Phytochemical Analysis and Evaluation of Antioxidant, Antidiabetic, and Anti-inflammatory Properties of Aegle marmelos and Its Validation in an In-Vitro Cell Model

Abstract

Introduction: Persistent hyperglycemia significantly increases oxidative stress and inflammation resulting in multiple cellular and molecular alterations which further exacerbate the diabetes associated complications. Aegle marmelos (L.) Corrêa is a medicinal plant used in the Indian system of medicine for treating various disorders including diabetes. However, studies on phytoconstituents and their pharmacological activity of this plant are limited. Therefore, we aimed to determine the phytochemical components, evaluate the antidiabetic activity, anti-inflammatory activity, and antioxidant activity of A. marmelos leaf extract, and validate its mechanistic effects in an in vitro cell model.

Methods: The qualitative and quantitative analysis of the different phytoconstituents in the extract was determined using standardized protocols. The antioxidant activity of the extract was evaluated by 2,2-di-phenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity assay and ferric reducing antioxidant power (FRAP) assay. The antidiabetic activity of the extract was evaluated by α-amylase inhibition and α-glucosidase inhibition assay. The anti-inflammatory activity was studied using an albumin denaturation assay. In addition, the pharmacological effect(s) of leaf extract was checked in the normal rat kidney fibroblast cells (NRK-49F) under high glucose conditions. Intracellular reactive oxygen species (ROS) generation was measured by fluorometry using fluorescence probe 2',7'-dichlorodihydrofluorescin diacetate (DCF-DA). mRNA expression of inflammatory markers including inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α) was studied using real-time quantitative polymerase chain reaction (RT-qPCR). Cell migration was studied using cell scratch assay. Statistical analysis was performed using GraphPad Prism version 8.0.

Results: The phytochemical analysis of A. marmelos leaf extract revealed the presence of alkaloids, phenols, flavonoids, and saponins. The extract showed higher antioxidant activity in the DPPH (IC₅₀=258.21 µg/mL) and FRAP assay (IC₅₀=293.83 µg/mL). The extract exhibited prominent antidiabetic activity by inhibiting enzymes α-Amylase (IC₅₀=73.2 µg/mL) and α-glucosidase (IC₅₀=43.9 µg/mL). In addition, the extract showed effective anti-inflammatory activity by significantly inhibiting the denaturation of egg albumin (IC₅₀=102.8 µg/mL). Further, the leaf extract significantly decreased the high glucose-induced ROS generation as well as inflammatory markers in rat fibroblast cell lines in a dose-dependent manner. Additionally, high glucose-induced cell migration as the measure of cell injury was effectively reduced by the extract treatment.

Conclusion: A. marmelos leaf extract was quantified to possess a substantial amount of important phytoconstituents that have promising pharmacological properties. Besides showing antidiabetic activity, the extract significantly combats the high glucose-induced ROS generation, inflammatory markers expressions, and cell migration. Further, in-depth studies and clinical trials are warranted so as to position these traditional remedies for the treatment of metabolic disorders.

Keywords: aegle marmelos; anti-inflammatory; antioxidant; hyperglycemia; phyto pharmacology.

Figure 1. The antioxidant activity of the extract

In both antioxidant activity assays (DPPH (A) and FRAP (B) assays), the extract showed dose-dependent inhibition and results were compared with the standard drug, ascorbic acid. Data were represented as mean ± SEM (n=3). *p<0.05 compared with standard drug (100 µg/mL), #p<0.05 compared with extract (100 µg/mL). DPPH: 2,2-di-phenyl-1-picrylhydrazyl, FRAP: ferric reducing antioxidant power

Figure 2. The antidiabetic activity of the extract

In both antidiabetic assays (α-amylase inhibition assay (A) and α-glucosidase inhibition assay (B)), the extract showed dose-dependent inhibition of both enzymes, and results were compared with the standard drug acarbose. Data were represented as mean ± SEM (n=3). *p<0.05 compared with standard drug (50 µg/mL), #p<0.05 compared with extract (50 µg/mL).

Figure 3. The anti-inflammatory activity of the extract

The extract showed significant dose-dependent denaturation of protein albumin and results were compared with diclofenac sodium. Data were represented as mean ± SEM (n=3).*p<0.05 compared with standard drug (50 µg/mL), #p<0.05 compared with extract (50 µg/mL).

Figure 4. The effect of the extract on cell viability in NRK-49F cell line

The extract did not significantly reduce the cell viability till 2000 µg/mL. Data were represented as mean ± SEM (n=3). *p<0.05 compared to without treatment condition.

Figure 5. The effect of the extract on high glucose-induced ROS generation in NRK-49F cell line

The extract treatment significantly decreased the ROS generation under high glucose in a dose-dependent manner. Data were represented as mean ± SEM (n=3). *p<0.05 compared to control; #p<0.05 compared to HG. HG: high glucose; RFU: relative fluorescence units

Figure 6. The effect of the extract on inflammatory markers in NRK-49F cell line

Extract treatment decreased the inflammatory markers iNOS (A) and TNF-α (B) in a dose-dependent manner under high glucose conditions. Data were represented as mean ± SEM (n=3). *p<0.05 compared to control; #p<0.05 compared to HG. HG: high glucose; iNOS: inducible nitric oxide synthase; TNF-α: tumor necrosis factor alpha

Figure 7. The effect of the extract on cell migration in NRK-49F cell line

Extract treatment significantly decreased the cell migration when compared with the high glucose condition in a dose-dependent manner. Data were represented as mean ± SEM (n=3). *p<0.05 compared to control; #p<0.05 compared to HG. HG: high glucose