Ethanol extract of Annona muricata Linn fruit perform antidiabetic effect on type 2 diabetic mice through α-glucosidase inhibition

QIN Hai-long, YAO Jia-hui, WANG Yan, XU Qi-peng, LIU Zheng, LI You-bin, GONG Jing-wen

Hainan Provincial Key Laboratory for Research and Development of Tropical Herbs; Key Laboratory of Tropical Translational Medicine of Ministry of Education; Haikou Key Laboratory of Li Nationality Medicine; School of Pharmacy, Hainan Medical University, Haikou 571100, China

ABSTRACT Objective: To explore the anti-diabetic effects and its underlying mechanism of Annona muricata Linn fruit ethanol extract (AME).Methods: Streptozotocin-induced type 2 diabetic(T2DM) mouse model was constructed.Those diabetic mice were randomly grouped and given 50 mg/kg acarbose or AME (200 mg/kg, 100 mg/kg or 50 mg/kg) for four weeks.The body weight, postprandial blood glucose and glycosylated hemoglobin levels were measured during the administration.After the administration, a glucose tolerance test was performed,and the levels of triglycerides, cholesterol and low-density lipoproteins in mice were detected by biochemical test kits.The inhibitory activity of AME on α-glucosidase in vivo and in vitro was determined by enzyme inhibition tests.Results: AME significantly reduced weight gain, postprandial blood glucose, glycosylated hemoglobin and low-density lipoprotein levels in T2DM mice; enhanced glucose tolerance and pancreatic β-cell function of T2DM mice;inhibited α-glucosidase activity in mouse intestine in an noncompetitive manner.Conclusion:AME may noncompetitive inhibit α-glucosidase activity and reduce postprandial glucose intake to achieve a therapeutic and regulatory effect on type 2 diabetes.

Keywords:

Annona muricata Linn

Type 2 diabetes mellitus

α-glucosidase

Postprandial blood glucose

1.Introduction

Diabetes mellitus is a metabolic disorder that attracts worldwide attention, which causes a high medical burden and affects the life quality of patients.It is predicted that the number of people with diabetes will climb to about 700 million in about 20 years, and people with type 2 diabetes mellitus (T2DM) will account for about 90% of them[1].Currently, the management of T2DM focuses on increasing exercise and controlling blood glucose, thus preventing the progression of diabetes and other complications.Poor glycemic control in T2DM patients is likely to lead to cardiovascular disease or other diabetic complications, thus deepening the physical and financial burden of the patient[2].In addition, there is growing evidence that postprandial glucose fluctuations are highly correlated with microvascular and macrovascular morbidity or cardiovascular mortality in T2DM patients, and that postprandial hyperglycemia is an important factor in the development of T2DM disease and complications[3].Therefore, one of the main strategies for the treatment of T2DM is to lower postprandial glucose levels, and α-glucosidase is mainly related to gastrointestinal carbohydrate catabolism and glucose absorption functions[4], the related α-glucosidase inhibitors have also been developed by this mechanism.

Currently, chemically synthesized drugs such as acarbose and voglibose, which inhibit α-glucosidase activity to treat T2DM,are commonly associated with side effects such as gastrointestinal dysfunction[4,5].For this reason, patients are not too happy to take them regularly despite their efficacy.Therefore, many researchers are gradually shifting their attention from chemosynthetic drugs to natural drugs, hoping to take advantage of their natural origin,reliable and stable efficacy[6], and low side effects, so that more efficient and safe natural active ingredients can be developed from them.

Annona muricata Linn (AML) is known for its many medicinal properties.According to traditional books, AML is used to treat diabetes, headache, insomnia, cystitis, liver disease, anti-tumor,and anti-inflammatory diseases[7].Modern pharmacological studies have shown that AML has anti-tumor, anti-bacterial and anti-oxidant effects[8].In recent years, in vitro experiments and blood experiments on healthy rats have found that the fruit extracts of AML can significantly inhibit α-glucosidase and α-amylase activities[9,10].However, in vivo antidiabetic studies on the fruit extracts of AML are less reported, focusing mostly on the leaf extracts of AML[11,12].The fruit extract of AML was only reported to improve glucose tolerance in normal mice[13], but the underlying mechanism of action is not clear, and the real effect of its action on the treatment of diabetes has not yet been studied.Therefore, in this paper, we investigated the real hypoglycemic effect of the ethanolic extract of the fruit of Annona muricata Linn (AME) by examining the postprandial glucose, body weight changes and some other serological indicators in the T2DM model mice.At the same time, we will try to detect the inhibitory activity of AME on α-glucosidase ether in vitro or in vivo, and study the possible mechanism of action of AME in T2DM model mice, so as to provide scientific basis for the development and utilization of AML fruits.

2.Materials and Methods

2.1 Materials

2.1.1 Drugs and reagents

Ethanol extract of Annona muricata Linn (AME): The extraction method was similar to that used in the literature by Jingwen Gong et al [14,15].128 g of infusion was extracted from each 1 Kg of AML fruit.Before using these extracts, they were dissolved with ultrapure water containing 1% DMSO, thus configuring the sample solution to the appropriate concentration.

All of the compounds used in this article were purchased from Solarbio, except for streptozotocin (Sigma, USA).The serum biochemical index detection kit was purchased from Jiufubio,BioSino Bio and J&L Biological; the α-glucosidase activity detection kit was purchased from Solarbio.Both basic feed and high-fat feed were purchased from Changsha Tianqin Biotechnology Company.

2.1.2Animals

SPF ICR mice (male, weight 18 ± 2 g) were fed in SPF-grade animal holding rooms and they can drink and feed freely.This batch of experimental mice were purchased from Changsha Tianqin Biotechnology Company.(License No.: SCXK (Xiang) 2019-0013).The experimental design and implementation were in accordance with the ethical and welfare principles of experimental animals,and the experimental protocol was reviewed and approved by the Laboratory Animal Management Committee of Hainan Medical University (HYLL-2019-024).

2.1.3 Apparatus

Yuwell 580 Blood Glucose Meter (Jiangsu Yuwell Medical Group);Spectra Max 190 full wavelength microplate reader (Molecular Devices Company), Indiko?automatic biochemical analyzer(Thermo Fisher Scientific).

2.2 Methods

2.2.1 Model building of experimental animals

The T2DM mouse model was constructed using the modeling method previously reported by Jingwen Gong et al (but not using nicotine)[14,16].After 4 weeks of high-fat diet, mice in the experimental group were injected with streptozotocin (STZ) at the rate of 150 mg/kg for modeling The control group was fed with ordinary feed for 4 weeks, and the rest operations was same to the above mentioned literature.

2.2.2 Screening and drug administration of the model mice

After STZ modeling, mice with fasting blood glucose value greater than 11.1 mmol/L were selected for the subsequent trial and rerandomized into Model group (MOD), Acarbose (Acarbose, 50 mg/kg), AME high-dose (AME-H, 200 mg/Kg), medium-dose(AME-M, 100 mg/Kg) and low-dose (AME-L, 50 mg/Kg) group,10 mice per group.Mice in the common feed group were set to the blank control group (CON).Mice in each group were administered by gavage once a day for 4 weeks.The model group and the control group were given the corresponding vehicle, and the body weight and postprandial blood glucose values of the experimental mice were monitored regularly during the experiment.

2.2.3 Determination of hemoglobin levels

On the seventh day before the end of the administration, the glycosylated hemoglobin (HbA1c%) levels of each group of mice were measured using the HbA1c% test kit.

2.2.4 OGTT experiment

At the end of drug administration, OGTT experiments were performed on each group of mice according to the OGTT experimental method in the literature of Jingwen Gong et al[14].Calculate the area under the drug-time curve (AUC) for each group of mice according to the time and blood glucose value curves.

2.2.5 Measurement method of each biochemical index

After the administration, the mice in each group were fasted for 12 h, and then tested for biochemical indicators.After drawing and collecting blood from the eye of each mouse, putting the blood stand for 2 h and centrifuged for 10 min (4℃, 5 000 r/min) in the cryogenic centrifuge.After blood collection, all experimental mice were executed using the cervical dislocation method, and the small intestinal endothelial tissue was removed and stored frozen at -80 ℃in a refrigerator.The levels of triglycerides (TG), total cholesterol(TC) and low density lipoprotein (LDL) were measured in the serum,and fasting insulin levels (FINS) were measured in each group of mice using a microplate reader.Relevant data were analyzed by statistical methods.Calculate the HOMA-IR index and HOMA-β index, and comprehensive glucose and lipid metabolism parameters were used to comprehensively evaluate the effect of each treatment group on T2DM mice.The calculation method is the same as the one used in Gong Jingwen’s literature [14].

2.2.6 Inhibition experiments of α-glucosidase activity in vivo

Take 0.1 g of frozen small intestinal endothelial tissue from each group and prepare samples to be tested according to the instructions of α-glucosidase activity detection kit.The α-glucosidase activity of mice in the control group was seen as 100%, and the inhibition rate of α-glucosidase was calculated for each administration group.

2.2.7 Activity inhibition experiment of α-glucosidase in vitro

Take 50 μL fruit extracts which has diluted to corresponding concentrations (0.2, 0.4, 0.6 and 0.8 mg/mL), added to the prepared solution of 1.0 M phosphate buffer (pH 6.9) with 100 μL α-glucosidase solution (1.0 U/mL) and culture at 25℃ for 10 min.Use 0.1 M phosphate buffer (pH 6.9) to dissolve few p-nitrophenylβ-D-glucopyranoside (pNPG) into 5 mM solution.Add another 50 μL pNPG solution to the mixed enzyme solution, which has already incubated for 10 min, and then continue incubated at 25℃ for 8 minutes.After completing the above steps, put the 96-well plate placed in the enzyme marker to measure the absorbance value at the wavelength of 405 nm.Acarbose (10, 20, 30 and 40 μg/mL)was used as a positive control, thus calculating the inhibition of α-glucosidase by different concentrations of AME..The calculation formula is:

2.2.8 Inhibition type experiment of α-glucosidase

The experimental method in the literature of Qinan Wu et al.is referred to and modified to complete the experiment[17].50 μL of AME at 0.2 mg/mL and 0.8 mg/mL were pipetted into a 96-well plate, and 100 μL of α-glucosidase solution at a concentration of 1 U/mL was added to each well, and then mixed well and reacted at 25℃ for 10 min.Then, 50 μL of pNPG solutions with concentrations of 0.625, 1.25, 2.5, 5.0 and 10.0 mM were added, and mixed evenly again.The absorbance (at 405 nm) was then measured every 5 min at 25℃ and the Lineweaver-Burk double inverse curve was plotted to determine the type of inhibition of α-glucosidase by AME.

2.2.9 Data processing

The results data were expressed as mean ± sample standard deviation (±s), and were statistically analyzed by GraphPad prism 6.5 according to one-way ANOVA or Kruskal-Walls H test (P＜0.05 was considered significant).

3.Experimental results

3.1 Effects on body weight

During the 4 week experiment, the weight growth rate of mice in model group was significantly higher than that in control group(Table 1, P＜0.01).Acarbose significantly controlled the weight growth of mice, and the growth rate was slow.Acarbose showed significant difference with the model group due to the rapid weight growth of mice since the first week of administration.

The body weight of mice in the three AME dose groups increased slowly during administration, showing significant differences from the first week compared to the model group (P＜0.01 or P＜0.001).Compared with AME-L group, AME-H and AME-M groups showed better weight control in T2DM mice.

3.2 Effects on blood sugar

As can be seen from Table 2, the post-prandial blood glucose changes of the mice in the control group fluctuated little and tended to be stable under the feeding of sufficient feed and water.Mice in the model group remained hyperglycemic (P＜0.001 vs control group).Post-prandial blood glucose levels in T2DM mice began to decrease gradually after 7 d of acarbose administration (P＜0.05 vs model group), and the inhibitory effect was significantly enhanced after one week (P＜0.01 vs model group).200 mg/kg AME also inhibited post-prandial blood glucose on day 7 (P＜0.05 vs model group), and AME at all three concentrations showed significant hypoglycemic effects from day 14, showing a dose-dependentinhibitory relationship.

Tab 1 Body weights of T2DM mice during 4 weeks(n=10,±s)

Tab 1 Body weights of T2DM mice during 4 weeks(n=10,±s)

Note:comparison with control group: #P＜0.05, ##P＜0.01, ###P＜0.001; comparison with model group: *P＜0.05, **P＜0.01, ***P＜0.001.

Weight/(g)Day 1 Day 7 Day 14 Day 21 Day 28 Control - 32.13±0.42 33.37±0.33 34.67±0.29 36.61±0.35 38.54±0.31 Model - 35.95±0.36### 39.10±0.47### 41.60±0.838### 44.06±0.33### 46.14±0.40###Acarbose 50 35.66±0.47### 35.77±0.39***,# 37.18±0.43***,##9 37.69±0.44*** 38.61±0.43***AME-H 200 35.39±0.50## 35.85±0.55***,## 37.04±0.52***,## 37.85±0.52*** 38.60±0.50***AME-M 100 35.42±0.67## 35.25±0.57*** 36.44±0.48*** 37.33±0.47*** 38.26±0.49***AME-L 50 35.35±0.78## 35.75±0.57***,# 37.39±0.52***,## 39.00±0.48***,## 40.17±0.43***Group Dose(mg/kg)

Tab 2 Postprandial blood glucose of T2DM mice during 4 weeks(n=10,±s)

Tab 2 Postprandial blood glucose of T2DM mice during 4 weeks(n=10,±s)

Note: During the whole administration period, the blood glucose values of the three AME groups were consistently significantly different from those of the control group, and the labeling is omitted here.Comparison with control group: ###P＜0.001; comparison with model group: *P＜0.05, **P＜0.01, ***P＜0.001.

Post-prandial blood glucose/(mmol/L)Day 1 Day 7 Day 14 Day 21 Day 28 Blank - 7.77±0.11 7.85±0.13 7.83±0.10 7.88±0.08 7.86±0.10 Model - 23.02±0.84### 23.13±0.88 ### 24.42±0.63### 25.25±0.57### 26.34±0.39###Acarbose 50 23.87±0.77### 20.43±0.38* 18.84±0.31*** 17.21±0.44*** 15.68±0.51***AME-H 200 23.20±0.41### 20.12±0.42** 18.95±0.39*** 16.96±0.33*** 14.98±0.24***AME-M 100 22.83±0.64### 20.51±0.66 18.57±0.59*** 16.51±0.62*** 15.96±0.58***AME-L 50 23.87±0.67### 21.21±0.60 19.47±0.57*** 17.56±0.54*** 16.48±0.55***Group Dose(mg/kg)

3.3 Effect on glycosylated hemoglobin levels

Glycated hemoglobin (HbA1c) could reflect the body’s blood glucose level 2 to 3 months prior to the blood being taken, and is the most valuable indicator to evaluate the blood glucose control ability [18].As shown in Figure 1, the HbA1c% level [(8.18±0.34)%]of mice in the model group was significantly higher than that in the control group [(3.93±0.21)%] (P＜0.001).Compared with the model group, acarbose and AME-H groups could significantly reduce the HbA1c% level, which was [(5.47±0.20)%] (P＜0.01)and [(4.92±0.31)%] (P＜0.001), respectively.In addition, only the HbA1c% level of AME-H group was close to the control group (P＞0.05).In conclusion, AME can significantly reduce the level of glycosylated hemoglobin in model mice, and the level of glycosylated hemoglobin in T2DM mice is close to the normal level.

Fig 1 HbAlc level in each drug group(n=10, ±s)

3.4 Effect on glucose tolerance

Glucose tolerance reflects the body’s ability to regulate blood glucose after eating glucose, and can evaluate whether the function of pancreatic beta cells is impaired[19].As shown in Figure 2, the area under the curve of the model group (3 600.00±71.40 mg/mL*min) was significantly larger than that of the control group (1 386.00±186.81 mg/mL*min) (P＜0.001), indicating that the model group mice had poor regulation of glucose.The area under the curve of the acarbose group (2 926.00±85.64 mg/ml*min) and the AME-H group (2963.00±98.05 mg/mL*min) was significantly lower than that of the model group (P＜0.05, P＜0.05), indicating that AME in the high concentration group, like acarbose, was able to suppress the oral glucose-induced increase in blood glucose.In other words,AME-H regulate the glucose metabolism well in mice, and enhance the glucose tolerance of the mouse organism, which is beneficial to the regulation of mouse blood glucose.The above results indicate that AME in the high concentration group has a good ability to regulate glucose metabolism in T2DM mice, which is beneficial to control the blood glucose of diabetic mice.

Fig 2 AUC of time-postprandial blood glucose curve in each group(n=10,±s)

3.5 Effect on TG、TC and LDL levels

Diabetic patients often suffer from hyperlipidemia due to poor dietary habits leading to excessive fat intake, which increases fatty acid oxidative stress and decreases glucose uptake and utilization,resulting in insulin resistance [20].TG, TC and LDL are important indicators for the evaluation of lipid metabolism disorders in diabetes.The normal range for TG is 0.22-1.65 mmol/L, for TC is 2.9-6.0 mmol/L, and for LDL is 0.78-2.2 mmol/L.As shown in Table 3, TG, TC and LDL levels of mice in the model group were higher than those of mice in the control group (P＜0.05), while acarbose could significantly reduce LDL levels (P＜0.05) and also TG levels in mice, but there was no significant difference, and there was no significant effect on TC levels.The AME-H group significantly reduced LDL levels in mice (P＜0.05), and the AME-M and AME-L groups also had reduced LDL levels compared to the model group,but failed to show significant differences.The AME-H group reduced the TG level to be within the normal range, but failed to show a significant difference, while the AME-M and AME-L groupshad different degrees of elevated TG levels compared to the model group, beyond the normal range.

Tab 3 TG、TC and LDL levels in each group(n=10, ±s)

Tab 3 TG、TC and LDL levels in each group(n=10, ±s)

Note: Comparison with control group (CON): #P＜0.05; comparison with model group (MOD): *P＜0.5.

Serologic parameters Blank Model Acarbose AME-H AME-M AME-L TG 0.74±0.08 2.10±0.15# 1.52±0.32 1.32±0.21 2.00±0.28 2.02±0.26 TC 2.26±0.17 4.96±0.49# 5.26±0.31# 5.56±0.34# 6.83±0.94# 5.62±0.61#LDL 0.84±0.12 1.51±0.24# 0.92±0.02* 0.86±0.10* 0.94±0.14 1.05±0.16

3.6 Inhibition of α-glucosidase in the intestinal tract of T2DM mice

As shown in Table 4, the inhibitory effect of AME on α-glucosidase in the intestine of T2DM mice gradually increased with increasing drug concentration (P＜0.001), showing a dosedependent inhibitory effect.The inhibition rate of 200 mg/kg AME was 84.10±3.0%, while that of acarbose was 82.11±1.1%.It indicates that AME may regulate blood glucose by inhibiting the intestinal uptake of carbohydrates in T2DM mice, and the inhibitory effect is close to that of acarbose.

Tab 4Inhibition rate of each drug on α-glucoside in T2DM mice(n=10,±s)

Tab 4Inhibition rate of each drug on α-glucoside in T2DM mice(n=10,±s)

Note: Comparison with model group (MOD): ***P＜0.001.

Group Inhibition rate on α-glucoside (%)Blank 0 Model 17.80±2.2 Acarbose 82.11±1.1***AME-H 84.10±3.0***AME-M 75.61±2.8***AME-L 58.64±4.8***

3.7 Inhibition of α-glucosidase by extracts from various parts of AML

As can be seen from Figure 3, the inhibitory effect of extracts from various parts of AML on α-glucosidase was gradually enhanced with increasing concentration.In contrast, at the same concentration,the fruit extract showed the strongest inhibitory effect, followed by the leaf extract and the root extract was the weakest.The same conclusion is supported by the EC50values of each extract in Table 5.The EC50 value of the fruit extract was the smallest among the three extracts (164.8±7.3 μg/mL), which indicated that the fruit extract had the strongest inhibitory effect on α-glucosidase compared to the root and leaves, but was significantly weaker than the positive inhibitor acarbose (4.0±0.0 μg/mL).

Fig 3 inhibition rate (%) of each extract and acarbose on αglucosidase(n=10,±s)

Tab 5 EC50 of each drug on α-glucoside(n=10, ±s)

Tab 5 EC50 of each drug on α-glucoside(n=10, ±s)

Note: Letters a, b, c, and d in the table indicate significant differences between the groups (P＜0.001).

Classification EC50(μg/ml)the ethanol extract of the fruit of AML(AME) 164.8±7.3a the ethanol extract of the root of AML 767.3±20.9b the ethanol extract of the leaves of AML 323.9±5.0c acarbose 4.0±0.0d

3.8 Types of α-glucosidase inhibition by AME

The results in Figure 4 show that the double inverse curves of AME at different concentrations intersect at a point on the horizontal axis, indicating that they have equal values of the Km.As the AME concentration increases, the slope increases, which indicates that the reaction rate (Vmax) decreases.These characteristics are consistent with the action characteristics of a non-competitive inhibitor of this enzyme, and it is hypothesized that AME exerts its hypoglycemic effect by non-competitive inhibition of α-glucosidase activity, a conclusion that is consistent with the results already reported[21].

Fig 4 Lineweaver-Burk plots of the reactions of α-glucosidase with AMEs

4.Discussion and conclusion

Diabetes mellitus is a chronic metabolic disease that plagues countries around the world.Its pathogenesis is complex, and the pathogenesis has not been fully studied.Patients commonly suffer from a variety of symptoms such as disorders of glucose and lipid metabolism and insulin resistance.Most current treatments for the disease are glycemic control and increased exercise.However, there are problems with drug resistance to the relevant therapeutic drugs,and diabetics are less willing to use them, most of them often live with a poor quality of life and a high financial burden.If the blood glucose of a T2DM patient is not well controlled, it usually leads to an exacerbation of diabetes, as well as to cardiovascular disease and other complications.But measuring fasting blood glucose and glycated hemoglobin alone does not accurately reflect glycemic control because they do not reflect what may occur after meals and throughout the day when the patient is living in free-living conditions[19].Researchers found that postprandial glucose fluctuations can affect micro/macrovascular morbidity or cardiovascular mortality in T2DM patients more than HbA1c or fasting glucose levels[3].Therefore, in the treatment of diabetes, it is not only necessary to lower the patient’s HbA1c level and fasting blood glucose level,but also to regulate the patient’s postprandial blood glucose level.Therefore, it is especially important to develop effective drugs and therapies that can regulate patients’postprandial blood glucose.

There are many models for studying diabetes, and drug-testing models for studying postprandial blood glucose levels should simulate the free-living state of patients with T2DM so that the effect of drugs on the patient’s postprandial and all-day blood glucose regulation can be determined.The present study showed that the T2DM model mice were successfully modeled and their blood glucose remained high for a long time, and during the modeling period, their body weight increased rapidly and the TG, TC and LDL levels increased significantly.During the 4 weeks of drug administration, T2DM mice had free access to food and water from 7 pm to 7 am, and we measured postprandial glucose values at 7 am daily to mimic the daily diet of T2DM patients in order to test the effect of AME on postprandial glucose regulation.The positive drug acarbose was able to suppress the postprandial blood glucose and body weight gain in the model mice stably, maintaining them in a reasonable range.The AME-H group significantly reduced postprandial glucose, glycated hemoglobin and low-density lipoprotein levels and controlled body weight gain in mice.In addition, the glucose tolerance test showed that the AME-H group was able to enhance the ability of the mouse organism to tolerate sugar.The above data indicate that AME can effectively regulate postprandial glucose and improve diabetic symptoms in T2DM mice.In conclusion, Annona muricata Linn fruit extract demonstrated effective ability to lower and control postprandial glucose in the treatment of T2DM.

α-glucosidase is a key enzyme that promotes starch hydrolysis,and α-glucosidase inhibitors are commonly used in the treatment or management of diabetes to control postprandial glucose by inhibiting the hydrolysis of starch to reduce the concentration of glucose absorbed into the blood[22].The in vitro and in vivo α-glucosidase inhibition experiments in this study showed that AME had an extremely significant enzyme inhibition effect, which was better than that of the leaf and root extracts of the same plant.Furthermore, the type of inhibition of AME on α-glucosidase is non-competitive, which indicates that AME has no effect on the binding of α-glucosidase to its substrate.This suggests that the inhibitory effect of AME cannot be alleviated by increasing the substrate concentration (intestinal carbohydrates).This means that AME can effectively inhibit α-glucosidase regardless of how many carbohydrates the patient eats, thus controlling post-meal blood glucose levels at low values.This can undoubtedly provide a great convenience to diabetic patients’meals, thus reducing the impact on their quality of life.

α-glucosidase inhibitors can also be used to treat prodromal diabetes.Prodromal diabetes is the stage when blood glucose levels have been above normal for a long time but diabetes has not yet developed, and this is the best time for drug intervention[23].By controlling postprandial blood glucose, there is a certain chance that the patient’s blood glucose level can return to normal.Therefore,AME can also be tried for the treatment of prodromal diabetes.

In summary, the extract of the fruit of Annona muricata Linn was able to regulate postprandial glucose in a T2DM mouse model,reversing various symptoms of type 2 diabetes, such as high HbA1c% levels and LDL levels.The mechanism was most likely related to the inhibition of α-glucosidase activity.The ability of AME to suppress postprandial blood glucose may also enable its attempted application in the treatment of prodromal diabetes.This study provides the experimental data and theoretical basis for the prevention and treatment of AME on type 2 diabetes, and lays the scientific foundation for the further development and utilization of the fruit of Annona muricata Linn.

Author Contributions

Data curation: Hailong Qin, Jingwen Gong.

Software: Hailong Qin, Jingwen Gong.

Supervision: Youbin Li.

Validation: Youbin Li.

Writing - original draft: Hailong Qin, Jiahui Yao, Yan Wang, Qipeng Xu, Zheng Liu.

Writing - review & editing: Youbin Li, Jingwen Gong.There is no conflict of interest in this article.