Original Article

Exploration of Antidiabetic Activity of the Whole Plant Extract of Eclipta prostrata by In Silico, In Vitro, and In Vivo Methods

Please cite this article as follows: Muni M, Mim MMA, Aktar F, Afrin SS, Alam S, Sikder AA, et al. Exploration of antidiabetic activity of the whole plant extract of eclipta prostrata by in silico, in vitro, and in vivo methods. Avicenna J Med Biochem. 2024; 12(2):64-76. doi:10.34172/ajmb.2531

Background

Diabetes is one of the major chronic diseases affecting the world. According to the World Health Organization (WHO), approximately 422 million people have diabetes worldwide. In Bangladesh alone, there are about 25 million instances of diabetes, and this number is steadily rising. The prevalence rate increased from 5% to 13% between 2001 and 2018 (1). Approximately 8.1% of adults, or 8.4 million individuals aged 20 to 79, have diabetes (2). Diabetes occurs either when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces. Insulin is a hormone that regulates blood sugar by increasing its uptake into tissues from the blood (3). As a result of insulin deficiency, glucose levels rise in the blood, leading to harmful consequences in various organs of the body. Left uncontrolled, diabetes eventually causes disability and death. Diabetes is a lifelong condition that cannot be cured but only be managed. Despite the availability of several antidiabetic drugs in the market, the search for newer antidiabetic agents from natural sources persists. The side effects and the development of resistance due to the long-term use of modern medications encourage people to seek natural alternatives with fewer side effects (4). In this quest for natural antidiabetic agents, native plants known for their medical properties since ancient times (5-9) have attracted significant attention. Eclipta prostratais one such plant.

Eclipta prostrata is a member of the Asteraceae family and is widely distributed in tropical and subtropical regions. It is commonly found in Asia, North America, and parts of Africa, Europe, Oceania, and Central America. It can survive in a wide temperature range (20-38 °C) and a pH range of 4-8 (10). Commonly known as the False daisy. E. prostrata contains various natural compounds, including flavonoids, alkaloids, triterpenoids, saponins, phenolic compounds, essential oils, thiophenes, and steroids. It also has a high concentration of saponins and tannins. Many of the plant’s compounds remain unidentified. Due to its diverse compounds, E. prostrata has been used in the treatment of several diseases, including infectious hepatitis, snake venom poisoning, gastritis, and respiratory diseases such as cough and asthma (11). It is also used to treat hemorrhagic diseases, respiratory disorders, skin diseases, microbial infections, heart disease, vitiligo, snake bites, hair loss, hepatic disorders, dizziness, and renal diseases (12).

Eclipta prostrata has exhibited antidiabetic and antihyperlipidemic activity. When administered at a dose of 300 mg/kg body weight, its extract has been shown to significantly reduce glucose levels in diabetic rats when compared to untreated diabetic rats (13). The secretion of pancreatic beta cells in diabetic rats has been brought back to normal levels when treated with the medicinal plant (14). Despite a few in vivo studies, there is a notable lack of in vitro or in silico studies exploring the extent or mechanisms of E. prostrata’s antidiabetic activity (15); therefore, the exact mechanisms behind its antidiabetic activity remain unclear. As such, the current study aimed to establish the antidiabetic activity of E. prostrata both qualitatively and quantitatively by applying in silico, in vitro, and in vivo methods, with a focus on shedding further light on the mechanism of action behind its activity.

Materials and Methods

In Silico Study Assessing the Antidiabetic Potentialities of Selected Phytocompounds

Selection, Optimization, and Ligand Preparation

A ligand library containing 115 constituents of E. prostrataextract was prepared through literature mining. The 3-dimensional structures of the ligands were downloaded in the sdf format from the ‘PubChem’ database, and the structures not available were drawn using the ‘Avogadro’ software package (16,17). All ligands were optimized under the MMFF94 force field using the steepest descent algorithm, with the convergence value set to 10e-7, in the ‘Avogadro’ software package and saved in pdb format.

Protein Preparation and Molecular Docking Study

Five common antidiabetic drug macromolecular targets, namely, α-amylase, α-glucosidase, AMP-activated protein kinase (AMPK), dipeptidyl peptidase-IV (DPP-IV), and peroxisome proliferator-activated receptor-γ (PPAR-γ) were downloaded from the ‘Protein Data Bank’ database in pdb format. Their respective PDB IDs are 3OLG, 2ZE0, 6C9F, 2G5T, and 4EMA (18). The macromolecules were prepared using the PyMol software package, and energy minimization was carried out in vacuo under the ‘GROMOS96’ force field, using the 43B1 parameters set via the Swiss-PdbViewer 4.1.0 software (19-21).

The PyRx software, using its AutoDock Vina component, was used to perform molecular docking. The binding sites were obtained from previous literature and specified in the docking procedure. The results were then analyzed and visualized using the PyMol and Discovery Studio Visualizer 2020 software packages (20,22).

In Vitro and In Vivo Activity Assessment of Eclipta prostrata

Plant Collection and Preparation

Fresh herbs of E. prostrata, weighing around 10 kg, were purchased from the Bangladesh National Herbarium, Dhaka. The plants were washed thoroughly and shed-dried for two weeks. Afterward, they were dried in a temperature-controlled oven at 40 to 50 °C to reduce moisture content. Once fully dried, the herbs were converted into fine powder and weighed, with the final weight of E. prostrata powder being around 650 g.

Botanical Authentication

Each part of the plant was deposited at the National Herbarium for botanical authentication according to their guidelines. The herbarium authority provided the accession number DACB 90637 for the plant specimen.

Ethical Clearance

Ethical approval was obtained on 18 July, 2023 from the Ethical Review Committee of the Faculty of Pharmacy, University of Dhaka (Reference No. Fa. Ph. E/017/23).

Extract Preparation

The dried plant powder was macerated with a 70% ethanol solution for 20 days (23). The mixture was shaken daily and filtered every 7 days, with the residue being redissolved in a fresh solvent. The resulting extract was collected in a beaker, and the solvent was evaporated under reduced pressure using a rotary evaporator. After evaporation was completed, the dried extract was collected and stored in a container with proper labeling.

In Vitro Activity Assessment

α-Amylase Inhibitory Test

To perform the α-amylase inhibitory test, 0.5 mL of each extract or the standard (acarbose), 0.5 mL of α-amylase solution, and 1 mL of sodium phosphate buffer were combined in test tubes. The mixture was incubated at room temperature (25 °C) for 10 minutes. After 10 minutes, 0.5 mL of starch solution was added to each test tube, and the mixture was incubated at room temperature (25 °C) for 10 minutes. The reaction was then terminated using 1 mL of dinitrosalicylic acid color reagent. The test tubes were immediately placed in a water bath at 100 °C for 5 minutes. After 5 minutes, the test tubes were removed from the water bath and allowed to cool until room temperature was attained. The mixture was then diluted with 10 mL of distilled water, and the absorbance was measured at 540 nm. Absorbance readings for the blank (buffer instead of extract and amylase solution) and control (buffer instead of extract) samples were also determined. α-amylase inhibition potentiality and IC₅₀ was calculated (24). The α-amylase inhibition potential was expressed as percentage inhibition, which was calculated using the following equation:

% α-amylase inhibition = 100% × (∆ A_control - ∆ A_sample)/ ∆ A_control

where,

∆A _control= A _control – A _blank

∆A _sample = A _extract – A _blank or, A _standard – A _blank

Where A_control is the absorbance of the solution without extract (buffer instead of extract) and with α-amylase solution; A_extract is the absorbance of the solution with extract and α-amylase solution; A_standard is the absorbance of the solution with acarbose and α-amylase solution; and A _blank is the absorbance of the solution without drug/extract and α-amylase solution.

In Vivo Antidiabetic Activity Assessment

Experimental Design and Dose Determination

A total of 35 Wister albino rats, aged 4-6 weeks, were purchased from the animal house of the Department of Pharmacy, Jahangirnagar University and kept uninterrupted for one week to acclimatize to the new environment. Their weight and blood glucose levels were measured before and after glucose administration to ensure they were healthy, with appropriate body weights (100-150 g) and blood glucose levels within a normal range (3.0-6.0 mmol/L). The rats were then divided into 7 groups, each containing 5 rats. They were then housed groupwise in a clean and well-ventilated experimental animal house. Temperature and humidity were maintained at 25 °C and 30%, respectively, throughout the experiment. Food and water were provided ad libitum. All experiments on rats were conducted properly, complying with the ethical guidelines for the care and use of laboratory animals, as approved by the Ethical Review Committee, Faculty of Pharmacy, University of Dhaka.

Based on the literature review, three doses of E. prostrata, namely, 250 mg/kg, 500 mg/kg, and 750mg/kg were chosen as low, medium, and high doses, respectively for administration to the experimental rats (14-16). The test substances were administered daily as a single dose via oral gavage needle (25). The standard drug, Vildagliptin (brand name Vildus from Opsonin Pharma, 50 mg), was used at a dose of 6mg/kg in the standard group of rats (Table 1).

Alloxan Administration and Induction of Diabetes

All rats, except groups 1 and group 7, were administered alloxan monohydrate intraperitoneally at a dose of 150 mg/kg after an overnight fast by dissolving into normal saline (26). Then, a 2 g/kg dose of glucose solution was provided instead of water during the first 24 hours post-injection to prevent death due to hypoglycemia. After 7 days, fasting and postprandial blood glucose levels were measured again after an overnight fast to check if glucose levels had increased above the normal range, which indicates the onset of diabetes mellitus.

Administration of Ecliptaprostrata Extract to Experimental Rats

After confirming diabetes successfully in the selected groups, the aqueous E. prostrata extract was administered orally at the specified concentrations using an oral gavage needle. The dried extract was dissolved in water with Tween 20 to prepare the oral solution. The treatment duration was four weeks, during which fasting blood glucose levels were measured weekly (15).

Measurement of Fasting and Postprandial Blood Glucose Levels

The rats were weighed, and their tails were sanitized with an alcohol pad. Then, blood was collected from the tip of their tails using lancets, and the drop of blood was placed on a strip attached to a glucometer. The glucometer immediately provided the blood glucose reading in mmol/L, which was recorded in the datasheet. Life Chek TD-4141 Blood Glucose Test Strips and glucometers were purchased from the Bangladesh Medical Association (BMA), Dhaka, for conducting this procedure.

Sample Collection and Plasma Preparation

At the end of the four-week treatment period, the rats were euthanized in the post-prandial condition using chloroform vapor and cervical dislocation. Blood was collected immediately after death through a heart puncture using a 3 mL sterile syringe, and the collected blood was placed in EDTA-coated tubes with proper labeling. Then, the tubes containing blood were centrifuged for 15 minutes at 1000 × g and 4 °C temperature. The supernatant (i.e., plasma) was then aspirated using a micropipette and transformed into another blood collection tube. The plasma was stored at -20 °C for further analysis.

Histopathological Study of Liver Tissue

The rat liver was cut out from the abdomen after dissection and stored in a conical flask containing 10% formalin solution. The liver samples were sectioned and subjected to histopathological analysis following standard procedures (27).

Measurement of Glucagon-Like Peptide-1 Protein in Plasma Using Enzyme-linked Immunosorbent Assay

To quantitatively determine the concentration of glucagon-like peptide-1 (GLP-1), an antidiabetic marker protein that helps lower blood glucose by stimulating insulin secretion, enzyme-linked immunosorbent assay (ELISA) was carried out on post-prandial plasma samples using Rat GLP-1 ELISA kit (48T) purchased from FineTest company. The test was carried out in a single day by meticulously following the instructions provided in the test kit by the manufacturer (28).

Statistical Analysis

The rat body weight, fasting blood glucose (FBG), and post-prandial blood glucose (PPG) data were analyzed using an independent sample t-test, while the ELISA data were analyzed via one-way ANOVA followed by Tukey’s multiple comparison test using the Statistical Package for Social Science (SPSS) Statistics 16 package (SPSS Inc., Chicago, IL). All values are expressed as mean ± Standard error of the mean (SEM), and a P value of< 0.05 was considered statistically significant.

Results

In Silico Study Results

List of Compounds Obtained by Literature Mining

The list of phytoconstituents present in E. prostratais presented in Table 2.

Molecular Docking

A comprehensive literature analysis was conducted to identify appropriate antidiabetic macromolecular targets for the study. Five molecular targets were selected, along with their therapeutic activity ligands. They were categorized into two groups: the first group for molecular targets and the second for respective controls. The binding affinity and the amino acid interactions between ligands and macromolecules are provided in Table 3 and Figure 1.

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Figure 1.

Interaction and Binding Poses of the High-Affinity Ligand Compared to Standard Drug With Antidiabetic Macromolecules. Note.A: Interaction and binding pose of C82 and control (acarbose) with α-Amylase (3OLG); B: Interaction and binding pose of C76 and control (acarbose) with α-Glucosidase (2ZE0); C: Interaction and binding pose of C49 and control (PT1) with AMP-activated protein kinase (6C9F). D: Interaction and binding pose of C29 and the control (Sitagliptin) with dipeptidyl peptidase-IV (2G5T); E: Interaction and binding pose of C81 and the control (Rosiglitazone) with peroxisome proliferator-activated receptor-γ (4EMA)

Figure 1.

Interaction and Binding Poses of the High-Affinity Ligand Compared to Standard Drug With Antidiabetic Macromolecules. Note.A: Interaction and binding pose of C82 and control (acarbose) with α-Amylase (3OLG); B: Interaction and binding pose of C76 and control (acarbose) with α-Glucosidase (2ZE0); C: Interaction and binding pose of C49 and control (PT1) with AMP-activated protein kinase (6C9F). D: Interaction and binding pose of C29 and the control (Sitagliptin) with dipeptidyl peptidase-IV (2G5T); E: Interaction and binding pose of C81 and the control (Rosiglitazone) with peroxisome proliferator-activated receptor-γ (4EMA)

With α-Amylase

Out of 115 ligands, 62 displayed higher binding affinity than the control acarbose with α-amylase (3OLG) in molecular docking studies. Among the top 10 compounds displaying superior binding affinity compared to acarbose (-7.9), 3 were triterpenoids and 7 were steroids, with compound 82 exhibiting the highest binding affinity (-11.2). Most ligands shared common amino acid interaction residues with the control.

With α-Glucosidase

Out of 115 ligands, 56 displayed higher binding affinity than the control acarbose with α-glucosidase (2ZE0) in the molecular docking studies. As observed with α-amylase, triterpenoids and steroids performed well in this case. Compound 76 showed the highest binding affinity at -11.6 (acarbose: -9.4).

With AMP-Activated Protein Kinase

Out of 115 ligands, 6 displayed higher binding affinity than the control PT1 with AMP-activated protein kinase (6C9F) in molecular docking studies. Three were triterpenoids, 2 were flavonoids, and 1 was a saponin. These compounds shared multiple interacting residues with the control PT1, and compound 49 demonstrated the highest binding affinity at -8.9.

With Dipeptidyl Peptidase IV

Out of 115 ligands, 46 displayed a higher binding affinity with dipeptidyl peptidase IV (2G5T) than the control sitagliptin, which had a binding affinity of -8.5. Among them, 27 were triterpenoids, including the ligand with the highest binding affinity, compound 29 (-10.2). All ligands had two or more common interacting residues with the control, except for compound 39, which had only one.

With Peroxisome Proliferator-activated Receptor-γ

Out of 115 ligands, 13 displayed a higher binding affinity than the control rosiglitazone, which had a binding affinity of -8, with peroxisome proliferator-activated receptor-γ (PPAR-γ) or 4EMA in the molecular docking studies. Eight of these ligands were steroids, including compound 81, which had the highest binding affinity of -8.5. All ligands shared interacting residues with the control.

In Vitro Study Results

Alpha-Amylase Inhibitory Activity Assessment

The plant extract inhibited the alpha-amylase enzyme in a dose-dependent manner, which was comparable to the inhibition observed with a standard alpha-amylase inhibitor (acarbose).

IC₅₀ Determination From Standard Curve

The linear equation for commercial acarbose is presented in Figure 2A:

y = 2.6273x + 2.2981

For 50% inhibition, y = 50. Therefore, x = (50 - 2.2981)/ 2.6273 = 18.156 mg/mL.

Hence, the IC₅₀ of commercial acarbose is18.156 mg/mL.

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Figure 2.

Standard Curve for Alpha-Amylase Inhibition by Acarbose (A) and Extract (B)

Figure 2.

Standard Curve for Alpha-Amylase Inhibition by Acarbose (A) and Extract (B)

The linear equation for the extract is provided in Figure 2B:

y = 2.2174x + 0.7453

For 50% inhibition, y = 50. Therefore, x = (50-0.7453)/ 2.2174 = 22.213 mg/mL.

Hence, the IC₅₀ for E. prostrata extract is 22.213 mg/mL.

In Vivo Study Results

Blood Glucose Level

All rats had FBG levels within a normal range (3-6 mmol/L) before any intervention (Day 0). After administration of alloxan into the diabetic (D), standard (S), and treatment groups (LD, MD, and HD), the FBG level increased on Day 1. The FBG remained significantly higher than the normal control group on Day 28 in the diabetic control group, which received no treatment. However, after 28 days of treatment, the FBG in the treatment and standard groups decreased compared to Day 1. These variations are shown in Figure 3A. A statistically significant difference was observed between group D and group MD, as presented in Table 4 and Figure 4.

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Figure 3.

Comparison of Fasting Blood Glucose (A) and Post-prandial Blood Glucose Levels (B) in Rats Across Different Test Groups Before and After Diabetes Induction and After 28 Days of Treatment. Note. N: Normal control; D: Diabetic control; S: Diabetic treated with standard drug (vildagliptin 6 mg/kg); NM: Normal rat treated with extract (500 mg/kg); LD: Diabetic treated with Low dose of extract (250 mg/kg); MD: Diabetic treated with medium dose of extract (500 mg/kg); HD: Diabetic treated with high dose of extract (750 mg/kg). Day 0 denotes the time before induction of diabetes; Day 1 indicates the start of treatment; Day 28 denotes the end of treatment

Figure 3.

Comparison of Fasting Blood Glucose (A) and Post-prandial Blood Glucose Levels (B) in Rats Across Different Test Groups Before and After Diabetes Induction and After 28 Days of Treatment. Note. N: Normal control; D: Diabetic control; S: Diabetic treated with standard drug (vildagliptin 6 mg/kg); NM: Normal rat treated with extract (500 mg/kg); LD: Diabetic treated with Low dose of extract (250 mg/kg); MD: Diabetic treated with medium dose of extract (500 mg/kg); HD: Diabetic treated with high dose of extract (750 mg/kg). Day 0 denotes the time before induction of diabetes; Day 1 indicates the start of treatment; Day 28 denotes the end of treatment

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Figure 4.

Change in Blood Glucose Level After 28 Days of Treatment (with the 0 line indicating the initial blood glucose level). Note. N: Normal control; D: Diabetic control; S: Diabetic treated with standard drug (vildagliptin 6 mg/kg); NM: Normal rat treated with extract (500 mg/kg); LD: Diabetic treated with low dose of extract (250 mg/kg); MD: Diabetic treated with medium dose of extract (500 mg/kg); HD: Diabetic treated with high dose of extract (750 mg/kg). * indicates statistical significance (P < 0.05) compared to group D, as determined by an independent t-test

Figure 4.

Change in Blood Glucose Level After 28 Days of Treatment (with the 0 line indicating the initial blood glucose level). Note. N: Normal control; D: Diabetic control; S: Diabetic treated with standard drug (vildagliptin 6 mg/kg); NM: Normal rat treated with extract (500 mg/kg); LD: Diabetic treated with low dose of extract (250 mg/kg); MD: Diabetic treated with medium dose of extract (500 mg/kg); HD: Diabetic treated with high dose of extract (750 mg/kg). * indicates statistical significance (P < 0.05) compared to group D, as determined by an independent t-test

Post-prandial Blood Glucose

All the rats had PPG levels within the normal range (3-6 mmol/L) two hours after administration of 2 mg/kg dose of glucose before any intervention (Day 0). After the administration of alloxan into the D, S, and treatment groups (LD, MD, and HD), the PPG level increased on Day 1. The PPG remained significantly higher than the normal control group on Day 28 in the diabetic control group, which received no treatment. However, after 28 days of treatment, the FBG in the treatment and standard groups decreased compared to Day 1. These variations are presented in Figure 3B. A statistically significant difference was noted between Group D and Group MD, as presented in Table 4 and Figure 4.

Histopathology

The histoarchitecture of each of the seven groups of rats is shown in Figure 5:

• (N) Normal control rat liver shows normal hepatocytes with normal radial arrangements around hepatic cords.

• (D) Diabetic control rat liver shows increased vacuolation in the cytoplasm of hepatocytes, appearing as indistinct clear vacuoles, indicating glycogen infiltration in diabetes.

• (S) The liver of diabetic rats treated with the standard drug Vildagliptin exhibits normal hepatocytes and hepatic architecture with mild vacuolation of hepatocytes (H&E, x100).

• (LD) The liver of diabetic rats treated with 250 mg/kg E. prostrata extract shows slightly improved liver architecture with normal hepatocytes but disrupted sinusoids.

• (MD) The liver of diabetic rats treated with 500 mg/kg E. prostrata extract shows similar liver architecture as LD.

• (HD) The liver of diabetic rats treated with 750 mg/kg E. prostrata extract displays normal hepatocytes and hepatic architecture.

• (NM) Liver of normal rats treated with 500 mg/kg E. prostrata extract shows normal hepatocytes with normal radial arrangements around hepatic cords just, similar to the normal control group.

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Figure 5.

Comparison of the Histological Images of Rat Livers From Different Groups. (H& E, × 100). Note. E. prostrata: Eclipta prostrata; N: Normal control rat liver showing normal hepatocytes with normal radial arrangements around hepatic cords; D: Diabetic control rat liver showing increased vacuolation in the cytoplasm of hepatocytes; S: Standard rat treated with Vildagliptin (6 mg/kg) showing normal hepatocytes and normal hepatic architecture, with mild vacuolation of hepatocytes; LD: Liver of diabetic rat treated with 250 mg/kg E. prostrata extract showing slightly improved liver architecture with normal hepatocytes but disrupted sinusoids; MD: Liver of diabetic rat treated with 500 mg/kg E. prostrata extract showing similar liver architecture as LD; HD: Liver of diabetic rat treated with 750 mg/kg E. prostrata extract showing normal hepatocytes and normal hepatic architecture; NM: Liver of normal rats treated with 500 mg/kg E. prostrata extract showing normal hepatocytes with a normal radial arrangement around hepatic cords

Figure 5.

Comparison of the Histological Images of Rat Livers From Different Groups. (H& E, × 100). Note. E. prostrata: Eclipta prostrata; N: Normal control rat liver showing normal hepatocytes with normal radial arrangements around hepatic cords; D: Diabetic control rat liver showing increased vacuolation in the cytoplasm of hepatocytes; S: Standard rat treated with Vildagliptin (6 mg/kg) showing normal hepatocytes and normal hepatic architecture, with mild vacuolation of hepatocytes; LD: Liver of diabetic rat treated with 250 mg/kg E. prostrata extract showing slightly improved liver architecture with normal hepatocytes but disrupted sinusoids; MD: Liver of diabetic rat treated with 500 mg/kg E. prostrata extract showing similar liver architecture as LD; HD: Liver of diabetic rat treated with 750 mg/kg E. prostrata extract showing normal hepatocytes and normal hepatic architecture; NM: Liver of normal rats treated with 500 mg/kg E. prostrata extract showing normal hepatocytes with a normal radial arrangement around hepatic cords

Enzyme-Linked Immunosorbent Assay

The standard curve was prepared using the absorbance value of different concentrations of the supplied standard. Using the equation from the standard curve (y = 0.0014x + 0.0597) and the absorbance value of the samples (y), the concentration of GLP-1 in each plasma sample (x) was determined and converted into pM or pmol/L (1 pmol/L = 3.297 pg/mL). The resulting concentrations are presented in Figure 6.

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Figure 6.

Comparison of the Concentration of GLP-1 in pM Level of Rats From Different Test Groups. Note. GLP-1: Glucagon-like peptide-1; N: Normal control; D: Diabetic control; S: Diabetic treated with standard drug (vildagliptin 6 mg/kg); NM: Normal rat treated with extract (500 mg/kg); LD: Diabetic treated with low dose of extract (250 mg/kg); MD: Diabetic treated with medium dose of extract (500 mg/kg); HD: Diabetic treated with high dose of extract (750 mg/kg). One-way ANOVA was used to test statistical significance between diabetic (D) and other groups. *indicates P < 0.05, **indicates P < 0.01, and ***indicates P < 0.005 level of statistical significance

Figure 6.

Comparison of the Concentration of GLP-1 in pM Level of Rats From Different Test Groups. Note. GLP-1: Glucagon-like peptide-1; N: Normal control; D: Diabetic control; S: Diabetic treated with standard drug (vildagliptin 6 mg/kg); NM: Normal rat treated with extract (500 mg/kg); LD: Diabetic treated with low dose of extract (250 mg/kg); MD: Diabetic treated with medium dose of extract (500 mg/kg); HD: Diabetic treated with high dose of extract (750 mg/kg). One-way ANOVA was used to test statistical significance between diabetic (D) and other groups. *indicates P < 0.05, **indicates P < 0.01, and ***indicates P < 0.005 level of statistical significance

Discussion

The molecular docking study was conducted to examine the binding affinity between the phytoconstituents present in the E. prostrata plant, as reported in previous research (Table 2), and the macromolecules involved in the pathogenesis of diabetes. Further research can focus on isolating the compounds that showed promising activity (Figure 1) from the plant or synthesizing them in the laboratory. These compounds can then be tested for their activity using in vitro or in vivo methods.

Calcium metalloenzymes such as alpha-amylases are inactive in the absence of calcium. Humans possess a variety of digestive enzymes, but the most significant one is pancreatic alpha-amylase (EC 3.2.1.1), which catalyzes the breakdown of alpha-1,4 glycosidic bonds, which hold starch, amylopectin, amylose, glycogen, and several maltodextrins together. This reaction is essential for the breakdown of starch. Another significant enzyme is alpha-glucosidase (also known as maltase, EC 3.2.1.20), which acts on 1,4-alpha bonds to catalyze the final stage of carbohydrate digestion, primarily starch, resulting in the production of glucose.

Alpha-amylase breaks down large molecules such as starch into smaller sugar fragments, which can then pass through the blood-brain barrier. Since large molecules such as starch cannot cross the blood-brain barrier, so glucose must first be broken down into smaller components before reaching the brain. A higher blood sugar level results from an excess conversion of starch to sugars. Insulin plays a role in this situation by instructing cells to metabolize the extra sugar molecules and store them as glycogen, which is used as an energy source. In a healthy individual, this cycle never ends. However, in certain cases, high blood glucose levels occur due to amylase enzyme overactivity and insulin insufficiency or resistance. In our study, type 1 diabetes was induced by injecting alloxan, which causes cellular necrosis of beta cells and results in insulin deficiency. These conditions may lead to hyperglycemia (55).

The alpha-amylase inhibitory assay was conducted to test whether the extract can inhibit the alpha-amylase enzyme, which is responsible for the breakdown of starch into maltose in the human gut (56). The results (Figure 2) suggest that the extract is a less potent inhibitor of the alpha-amylase enzyme than the standard drug Acarbose. While the concentration required for 50% enzyme inhibition (IC₅₀) is 18.156 mg/mL for commercial acarbose, the IC₅₀ value of the extract is 22.213 mg/mL, indicating that alpha-amylase inhibition might be one of the mechanisms by which the plant exerts its hypoglycemic actions.

It has recently been established that poor alpha-cell function contributes to the etiology of type 2 diabetes (57). This defect prevents the hepatic glucose increase that occurs after eating and the usual reduction of fasting glucagon. Insulin resistance and low insulin levels combine to cause hyperglycemia. As gastrointestinal mediators of insulin secretion and, more specifically, glucagon suppression in the context of GLP-1, incretins are essential. Gastrointestinal peptide (GIP) activity is compromised in people with type 2 diabetes (58). GLP-1 continues to exert its insulinotropic effects, suggesting that GLP-1 could be a useful therapeutic intervention. GLP-1 is rapidly deactivated by DPP-IV in vivo, similar to GIP (59).

The current study aimed to examine the antidiabetic activity of E. prostrata extract. The results of the in vivo experiments indicate that the plant extract was effective in improving the diseased condition in a dose-dependent manner, which is comparable to the previous in vivo studies (15,54).

The FBG and PPG level data, presented in Table 4 and Figure 3, demonstrate that the medium dose of the extract (500 mg/kg) is the most effective in reducing elevated blood glucose levels in diabetic rats. The rate of blood glucose reduction was significant (P = 0.019 < 0.05 and 0.044 < 0.05, respectively) compared to the untreated diabetic group. These results are consistent with previous research conducted by Sharma et al, which found the most effective dose to be 400 mg/kg/d (25). In that study, both 200 mg and 400 mg doses of the extract demonstrated significant therapeutic activity. However, we used three distinct doses (250 mg, 500 mg, and 750 mg) to explore whether the 400 mg dose can lead to receptor saturation. If receptor saturation occurs, therapeutic activity will not increase with the 750 mg dose compared to the 500 mg dose. On the contrary, an increase in the activity suggests that 400 mg would not be the highest effective dose of extract and higher extract is necessary to achieve the highest therapeutic effects. When healthy rats were administered the extract, the blood glucose level did not decrease significantly, indicating that the plant itself was not hypoglycemic.

The histopathological data shown in Figure 5 suggest that the plant extract is not toxic to the liver tissue, as no abnormalities were observed in the liver architecture of the rats treated with different doses of the extract (60). In fact, the extract improved the condition of the liver tissues that were damaged due to the induction of diabetes.

The GLP-1 ELISA assay data presented in Figure 6 indicate that the GLP-1 levels in plasma significantly increase with the administration of both the medium and high doses of the extract compared to the untreated group. This suggests that the antidiabetic effect exhibited by E. prostrata may be associated with its ability to inhibit DPP-IV as DPP-IV is responsible for the breakdown of GLP-1 and the inhibition of glucose uptake from serum to tissue (61). The control drug, Vildagliptin, used in the standard group is a DPP-IV inhibitor, and our test drug exhibited better activity compared to this standard in the experimental studies.

Conclusion

Eclipta prostrata was studied to provide a scientific basis for its renowned antihyperglycemic activity and to examine its potential as an antidiabetic medication. This detailed comprehensive study on the tropical herb E. prostrata included a combination of in silico, in vitro, and in vivo methods. The in-silico study identified constituents that are most likely responsible for the plant’s antidiabetic activity. The in vitro study highlighted its alpha-amylase inhibitory effect, which can be considered an auxiliary mechanism contributing to its activity. Furthermore, the in vivo study confirmed the plant’s antidiabetic effect as well as its most effective dose, which is 500 mg/kg body weight for rats. We also proposed a mechanism of action involving the inhibition of the DPP-4 enzyme and subsequent upregulation of plasma GLP-1 levels, which was validated through the ELISA. The safety of the plant extract was also confirmed by the absence of adverse effects in healthy rats treated with the extract and through histological studies. Hence, it can be inferred that this study will guide future researchers in finding an effective marketable drug for diabetes from the valuable herb E. prostrata.

Acknowledgements

We would like to express our gratitude to all open-access journals that provided free access to articles, the laboratory technicians for their immense support, and the government of Bangladesh for funding this project.

Authors’ Contribution

Conceptualization: Md. Shah Amran.

Data curation: Maniza Muni, Mst. Morsheda Akter Mim.

Formal analysis: Maniza Muni, Mst. Morsheda Akter Mim, Syeda Sadia Afrin.

Funding acquisition: Md. Shah Amran.

Investigation: Maniza Muni, Mst. Morsheda Akter Mim, Safaet Alam.

Methodology: Al Amin Sikder, Md. Zakir Sultan.

Project administration: Md. Shah Amran.

Resources: Md. Shah Amran.

Software: Jakir Ahmed Chowdhury, Abu Asad Chowdhury, Shaila Kabir.

Supervision: Fahima Aktar, Md. Shah Amran.

Validation: Md. Shah Amran.

Visualization: Md. Shah Amran.

Writing–original draft: Maniza Muni.

Writing–review & editing: Fahima Aktar.

Competing Interests

The authors declare no competing interests.

Ethical Approval

Ethical approval for the animal model study was obtained on July 18, 2023, from the Ethical Review Committee of the Faculty of Pharmacy, University of Dhaka (Reference No. Fa. Ph. E/017/23).

Funding

This project was funded by the Ministry of Science and Technology (MOST), Bangladesh, under the DU-UGC grants programs.

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