Propolis samples

Samples of 100 g of raw propolis were obtained of three apiaries of different locality from Yucatán (México); Maxcanú (west of the state) coordinates 20° 35’ latitude (North) and 90° 0’ longitude (West), Tizimín (east) coordinates 20° 57’ latitude (North) and 87° 31’ longitude (West), and Huhí (center) coordinates 20° 38’ latitude (North) and 89° 15’ longitude (West). The three localities were selected according to the presence of apiaries and number of hives of Apis mellifera. Samples were collected of thirteen hives using the traditional scraping method with stainless steel spatulas, the sample stuck in the lid of each hive box was removed and stored in a glass container for its transfer. Samples of raw propolis were cleaned removing pieces of wood, small stones, and parts of insects. These samples were maintained in freezing at 0 °C for further study and analysis.

Chemicals reagents

Catechin, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu, ferric chloride, 2,4,6-Tri(2-pyridyl)-s-triazine(TPTZ), acetic acid (glacial), aluminum chloride anhydrous, gallic acid, ethanol, sodium carbonate and phenols and flavonoids used as standards compounds were reagents analytical grade and purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Propolis ethanolic extract (PEE) preparation

Raw propolis (6 g) was mixed in 95% (v/v) ethanol (20 mL) and stirred every day for twenty-eight days at 25 °C. The extract solution was filtered and dried in a vacuum rotary evaporator (40 °C). The dry extract was lyophilized and preserved at -20 °C until further study and analysis. The extract of each sample was made in triplicate. All analyses were carried out in triplicate.

Phenolic and flavonoids compounds by HPLC

An aliquot of the PEE was dissolved with HPLC grade methanol (20 mg mL^-1), the solution was filtered (0.45 μm Teknokroma filters), 20 𝜇L of the filtrate was injected into an Agilent HPLC-1220 using a Nucleosil C18 column (250 x 4.6 mm, 5 𝜇m); and detected at 280 nm. The mobile phase consisted of 1% formic acid in water (Phase A) (98%) and acetonitrile (Phase B) (2%) a flow of 0.5 mL min^-1 was used. The separation was performed with linear gradient elution from 2 to 100% of phase B, from 0 to 70 min (^{Yahia et al. 2011}). Identification was performed by comparison of retention times of injection of known compound standards (50 ppm).

Total phenols and flavonoids content

An aliquot of 100 𝜇L of PEE (10 mg mL^-1) or each solution of standard (gallic acid, GA), was mixed with 500 𝜇L of Folin-Ciocalteu reagent, shaken for 30 s and left to stand for 2 min. Then, 400 𝜇L of Na₂CO₃ (75 mg mL^-1) were added, incubated for 15 min at 50 °C and allowed to cool at room temperature. The absorbance was measured at 760 nm and expressed as gallic acid equivalent (GAE), (mg GAE g^-1 of dry extract) (^{Brat et al. 2006}) with a calibration curve of GA (20 -100 𝜇g mL^-1). Total flavonoids content was determined using a colorimetric assay (^{Lee et al. 2003}) using catechin as standard. An aliquot of 250 𝜇L of PEE (10 mg mL^-1) or each standard solution was mixed with 250 𝜇L of 5% sodium nitrite; after 5 min, 125 𝜇L of 10% aqueous solution of aluminum chloride (AlCl₃·6H₃0) was added. Subsequently, 124 𝜇L of sodium hydroxide (1M) was added 6 min after. The absorbance was measured at 490 nm. Total flavonoids content was expressed as catechin equivalent (CE), (mg CE/g of dry extract) (^{Brat et al. 2006}) with a calibration curve (160 - 640 𝜇g mL^-1).

DPPH* free radical-scavenging assay

With modifications DPPH* method was used (^{Shimada et al. 1992}), PEE (100 𝜇L) (10 mg mL^-1) was added to 1.0 mL of DPPH* 0.1 mM in 95% ethanol. The mixture was shaken for 30 min at room temperature and absorbance was measured at 517 nm. Ethanol was utilized as negative control and gallic acid (10 mg mL^-1) as positive control. Antioxidant activity was expressed as the percentage inhibition of DPPH* free radical scavenging (% of DPPH*) calculated using the equation:

Where AC is the absorbance of DPPH* solution in absence of a sample added and AE is the absorbance of DPPH* in the presence of either the sample or standard after 30 min of reaction.

ABTS free radical-scavenging assay

The ABTS •+ radical cation method was used, reacting 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS) with potassium persulfate (^{Pukalskas et al. 2002}). The ABTS •+ radical cation was produced reacting 10 mL ABTS stock solution (2 mM) with 40 𝜇L K₂S₄O₈ solution 70 mM and allowing the mixture to stand in darkness at room temperature for 16-17 h before use. An aliquot of this ABTS solution was diluted with PBS (phosphate buffered saline pH 7.3) solution adjusting its absorbance at 0.800 ± 0.030 AU at 734 nm. For the analysis, to 990 𝜇L of this ABTS •+ solution, 10 𝜇L of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) standard solution of different concentrations (0.5-3.5 mM) in PBS was added, then absorbance was read 6 min after mixing. Percentage decrease in absorbance at 734 nm was calculated and plotted as a function of Trolox solution concentration. Similarly, with the PEE extract a plot of percentage decrease in absorbance at 734 nm was obtained by reacting solutions of different concentration of PEE with ABTS •+ solution. In this method the total antioxidant activity was expressed as the Trolox Equivalent Antioxidant Capacity (TEAC) (mM Trolox/g dry PEE) which was calculated based on slopes of curves obtained by reaction different concentrations of PEE and Trolox (reference antioxidant compound). To calculate TEAC of PEE, the slope of the plot of the percentage inhibition of absorbance vs PEE concentration was divided by the slope of the Trolox plot (percentage inhibition of absorbance vs Trolox concentration). This produce the TEAC at the specific time. TEAC is one of the assays widely used to evaluate antioxidant activity of several types of compounds. This method was first developed as a simple and convenient method to determination of total antioxidant capacity (Miller et al. 1993).

ACE-I (angiotensin converting enzyme) inhibitory activity

With some modifications from ^{Hayakari et al. (1978)}. Hippuryl-L-histidyl-L-leucine (HHL) is hydrolyzed by ACE to yield hippuric acid and histidyl-leucine. This method relies on the colorimetric reaction of hippuric acid with 2,4,6-trichloro-s-triazine (TT) in a solution (0.5 mL) containing 40 𝜇mol potassium phosphate buffer (pH 8.3), 300 𝜇mol sodium chloride, 40 𝜇mol 3% HHL in potassium phosphate buffer (pH 8.3), and 100 mU/ml ACE-I. The mixture was incubated at 37 °C for 45 min and the reaction was terminated by adding TT (3% v/v) in dioxane and 3 mL of 0.2 M potassium phosphate buffer (pH 8.3). After centrifuging the reaction mixture at 10,000 g for 10 min, enzymatic activity was determined in the supernatant by measuring absorbance at 382 nm. ACE-I inhibitory activity (%) was quantified in a concentration of 40 mg mL^-1 and calculated as follow:

where, A represents absorbance in presence of ACEI sample, B absorbance of the control, and C absorbance of the reaction blank.

Anti-diabetic activity. In vitro α-amylase inhibition

Starch (2 mg) (substrate) (^{Dineshkumar et al. 2010}) was suspended in a tube with 0.2 mL of 0.5 M Tris-HCl buffer (pH 6.9) containing 0.01 M CaCl₂. The tube was boiled for 5 min and incubated at 37 °C for 5 min; 200 𝜇L PEE at 150 mg mL^-1 were dissolved with 1 mL of 0.1% of dimethyl sulfoxide. Then 0.2 mL of each PEE was put in the tube containing the substrate solution, and 0.1 mL of porcine pancreatic amylase in Tris-HCl buffer (2 units mL^-1) were added. The reaction was carried out at 37 °C for 10 min. The reaction was stopped adding 0.5 mL of 50% acetic acid in each tube. The reaction mixture was then centrifuged at 2 000 g for 5 min at 4 °C. The absorbance was measured at 595 nm. The results were expressed as percent inhibition of 𝛼-amylase through equation:

where, Ac⁺, Ac^-, As, Ab are defined as the absorbance of 100% enzyme activity (only solvent with enzyme), 0% enzyme activity (only solvent without enzyme), a test sample (with enzyme) and a blank (a test sample without enzyme), respectively.

In vitro α-glucosidase inhibition

The α-glucosidase (50 μL, 2 U mL^-1) (^{Dineshkumar et al. 2010}) was premixed with 50 μL of PEE (at concentration of 0.5 mg mL^-1) and incubated for 5 min at 37 ⁰C. Then 1 mM para-nitrophenyl gluco-pyranoside (pNPG) (50 μL) in 50 mM of phosphate buffer (pH 6.8) was added to initiate the reaction. The mixture was further incubated at 37 ⁰C for 20 min. The reaction was terminated by the addition of 125 μL of 1 mM sodium carbonate. The α-glucosidase activity was determined at 405 nm. The results were expressed as percent inhibition of α-glucosidase through equation:

where, Ac⁺, Ac^-, As, Ab are defined as the absorbance of enzyme solution 100% enzyme activity (only solvent with enzyme), 0% enzyme solution activity (only solvent without enzyme), a test sample (with enzyme) and a blank (a test sample without enzyme), respectively.

Statistical analysis

The results were evaluated by analysis of variance, to determine significant differences between means (p < 0.05) which was determined by Tukey’s multiple range test with the statistical package Statgraphics® Centurion version 16.1.15. The correlation coefficient (r) between results (total phenolics, flavonoids, antioxidant and pharmacological activities) was determined from the Pearson correlation coefficient, which quantifies the linear association between two quantitative variables. This correlation was determined using Statistica 7.0 software.

Total phenols and flavonoids content

The percentage obtained of dry PEE from each one of the three raw propolis samples was 9.97, 5.56 and 6.66% from Tizimín, Huhí and Maxcanú, respectively. Table 1 shows the average of total content of phenols and flavonoids of the PEE of the three samples, collected in three different places in Yucatán Peninsula. A significant difference was found (p < 0.05) in both type of BC, in the three samples. Huhí sample had 85.64 mg CE g^-1 dry extract of flavonoids, which was the highest among all three samples. Regarding total phenols content, also Huhí sample showed the highest amount with 25.95 mg GAE g^-1 dry extract. The sample of Tizimín has the lowest content of phenols (1.27 ± 0.02 mg GAE g^-1 dry extract) and Maxcanú the lowest of flavonoids (65.06 ± 3.48 mg CE g^-1 dry extract).

Antioxidant activity

Figure 1 shows antioxidant activity, (ability scavenging free radicals) of propolis analyzed by two methods DPPH* and ABTS+ (TEAC). The three extracts possessed potent and diverse antioxidant activity with significant differences (p < 0.05). Propolis extract from Huhí exhibited the higher antioxidant potential in both methods (Fig. 1) 67.32 and 64.29%, respectively. Likewise, the PEE from Tizimín showed the lower DPPH* and TEAC antioxidant activity. Differences in antioxidant activity indicate that composition of propolis is significantly different.

Figure 1: Means ± Standard deviation of antioxidant activity of PEE (10 mg mL^-1). 1A) DPPH* free radical scavenging assay, 1B) Trolox Equivalent Antioxidant Capacity assay. Data are presented as means (n = 3). 𝑎−𝑐 Different letters indicate significant difference (p < 0.05). 

Composition and chromatographic profile of PEE

Chromatographic profile of PEE of three samples is represented in Figure 2 and some identified compounds are in Table 2. The profile of PEE from Huhí has several compounds significantly higher concentration than the Maxcanú and Tizimín PEE. Compounds identified by HPLC reveal the occurrence of at least six flavonoids and ten phenolic acids (phenols). Five of them, epicatechin, sinapic acid, trans-cinnamic acid, kaempferol and isorhamnetim were identified in the three PEE samples, where epicatechin and p-coumaric acid were major constituent flavonoid and phenol observed in Huhí PEE. These results corroborate the difference in the chemical composition of PEEs.

Figure 2 HPLC chromatograms at 280 nm of propolis ethanolic extract from: 2A, Huhí; 2B, Maxcanú; 2C, Tizimín respectively. 

In vitro ACE-I inhibitory activity

The Figure 3 shows a range of inhibition of ACE-I between 29.48 and 63.99%, while bioactive compounds present in Huhí PEE showed highest activity (63.99%) of the samples studied.

Figure 3: Means ± standard deviation of ACE-I inhibitory activity of propolis of three localities from Yucatan. Data are presented as means (n = 3). ^a-c Different letters indicate significant difference (p < 0.05). 

In vitro anti-diabetic activity

The bioactive compounds present in PEE, showed inhibition for a-amylase enzyme values of 7.49 ± 0.03%, 83.3 ± 0.04% and 100 ± 0.4% in Tizimín, Maxcanú and Huhí extracts, respectively (Figure 4), the significant difference in the inhibitory activity of the extracts is evident. Huhí PEE exhibited a higher inhibition percentage than that found in samples from India, Kerala (74.67 ± 2.57%) and Tamil Nadu (72.85 ± 3.89%) (^{Ramnath and Venkataramegowda 2017}). Likewise, the Huhí PEE showed the higher inhibition of α-glucosidase activity value (29.97 ± 1.5%). Inhibitory activity of this enzyme of Maxcanú (2.53 ± 0.05%) and Tizimín (2.84 ± 0.06%) PEE were significantly lower.

Figure 4: Means ± standard deviation of anti-diabetic activity of PEE of three localities from Yucatan. 4A)α-amylase, 4B) α-glucosidase enzyme inhibition percentages. Data are presented as means (n = 3). ^a-c Different letters indicate significant difference (p < 0.05). 

Correlation

Antioxidant activity of PEE partly is due to its content of phenolic and flavonoids compounds, which is evident by the high significant values of linear correlation r (Perason’s correlation coefficient) found between phenolic and flavonoids concentration with antioxidant activity evaluated (Table 3), r = 0.99 and 0.75 with ABTS •+ and r = 0.99 and 0.82 for DPPH* respectively. The Inhibition of the enzyme activities (α-amylase, α-glucosidase and ACE-I) is significantly correlated with concentration of phenols and flavonoids. The significantly correlation suggests that antioxidant activity and inhibition of the enzymes are closely related to the phenol and flavonoid content of PEE.