In vitro wound healing potential and identification of bioactive compounds from Moringa oleifera Lam - PubMed (original) (raw)

In vitro wound healing potential and identification of bioactive compounds from Moringa oleifera Lam

Abubakar Amali Muhammad et al. Biomed Res Int. 2013.

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Abstract

Moringa oleifera Lam. (M. oleifera) from the monogeneric family Moringaceae is found in tropical and subtropical countries. The present study was aimed at exploring the in vitro wound healing potential of M. oleifera and identification of active compounds that may be responsible for its wound healing action. The study included cell viability, proliferation, and wound scratch test assays. Different solvent crude extracts were screened, and the most active crude extract was further subjected to differential bioguided fractionation. Fractions were also screened and most active aqueous fraction was finally obtained for further investigation. HPLC and LC-MS/MS analysis were used for identification and confirmation of bioactive compounds. The results of our study demonstrated that aqueous fraction of M. oleifera significantly enhanced proliferation and viability as well as migration of human dermal fibroblast (HDF) cells compared to the untreated control and other fractions. The HPLC and LC-MS/MS studies revealed kaempferol and quercetin compounds in the crude methanolic extract and a major bioactive compound Vicenin-2 was identified in the bioactive aqueous fraction which was confirmed with standard Vicenin-2 using HPLC and UV spectroscopic methods. These findings suggest that bioactive fraction of M. oleifera containing Vicenin-2 compound may enhance faster wound healing in vitro.

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Figures

Figure 1

Figure 1

Percentage of yield of crude extracts and fractions of M. oleifera obtained following complete extraction of M. oleifera leaves. DCM: dichloromethane; EA: ethyl acetate. Values were obtained per 100 g of powdered leaves sample for the crude extracts and 10 g of dried methanolic extract for the aqueous fraction.

Figure 2

Figure 2

Effect of different crude extracts of M. oleifera on cell count and viability of human dermal fibroblast (HDF) administered at 12.5 _μ_g/mL. The data were expressed as mean ± SD of triplicate values. ANOVA was used for statistical analysis. **P < 0.05 (methanolic extract treated versus other solvent extracts and control).

Figure 3

Figure 3

Effects of different fractions of M. oleifera on cell count and percentage viability of HDF administered at 25 _μ_g/mL of each fraction and readings were taken after 72 hrs. Percentage viability was estimated using standard formula as % cell viability = total viable cells/total cells (dead + viable) × 100. Data were expressed as mean ± SD of triplicate values. **P < 0.05 (methanolic extract versus other solvent extracts and control).

Figure 4

Figure 4

Digital image showing the effect of different fractions of M. oleifera on human dermal fibroblast migration in a wound scratch test assay: (a) (i): control without treatment; (ii): methanol; (iii): ethanol; (iv): aqueous extracts; (b) (i): control; (ii): n-hexane; (iii): dichloromethane; (iv): ethyl acetate; (v): n-butanol; (vi): aqueous fractions. A confluent monolayer of human dermal fibroblast (HDF) was scratched using a sterilised 200 _μ_L pipette tip. Different fractions were applied as treatments to the wounded (open gap) and fibroblast media served as control. Migration of fibroblast cells were captured and measured using light microscope attached to a digital camera. Magnification (4x).

Figure 5

Figure 5

Effect of different crude extracts and fractions of M. oleifera on human dermal fibroblast in a wound scratch test. The enhanced migratory HDF cells that completely closed the gap created after 72 hrs were seen in methanolic crude extract and aqueous fraction compared to other solvent extracts. Values are expressed as mean ± SD of triplicate determinations. **P < 0.05 (methanolic extract versus control and other extracts in (a)). **P < 0.05 (aqueous fraction versus control and other fractions in (b)).

Figure 6

Figure 6

Effects of aqueous fraction treated human dermal fibroblasts (HDF) on cell proliferation and viability of various concentrations at 48 (a) and 72 hrs (b), respectively. The value from baseline control group was set at 100%. The proliferation activity was estimated by MTT assay and calculated by comparing the values from the aqueous fraction treated group with that of control group. Data were expressed as mean ± SD of triplicate values. **P < 0.05 (aqueous fraction treated groups versus control group).

Figure 7

Figure 7

HPLC-DAD chromatogram of (a) methanolic crude extract and (b) aqueous fraction of M. oleifera. The peaks represent general profiling of compounds and the pattern of distribution showed a combination of polar, intermediate polar, and nonpolar compounds as represented in methanolic crude extract, while only polar region was observed in aqueous fraction.

Figure 8

Figure 8

HPLC-DAD chromatogram for reference standard Vicenin-2 (a) and aqueous fraction sample of M. oleifera (b). Both peaks have similar retention time of about 11 mins indicating that our aqueous fraction sample contains Vicenin-2 as the major active compound.

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