Phenolic Compounds In PROPOLIS Research Articles

Effect of wax separation on macro- and micro-elements, phenolic compounds, pesticide residues, and toxic elements in propolis.

Propolis, a natural product with many biological activities, is a resinous material produced by honeybees. It contains not only valuable components but also some possible contaminants in varying amounts. Hence, this study aimed to examine how the process step of wax separation affects certain elements, pesticide residues, and phenolic compounds in propolis. Total phenolics, elements, and some pesticide residues were analyzed in the crude propolis (CP samples), wax portion (W samples), and remaining propolis fraction (PF samples) after wax separation. Total phenolics of the CP samples were determined in the range of 31.90-45.00 mg GAE g-1 sample, while those of the PF samples were in the range of 54.97-162.09 mg GAE g-1 sample. Loss/reduction values by means of wax separation for phenolics were calculated as 10.88% and 17.89%, respectively. Pb contents of all PF samples were low (0.232-1.520 mg kg-1), but it was also noteworthy that nearly 40% or even more of Cr, As, Cd, and Pb were removed by wax separation. Removal of significant amounts of carbendazim (38.09%-67.35%), metalaxyl (81.57%-72.67%), tebuconazole (65.99%-78.36%), and propargite (88.46%-83.05%) was also achieved. Wax separation enables the removal of toxic substances from crude propolis without causing huge losses in phenolic compounds.

Simultaneous determination of 20 phenolic compounds in propolis by HPLC-UV and HPLC-MS/MS

Propolis is rich in flavonoids and phenolic acids, which has strong antioxidant capacity. Two methodologies for simultaneous determination of 20 phenolic compounds in propolis by HPLC-UV and HPLC-MS/MS were developed. The sample extraction and chromatographic separation conditions, and ultraviolet detection wavelength, as well as mass spectrometry parameters were selected and optimized. Under the optimized conditions, good linearities (R2≥0.998) were obtained for all analytes. The limits of quantification (LOQs) of the proposed methods by HPLC-UV and HPLC-MS/MS for all analytes were in the range of 0.02–0.1 mg/g and 0.05–0.2 mg/g, respectively, and the recoveries were in the range of 95.4 %− 109.5 % and 94.0 %− 106.8 % with the relative standard deviations (RSDs) in the range of 1.1 %− 7.5 % and 0.4 %− 11.1 %, respectively. The proposed methods were applied for the determination of 20 phenolic compounds in 13 propolis samples collected from China and Brazil. The detection results of the two methods are almost consistent with each other, indicating that both methods are applicable, and the inter-confirmation of the two methods can improve the accuracy of the determination of phenolic components in propolis.

Anti-fungal and antioxidant properties of propolis (bee glue) extracts

This study aimed to identify the phenolic compounds contained in propolis and to evaluate the effect of propolis and its extracts on the antifungal activity, pH, color, and sensory analysis of creamy cheese with thyme (thyme labneh). Ethanolic and water extracts of propolis were evaluated to determine its phenolic compound content and antioxidant activity. Phenolic compounds in propolis were identified and quantified using gas chromatography–mass spectrometry (GC–MS). Antifungal activities, color, pH, and sensory evaluation of propolis and its extracts (water and ethanolic) were investigated at concentrations of 0.5%, 1%, and 1.5%. The results showed 11 phenolic compounds in the propolis extract. Our findings revealed a significant difference in phenolic content and antioxidant activity in the ethanolic extract of propolis when compared with the water extract (P < 0.05). Microbial counts in thyme labneh treated with propolis powder and its extracts (ethanolic and water) showed significant differences compared with the control at all concentrations (0.5%, 1%, and 1.5%). Propolis powder and ethanolic extracts at concentrations of 1% and 1.5% were limited the rapid growth of mold and yeast, so the results showed no significant difference between 14 and 21 days for these samples. In addition, the 1.5% water extract did not show a significant difference (P > 0.05) between Days 14 and 21. The sensory panel did not detect a significant difference in any sensory attribute in the thyme labneh treated with propolis extracts. This study identified the significant antioxidant and antimicrobial effectiveness of using propolis in dairy products, suggesting its potential as a natural preservative.

Potential hepatoprotective effects of flavonoids contained in propolis from South Sulawesi against chemotherapy agents.

The use of doxorubicin and epirubicin as chemotherapy agent causes side effects such as liver damage due to oxidative stress by reactive oxygen species (ROS) that cause increased of ALT and AST level as liver parameter. One source of natural antioxidants as ROS neutralizer comes from flavonoid that contain in propolis. Most researchers claim that flavonoid can be used to protect the liver. The aim of this study was to test the hepatoprotective effect of flavonoid in propolis from South Sulawesi against doxorubicin and epirubicin. The experiment included male Sprague dawley rats divided into nine groups. The rats received the microcapsule propolis or the quercetin orally for 15 days. The hepatotoxicity was promoted by injection epirubicin and doxorubicin (i.v.) with a cumulative dose of 9 mg/kg. In this study, total polyphenol and flavonoid tests of propolis have been carried out, there were 1.1% polyphenols and 2.7% flavonoids, the antioxidant activity tests showed IC50 value of 9849 ppm and LCMS/MS tests supported the presence of phenolic compounds in propolis from South Sulawesi. Liver parameter was measured and the results showed that the propolis 200 mg/kg group produced the lowest ALT and had potential protective effect against doxorubicin and epirubicin-induced hepatotoxicity.

High-throughput subzero-temperature assisted homogenous liquid-liquid extraction for the fast sample preparation of multiple phenolic compounds in propolis

In the present study, a high-throughput homogenous liquid-liquid extraction method was developed for fast sample preparation of multiple phenolic compounds in propolis. This method was proposed based on cooling samples array in subzero temperature to induce phase separation of ACN-H2O extractant. Due to the high-throughput ability, optimization of extraction parameters was rapidly achieved by using a 5 × 4 × 3 samples array. In addition, multiple arrays were investigated for evaluating the analytical performance of the high-throughput method, which indicated that limits of detection and quantification were ranged from 0.04 to 0.35 µg/mL and 0.12 to 1.05 µg/mL, respectively. Recoveries and precisions in inter-day high-throughput studies were in the range of 90.55–105.50% and 2.58–4.30%, respectively. Comparing with the conventional liquid extraction method, this ecofriendly high-throughput method presented remarkable advantages in reducing sample and chemical consumption, as well as saving labor and time cost. The proposed method might provide a valuable strategy for the design of high-throughput extraction procedures.

Multivariate calibration: Identification of phenolic compounds in PROPOLIS using FT‐NIR

AbstractThe chemical composition of propolis is complex and varies according to location, seasonality, and vegetation where the hives are located. Raw and macerated propolis produced in different regions of Brazil were assessed and produced iPLS models from FTNIR combined to multivariate calibration to determine total phenolic content (TPC). The spectra of raw propolis were obtained analyzing samples directly, without treatment or processing, while macerated propolis was submitted to the milling process using liquid nitrogen. Concerning the results, the TPC ranged from 3.61 to 90.4 mg GAE g‐1, which indicates a significant difference among samples. It was possible to conclude that macerated propolis presented the best multivariate calibration obtained from the second derivative and SNV pre‐processing. The error values RMSECV and RMSEP were 3.79 and 6.17 mg GAE g‐1 respectively, while the determination coefficients in the calibration, cross‐validation, and external validation were 0.97, 0.87, and 0.82, respectively.

Effect of propolis phenolic compounds on free fatty acid receptor 4 activation.

Propolis is known to have multiple biological and pharmacological properties including the regulation of energy homeostasis. Although phenolic compounds are considered to be the major active components in propolis, there is little information available about their mechanisms underlying the regulation of energy homeostasis. In this study, the effects of five phenolic compounds in propolis, chrysin, pinocembrin, galangin, pinobanksin, and caffeic acid phenethyl ester (CAPE) were evaluated on the activation of free fatty acid receptor 4 (FFA4), which are involved in the control of energy homeostasis by enhancing insulin signaling, increasing glucose uptake, and regulating adipogenesis. The results showed that three phenolic compounds exhibited the activation of FFA4, which were ranked in the order of pinocembrin, CAPE and pinobanksin in FFA4-expressing cells. These results suggest that some phenolic compounds in propolis, particularly pinocembrin, may affect the control of energy homeostasis via the activation of FFA4.

Determination of biologically active phenolic compounds in propolis by LC–MS/MS according to seasons and altitudes

The present study aimed to determine the effect of season and altitude to amount and prevalence of phenolic compounds in propolis. For the aim, 174 propolis samples were collected from 58 stationary apiaries from altitudes of 0–200 m, 200–500 m and above 500 m in spring, summer, and autumn distributed in the Marmara region of Turkey. Ethanol-extracted propolis samples were analyzed by LC–MS/MS to determine nine flavonoids and seven phenolic acids. Significant differences were found in average levels of galangin, naringenin, pinocembrin, quercetin, luteolin, caffeic acid, trans-cinnamic acid, p-coumaric acid, m-coumaric acid, and CAPE across the seasons and altitudes (P < 0.05). However, no differences were found levels of rutin, apigenin, kaempferol, ferulic acid and gallic acid (P < 0.05). Most of the significant differences were found between spring and autumn (10 differences). The prevalence of flavonoids was generally lower than phenolic acids. CAPE and pinocembrin had majority of the significant differences in phenolic compounds according to season and altitude. The prevalence of pinocembrin 100%, m-coumaric acid 99.4%, CAPE 99.4%, naringenin 99.4%, caffeic acid 98.9%, p-coumaric acid 98.3%, quercetin 96% and luteolin 95.4% were determined that dominant phenolic compound of Marmara Region of Turkey’s propolis. Furthermore, Salix spp. 75.8%, Quercus spp. 74.4%, Populus spp. 72.4%, and Pinus spp. 53.5% were mainly determined source of propolis around the apiaries. These results show that different sampling of seasons and altitudes effect chemical composition of propolis even from the same area. The study indicated that seasons and altitudes are important factors for standardization of propolis.

Comparison of the Partition Efficiencies of Multiple Phenolic Compounds Contained in Propolis in Different Modes of Acetonitrile⁻Water-Based Homogenous Liquid⁻Liquid Extraction.

Homogeneous liquid–liquid extraction (HLLE) has attracted considerable interest in the sample preparation of multi-analyte analysis. In this study, HLLEs of multiple phenolic compounds in propolis, a polyphenol-enriched resinous substance collected by honeybees, were performed for improving the understanding of the differences in partition efficiencies in four acetonitrile–water-based HLLE methods, including salting-out assisted liquid–liquid extraction (SALLE), sugaring-out assisted liquid–liquid extraction (SULLE), hydrophobic-solvent assisted liquid–liquid extraction (HSLLE), and subzero-temperature assisted liquid–liquid extraction (STLLE). Phenolic compounds were separated in reversed-phase HPLC, and the partition efficiencies in different experimental conditions were evaluated. Results showed that less-polar phenolic compounds (kaempferol and caffeic acid phenethyl ester) were highly efficiently partitioned into the upper acetonitrile (ACN) phase in all four HLLE methods. For more-polar phenolic compounds (caffeic acid, p-coumaric acid, isoferulic acid, dimethoxycinnamic acid, and cinnamic acid), increasing the concentration of ACN in the ACN–H2O mixture could dramatically improve the partition efficiency. Moreover, results indicated that NaCl-based SALLE, HSLLE, and STLLE with ACN concentrations of 50:50 (ACN:H2O, v/v) could be used for the selective extraction of low-polarity phenolic compounds. MgSO4-based SALLE in the 50:50 ACN–H2O mixture (ACN:H2O, v/v) and the NaCl-based SALLE, SULLE, and STLLE with ACN concentrations of 70:30 (ACN:H2O, v/v) could be used as general extraction methods for multiple phenolic compounds.

Ultrasound-assisted ionic liquid-based micellar extraction combined with microcrystalline cellulose as sorbent in dispersive microextraction for the determination of phenolic compounds in propolis

An ionic liquid-(IL) based micellar extraction combined with microcrystalline cellulose- (MCC) assisted dispersive micro solid-phase extraction method was developed to extract phenolic compounds from propolis. A total of 20 target compounds were identified by ultra-high- performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. The main extraction parameters were optimized and included the ultrasonic power, ultrasonic time, sample pH, type of IL, the concentration of [C12mim]Br, extraction time, concentration of MCC, type of sorbent and type of elution solvents. Under the optimum conditions, the proposed method exhibited good linearities (r2 ≥ 0.999) for all plant phenolic compounds with the lower limits of detection in the range of 0.21–0.41 ng/mL. The recoveries ranged from 82.74% to 97.88% for pinocembrin, chrysin and galangin. Compared with conventional solvent extraction, the present method was simpler and more efficient and required less organic solvent and a shorter extraction time. Finally, the methodology was successfully used for the extraction and enrichment of phenolic compounds in propolis.

Effects of phenolic compounds in propolis on digestive and ruminal parameters in dairy cows

Four rumen-cannulated primiparous lactating cows were studied in a 4 × 4 Latin square design experiment to evaluate the effects of propolis-based products (PBP) with different concentrations of propolis and alcohol levels on total digestibility, (TD), ruminal digestibility (RD), intestinal digestibility (ID), pH, ruminal ammonia-nitrogen production (NH3-N), rumen microbial synthesis, and blood parameters. The feed consisted of 591.9 g/kg corn silage and 408.1 g/kg concentrate (dry matter [DM] basis), and treatments differed with regard to the inclusion (via ruminal cannula) or exclusion of PBP as follows: control (without the PBP), PBP B1 (3.81 mg of phenolic compounds/kg of ingested DM), PBP C1 (3.27 mg of phenolic compounds/kg of ingested DM), and PBP C3 (1.93 mg of phenolic compounds/kg of ingested DM). Inclusion of PBP reduced the RD of dietary crude protein (CP). Treatment PBP C1 reduced ruminal NH3-N production, while PBP B1 increased the ID of CP relative to that in the control. These findings indicate that propolis had a positive effect on rumen nitrogen metabolism. Rumen pH, efficiency of microbial protein synthesis, and blood parameters were not affected by addition of PBP, but there were significant effects on the other parameters when the treatments containing propolis were contrasted. Higher TD of DM (0.717 vs. 0.685), OM (0.737 vs. 0.703), and CP (0.760 vs. 0.739), as well as higher NDF (0.622 vs. 0.558) and TDN (0.747 vs. 0.712) were observed when comparing PBP C1 with C3. Inclusion of propolis in diets for dairy cows have positive effects on protein metabolism in the rumen. Variation in the amounts of phenolic compounds in the different PBP may explain the diverse effects on the digestive parameters evaluated.

Pollen types and levels of total phenolic compounds in propolis produced by Apis mellifera L. (Apidae) in an area of the Semiarid Region of Bahia, Brazil

Twenty-two propolis samples produced by Apis mellifera L. in an area of the Semiarid region the the State of Bahia (Agreste of Alagoinhas), Brazil, were palynologically analyzed and quantified regarding their levels of total phenolic compounds. These samples were processed using the acetolysis technique with the changes suggested for use with propolis. We found 59 pollen types belonging to 19 families and 36 genera. The family Fabaceae was the most representative in this study with nine pollen types, followed by the family Asteraceae with seven types. The types Mikania and Mimosa pudica occurred in all samples analyzed. The types Mimosa pudica and Eucalyptus had frequency of occurrence above 50% in at least one sample. The highest similarity index (c. 72%) occurred between the samples ER1 and ER2, belonging to the municipality of Entre Rios. Samples from the municipality of Inhambupe displayed the highest (36.78±1.52 mg/g EqAG) and lowest (7.68 ± 2.58 mg/g EqAG) levels of total phenolic compounds. Through the Spearman Correlation Coefficient we noticed that there was a negative linear correlation between the types Mimosa pudica (rs = -0.0419) and Eucalyptus (rs = -0.7090) with the profile of the levels of total phenolic compounds of the samples.

High Performance Liquid Chromatographic Determination of Phenolic Compounds in Propolis

Purpose: To develop a simple, precise and rapid high-performance liquid chromatographic technique coupled with photodiode array detection (DAD) method for the simultaneous determination of rutin, quercetin, luteolin, genistein, galangin and curcumin in propolis.Methods: Ultrasound-assisted extraction was applied to isolate six antioxidant phenolic compounds from propolis samples. Chromatographic separation was performed using an Eclipse XDB-C18 column (150 mm×4.6 mm, 5μm) by isocratic elution at 25oC The mobile phase consisted of phosphate buffered saline (pH = 4.5) and methanol (40/60, v/v). The flow rate was set at 0.8 ml/min and the effluent was monitored using a DAD set at 260 nm.Results: Excellent linear correlation coefficient was observed in the range of 0.9977 to 0.9997. The reproducibility of the method was 1.58 % (RSD) and accuracy was confirmed with average recovery ranging from 95.71 to 104.26 %. Six antioxidant phenolic compounds in propolis samples were successfully determined within 50 min.Conclusion: The proposed isocratic LC method gave good separation and accurate determination of rutin, quercetin, luteolin, genistein, galangin and curcumin in propolis. The proposed method showed good sensitivity, linearity, repeatability and accuracy.Keywords: Propolis, Rutin, Quercetin, Luteolin, Genistein, Galangin, Curcumin, HPLC.

DART-Orbitrap MS: a novel mass spectrometric approach for the identification of phenolic compounds in propolis

This is the first direct analysis in real-time mass spectrometry (DART-MS) study of propolis and a first study on the analysis of bee products using high-resolution DART-MS (DART-HRMS). Identification of flavonoids and other phenolic compounds in propolis using direct analysis in real-time coupling with Orbitrap mass spectrometry (DART-Orbitrap MS) was performed in the negative ion mode for minimizing the matrix effects, while the positive ion mode was used for the confirmation of selected compounds. Possible elemental formulae were suggested for marker components. The duration of one sample analysis by DART-MS analysis lasted ca. 30 s, and all benefits of high-resolution mass spectrometry were used upon data processing using the coupling of DART with the Orbitrap mass spectrometer. The possibility for scanning analysis of dried propolis extract spots on a planar porous surface was investigated in the heated gas flow of the DART ion source with adjustable angle. As an independent method, the approach of scanning analysis is of high interest and of future potential for confirmation of the results obtained from liquid sample analysis. Scanning analysis is highly promising for further development in the bioanalytical field due to the convenience of the storage and transportation of dried sample spots.

A reliable quantitative method for the analysis of phenolic compounds in Brazilian propolis by reverse phase high performance liquid chromatography

A sensitive and reliable RP-HPLC method was developed using a C18 CLC-ODS (M) - 4.6x250 mm(2)column and gradient elution for the analysis of phenolic compounds in propolis raw material and its products. A procedure for the extraction of phenolic compounds using aqueous ethanol (90%) with the addition of veratraldehyde as the internal standard (IS) was developed allowing to quantify ten compounds: caffeic acid, coumaric acid, ferulic acid, cinnamic acid, aromadendrin-4'-methyl ether (AME), isosakuranetin, drupanin, artepellin C, baccharin, and 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran acid (DCBEN). The developed method gave good detection response and linearity in the range of 20.83-533.33 microg/mL.

Selective analysis of phenolic compounds in propolis by HPLC‐MS/MS

Phenolic compounds (flavonoids and phenolic acid derivatives) are major active constituents of the resinous fraction of propolis, and also represent its allergenic principles. We have developed a chromatography electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) method to characterise the polyphenolic fraction of propolis rapidly and quali-quantitatively. With precursor ion scanning, selective detection of caffeic esters was easily achieved, confirming the identification of prenyl caffeate, benzyl caffeate and phenylethyl caffeate by comparison with synthetic standards. The ionisation and fragmentation behaviour of the major propolis flavonoids was rationalised and applied to selected real samples. Taken together, the results of this study show that the introduction of precursor ion analysis leads to a significant improvement in the characterisation of the phenolic fraction of propolis, paving the way to the establishment of a better quality control for this important natural remedy.