Chemical characterization and toxicological studies of Brazilian red propolis and its botanical source Dalbergia ecastaphyllum (L.) Taub

Author(s)

Jennyfer Andrea Aldana Mejía

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-09-28

Examining board members

Jairo Kenupp Bastos; Juliana de Carvalho da Costa; Bettina Monika Ruppelt; Raquel Alves dos Santos

• Advisor: Jairo Kenupp Bastos

Abstract

Brazilian red propolis results from the interaction between Apis mellifera bees and two plant species, being Dalbergia ecastaphyllum (L.) Taub. (Fabaceae), the primary source of isoflavonoids and other characteristic phenolic compounds. This variety of propolis has wide traditional use, and several biological activities reported, presenting a high potential in the development of pharmaceutical products. However, few studies guarantee safety in its use. In this sense, this work aimed to carry out the chemical characterization, the validation of the analytical method, and the evaluation of the in vitro and in vivo toxicological potential of red propolis from Canavieiras (Bahia). For that, some chromatographic techniques were used to purify compounds from the hydroalcoholic extract of red propolis (BRP), and spectroscopy to identify the compounds. Among these techniques is the nuclear magnetic resonance, identifying compounds such as liquiritigenin, calycosin, isoliquiritigenin, vestitol, neovestitol, medicarpin, biochanin A, 7-O-methylvestitol, oblongifolin B and a mixture of gutiferone E / xanthochymol isomers. Subsequently, an analytical method by HPLC-DAD was developed and validated to analyze the extract, including nine isolated chemical markers, prioritizing isoflavonoids. The method showed linearity, selectivity, precision, accuracy, and robustness according to the validation guides\' designs. A seasonal analysis of samples of this variety of propolis was also performed, showing a higher concentration of markers used in periods of the rainy season with higher humidity and lower solar radiation. With regard to biological assays, the cytotoxic activity of BRP, leaf extract (DLE), and stem extract (DSE) of D. ecastaphyllum in a human fibroblast lineage (GM07492A) was evaluated employing the XTT colorimetric assay. Significant reduction in cell viability was observed at concentrations greater than or equal to 78 µg/mL. The genotoxic potential of BRP, DLE, and DSE was evaluated through the micronucleus test in V79 cells (Chinese hamster lung fibroblasts) and BRP in peripheral blood of mice. The results revealed the absence of the extracts\' genotoxicity in vitro and in vivo. A single dose acute toxicity test was also performed. This trial showed that the mean lethal dose (LD50) of BRP by oral administration in Wistar Hannover rats (female) is more significant than 2000 mg/kg. Based on the results obtained, a subchronic toxicity test of BRP was carried out in Wistar Hannover rats (male and female) at repeated doses of 1000 mg/kg for 90 days. No symptoms of immediate toxicity were observed in animals treated with BRP, although some changes were observed in the relative weight of some tissues, such as the liver, thyroid, and prostate, and some of the parameters related to renal and hepatic function in males. These results reinforce the importance of further studies to investigate possible toxicity mediated by prolonged consumption of red propolis. (AU)

• FAPESP's process: 17/26252-7 - No clinical toxicology studies of red propolis and its botanical source Dalbergia ecastophyllum (L.) Taub.
• Grantee: Jennyfer Andrea Aldana Mejía
• Support Opportunities: Scholarships in Brazil - Doctorate