Antioxidant and Anti-inflammatory Compounds of Mexican Propolis

Propolis is made by bees from a combination of plant resins, wax, and saliva. It is used in the hive as a building material, disinfectant, and embalming agent. Propolis has many reported health benefits for humans, due to its antimicrobial, antioxidant, and anticancer properties. Multiple factors impact the composition of propolis, including location, the season of harvest, local flora, and species of bee. Mexico is a major honey producer, with production in Yucatán, Campeche, Jalisco, and Chiapas; each region producing propolis which contains a variety of active compounds. This study assessed the antioxidant properties of five samples of propolis from two states in Mexico.

Propolis samples were collected from five regions in Mexico; four from the state of Chiapas (1-4), and one from the state of Yucatán (5). Samples 1, 2, 3, and 5 were collected from Apis mellifera bee breeders, while sample 4 was collected from a wild colony. Samples were collected, ground in a coffee mill, and extracted at room temperature along an increasing polarity gradient. All samples, except sample 4, were sequentially extracted by hexane, ethyl acetate, and methanol. Sample 4 was insoluble in hexane but completely dissolved in ethyl acetate, so that sample only received one treatment.

The inhibitory lipid peroxidation properties of each sample were assessed using a thiobarbituric acid reactive substances (TBARS) assay. Samples 2, 3, and 4 from Chiapas showed activity at 10 µg/mL. Since sample 4 showed the most IC50activity, it was selected for further analysis. It was fractionated using a hexane-ethyl acetate gradient (CC), which resulted in the isolation of 12 compounds. These compounds were analyzed by high-performance liquid chromatography (HPLC), TBARS assay, reactive oxygen species (ROS) assay, 2,2-diphenyl-1-picrylhydrazyl-hydrate (DPPH) antioxidant assay, tissue-type plasminogen activator (tPA), inhibition of myeloperoxidase assay, and an anti-mycobacterial activity assay.

Data were represented as a mean ± standard error (SEM) and analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test to find significant differences in means. IC50 was estimated by linear regression.

Sample 4 (4-EAEP) was found to have the most potent IC50 (1.42 ± 0.07 µg/mL and 99.28% inhibition) in the TBARS assay. Samples 2 and 3 showed significantly less activity, and samples 1 and 5 showed no activity.

Fractionation of 4-EAEP by CC resulted in the isolation of 12 compounds (six flavonoids, five phenylpropanoids, and one fatty acid lactone), two of which were previously unreported (epoxypinocembrin chalcone and a ɛ-caprolactone derivative). HPLC analysis revealed the flavanone pinocembrin and the flavanol pinostrobin to be the most abundant, and the two newly identified compounds being the least abundant. TBARS analysis revealed that the antioxidant activity of the entire 4-EAEP sample was more potent than its isolated compounds. The ROS assay confirmed the presence of antioxidant compounds within 4-EAEP. The DPPH assay found three of the compounds in 4-EAEP to have free radical scavenging ability. The DPPH assay demonstrated that 4-EAEP had an IC50 = 16.55 ± 0.87 μg/mL. Histological sections from mouse ears subjected to tPA were used to assess the ability of 4-EAEP and its compounds to inhibit inflammation and myeloperoxidase. One mg/ear of 4-EAEP was effective at lowering myeloperoxidase activity by 95.46 ± 0.002%. Finally, 4-EAEP had a minimum inhibitory concentration of 250 µg/mL for anti-mycobacterial activity.

The authors conclude that the propolis sample gathered from a wild bee colony in Chiapas, Mexico, contained the promising antioxidant, anti-inflammatory, and lipid peroxide reduction properties. They also identified two new active compounds in the propolis. This study suggests propolis might be used as a possible treatment to explore degenerative and inflammatory diseases. Certainly, in vivo and human trials are needed to verify efficacy in more robust studies. Standardization of propolis may also be required, as the study indicates that the location of origin can affect the active compounds present.

The authors declare no conflict of interest.

Resource:

Guzmán-Gutiérrez SL, Nieto-Camacho A, Castillo-Arellano JI, et al. Mexican propolis: a source of antioxidants and anti-inflammatory compounds, and isolation of a novel chalcone and ε-caprolactone derivative. Molecules. February 2018;23(2):334. doi:10.3390/molecules23020334.

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