA from the supernatant was precipitated using an equivalent amount of isopropanol (Burgoyne Urbidges & Co, India, Cat: 67-63-0). The RNA pellet was washed two times in 70% ethanol (70 ml of absolute ethanol (BDH Analytical Chemicals, Poole, England, Cat: 10107-7Y) in 30 ml of nuclease-free water (Inqaba Biotec, West Africa, Lot no: 0596C320, code: E476-500ML)). The pellets were air-dried for 5 min and solubilized in RNA buffer (1 mM sodium citrate, pH 6.4).
cDNA conversion.
Before cDNA transformation, absolute RNA amount (concentration (μg/ml) = 40 * A260) and quality (≥ 1.8) were evaluated using the proportion of A260/A280 (A = absorbance) read via a spectrophotometer. DNA contamination was removed from RNA following DNAse I treatment (NEB, Cat: M0303S) as itemized by the manufacturer. 2 μl solution comprising 100 ng DNA-free RNA was changed to cDNA via M-MuLV Reverse transcriptase Kit (NEB, Cat: M0253S) in 20 μl final volume (2 μl, N9 random primer mix; 2 μl, 10X M-MuLV buffer; 1 μl, M-MuLV RT (200 U/μl); 2 μl, 10 mM dNTP; 0.2 μl, RNase Inhibitor (40 U/μl), and 10.8 μl nuclease-free water). The reaction process continued at 25°C O/N. Inactivation of M-MuLV reverse transcriptase was achieved at 65°C/20 min.
PCR amplification and agarose gel electrophoresis.
We used the protocol illustrated in Ojo et al. to perform PCR intensification for the evaluation of genes whose primers (Primer3 software) are listed below [24, 44]. In a 25 μl volume mixture of 2 μl cDNA (10 ng), 2 μl primer (100 pmol), and 2 μl water, PCR enhancement was achieved. 12.5 μl Ready Mix Taq PCR master mix (One Taq Quick-Load 2x, master mix, NEB, Cat: M0486S) and 8.5 μl nuclease-free water. An initial denaturation at 95°C for 5 minutes was followed by 20 cycles of amplification (denaturation at 95°C for 30 seconds, annealing for 30 seconds, and extension at 72°C for 60 seconds) and a final extension at 72°C for 10 minutes). In all tests, we incorporated negative controls, where the mixture has no cDNA. The amplicons were sorted out on a 1.5% agarose gel (Cleaver Scientific Limited: Lot: 14170811) in Tris (RGT reagent, China, Lot: 20170605). -Borate (JHD chemicals, China, Lot 20141117) EDTA buffer (pH 8.4). The primer information can be found in the S1 File.
Amplicon image processing.
We caught the in-gel amplicon bands pictures on camera and treated them on the Keynote platform as revealed by Ojo et al. and evaluated them via image-J software [24, 44].
Histological studies.
After paraffin embedding, the fixed pancreatic tissues were stained with hematoxylin and eosin. A Leica slide scanner was used to view the slides (SCN 4000, Leica Biosystems, Germany) [45].
Statistical analysis
The study was conducted in triplicates for in vitro studies, and results are expressed as numbers, percentages, and mean ± SD. The data were analyzed using one-way analysis of variance (ANOVA) with Tukey’s post hoc test. We used the GraphPad Prism 9 edition to plot the graphs. A p value of < 0.05 is considered significant.
Results
High-performance liquid chromatography (HPLC-DAD) of AEAMP
The HPLC-DAD analysis of AEAMP (Fig 1) revealed several constituents present as revealed by the chromatograms. The compounds are luteolin, resorcinol, vanillin, benzoic acid, cyanidin, and quercetin (Table 1).
Cyanidin (peak 1), Vanillin (peak 2), Benzoic acid (peak 3), Luteolin (peak 4), Resorcinol (peak 5) and Quercetin (peak 6). AEAMP: aqueous extract of Annona muricata peel.
In vitro antidiabetic study (α-amylase and α-glucosidase activities)
Fig 2 shows that acarbose had the highest inhibitory efficacy against α-glucosidase when compared to AEAMP. AEAMP inhibited α-glucosidase at a maximum of 71.56% and acarbose at a maximum of 76.41%. Furthermore, there was a significant difference in AEAMP’s α-amylase inhibitory property (60.46%) compared to acarbose (76.41%).
Data are expressed as mean ±SD of triplicate determinations. AEAMP: aqueous extract of Annona muricata peels.
In vivo experimental studies
Rats administered with alloxan had an increase in FBG level. In contrast, FBG levels decreased after AEAMP administration at 6.76 mg/kg, 13.53 mg/kg, and 27.06 mg/kg b.wt. The diabetic groups treated with 27.06 mg/kg body weight had FBG levels that were comparable to the control group. At 27.06 mg/kg b.wt., the most pronounced effect was observed (Table 2). The induction of alloxan led to a significant decrease in the animals’ bodyweight (Table 3). The rats’ body weight increased after being treated with AEAMP at a dose of 26.06 mg/kg b. wt. The organ-to-body weight ratios revealed that the diabetic control pancreas values were higher than the other groups. The liver values revealed that the AEAMP-treated groups’ values were higher than the normal control rats and the metformin-treated group (Table 4).
Serum insulin and HOMA-β levels decreased significantly in diabetic untreated rats, whereas HOMA-IR levels increased (Table 5). The administration of AEAMP at 6.76 mg/kg, 13.53 mg/kg, and 27.06 mg/kg increased these parameters while decreasing HOMA-IR values. When AEAMP and metformin were administered to diabetic rats, the activities of GST, CAT, SOD, and GSH levels in the liver (Table 6) and pancreas (Table 7) were higher than in untreated rats. MDA levels were, however, depleted after administration of metformin and AEAMP, contrary to the increased levels in diabetic rats. The results were observed to be dose-dependent.
Alloxan administration raised TG, CRI, AI, VLDL-c, LDL-c, and total cholesterol (TC) concentrations while decreasing HDL-c levels (Table 8). AEAMP at 6.76 mg/kg, 13.53 mg/kg, and 27.06 mg/kg decreased the levels of these parameters when compared to diabetic rats. In contrast, AEAMP dosages increased serum HDL-c levels in diabetic rats. This is similar to metformin treatment, which resulted in lowering the parameters. Alloxan induction caused a decrease in the amounts of liver glycogen and the glycolytic enzyme, hexokinase, while administration of AEAMP and metformin increased these parameters (Table 9). Furthermore, G6P and F-1,6-BP activities were increased in diabetics untreated while treatment with AEAMP and metformin resulted in a decrease in activity (Table 9). In comparison to control rats, alloxan administration increased serum concentrations of IL-6, TNF-α, and NF-κB (Fig 3). AEAMP, like metformin, reduced IL-6, TNF-α, and NF-κB levels in diabetic rats. AEAMP at 27.06 mg/kg b.wt demonstrated the most pronounced reversal of the increases observed.
Data are expressed as mean ±SD (n = 6); AEAMP: aqueous extract of Annona muricata peels; CT: control group; DC: Diabetic control; DM: Diabetic metformin; AM-1: Diabetic + AEAMP (6.76 mg/kg); AM-2: Diabetic + AEAMP (13.53 mg/kg); AM-3: Diabetic + AEAMP (27.06 mg/kg); IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-alpha; NF-κB: Nuclear factor-kappa B; #: significantly different from normal control (p < 0.05); * is significant at p< 0.05 and ** is significant at p< 0.01, *** is significant at p< 0.001 versus diabetic control.
Gene expression.
Fig 4 depicts the PI3K, AKT, Bcl-2, and PCNA mRNA levels in the liver and pancreas. Liver PI3K, liver PCNA and pancreas PCNA were not significantly different in untreated diabetic rats when compared to normal rats suggesting alloxan induction of diabetes did not downregulate the mRNA expression of these genes. The expression of PI3K was shown to be down-regulated in the pancreas of diabetic rats. Diabetic rats, on the other hand, had reduced levels of mRNA expression for AKT, Bcl-2, and PCNA. Treatment with 6.76, 13.53, and 27.06 mg/kg of AEAMP and metformin upregulated pancreas PI3K and AKT mRNA levels, as well as Bcl-2 both in liver and pancreatic tissues (Fig 4).
GAPDH and cyclophilin were used as loading control for liver and pancreas PI3K/ATK, Bcl2, and PCNA. Data are expressed as mean ±SD (n = 6); AEAMP: aqueous extract of A. muricata peels; PCNA: proliferating cell nuclear antigen; CT: control group; DC: Diabetic control; DM: Diabetic metformin; AM-1: Diabetic + AEAMP (6.76 mg/kg); AM-2: Diabetic + AEAMP (13.53 mg/kg); AM-3: Diabetic + AEAMP (27.06 mg/kg).
Histological examination of the pancreas.
Fig 5 shows the histological evaluation of the pancreas of experimental animals after the induction of alloxan and administration of AEAMP. In contrast to the normal architecture displayed by the pancreas of normal control rats, the pancreas of alloxan-induced rats had significant necrosis, inflammatory infiltrates, and nuclear pycnosis. The administration of metformin and AEAMP decreased the occurrence of these changes in the pancreas of rats and revealed near-normal architecture comparable to the alloxan alone rats. It also improves and restores the impaired pancreatic islets cells at doses investigated.
MG X100; H & E stain. (A) CT: control group showing normal histomorphological presentation of the islet cells with characteristic normal staining intensity. There are no histopathological alterations (yellow arrow); (B) DC: Diabetic control showing normal general histomorphological presentation with conspicuous metallic deposit (yellow circles) suggesting possible mild pathological alteration, mild infiltration of inflammatory cells; (C) DM: Diabetic metformin; (D) AM-1: Diabetic + AEAMP (6.76 mg/kg); (E) AM-2: Diabetic + AEAMP (13.53 mg/kg); (F) AM-3: Diabetic + AEAMP (27.06 mg/kg) shows the restoration of the islet of Langerhans (IL) (yellow arrows).
Discussion
The need for qualitatively extensive research for the advancement of effective antidiabetic agents is on the rise, as the increased prevalence of T1DM poses a threat to human life. Hence, plants possessing pharmacological properties are continually evaluated with the expectation of establishing plant-based antidiabetic products that are relatively safe [46, 47]. The potential of the AEAMP was revealed in this study as it was shown to elicit a decline in the heightened plasma glucose level and diabetic complications.
The use of inhibitors of the α-glucosidase and α-amylase enzymes involved in carbohydrate metabolism is a recognized technique in the management of DM for decreasing postprandial hyperglycemia [48]. Efficient control of the glycemic index in diabetes requires moderate levels of the α-amylase inhibitors and potent levels of α-glucosidase inhibitors, which aid in efficiently regulating the available dietary sugar necessary for absorption in the small intestine [49]. The inhibitory effect of AEAMP on the activity of α-glucosidase and α-amylase was demonstrated to be concentration-dependent, which could be an indication of the extract’s antidiabetic action. Similarly, some fruits, vegetables, and mushrooms, as well as phenolics, flavonoids, and their glycosides, all from plant origin, have been reported to inhibit both α-glucosidase and α-amylase [50–53].
Alloxan, a diabetogenic agent, has been shown to cause extensive selective damage to pancreatic β-cells via reactive oxygen species generation, resulting in a partial or complete defect in insulin action and hyperglycemia. In the long term, metformin has demonstrated its capability for blood glucose level reduction, mediated majorly via suppressed glucose production in the liver, with improved insulin action in peripheral tissues [54]. Fasting blood glucose is a key clinical indicator of DM. An elevation in the blood glucose levels of the diabetic rats was confirmed, validating the successful induction of DM. The reduction in fasting blood glucose levels caused by AEAMP at various dosages could be due to pancreatic β-cell restoration, and the findings are consistent with previous research indicating the extract’s potential for hypoglycemic activity [55, 56].
A decline in the body weight of diabetic rats could be associated with structural protein degradation resulting from the occurrence of diabetes as proteins play a contributory role in body weight. The reduction in body weight observed in diabetic rats could be due to a reduction in carbohydrate metabolism, increased lipid metabolism, and degradation of proteins [57]. The increase in body weight of diabetic rats given the highest dose of AEAMP could be attributed to better glycemic control via enhanced insulin secretion. The effect of AEAMP on bodyweight suggests that it has the potential to improve the weight defect, which is a feature of DM due to the decreased hyperglycemia [58]. This is in accordance with the effects of known medicinal plants, such as Carica papaya, Garcinia kola, Muntingia calabura, Artemisia herba alba, and Dryopteris dilatata, used in the management of diabetes [59–62].
A defect in the action of the insulin hormone is one of the outcomes of DM, usually manifested in the form of inadequate or no insulin secretion [17]. A decline in the levels of serum insulin and deterioration in the function of the β-cells of the diabetic control group were evident in this study. In the AEAMP and metformin-treated groups, a notable improvement was observed as the β-cells were restored to levels close to the normal range. Serum insulin levels may have increased in the treatment groups due to the possibility of β-cell regeneration, as indicated by the higher HOMA-β score values in the treatment group. A decrease in the HOMA-IR scores of the treatment groups in contrast to the diabetic control group might be indicative of improved insulin sensitivity aside from stimulation of glucose absorption [17, 24]. Our results are in consistent with the findings of Ojo et al. [24] who reported an increase in insulin level and decrease in HOMA-IR scores the effect of in alloxan-induced diabetic rats treated with Persea americana seeds.
Non-enzymatic antioxidants (MDA) and enzymatic antioxidants (CAT, SOD, GPx, GSH, and GST) play critical roles in preserving the physiological levels of H2O2 and O2 by enhancing the dismutation and cleaning of O2 radicals and peroxides created by alloxan induction [63, 64]. This investigation found that alloxan-induced stress interfered with the function of hepatic and pancreatic enzymes [65]. The observed decrease in CAT, SOD, and GST activities, as well as GSH levels in diabetic rats’ liver and pancreas, could be attributed to the reduction of alloxan to dialuric acid, resulting in the production of free radicals [66]. AEAMP increased the activities of these enzymes, which reduced the disparity between ROS generation and the activities of CAT, SOD, and GST in diabetic rats. This could be because AEAMP contains phenolics and flavonoids, both of which have been shown to have antioxidant properties. Our results correlate with the reports documented by Okoduwa et al. [46] who reported that Ocimum gratissimum leaf fractions improved the antioxidant activities in type-2 diabetes rat model.
The phenomenon of dyslipidemia occurring in DM has been described by alterations in plasma lipoprotein regulated by the inadequacy of the pancreatic β-cells and hyperglycemia. Insulin modulates the activity of lipoprotein at specific levels, such as gene expression and protein synthesis. Hence, a decline in lipoprotein is observed in the presence of insulin resistance occurring in a diabetic state, resulting in increased triglyceride (TG), VLDL-c, and decreased HDL-c [67]. The observed levels of VLDL-c, triglycerides (TG), LDL-c, and total cholesterol (TC) observed in the diabetic control group corroborate prior reports [62, 67]. The elevated atherogenic index and LDL-c are suggestive of susceptibility to cardiovascular diseases [17, 24]. However, administration of AEAMP and metformin led to a significant decline in the VLDL-c, TG, LDL-c, and TC levels, with a resultant increase in the HDL-c levels. This is indicative of the potential of the extract to improve dyslipidemia due to the lipase activation required for lipid hydrolysis [68]. Our results agree with those of Ojo et al. [47] who documented that the extract of Blighia sapida bark improved the lipid parameters in alloxan-induced diabetic rats.
Glycogen, synthesized by glycogen synthase, is a storage disposition of glucose intracellularly within the liver, and its quantification is regarded as a crucial indication of DM [69]. Physiologically, mammals respond to elevations in blood glucose levels occurring after a meal by depositing glycogen in the liver and other adjoining tissues [70]. The amount of glycogen in the liver was shown to decrease in diabetic rats, which may be because of the absence of insulin, as DM has been reported to cause impairment of the liver’s capacity to appropriately synthesize glycogen. Oral administration of AEAMP and metformin was shown to elevate the glycogen levels significantly, and this could be owing to an improvement in the activity of insulin [69, 70]. Our findings correlate with some researchers who reported that Cenchrus purpureus improved glycogen levels in alloxan-induced diabetic rats [44].
Hence, the liver is the location for glucose production. The two major complementary metabolic pathways responsible for creating a balance for the glucose deposit in the body, which is usually identified by a limited or complete defect in insulin activity, are gluconeogenesis and glycolysis. These pathways play crucial roles during disorders in the metabolism of glucose, resulting in elevated glucose levels. Insulin aids in preventing hyperglycemia partly by subduing glycogenolysis and gluconeogenesis while promoting glycogenesis [70]. Hexokinase, a key enzyme in glucose catabolism, is responsible for mediating phosphorylation of glucose to glucose-6-phosphate. Impairment of glucose oxidation through glycolysis could occur as a result of decreased hexokinase activity, leading to a decline in the production of ATP and hyperglycemia [69]. Hexokinase activity was detected to decline in the diabetic control group, with a notable increase after the administration of AEAMP and metformin. Suppression of blood glucose levels is suggested to arise owing to the acceleration of the glycolytic pathway [70]. The enhanced activity by AEAMP can expedite glucose use for energy production [24, 44, 62].
Glucose-6-phosphatase (G-6-Pase) is an enzyme that is essential in the maintenance of blood glucose homeostasis and delivers glucose during hyperglycemia. A decrease in G-6-Pase activity causes metabolic alterations characterized by low blood glucose triggered by cAMP. In diabetes, insulin insufficiency raises G-6-Pase activity, resulting in a higher blood glucose level. In this study, AEAMP and metformin were found to reduce G-6-Pase activity in diabetic rats. G-6-Pase activity decreases glucose synthesis [24, 71].
In a diabetic state, fructose-1,6-bisphosphatase (F-1,6-BPase) is essential for glucose release into circulation. Its activity was found to increase in diabetic rats, most likely due to a lack of insulin. Treatment with AEAMP and metformin aided in the lowering of excessive F-1,6-BPase levels in diabetic rats. This enzyme’s activity may be altered by AEAMP and metformin due to their reduction of glycolytic and glucogenic pathways [44, 70].
The flavonoids (luteolin, resorcinol, cyanidin, vanillin, and quercetin) and benzoic acid were compounds identified to be present in AEAMP. These compounds occur abundantly in plants and have been established to exhibit numerous therapeutic activities, namely antioxidant, antiviral, antifungal, anticancer, anti-inflammatory, antimicrobial, anti-allergic, analgesic, neuroprotective, antidiabetic, antihypertensive, cardioprotective, anti-obesity, antitumor, immunomodulatory, chemopreventive, as well as regulation of the PI3K/AKT signaling pathway, amongst others [72–76]. Therefore, the therapeutic activities exhibited by AEAMP, as evidenced by the results, could be because of these compounds.
The PI3K/Akt pathway appears to be a key player in mediating cell survival in a variety of situations. A number of signaling cascades, such as the phosphatidylinositol-3-OH kinase (PI3K)/Akt ([also known as protein kinase B]), the Ras-mitogen-activated protein kinase (MAPK), and the cAMP/protein kinase A (PKA) pathways, are activated by trophic factors such as NGF, insulin-like growth factor I, or BDNF. These aforementioned pathways help cells survive in specific circumstances [77]. According to Ojo et al. [24], the PI3K/AKT pathway is important in carbohydrate metabolism, which is required for glucose uptake enhanced by insulin in the liver [24, 78–80]. By phosphorylation, AKT activation enhances cell survival [81]. Insulin action is facilitated via the stimulation of PI3K and its effectors. AEAMP could halt oxidative injury caused by alloxan via an anti-apoptotic mechanism and boost the expression of phosphorylation of AKT [82]. AEAMP restored the decreased levels of PI3K and AKT in diabetic rats (Fig 6). This is in agreement with the study reported on the antidiabetic activity of avocado seeds in diabetic rats via upregulation of PI3K/AKT [24].
The proposed mechanism of action of A. muricata peels in diabetic rats on improving insulin binding and increase glucose metabolism. A. muricata, upregulates mRNA expression of the pancreatic PI3K, Akt, and Bcl2 genes and liver Akt, and Bcl2 genes while liver PI3K, PCNA, and pancreas PCNA were not downregulated by alloxan. This leads to increase in the insulin sensitivity and reduce the blood glucose.
BCL-2, an anti-apoptotic molecule, is a key player involved in cell death signaling. Alloxan induction caused a lower expression of BCL-2 in hepatic and pancreatic tissues, whereas AEAMP significantly changed the equilibrium of anti-apoptotic molecules and averted apoptosis in these tissues at 6.76 and 13.53 mg/kg, respectively, as did metformin. This is consistent with the studies reported on the effect of Cenchrus purpureus in diabetic rats and the comparison of streptozotocin-induced diabetic and insulin resistant effects on spermatogenesis with proliferating cell nuclear antigen (PCNA) immunostaining of adult rat testis [44, 83].
At the start of cell propagation, proliferating cell nuclear antigen (PCNA), a vital proliferation marker, facilitates DNA polymerase. In addition to antibodies against foreign substances, PCNA plays an important role in the eukaryotic cell cycle [24, 44, 82, 84]. PCNA mRNA expression was found to be upregulated in normal rat hepatocytes and pancreatic tissues but downregulated in diabetic rats. AEAMP increased PCNA expression in diabetic rats’ liver and pancreatic tissues. This is consistent with earlier studies reported on the effect of Persea americana seeds and Cenchrus purpureus on PCNA expression in diabetic rats [24, 44].
Conclusions
Taken together, AEAMP reduces insulin resistance in diabetic rats. In diabetic rats, the probable molecular processes include an increase in glucose absorption, improved hexokinase activity, and a reduction in pancreatic β-cell apoptosis via the PI3K/Akt gene upregulation (Fig 6). Because it reveals a regulated high blood glucose level and other related biochemical parameters, AEAMP could be a valuable source of antidiabetic drugs. Liver PI3K, liver PCNA and pancreas PCNA were not significantly different in untreated diabetic rats when compared to normal rats suggesting alloxan induction of diabetes did not downregulate the mRNA expression of these genes. AEAMP significantly up-regulated expression of AKT and Bcl2 in the liver and pancreatic tissue.
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