Phytochemical study, development of analytical methods and biological evaluation of brown propolis from southeastern Brazil produced by Apis mellifera

Author(s)

Victor Pena Ribeiro

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-03-03

Examining board members

Jairo Kenupp Bastos; Juliana de Carvalho da Costa; Leandro Machado Rocha; Jose Mauricio Sforcin

• Advisor: Jairo Kenupp Bastos

Abstract

Propolis has been the subject of several pharmacological studies due to important biological properties such as antioxidant and anti-inflammatory, among others, being an important therapeutic alternative, from the economic point of view, for being pharmacologically efficient and easy to obtain. The main types of Brazilian propolis are: green, red, yellow and brown. Despite the commercial, historical and economic importance of Brazilian propolis, the chemical composition of brown propolis has been scarcely investigated. All biological properties attributed to propolis are strongly dependent on the concentration of active constituents. Thus, chemical investigation and standardization of propolis products are fundamental due to the variability in the chemical profiles of the extracts. Therefore, the objective of this work was to investigate the chemical composition, develop and validate chromatographic methods to quantify the markers and evaluate the biological activity of samples of brown propolis produced by Apis mellifera collected in the southeastern region of Brazil. Samples of brown propolis were collected in the apiary Sol, located in the municipality of Cabo Verde - MG, using wooden collectors hollowed in the lids of Apis Mellifera hives. From the propolis, the crude extract was obtained through maceration with hydroalcoholic solutions. The crude extract was partitioned with solvents of different polarity and was subsequently subjected to different chromatographic techniques to isolate the major compounds. The isolated substances had their structures determined by means of techniques such as NMR. From the crude extract, 15 substances were isolated and identified, which were used in the development and validation of a method by HPLC/DAD. The validation took into account parameters established in the guides of agencies such as ANVISA and ICH. The crude extract was evaluated for cell cytotoxicity against normal and tumor lines, activity against Leishimania amazonenses and Plasmodium falciparum. The cytotoxic evaluation of the hydroalcoholic extract did not indicate an effect on the viability of normal cell lines (VERO and LLC-PK1), however, brown propolis exhibited a toxic effect on tumor cells, with the following IC50 values: 80 µg/mL for SK-MEL; 82 µg/ml for KB; 71 µg/ml for SK-OV-3; and 64 µg/ml for BT-549. Brown propolis showed promising leishmanicidal activity, with an IC50 of 1.8 µg/mL against the promastigote form of L. amazonensis and an IC50 of 2.4 µg/mL against the amastigote form. Regarding antiplasmodic activity, brown propolis showed activity against P. falciparum strains, with IC50 of 18.1 µg/mL and 15.6 µg/mL, against D6 and W2 strains, respectively. The volatile fraction of brown Propolis was also considered in this work, for which the propolis was submitted to hydrodistillation to obtain the volatile fraction. This fraction was fractionated by spinning band distillation and two fractions were subjected to open column chromatography to isolate the major compounds. The isolated substances had their structures determined by means of NMR and mass spectrometry. Eight volatile substances that were used in the development and validation of the method by GC-DIC were isolated and identified, which were considered the parameters established in the validation guides of ANVISA and ICH. The volatile fraction showed significant leishmanicidal activity, with IC50= 21.3 µg/mL against amastigote forms and IC50= 25.1 µg/mL against promastigote forms of Leishmania amazonensis. This fraction also showed an antibacterial effect by inhibiting the growth of Streptococcus mutans and Staphylococcus aureus at 25 µg/mL and 50 µg/mL, respectively, but was not cytotoxic against AGP-01, He-La and CHO-K1 strains, with IC50 > 100 µg/mL. The crude extract and the volatile fraction of brown propolis had their analgesic XIII and anti-inflammatory properties evaluated by means of the formalin test and mechanical hypernociception induced by carrageenan. The hydroalcoholic extract and the volatile fraction were evaluated at doses of 50, 100 and 200 mg/kg. In the formalin test, the crude extract reduced the response at 73 ± 7%, 83 ± 8%, and 89 ± 3% for the first phase and 48 ± 13%, 65 ± 9%, and 75 ± 6% for the second phase, respectively. The volatile fraction reduced the response by 75 ± 7%, 94 ± 5% and 99 ± 1% and 30 ± 7%, 53 ± 5%, and 91 ± 5% for the first and second phases. For the carrageenan test, the hydroalcoholic extract and the volatile fraction showed reductions in mechanical sensitization of 57 ± 7%, 67 ± 6% and 79 ± 5% and 62 ± 7%, 79 ± 9% and 88 ± 4% respectively, after 48 h. Indomethacin and morphine showed inhibitions of 42 ± 4% and 88 ± 2%, respectively. In the tail flick test, the results showed evidence of analgesic activities; animals treated with crude extract increased the nociceptive threshold by 58±8% and 70±6% and 77±10%, respectively, for the doses evaluated, and 55±6% and 73±7% and 82±14% for the volatile fraction, compared to the control. Co-administration of naloxone altered the antinociceptive effect of morphine and propolis, suggesting that brown propolis has an action on the central nervous system. A brown propolis collected in the city of Angatuba - SP had its chemical composition elucidated, for that, the crude extract was obtained through maceration with hydroalcoholic solutions that was subjected to partitions and later to different chromatographic techniques for isolation of its constituents. The isolated substances had their structures determined by means of NMR. The chemical composition of this propolis was complex and different from those reported in the literature. Sixteen substances from this brown propolis were isolated and identified, including seven phenolic compounds, one flavanonol, two lignans and six diterpenic acids/alcohol. The chemical profile of this propolis indicates that Pinus spp., Eucalyptus spp. and Araucaria angustifolia may be its main plant source. Brown propolis showed significant activity against Plasmodium falciparum D6 and W2 strains with IC50 of 5.3 and 9.7 µg/mL, respectively. The volatile fraction of this propolis was also active with IC50 of 22.5 and 41.8 µg/mL, respectively. Among the compounds, 1-O,2-O-digalloyl-6-O-trans-p-coumaroyl-β-D-glucopyranoside presented IC50 of 3.1 and 1.0 µg/mL against strains D6 and W2, respectively. , while comunic acid showed an IC50 of 4.0 µg/mL against the W2 strain. Cytotoxicity was determined in four tumor cell lines (SK-MEL, KB, BT-549 and SK-OV-3) and two normal renal cell lines (LLC-PK1 and VERO). Matairesinol, 7-O-methyl aromadendrin and isopimaric acid showed an IC50 range of 1.8 - 0.78 µg/mL, 7.3 - 100 µg/mL and 17-18 µg/mL, respectively, against the strains of tumor cells, but were not cytotoxic against normal cell lines. The brown propolis crude extract showed antimicrobial activity against C. neoformans, methicillin-resistant Staphylococcus aureus and P. aeruginosa at 29.9 µg/mL, 178.9 µg/mL and 160.7 µg/mL, respectively. The volatile fraction inhibited the growth of C. neoformans by 53.0 µg/mL. Brown propolis has a greater chemical variety than other types of Brazilian propolis, as several possible botanical sources are listed. Therefore, Brazilian brown propolis has great potential and should be further investigated to enable its production and commercialization as an effective and safe product for health. (AU)

• FAPESP's process: 17/08386-6 - Phytochemical study, development of analytical method and biological evaluation of brown propolis produced by Apis mellifera from Morus Alba
• Grantee: Victor Pena Ribeiro
• Support Opportunities: Scholarships in Brazil - Doctorate