Chemical Composition of Moringa oleifera Ethyl Acetate Fraction and Its Biological Activity in Diabetic Human Dermal Fibroblasts

Abstract

Background: Moringa oleifera (MO), commonly known as the drumstick tree, is used in folklore medicine for the treatment of skin disease.

Objective: The objective of this study is to evaluate the ethyl acetate (EtOAc) fraction of MO leaves for in vitro antibacterial, antioxidant, and wound healing activities and conduct gas chromatography-mass spectrometry (GC-MS) analysis.

Materials and methods: Antibacterial activity was evaluated against six Gram-positive bacteria and 10 Gram-negative bacteria by disc diffusion method. Free radical scavenging activity was assessed by 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical hydrogen peroxide scavenging and total phenolic content (TPC). Wound healing efficiency was studied using cell viability, proliferation, and scratch assays in diabetic human dermal fibroblast (HDF-D) cells.

Results: The EtOAc fraction showed moderate activity against all bacterial strains tested, and the maximum inhibition zone was observed against Streptococcus pyogenes (30 mm in diameter). The fraction showed higher sensitivity to Gram-positive strains than Gram-negative strains. In the quantitative analysis of antioxidant content, the EtOAc fraction was found to have a TPC of 65.81 ± 0.01. The DPPH scavenging activity and the hydrogen peroxide assay were correlated with the TPC value, with IC₅₀ values of 18.21 ± 0.06 and 59.22 ± 0.04, respectively. The wound healing experiment revealed a significant enhancement of cell proliferation and migration of HDF-D cells. GC-MS analysis confirmed the presence of 17 bioactive constituents that may be the principal factors in the significant antibacterial, antioxidant, and wound healing activity.

Conclusion: The EtOAc fraction of MO leaves possesses remarkable wound healing properties, which can be attributed to the antibacterial and antioxidant activities of the fraction.

Summary: Moringa oleifera (MO) leaf ethyl acetate (EtOAc) fraction possesses antibacterial activities toward Gram-positive bacteria such as Streptococcus pyogenes, Streptococcus faecalis, Bacillus subtilis, Bacillus cereus and Staphylococcus aureus, and Gram-negative bacteria such as Proteus mirabilis and Salmonella typhimuriumMO leaf EtOAc fraction contained the phenolic content of 65.81 ± 0.01 and flavonoid content of 37.1 ± 0.03, respectively. In addition, the fraction contained 17 bioactive constituents associated with the antibacterial, antioxidant, and wound healing properties that were identified using gas chromatography-mass spectrometry analysisMO leaf EtOAc fraction supports wound closure rate about 80% for treatments when compared with control group. Abbreviations used: MO: Moringa oleifera; EtOAc: Ethyl acetate; GC-MS: Gas Chromatography-Mass Spectrometry; HDF-D: Diabetic Human Dermal Fibroblast cells.

Keywords: Diabetic wound healing; gas chromatography-mass spectrometry; migration rate; phenolic content; scratch assay; skin pathogen.

Figure 1

1-diphenyl-2-picryl hydrazyl scavenging activity of the ethyl acetate fraction of Moringa oleifera leaves at various concentrations. Values are mean ± standard deviation (n = 3). The 1-diphenyl-2-picryl hydrazyl scavenging activity of ethyl acetate fraction increased in a concentration-dependent manner comparable to ascorbic acid

Figure 2

Hydrogen peroxide scavenging activity of Moringa oleifera leaf ethyl acetate fraction at various concentrations. Values are mean ± SD (n = 3). The H₂O₂ radical inhibition activity of ethyl acetate fraction increased in a concentration-dependent manner comparable to ascorbic acid

Figure 3

The cytotoxicity of Moringa oleifera leaf ethyl acetate fraction treatment in diabetic human dermal fibroblast cells was assessed. Cells were treated with various concentrations of isolated fractions (15.62, 31.25, 62.5, 125, 250, and 500 μg/mL) for 24 h. After the incubation period, the viability of fraction-treated cells was evaluated by 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide assay. Values are presented as the mean percentage ± standard deviation of three replicates. ***P < 0.001 versus control group

Figure 4

The proliferative effect of the Moringa oleifera leaf ethyl acetate fraction on diabetic human dermal fibroblast cells. Cells were seeded in a 96-well plate, different concentrations of Moringa oleifera ethyl acetate fraction were added, and the cells were left to stand for 24 h. The proliferative effect was measured by Cell Counting Kit-8 assay kit and calculated by a comparison of the values from the ethyl acetate fraction treatment group with the control group. Data are expressed as mean ± standard deviation from three individual experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group

Figure 5

The percentage of cell migration rate of Moringa oleifera leaf ethyl acetate fraction treated wound in diabetic human dermal fibroblast. Cells were seeded into 6-well plates with the addition of different concentrations of Moringa oleifera ethyl acetate leaves fraction for 24 h. The migration rate of Moringa oleifera leaf ethyl acetate fraction-treated wounds in diabetic human dermal fibroblast cells was evaluated at 0 and 24 h after treatment and calculated by Image-J software. Results are expressed as mean ± standard deviation from each individual experiment. *P < 0.05, **P < 0.01***P < 0.001 versus control group

Figure 6

The migration rate of the fraction on diabetic human dermal fibroblast cells. The various concentrations (12.5, 25, and 50 μg/mL) of Moringa oleifera leaf ethyl acetate fraction were treated on a wound created in diabetic human dermal fibroblast observed by scratch assay. Human dermal fibroblast-diabetic cells were scratched with p200 pipette tips, and photos were captured at 0 h. After the treatment with various concentrations of Moringa oleifera leaf ethyl acetate fraction, the cells were photographed after 24 h incubation (a) Control, (b) Moringa oleifera leaf ethyl acetate fraction 12.5 μg/mL, (c) Moringa oleifera leaf ethyl acetate fraction 25 μg/mL, (d) Moringa oleifera leaf ethyl acetate fraction 50 μg/mL, (e) allantoin 50 μg/mL

Figure 7

Gas chromatogram obtained for ethyl acetate fraction of Moringa oleifera leaf extract