Propolis standardized extract (EPP-AF®), an innovative chemically and biologically reproducible pharmaceutical compound for treating wounds

Andresa Aparecida Berretta, Andresa Piacezzi Nascimento, Paula Carolina Pires Bueno, Mirela Mara de Oliveira Lima Leite Vaz, Juliana Maldonado Marchetti, Andresa Aparecida Berretta, Andresa Piacezzi Nascimento, Paula Carolina Pires Bueno, Mirela Mara de Oliveira Lima Leite Vaz, Juliana Maldonado Marchetti

Abstract

The aim of this study was to develop a formulation, containing the propolis standardized extract (EPP-AF(®)), which can assist in the healing of skin lesions. To achieve this objective the antimicrobial activity and chemical composition of the propolis extract was determined. The final product was subjected to in vitro and in vivo pre-clinical evaluation. The broth macrodilution method was used to determine the antimicrobial activity of the extracts and formulations against the microorganisms most commonly found in burns, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis. Wistar rats with puncture wounded skin were used to evaluate the wound healing properties of propolis. The results of chemical and biological characterization demonstrated the batch-to-batch reproducibility of the standardized extract which is an unprecedented result. The antimicrobial and wound healing activity of the pharmaceutical studied showed the best results when samples contain 3.6% propolis, suggesting that this is the most promising composition.

Keywords: EPP-AF®; antimicrobial activity; pharmaceutics; propolis; reproducibility; wound healing.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Chromatographic profile of (a) Propolis Standardized Extract (EPP-AF®) Batch 1440110 ; (b) Batch 1400410 and (c) Batch 010/08. Chromatographic profile includes the compounds: 1. Caffeic acid (around 15 min.); 2. p-coumaric acid (around 20 min.); 3. trans-cinnamic acid (around 36 min.), 4. aromadendrin (38 min.) 5. isosakuretin (around 48 min.) and 6.Artepillin C (around 61 min.). The chromatograms were plotted at 275 nm, using the RP-HPLC, C18 (Shim-pack, CLC-ODS (M), 25 cm x 4.6) column and gradient elution with methanol and acidic water (formic acid pH=2.7).
Figure 2
Figure 2
Photograph showing the healing process of the control group (A) and the group treated with gel in the absence of propolis (B and C) after 3 days of treatment. Slices of 6 μm were obtained and stained with Masson´s trichromic. a) Lesions treated with saline (control). Start of the healing process (100x magnification); b) Lesions treated with gel without propolis. Start of the healing process (100x magnification); c) Lesions treated with gel without propolis. Cream leukocitary cells (arrow) observed just below the initial clot (*) (400X magnification).
Figure 3
Figure 3
Photograph showing the healing process after three days of treatment with the gel containing 1.2% of dry extract of propolis. a) Lesions treated with gel containing standardized extract of propolis (1.2%) in the subcutaneous tissue. The reorganization of epithelial cells (c) and increased collagen fibers were observed just below the initial clot (*). Slices of 6 μm were obtained and stained with Masson´s trichromic (100x magnification); b) lesions treated with gel containing standardized extract of propolis (1.2%). There are a large number of fibroblasts synthesizing collagen (arrow). Slices of 6 μm were obtained and stained with Masson´s trichromic (400X magnification).
Figure 4
Figure 4
Photograph showing the healing process after three days of treatment with the gels containing either 2.4% or 3.6% propolis. Slices of 6 μm were obtained and stained with Masson´s trichromic. a) Lesions treated with the 2.4% propolis gel. Closing of the lesion in the epithelial layer (1) and subcutaneously (2) there is a large number of fibroblasts synthesizing collagen (arrow) (100x magnification); b) Lesion treated with the 2.4% propolis gel. Subcutaneously (2) there is an increase of collagen fibers and a large number of cells undergoing mitosis (arrow) (400X magnification); c) Lesions treated with the 3.6% propolis gel. Wound area completely rebuilt, the epithelium (1) is already presented in several layers forming the corneal region. Subcutaneously (2) thicker collagen fibers (arrow) were observed (400X magnification).

References

    1. Nasser S, Mabrouk A, Maher A. Colonization of burn wounds in Shams University Burn Unit. Burns. 2003;29:229–233.
    1. Gerding R L, Emerman CL, Effron D, Lukens T, Imbembo AL, Fratianne RB. Outpatient management of partial-thickness burns: Biobrane® versus 1% silver sulfadiazine. Ann Emerg Med. 1990;19:121–124.
    1. Nalbandian RM, Henry RL, Balko KW, Adams DV, Neuman NR. Pluronic F-127 gel preparation as an artificial skin in the treatment of third-degree burns in pigs. J Biomed Mater Res. 1987;21:1135–1148.
    1. Cabana A, Ait-Kadi A, Juhasz J. Study of the gelation process of polyethylene oxide-polypropylene oxide- polyethylene oxide copolymer (poloxamer 407®) aqueous solution. J Colloid Interf Sci. 1997;190:307–312.
    1. Simões LMC, Gregório LE, da Silva Filho AA, de Souza ML, Azzolini AECS, Bastos JK, Lucisano-Valim YM. Effect of Brazilian green propolis on the production of reactive oxygen species by stimulated neutrophils. J Ethnopharmacol. 2004;94:59–65.
    1. Park YK, Ikegaki M. Preparation of water and ethanolic extracts of propolis and evaluation of the preparations. Biosci Biotechnol Biochem. 1998;62:2230–2232.
    1. Burdock GA. Review of the biological properties and toxicity of bee propolis. Food Chem Toxicol. 1998;36:347–363.
    1. Borrelli F, Maffia P, Pinto L, Ianaro A, Russo A, Capasso F. et al. Phytochemical compounds involved in the anti-inflammatory effect of propolis extract. Fitoterapia. 2002;73:S53–S63.
    1. Diaz JCQ, Rodrigues OA, Velázquez MD, Miliá NML. Empleo de la tintura de propóleo en la cura de heridas sépticas faciales. Rev Cuba Estomatol. 1997;34:347–351.
    1. Koo H, Gomes BPFA, Rosalen PL, Ambrosano GMB, Park YK, Cury JA. In vitro antimicrobial activity of propolis and Arnica montana against oral pathogens. Arch Oral Biol. 2000;45:141–148.
    1. Reis CMF, Carvalho JCT, Caputo LRG, Patricio KCM, Barbosa MVJ, Chieff AL. et al. Atividade antiinflamatória, anti-úlcera gástrica e toxicidade subcrônica do extrato etanólico de própolis. Rev Bras Farmacogn. 2000;9-10:43–52.
    1. Barros MP, Sousa JPB, Bastos JK, Andrade SF. Effect of Brazilian green propolis on experimental gastric ulcers in rats. J Ethnopharmacol. 2007;110:567–571.
    1. Rezende GPSR, Pimenta FC, Costa LRRS. Antimicrobial activity of two Brazilian commercial propolis extracts. Braz J Oral Sci. 2006;5:967–970.
    1. De Castro PA, Savoldi M, Bonatto D, Barros MH, Goldman MHS, Berretta AA. et al. Molecular characterization of propolis-induced cell death in Saccharomyces cerevisiae. Eukaryotic Cell. 2011;10:398–411.
    1. Senedese JM, Rodrigues AR, Furtado MA, Faustino VD, Berretta AA, Marchetti JM. et al. Assessment of the mutagenic activity of extracts of Brazilian propolis in topical pharmaceutical formulations on mammalian cells in vitro and in vivo. Evid Based Complement Alternat Med. 2008 doi:10.1093/ecam/nen049.
    1. Tavares DC, Lira WM, Santini CB, Takahashi CS, Bastos JK. Effects of propolis crude hydroalcoholic extract on chromosomal aberrations induced by doxorubicin in rats. Planta Med. 2007;73:1531–1536.
    1. Czaja W, Krystynowicz A, Bielecki S, Brown Jr RM. Microbial cellulose—the natural power to heal wounds. Biomater. 2006;27:145–151.
    1. Sousa JPB, Bueno PCP, Gregório LE, Silva Filho AA, Furtado NAJC, Sousa ML. et al. A reliable quantitative method for the analysis of phenolic compounds in Brazilian propolis by reverse phase high performance liquid chromatography. J Sep Sci. 2007;30:2656–2665.
    1. Schmolka IV. Artificial skin I. preparation and properties of Pluronic f.-127 gels of treatment of burns. J Biomed Mat Res. 1972;6:571–82.
    1. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 6th ed. NCCLS document M7-A6. 2003.
    1. Hu RH, Yu YM, Costa DBS, Young VR, Ryan CM, Burke JF. et al. A rabbit model for metabolic studies after burn injury. J Surg Res. 1998;75:153–158.
    1. Magro-Filho O, de Carvalho AC. Application of propolis to dental sockets and skin wounds. J Nihom Univ Sch Dent. 1990;32:4–13.
    1. Junqueira LC, Carneiro J. Histologia Básica. Rio de Janeiro, Brasil: Guanabara Koogan; 2004.
    1. Sforcin JM, Bankova V. Propolis: Is there a potential for the development of new drug? J Ethnopharmacol. 2011;133:253–260.
    1. Almeida IF, Costa PC, Bahia MF. Evaluation of Functional Stability and Batch-to-Batch Reproducibility of a Castanea sativa Leaf Extract with Antioxidant Activity. AAPS PharmSciTech. 2010;11(1):120–125.
    1. Erkmen O, Özcan MM. Antimicrobial effects of Turkish propolis, pollen, and laurel on spoilage and pathogenic food-related microorganisms. J Med Food. 2008;11:587–592.
    1. Menna-Barreto RFS, Salomão K, Dantas AP, Santa-Rita RM, Soares MJ, Barbosa HS, De Castro SL. Review - Different cell death pathways induced by drugs in Trypanosoma cruzi: An ultrastructural study. Micron. 2009;40(2):157–168.
    1. Takaisi NB, Schilcher H. Electron microscopy and microcalorimetric investigations of the possible mechanism of the antibacterial action of a defined propole provenance. Planta Med. 1994;60:222–227.
    1. Urushisaki T, Takemura T, Tazawa S, Fukuoka M, Hosokawa-Muto J, Araki W, Kuwata K. Caffeoylquinic Acids Are Major Constituents with Potent Anti-Influenza Effects in Brazilian Green Propolis Water Extract. ECAM. 2011 doi:10.1155/2011/254914.
    1. Akarasereenont P, Mitchell JA, Bakhle C, Thiemermann C, Vane JR. Comparison of the induction of cyclooxigenase and nitric oxide synthase by endotoxin in endothelial cells and macrophages. Eur J Pharmacol. 1995;273:121–128.
    1. Shukla A, Rasik AM, Patnaik K. Depletion of reduced glutathione, ascorbic acid, vitamin E and antioxidant defence enzymes in a healing cutaneous wound. Free Radical Res. 1997;26:93–101.
    1. Amadeus TP, Braune AS, Porto LC, Desmoulière A, Costa AMA. Fribrillin-1 an elastin are differentially expressed in hypertrophic scars and keloids. Wound Repair Regen. 2004;12:169–174.
    1. Naito Y, Yasumuro M, Kondou K, Ohara N. Antiinflammatory effect of topically applied propolis extract in carrageenan-induced rat hind paw edema. Phytoth Res. 2007;21:452–456.
    1. Volpert R, Elstner EF. Interactions of different extracts of propolis with leukocytes and leukocytic enzymes. Arzn-Forsch/Drug Res. 1996;46:47–51.
    1. Bankova V, Castro SL, Marcucci MC. Propolis: recent advances in chemistry and plant origin. Apidol. 2000;31:3–15.
    1. Nayak S, Nalaboth P, Sandiford S, Bhogadi V, Adogwa A. Evaluation of wound healing activity of Allamanda cathartica. L. and Laurus nobilis. L. extracts on rats. BMC Complement Altern Med. 2006;6:12.

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