Chemical Characterization and Optimization of the Extraction Process of Bioactive Compounds from Propolis Produced by Selected Bees <i>Apis mellifera</i>

Oldoni, Tatiane Luiza Cadorin; Oliveira, Sheila C.; Andolfatto, Suelen; Karling, Mariéli; Calegari, Matheus A.; Sado, Ricardo Y.; Maia, Fabiana M. C.; Alencar, Severino M.; Lima, Vanderlei A.

doi:10.5935/0103-5053.20150186

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Articles • J. Braz. Chem. Soc. 26 (10) • Oct 2015 • https://doi.org/10.5935/0103-5053.20150186 copy

Chemical Characterization and Optimization of the Extraction Process of Bioactive Compounds from Propolis Produced by Selected Bees Apis mellifera

Authorship SCIMAGO INSTITUTIONS RANKINGS

The extraction process of bioactive compounds from propolis produced by selected bees was optimized using a 2³ factorial design planning to evaluate the effect of ethanol concentration, time and temperature on the extraction of total phenolic compounds and antioxidant activity. Analysis of total flavonoids, antioxidant activity and chemical characterization performed by high-performance liquid chromatography with photodiode array (HPLC-PDA) detection were carried out in optimal conditions of extraction. Optimizing results suggest that the best condition for extraction of bioactive compounds was obtained in higher levels: time (45 min), temperature (70 °C) and concentration of ethanol (80%). The extract evaluated in optimal conditions presented good antioxidant activity by the radical scavenging 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and ferric reducing antioxidant power (FRAP) methods and chromatographic analysis identified phenolic acids: para-coumaric, ferulic and caffeic, indicating that this material is a potential source of bioactive compounds.

Keywords:
factorial design; chemical characterization; phenolic compounds

Run	Factor 1 (X₁): ethanol concentration^a a Ethanol concentration in v/v; / %	Factor 2 (X₂): temperature / ºC	Factor 3 (X₃): time / min	Response1(Z₁): total phenolic content^b b total phenolic content in propolis extract as acid gallic equivalents (mg GAE g-1); / (mg GAE g^-1)	Response 2 (Z₂): DPPH^c c antioxidant activity expressed as equivalent of μmol trolox g-1. The numbers in parentheses, in the three columns of the factors, represent the levels of each variable used in the factorial design. / (μg trolox g^-1)
1	50 (−1)	30 (−1)	15 (−1)	2.79 ± 0.04	31.6 ± 0.12
2	80 (+1)	30 (−1)	15 (−1)	3.95 ± 0.28	47.0 ± 0.12
3	50 (−1)	70 (+1)	15 (−1)	3.33 ± 0.16	49.8 ± 0.13
4	80 (+1)	70 (+1)	15 (−1)	3.56 ± 0.01	58.5 ± 0.04
5	50 (−1)	30 (−1)	45 (+1)	3.37 ± 0.04	39.9 ± 0.14
6	80 (+1)	30 (−1)	45 (+1)	4.68 ± 0.09	58.4 ± 0.01
7	50 (−1)	70 (+1)	45 (+1)	2.42 ± 0.15	78.1 ± 0.06
8	80 (+1)	70 (+1)	45 (+1)	5.75 ± 0.24	87.5 ± 0.13
9	65 (0)	50 (0)	30 (0)	3.61 ± 0.01	57.9 ± 0.06
10	65 (0)	50 (0)	30 (0)	3.56 ± 0.02	56.0 ± 0.06
11	65 (0)	50 (0)	30 (0)	3.96 ± 0.02	58.2 ± 0.07

Term	DPPH coefficient	Phenolic compounds coefficient
β₀	56.5^a a significant at p < 0.001(***);	3.73^a a significant at p < 0.001(***);
β₁	6.5^a a significant at p < 0.001(***);	0.76^a a significant at p < 0.001(***);
β₂	12.1^a a significant at p < 0.001(***);	0.03^b b ns: not significant at p > 0.05;
β₃	9.6^a a significant at p < 0.001(***);	0.32^c c significant at p < 0.05 (*);
β₁₂	−2.0^a a significant at p < 0.001(***);	0.13^b b ns: not significant at p > 0.05;
β₁₃	0.47^b b ns: not significant at p > 0.05;	0.40^d d significant at p < 0.01 (**);
β₂₃	4.7^a a significant at p < 0.001(***);	−0.002^b b ns: not significant at p > 0.05;
r²	0.994	0.833
Main effects
Ethanol concentration	*^a a significant at p < 0.001(*);	*^a a significant at p < 0.001(*);
Temperature	*^a a significant at p < 0.001(*);	ns^b b ns: not significant at p > 0.05;
Time	*^a a significant at p < 0.001(*);	^c c significant at p < 0.05 ();
F value	454.71	24.93
F statistic table	3.03^e e 95% of confidence level;	3.29^e e 95% of confidence level;
F ratio^f f F ratio: (F-value/F-tabular value).	150.07	7.58

Phenolic compound	Retention time / min	Band UV / nm^a a λmax: maximum absorption wavelength;	Regression equation	r^b b r: correlation coefficient = √r2;	LOD^c c LOD: limit of detection in μg mL-1;	LOQ^d d LOQ: limit of quantification in μg mL-1;	Concentration / (mg 100g^-1 ± SD)^e e SD: standard deviation;	Precision / %
Galic acid	8.6	272	y = 0.393x − 0.558	0.999	1.10	3.32	nd^f f nd: not detected.	nd^f f nd: not detected.
Vanilic acid	23.8	260, 280	y = 0.329x − 0.594	0.994	1.18	3.57	nd^f f nd: not detected.	nd^f f nd: not detected.
Caffeic acid	24.3	323	y = 1.005x − 3.448	0.998	0.94	2.85	14.22 ± 0.53	3.72
Para-coumaric acid	28.3	309	y = 0.798x − 1.144	0.998	0.12	0.35	37.05 ± 1.90	2.27
Ferulic acid	29.3	322	y = 0.982x − 0.558	0.997	0.31	0.94	15.44 ± 0.35	5.14

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