Thin-layer chromatography in the authenticity testing of bee-products.

Quality control, nutritional value and the monitoring of hazardous residues in honey bee- products have become major topics for both producers and consumers. Due to its potential role in human health, bee-products rich in bioactive compounds are becoming increasingly popular. This review aims to provide an overview of thin-layer chromatography methods used in quality control,authenticity testing and chemical profiling of bee-products in order to help scientists engaged in the field of bee-products chemistry to utilize the advantages of this technique in the detection and elimination of fraudulent practices in bee-product manufacturing. Recently, hyphenation of thin-layer chromatography, image analysis and chemometrics support bee-products analysisbysimultaneousdeterminationofanalytes with different detection principles, identification of individual bioactive compounds as well as structure elucidation of compounds. Highlighted opportunities of thin-layer chromatography could encourage further investigations that would lead to improvements in the detection and elimination of marketing fraudulent practices.

The Effect of Oral Consumption of Propolis Alone and in Combination With Silver Nanoparticles on Wound Healing in Male Wistar Rats.

Research to identify and develop compounds that facilitate wound healing is important, especially for hard-to-heal chronic wounds. PURPOSE: This study was conducted to investigate the effects of orally administered propolis (a resinous substance found in beehives), alone and in combination with silver nanoparticles (SNPs), on the wound healing process in male rats. METHODS: Forty (40) male Wistar rats were randomly divided into 4 groups of 10 each: 1 control group received no treatment, and 3 study groups received a daily dose of 1) propolis (100 mg/kg), 2) propolis + 30 ppm SNPs, or 3) propolis + 60 ppm SNPs. Healing rate was determined by wound surface area reduction on days 4, 6, 8, and 10 post-surgery. On day 12 after wound creation, histological changes of wound healing, including number of new vessels, inflammatory cells (neutrophils, eosinophils, and mast cells) and fibroblasts were counted based on morphology using a 400x objective lens, and collagen deposition density was determined using hematoxylin and eosin and trichrome staining, respectively. The histological scores were based on a 0 to 4 scale from lowest to highest amount of improving tissue status and were analyzed using one-way analysis of variance, Tukey test, Kruskal-Wallis test, t test, and Mann-Whitney U test to examine differences among the groups. Significance was set at P <.05. RESULTS: The rate of wound healing was significantly different between the control and the treated groups on days 4, 6, 8, and 10 (percent change was not assessed on day 12) post-surgery, especially in the propolis + 30 ppm SNPs group compared to the control group. This difference was more significant on days 6 (wound healing percentage [WHP]: 75% and 45%) and 8 (WHP: 88% and 65% ) post-surgery (P <.001). Mean neutrophil count on day 12 was highest in the control (34.8 ± 2.97) and lowest in the propolis + 30 ppm SNPs group (16.55 ± 2.12). The number of eosinophils on day 12 was considerably higher in the control group (1.05 ± 4) compared to those in the propolis group (3 ± 0.70), propolis + 30 ppm SNPs group (60/0 ± 1/1), and propolis + 60 ppm SNPs group (0.5 ± 0.52) (P <.001). Mean propolis + 30 ppm SNPs scores for epithelialization and granulation tissue formation were 3 and 4, respectively; in the propolis + 60 ppm SNPs, scores were 2 and 3, respectively; in the propolis alone group scores were 2 and 3, respectively (statistical significance not computed for semiquantitative values). The highest fibroblast count was in the propolis + 30 ppm SNPs group (114.44 ± 3.90) compared to control group (73.2 ± 2.8); P <.001). The difference in collagen fiber density scores was also significant: 1.2 ± 0.42 in the control and 3.66 ± 0.50 in the propolis + 30 ppm SNPs group; (P <.001). The mean of collagen fiber density in the propolis + 60 ppm SNPs group was 2.63 ± 0.51. CONCLUSION: Oral propolis alone and in combination with 30 ppm SNPs appears to provide anti-inflammatory effects and increase fibroblast proliferation and collagen deposition in experimental wounds, which may explain the observed differences in healing. Propolis + 60 ppm SNPs appears to have a cytotoxic effect. Research confirming these results and that examines toxicity levels in animals and humans is needed.

Bioprofiling for the quality control of Egyptian propolis using an integrated NIR-HPTLC-image analysis strategy.

Quality control of propolis being a complex mixture of compounds that are very difficult to analyze and standardize is certainly challenging. Shown on the example of 35 Egyptian propolis samples, a strategy for an improved quality control was demonstrated in which efficacy-directed fingerprint analysis of high-performance thin-layer chromatography (HPTLC) fingerprints were evaluated in combination with Near Infra-Red spectrometry (NIRS) to realize precise discrimination and high quality assessment of chemical and effective consistency of propolis samples that are collected from various geographical locations in Egypt. The fingerprints obtained after derivatization and fluorescence detection (FLD) at λ 366 nm were analyzed by using multivariate data analysis and data were used to identify nine marker ingredients of the different propolis samples. These markers were then quantified by a new validated HPTLC method and an attempt to classify the samples by using a targeted approach was implemented. All investigated propolis samples were initially divided into two types in the untargeted and targeted HPTLC image multivariate analysis. The antimicrobial activities of propolis samples were then evaluated and the results showed that there was significant variation in the biological activities of the different samples. The quantitative data of marker compounds as well as the samples bioactivity results were then subjected to partial least squares regression (PLS-R) analysis which revealed that chrysin, galangin­5­O­methylether, pinostrobin, and islapinin were the main bioactive markers. Based on the efficacy-associated marker ingredients, pretreated spectral patterns of NIRS were selected as a complementary evaluation technique directed to the comprehensive efficacy-directed discrimination of the different Egyptian propolis samples which could successfully classify the propolis samples tested into three different types. Hierarchical cluster analysis and orthogonal projection to latent structures discriminant analysis (OPLS-DA) were applied to the quantitative results from the efficacy-associated markers only. From this systematic method, the 35 samples were subsequently divided into three types; orange, green and blue, displaying a more intricate division than OPLS-DA which is based only on the untargeted HPTLC analysis or the full NIR spectral data. This study initiated the research on the Egyptian propolis and confirmed the existence of three different types of Egyptian propolis, the blue, green and orange types. The study also emphasizes how it is critical to implement the fingerprint- efficacy relationship analysis in finding out the main health-relevant biomarkers for the quality assessment especially for samples displaying delicate differences in their chemical composition and bioactivity.

Analysis of the polyphenolic fraction of propolis from different sources by liquid chromatography-tandem mass spectrometry.

A reverse phase LC-DAD-MS method for quantification of phenolic acids and flavonoids in propolis raw materials was developed. The propolis samples from different geographical areas were extracted with ethanol for 2h at 70 degrees C and the resulting solutions analyzed on a 5 microm C18 symmetry 250 mm x 4.6mm column. The separation was performed by means of a linear gradient elution and DAD and MS data were acquired in the 200-450 nm and 100-1000 Da range, respectively. The identity of most of the compounds was assessed by comparing their chromatographic and UV behaviour with that of authentic standards. When the standards were not available, the identity was achieved by means of chromatographic and on-line UV data combined with mass spectrometry. European, Chinese and Argentinean propolis are characterized by the presence of phenolic acids and flavonoids and the most abundant were chrysin (2-4%), pinocembrin (2-4%), pinobanksin-acetate (1.6-3%) and galangin (1-2%). Some Brazilian propolis contains mainly artepillin C, different caffeoyl quinic acids and some flavonoids. When considering the total flavonoid content as quality index, we suggest that propolis with a content less than 11% should be considered of low quality, whereas propolis with a content of 11-14%, 14-17% or >17% should be classified as propolis of acceptable, good and high quality, respectively. The reported LC-DAD-MS analysis method may be applied for the phytochemical screening of raw propolis and its commercial formulations.

Artepillin C, is it a good marker for quality control of Brazilian green propolis?

As commercialisation of Brazilian green propolis is going on, quality evaluation and authenticity are important. The result demonstrated that artepillin C found by far in Brazilian green propolis by HPLC-ESI-MS/MS analysis, while a small interferent may be mistaken as artepillin C in some propolis from China. A new HPLC quality control method as artepillin C for marker was developed, which is the primary assessment criteria for quality control of Brazilian green propolis.

Chemical diversity of propolis and the problem of standardization.

Chemical variability of propolis is discussed with respect to the problem of standardization. Several chemical types of propolis are formulated, based on their plant source. Reliable criteria for chemical standardization of different propolis types are needed but such generally accepted criteria do not yet exist. The chemical profile of "poplar" propolis, typical for the temperate zone, can be characterized by the following parameters: total flavone and flavonol content, total flavanone and dihydroflavonol content, and total phenolics content. These parameters correlate better with the biological activity and are more informative that the quantification of individual components. There is still a lot of work to be done to achieve standardization of other propolis types. Working with standardized material will allow scientists to connect a particular chemical propolis type to a specific type of biological activity and formulate recommendations for mainstream practitioners.

Chemical Composition of the Same Brazilian Propolis Sample Analyzed in 1997 and in 2012: No Freezing Effect.

The same propolis sample that was analyzed by GC-MS in 1997 and 2012 was examined again after being frozen for 15 years. No qualitative changes were seen in its composition; artepilin C, prenyl-p-coumaric acid, dihydrocinnamic acid, caffeic acid were the major compounds.

Composition, labelling, and safety of food supplements based on bee products in the legislative framework of the European Union - Croatian experiences.

The European Union market is overflown by food supplements and an increasing number of consumers prefer those where bee products play an important part in their composition. This paper deals with complex European Union legislation concerning food supplements based on bee products, placing a special emphasis on their composition, labelling, and safety. Correct labelling of food supplements also represents a great challenge since, in spite of legal regulations in force, there are still open issues regarding the statements on the amount of propolis, which is not clearly defined by the legal framework. One of the key issues are the labels containing health claims from the EU positive list approved by the European Food Safety Authority. Emphasis will also be placed on informing consumers about food, as statements which imply the healing properties of food supplements and their capacity to cure diseases are forbidden. One of the key elements of product safety is HACCP based on the EU Regulations EC 178/02 and 852/2004. Health safety analyses of food supplements with bee products used as raw materials, which are standardised by legal regulations will also be discussed. In the future, attention should also be paid to establishing the European Union "nutrivigilance" system. Croatian experiences in addressing challenges faced by producers, supervisory entities, and regulatory and inspection bodies may serve as an example to countries aspiring to become part of the large European family.

International Regulations of Propolis Quality: Required Assays do not Necessarily Reflect their Polyphenolic-Related In Vitro Activities.

Propolis has been proposed as a polyphenolic-rich natural product potentially able to be used for human consumption or even for medicinal proposes. To guarantee a minimum phenolic and flavonoid content and as consequence of their related-biological activities, international requirements of propolis quality are commonly applied. In this work we assessed phenolic and flavonoid contents of propolis; the antioxidant capacity (toward peroxyl radicals and hypochlorous acid); the ability to generate nitric oxide (NO); and, finally the antimicrobial activity of 6 propolis samples from the VI region of Chile. Our results show that the total phenolic and flavonoid content of propolis samples are not always in agreement with their polyphenolic-associated in vitro activities. For example, P03 and P06 samples showed the lowest (25 ± 4 GAE/g propolis) and the highest (105 ± 3 GAE/g propolis) total phenolic content, respectively. This was in agreement with flavonoid content and their Oxygen Radical Absorbance Capacity (ORAC) activity. However, this dependence was not observed toward HOCl, NO release and antimicrobial activity. Based on our results, we consider that, in order to guarantee the antioxidant or antimicrobial in vitro effects, the international regulations of propolis quality should contemplate the convenience of incorporating other simple analytical test such as ORAC or antimicrobial tests.

Foreign matter contaminating ethanolic extract of propolis: a filth-test survey comparing products from small beekeeping farms and industrial producers.

Propolis is a resinous material collected by honeybees from the exudates and buds of plants. It has been widely used as a remedy by humans since ancient times, as well as for dietary supplements and cosmetics. European legislation recently focused on the quality and hygiene standards of foods, including beehive products, and extensive efforts have been made to avoid the presence of chemical contaminants, whilst in contrast few studies have investigated the magnitude of contamination by physical ones. We conducted a filth-test survey to evaluate the contamination of ethanolic extract of propolis by foreign materials. We also compared the abundance of contaminants in propolis extracts currently marketed by small beekeepers and industrial producers. We found different foreign materials in the ethanol extract of propolis. Contaminants differed in abundance, with a higher number of carbon particles (small beekeepers: 2.70 ± 0.63; industrial producers: 1.25 ± 0.49; mean (n/30 ml) ± SE) and other inorganic fragments (small beekeepers: 3.50 ± 0.31; industrial producers: 3.88 ± 1.11) than arthropod fragments (small beekeepers: 0.30 ± 0.21; industrial producers: 0.38 ± 0.26) and mammal hairs (small beekeepers: 0.10 ± 0.10; industrial producers: 0.38 ± 0.26). No differences in the abundance of foreign matter between propolis from small beekeepers and industrial producers were found, allowing us to point out an increased awareness by small producers of issues inherent in hygiene management. Contamination of propolis extracts by animal body parts, such as insect fragments, mites and rodent hairs, indicates poor management of hygiene in the production process and low effectiveness of the filtration phase. Animal-borne contaminants can act as pathogen vectors as well as introducing dangerous allergens when ingested or applied to human skin. The filth-test applied to ethanolic propolis extract quality control can be considered a promising tool, also for small beekeeper activities, since it is cheap and allows the quick interpretation of results.

Chrysin: a histone deacetylase 8 inhibitor with anticancer activity and a suitable candidate for the standardization of Chinese propolis.

Chinese propolis (CP) is a natural product collected by honeybees and a health food raw material. Previous studies have shown that CP exhibits a broad spectrum of biological activities including anticancer, antioxidant, antibacterial, anti-inflammatory, and antiviral activities. The focuses of the present study were the standardization of CP and the possible mechanisms of its active anticancer ingredients. Nine samples of CP were collected from different locations in China. Analyses of the CP samples revealed that all 9 had similar chemical compositions. Parameters analyzed included the CP extract dry weight, total phenolic content, and DPPH free radical scavenging activities. The active anticancer ingredient was isolated, characterized against human MDA-MB-231 breast cancer cells, and identified as chyrsin, a known potent anticancer compound. Chrysin is present at high levels in all 9 of the CP samples, constituting approximately 2.52% to 6.38% of the CP extracts. However, caffeic acid phenethyl ester (CAPE), another potent active ingredient is present in low levels in 9 samples of CP, constituting approximately 0.08% to 1.71% of the CP extracts. Results from analyses of enzymatic activity indicated that chrysin is a histone deacetylase inhibitor (HDACi) and that it markedly inhibited HDAC8 enzymatic activity (EC(50) = 40.2 µM). In vitro analyses demonstrated that chrysin significantly suppressed cell growth and induced differentiation in MDA-MB-231 cells. In a xenograft animal model (MDA-MB-231 cells), orally administered chrysin (90 mg/kg/day) significantly inhibited tumor growth. Despite the geographical diversity of the 9 samples' botanical origins, their chemical compositions and several analyzed parameters were similar, suggesting that CP is standardized, with chrysin being the major active ingredient. Overall, in vitro and in vivo data indicated that chrysin is an HDAC8 inhibitor, which can significantly inhibit tumor growth. Data also suggested that chrysin might represent a suitable candidate for standardization of CP.

Cuban and Brazilian red propolis: botanical origin and comparative analysis by high-performance liquid chromatography-photodiode array detection/electrospray ionization tandem mass spectrometry.

Chemical composition of propolis depends on the specificity of the local flora at the site of collection and thus on the geographic and climatic characteristics of this place. This paper describes a comparative analysis of Cuban red propolis (CRP), Brazilian red propolis (BRP), and Dalbergia ecastophyllum exudates (DEE) by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry. The aim of this study was to investigate the overall chemical profile and the botanical origin of red propolis and to suggest similarities and differences between samples collected in different tropical regions. Isoliquiritigenin (1), liquiritigenin and naringenin (2 and 17), isoflavones (3-4 and 16), isoflavans (5-7 and 18), and pterocarpans (8-13) were detected in CRP, BRP, and DEE, whereas polyisoprenylated benzophenones (PPBs) guttiferone E/xanthochymol (14a,b) and oblongifolin A (15) were detected only in BRP. Pigments responsible for the red color of DEE and red propolis were also identified as two C30 isoflavans, the new retusapurpurin B (19) and retusapurpurin A (20). PPBs and pigments were isolated and unambiguously characterized by 1D and 2D NMR analysis. These results show that red propolis samples from different tropical zones have a similar chemical composition. DEE is the main red propolis source, but the presence of PPBs in BRP suggests the contribution of different botanical sources for Brazilian samples. This chemical information is important for quality control of red propolis and its commercial products and for biological study.

Fatty acid composition of California grown almonds.

Eight almond (Prunus dulcis L.) cultivars from 12 different California counties, collected during crop years 2004 to 2005 and 2005 to 2006, were extracted with petroleum ether. The extracts were subjected to GC-MS analyses to determine fatty acid composition of soluble lipids. Results indicated palmitic (C16:0), oleic (C18:1), linoleic (C18:2), and alpha-linolenic (C18:3) acid, respectively, accounted for 5.07% to 6.78%, 57.54% to 73.94%, 19.32% to 35.18%, and 0.04% to 0.10%; of the total lipids. Oleic and linoleic acid were inversely correlated (r=-0.99, P= 0.05) and together accounted for 91.16% to 94.29% of the total soluble lipids. Statistically, fatty acid composition was significantly affected by cultivar and county.