Wound Healing Activity and Chemical Standardization of Eugenia pruniformis Cambess

Abstract

Background: Eugenia pruniformis is an endemic species from Brazil. Eugenia genus has flavonoids as one of the remarkable chemical classes which are related to the improvement of the healing process.

Aims: To evaluate of wound healing activity of E. pruniformis leaves and to identify and quantify its main flavonoids compounds.

Materials and methods: Wound excision model in rats was used to verify the hydroethanolic and ethyl acetate extracts potential. The animals were divided in four groups of six and the samples were evaluated until the 15° day of treatment. Hydroxyproline dosage and histological staining with hematoxilin-eosin and Sirius Red were used to observe the tissue organization and quantify the collagen deposition, respectively. Chemical compounds of the ethyl acetate extract were identified by chromatographic techniques and mass spectrometry analysis and total flavonoids content was determined by spectrophotometric method. The antioxidant activity was determined by oxygen radical absorbing capacity (ORAC) and 2,2-diphenyl-1-picrylhydrazylhydrate radical photometric (DPPH) assays.

Results: The treated group with the ethyl acetate extract showed collagen deposition increase, higher levels of hidroxyproline, better tissue reorganization and complete remodeling of epidermis. Quercetin, kaempferol and hyperoside were identified as main compounds and flavonoids content value was 43% (w/w). The ORAC value of the ethyl acetate extract was 0.81± 0.05 mmol TE/g whereas the concentration to produce 50% reduction of the DPPH was 7.05± 0.09 μg/mL.

Conclusion: The data indicate a wound healing and antioxidant activities of E. pruniformis. This study is the first report of flavonoids and wound healing activity of E. pruniformis.

Key messages: Eugenia pruniformis extract accelerates wound healing in skin rat model, probably due to its involvement with the collagen deposition increase, higher levels of hidroxyproline, dermal remodelling and potent antioxidant activity. Chemical standardization of the active wound healing extract was done. The total flavonoid content was 43% (w/w) and quercetin, kaempferol and hyperoside were identified as main compounds.

Summary: Wound excision model in rats showed the potential wound healing activity of E. pruniformis by collagen deposition increase, higher levels of hidroxyproline, better tissue reorganization and complete remodeling of epidermis.Flavonoids are the main compounds of the endemic E. pruniformis and quercetin, kaempferol and hyperoside were identified in ethyl acetate extract by TLC, HPLC-PDA and HRESI-MS analysis.The ethyl acetate extract of E. pruniformis showed a potent antioxidant activity by ORAC and DPPH assays Abbreviation used: NC: Negative control, PC: Positive control, CH: Crude hydroethanolic extract, EA: Ethyl acetate extract, TE: Trolox equivalent, mg: Milligram, mM: Millimolar, mL: Milliliter, HPLC-PDA: High performance liquid chromatography with a photodiode array detector, HRESI-MS: High-resolution electrospray ionization mass spectrometry analysis, TLC: Thin layer chromatography, ORAC: Oxygen radical absorbance capacity, w/v: Weight per volume.

Keywords: Eugenia pruniformis; flavonoids; myrtaceae; wound healing.

Figure 1

HPLC.PDA analysis of the EA extract from E. pruniformis leaves. UV 360 nm chromatogram. 408 C18 column, MeCN/0.1% aqueous formic acid, 10:90 to 60:40 in 30 min, 1.0 mL/min. Peaks are assigned to 409 the identified compound. H, hyperoside; K, kaempferol; Q, quercetin

Figure 2

The histological evaluation of the skin flaps revealed by HE coloration (10×). NC is the negative 412 control, PC is treated topically with collagenase ointment as positive control, CH is treated topically with 413 propylene glycol solution of 5% hydroethanolic extract, and EA is treated topically with propylene glycol solution of 5% EA extract. Day 0, 8, and 15 414 days after injury. Microscopic examination of the lesions treated 415 with EA indicated better epithelialization (arrow) and more re-organization of the dermis (arrowhead) 416 compared to the others groups on 8 and 15 days after injury

Figure 3

The evaluation of Sirius Red staining was made to recognize the total density of collagen. Each cut 419 was analyzed the percentage of area occupied by collagen fibers. NC is the negative control, PC is treated 420 topically with Collagenase ointment, CH is treated topically with propylene glycol solution of 5% crude 421 hydroethanolic extract, EA is treated topically with propylene glycol solution of 5% EA extract. Day 0, 8, and 15 days after injury. The distribution of collagen was more intense mainly in EA and PC groups on 8 422 and 15 423 days after injury (arrowhead)

Adriana Passos Oliveira