Antineoplastic Activity Evaluation of Brazilian Brown Propolis and Artepillin C in Colorectal Area of Wistar Rats

Abstract

Objective: The study's aim was to evaluate Brazilian Brown Propolis (BBP) and Artepillin C (ARC) chemopreventive action in Wistar rats' colons.

Methods: Fifty male Wistar rats were divided into ten experimental groups, including control groups, groups with and without 1,2-dimethylhydrazine (DMH) induction, and BBP, ARC, and ARC enriched fraction (EFR) treatments, for sixteen weeks. Aberrant crypt foci (ACF) were classified as hyperplastic or dysplastic, and proliferating cell nuclear antigen (PCNA) expression was quantified.

Result: ACF amounts in experimental groups (induced or not) decreased in both colon portions, while the isolated Aberrant Crypt (AC) number increased. Experimental groups of animals showed higher hyperplasia and dysplasia amounts compared with control groups. The ACF dysplastic amount present in groups induced and treated, in both colon portions, had similar values to IDMH (DMH induction group without treatment). In addition, DMH was effective in ACF inducing and there was positive staining for PCNA in basal and upper dysplastic foci portions in all experimental groups, in the mitotic index (MI) evaluation. To conclude, considering all the experimental groups, the one treated with EFR (fraction enriched with ARC) had the lowest rates of cell proliferation.

Conclusion: BBP and its derivatives prevented crypt cell clonal expansion.

Keywords: 1; 2-DIMETHYLHYDRAZINE (DMH); BRAZILIAN BROWN PROPOLIS (BBP); Chemoprevention; aberrant crypt foci (ACF).

Figure 1

Chemical Characterization of BBP and ARC Alcoholic Extract. A, ¹H NMR spectrum (300 MHz, CD3OD) of Artepillin C; B, Magnification of Artepillin C ¹H NMR spectrum (300 MHz, CD3OD)

Figure 1

Chemical Characterization of BBP and ARC Alcoholic Extract.C, . ¹³C NMR spectrum (75 MHz, CD3OD) of Artepillin C; D, DEPT 135º spectrum (75 MHz, CD3OD) of Artepillin C; E, UV and mass spectra (HPLC-DAD-MS) of Artepillin C

Figure 1

Chemical Characterization of BBP and ARC Alcoholic Extract. F, HPLC-DAD-MS chromatogram of Artepillin C and blank (solvent) at wavelength 210 to 400 nm

Figure 2

Representative Crypts and Aberrant Crypt Foci Photomicrographs of Rats' Distal Colon Exposed to 1,2-dimethylhydrazine (DMH). (A) isolated aberrant crypt (B,C,D) aberrant crypt foci with 2 (B), 9 (C), and more than 10 (D), characterized by intestinal lining epithelium dilated and protrusion towards the intestinal lumen. On panel, normal crypts (black arrow) are observed around aberrant crypt focus (red arrow) (Methylene Blue Stain). 20X objective

Figure 3

Illustrative Image of the Colon of Wistar Rats sStained with HE. (A) normal colon proximal region (B) proximal colon with gland hyperplasia and goblet cells (C) colon proximal region with dysplastic crypt (arrow). 20X objective

Figure 4

Number of Hyperplasia and Dysplasia in the Proximal and Distal Portions of the Groups after the HE Technique. Mean and standard deviation were calculations based on each group's number of animals. Results were expressed as means observed in animals of the same group ± standard deviation

Figure 5

Photomicrographs of a Colon Tagged with PCNA and Stained with DAB. Crypt cells in the crypt basal region (red arrow) and crypt apex (black arrow) with marked expression of PCNA; the presence of nuclear stratification, and mucin production reduced (*). 20X objective

Figure 6

Expression of PCNA-tagged Proteins. Results are expressed as percentages by calculating the mitotic index [(number of PCNA-labeled cells) / (total number of cells)] x100. *p < 0,05 by the oneway ANOVA test; **p < 0,05 by Kruskal-Wallis test. For Proximal, the difference between groups IDMH and IEFR, IDMH and IARC, IBBP and IEFR, IEFR and PBBP, IEFR and IARC, and PBBP and IARC (Tukey's test); For Distal, the difference between groups IDMH and IEFR, IBBP and IEFR, IBBP and PBBP, IEFR and PBBP, IEFR and IARC and PBBP and IARC (Nemeniy's test). a = IDMH; b = IBBP; c = IEFR; d = PBBP