Review on Bee Products as Potential Protective and Therapeutic Agents in Male Reproductive Impairment

Bee products are sources of functional food that have been used in complementary medicine to treat a variety of acute and chronic illnesses in many parts of the world. The products vary from location to location as well as country to country. Therefore, the aim of this review was to identify various bee products with potential preventive and therapeutic values used in the treatment of male reproductive impairment. We undertook a vigorous search for bee products with preventive and therapeutic values for the male reproductive system. These products included honey, royal jelly, bee pollen, bee brood, apilarnil, bee bread, bee wax, and bee venom. We also explained the mechanisms involved in testicular steroidogenesis, reactive oxygen species, oxidative stress, inflammation, and apoptosis, which may cumulatively lead to male reproductive impairment. The effects of bee pollen, bee venom, honey, propolis, royal jelly, and bee bread on male reproductive parameters were examined. Conclusively, these bee products showed positive effects on the steroidogenic, spermatogenic, oxidative stress, inflammatory, and apoptotic parameters, thereby making them a promising possible preventive and therapeutic treatment of male sub/infertility.     

Proteome analysis of <Emphasis Type="Italic">Apis mellifera</Emphasis> royal jelly

Royal jelly plays a pivotal role in the development of honey bee larvae. However, while various health promoting properties of royal jelly have been reported, most of the active substances within royal jelly that lead to these properties are still unknown. Since up to 50% (dry mass) of royal jelly is protein, royal jelly proteome analysis is a promising starting point for attempts to identify the proteins that provide health-promoting effects. However, the comprehensive analysis of royal jelly proteins is hampered by the enormous abundance of some proteins in the major royal jelly protein family, which constitutes 80–90% of the royal jelly proteome. The high heterogeneity of these proteins is an additional challenge for proteomic analysis, since it necessitates the use of analytical techniques that provide high resolution and a wide dynamic range. The application of individual methods such as 2D-PAGE or multidimensional chromatography can only yield certain subpopulations of a proteome due to the specific bias of each method. We applied different methods for the prefractionation and separation of royal jelly proteins in order to circumvent the shortcomings of the individual techniques and achieve a high coverage of the royal jelly proteome. In this way, we were able to identify 20 different proteins in total, as well as to show a very high degree of cleavage of different proteins of the major royal jelly protein family. Furthermore, we investigated the protein phosphorylation of royal jelly proteins, and identified and located two phosphorylation sites within venom protein 2. Electronic supplementary material The online version of this article doi:) contains supplementary material, which is available to authorized users.     

Bioactives from Bee Products and Accompanying Extracellular Vesicles as Novel Bioactive Components for Wound Healing

In recent years, interest has surged among researchers to determine compounds from bee products such as honey, royal jelly, propolis and bee pollen, which are beneficial to human health. Mass spectrometry techniques have shown that bee products contain a number of proven health-promoting compounds but also revealed rather high diversity in the chemical composition of bee products depending on several factors, such as for example botanical sources and geographical origin. In the present paper, we present recent scientific advances in the field of major bioactive compounds from bee products and corresponding regenerative properties. We also discuss extracellular vesicles from bee products as a potential novel bioactive nutraceutical component. Extracellular vesicles are cell-derived membranous structures that show promising potential in various therapeutic areas. It has been extensively reported that the use of vesicles, which are naturally formed in plant and animal cells, as delivery agents have many advantages. Whether the use of extracellular vesicles from bee products represents a new solution for wound healing remains still to be elucidated. However, promising results in specific applications of the bee products in wound healing and tissue regenerative properties of extracellular vesicles provide a good rationale to further explore this idea.     

Formulation of Ocular In Situ Gels with Lithuanian Royal Jelly and Their Biopharmaceutical Evaluation In Vitro

Royal jelly is a natural substance produced by worker bees that possesses a variety of biological activities, including antioxidant, anti-inflammatory, antibacterial, and protective. Although fresh royal jelly is kept at low temperatures, to increase its stability, it needs to be incorporated into pharmaceutical formulations, such as in situ gels. The aim of this study was to formulate in situ ocular gels containing Lithuanian royal jelly for topical corneal use in order to increase the retention time of the formulation on the ocular surface and bioavailability. Gels were evaluated for physicochemical characteristics (pH, rheological properties, refractive index) and in vitro drug release measuring the amount of 10-hydroxy-2-decenoic acid (10-HDA). An ocular irritation test and cell viability tests were performed using the SIRC (Statens Seruminstitut Rabbit Cornea) cell culture line. Results indicated that all the in situ gels were within an acceptable pH and refractive index range close to corneal properties. Rheology studies have shown that the gelation temperature varies between 25 and 32 °C, depending on the amount of poloxamers. The release studies have shown that the release of 10-HDA from in situ gels is more sustained than royal jelly suspension. All gel formulations were non-irritant according to the short-time exposure test (STE) using the SIRC cell culture line, and long-term cell viability studies indicated that the formulations used in small concentrations did not induce cell death. Prepared in situ gels containing royal jelly have potential for ocular drug delivery, and they may improve the bioavailability, stability of royal jelly, and formation of non-irritant ocular formulations.     

Multielemental Analysis of Bee Pollen,Propolis, and Royal Jelly Collected in West-Central Poland

Beehive products possess nutritional value and health-promoting properties and are recommended as so-called “superfoods”. However, because of their natural origin, they may contain relevant elemental contaminants. Therefore, to assess the quality of bee products, we examined concentrations of a broad range of 24 selected elements in propolis, bee pollen, and royal jelly. The quantitative analyses were performed with inductively coupled plasma-mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES) techniques. The results of our research indicate that bee products contain essential macronutrients (i.e., K, P, and S) and micronutrients (i.e., Zn and Fe) in concentrations depending on the products’ type. However, the presence of toxic heavy metals makes it necessary to test the quality of bee products before using them as dietary supplements. Bearing in mind that bee products are highly heterogenous and, depending on the environmental factors, differ in their elemental content, it is necessary to develop standards regulating the acceptable levels of inorganic pollutants. Furthermore, since bees and their products are considered to be an effective biomonitoring tool, our results may reflect the environment’s condition in west-central Poland, affecting the health and well-being of both humans and bees.     

Bee Venom: From Venom to Drug

Insects of the order Hymenoptera have a defensive substance that contains many biologically active compounds. Specifically, venom from honeybees (Apis mellifera) contains many enzymes and peptides that are effective against various diseases. Different research papers stated the possibility of using bee venom (a direct bee sting or in an injectable form) in treating several complications; either in vivo or in vitro. Other reports used the active fractions of bee venom clinically or at labratory scale. Many reports and publications have stated that bee venom and its constituents have multiple biological activities including anti-microbial, anti-protozoan, anti-cancer, anti-inflammatory, and anti-arthritic properties. The present review aims to refer to the use of bee venom itself or its fractions in treating several diseases and counteracting drug toxicities as an alternative protocol of therapy. The updated molecular mechanisms of actions of bee venom and its components are discussed in light of the previous updated publications. The review also summarizes the potential of venom loaded on nanoparticles as a drug delivery vehicle and its molecular mechanisms. Finally, the products of bee venom available in markets are also demonstrated.     

Simultaneous determination of seven fluoroquinolones in royal jelly by ultrasonic‐assisted extraction and liquid chromatography with fluorescence detection

A method for the quantitative determination of seven fluoroquinolone antibacterial agents (FQs) used in beekeeping, viz. ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, danofloxacin, enrofloxacin, and difloxacin, in royal jelly samples was developed on the basis of high performance liquid chromatography with fluorescence detection. Sample preparation included deproteination, ultrasonic‐assisted extraction with a mixed inorganic solution of monopotassium phosphate (KH₂PO₄) and ethylenediaminetetraacetic acid disodium salt (Na₂EDTA), and clean‐up on a solid‐phase extraction cartridge. The extraction procedure was optimized with regard to the amount of inorganic solvent and the duration of sonication for royal jelly as a complicated matrix. Overall recoveries for FQs ranged from 85.9 to 99.1% for royal jelly with standard deviations between 2.79 and 6.27%. Limits of quantification were 2–40 ng/g for seven FQs in royal jelly. A total of 57 real royal jelly samples collected from beekeepers and supermarkets were analyzed. The three most abundant honeybee‐use FQs, i. e. ofloxacin, ciprofloxacin, and norfloxacin, were determined in some royal jelly samples in concentrations ranging from 11.9 to 55.6 ng/g. Unexpectedly, however, difloxacin was found at concentrations of about 46.8 ng/g in one sample although it is rarely used in beekeeping. The presented method was successfully applied to quantify FQs in real royal jelly samples.     

Determination of tryptophan in bee pollen and royal jelly by high‐performance liquid chromatography with fluorescence detection

A method for tryptophan analysis in bee pollen and royal jelly was developed using HPLC with fluorescence detection. To determine the free tryptophan in bee pollen and royal jelly, ultrasonic extraction was performed using water (pH 6.3)–acetonitrile (10:1, v/v) as extraction solvent. While determining the total tryptophan in these bee products, the method involves alkaline hydrolysis of the proteins with 4 mol/L sodium hydroxide at 110°C for 20 h under anaerobic conditions. The operating conditions for the HPLC analysis were: Symmetry C₁₈ column (4.6 × 250 mm, 5 µm), 0.1% trifluoroacetic acid–methanol (75:25, v/v) as the mobile phase at a flow rate of 1.0 mL/min at 30°C. The fluorescence detector was operated at an excitation wavelength of 280 nm and an emission wavelength of 340 nm. A linear response (r² > 0.9998) was obtained in the range 0.0625–5.0 µg/mL. The method was successfully applied to the determination of the free and total tryptophan contents in bee pollens, which were 0.069 ± 0.003 and 2.693 ± 0.476 mg/g, respectively, while only the total tryptophan was detected in royal jelly, with a content of 1.743 ± 0.066 mg/g. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of trans-10-hydroxy-2-decenoic acid content in pure royal jelly and royal jelly products by column liquid chromatography.

In this research, several royal jellies and commercial products containing royal jelly were analysed for their trans-10-hydroxy-2-decenoic acid (10-HDA) content by using a column liquid chromatography technique. Ten samples claimed to be pure royal jelly, containing 10-HDA between 0.75 and 2.54%. Seven samples claimed to contain royal jelly as an ingredient which ranged from non-detectable to 0.054%. The technique was found to be rapid with high recovery.     

Antioxidant activity of honey supplemented with bee products

The aim of this work was to evaluate the influence of supplementation of multiflower honey with bee products on the phenolic compound content and on antioxidant activity. Average total phenolic and flavonoids contents in the multiflower honeys were 36.06 ± 10.18 mg GAE/100 g and 4.48 ± 1.69 mg QE/100 g, respectively. The addition of royal jelly did not affect significantly the phenolic compound content and antioxidant activity. Supplementation of honey with other bee products, i.e. beebread, propolis, pollen, resulted in significant increase in the total phenolic and flavonoids contents, and in antiradical activity and reducing power, with the largest effect found for addition of beebread. Significant linear correlations between the total phenolic and flavonoids contents and antiradical activity and reducing power were found.     

New multiresidue method using solid-phase extraction and gas chromatography-micro-electron-capture detection for pesticide residues analysis in royal jelly

Royal jelly, one of the most important bee products, can be contaminated with pesticide and/or antibiotic residues resulting from treatments applied either inside beehives or in the agricultural environment. A new multiresidue method was developed and validated for analysis of nine pesticides in royal jelly. Solid-phase extraction RP-C(18) cartridges were used for sample purification and isolation of analytes. Final solution was analyzed with GC and micro-electron-capture detection. Four synthetic acaricides used by beekeepers (bromopropylate, coumaphos, malathion and tau-fluvalinate), and moreover one pyrethroid, two organochlorine, and two organophosphate insecticides were tested. Linearity is demonstrated for the range of 0.0025-1mgkg(-1), with correlation coefficients ranging from 0.99991 to 0.99846, depending on the analyte. Overall recovery rates from royal jelly blank samples spiked at five fortification levels ranged from 80.8% (lindane) to 91.3% (ethion), well above the range defined by the SANCO/10232/2006 and EC/675/2002 documents. The limit of quantification was <0.003-0.005 mg kg(-1) depending on the analyte, and the reporting level of the method, defined as the lowest recovery level, was 0.005 mg kg(-1).     

Antibacterial polyethylene - Ethylene vinyl acetate polymeric blend by incorporation of zinc oxide nanoparticles

In this work, a Low-Density Polyethylene (LDPE) - Ethylene Vinyl Acetate (EVA) polymeric blend with antimicrobial activity was obtained. The main objective was to develop an antibacterial LDPE-EVA polymeric blend from the incorporation of antibacterial nanoparticles to increase the antimicrobial and sanitary safety of this polymeric blend when applied in the manufacture of medical products. The antibacterial activity was obtained from the incorporation of zinc oxide nanoparticles (ZnO-NPs) in the LDPE-EVA polymeric blends and the thermal properties were evaluated by differential scanning calorimetry and the mechanical properties by tensile stress tests for different percentages of ZnO-NPs. Scanning electron microscopy was used to study the morphological characteristics of the ZnO-NPs and also the characteristics of the distribution of nanoparticles in the polymer blends. The dispersive energy of x-ray fluorescence spectroscopy was used to study the chemical composition of the nanoparticles. Microbiological tests were performed to evaluate the antibacterial activity of the LDPE-EVA polymeric blends without and with ZnO-NPs against the bacteria Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative). The results obtained were excellent for the future application of the antibacterial LDPE-EVA polymeric blends to the manufacture of medical products. The Young's modulus values decreased and the tensile strength values showed small reductions and the thermal properties of the LDPE-EVA were not modified. However, the antibacterial activity of LDPE-EVA with 4 wt% of ZnO-NPs was excellent, eliminating the gram-positive bacteria in just 2 h and the gram-negative bacteria in just 2.5 h on their surfaces.     

Effects of Insecticides and Microbiological Contaminants on Apis mellifera Health

Over the past two decades, there has been an alarming decline in the number of honey bee colonies. This phenomenon is called Colony Collapse Disorder (CCD). Bee products play a significant role in human life and have a huge impact on agriculture, therefore bees are an economically important species. Honey has found its healing application in various sectors of human life, as well as other bee products such as royal jelly, propolis, and bee pollen. There are many putative factors of CCD, such as air pollution, GMO, viruses, or predators (such as wasps and hornets). It is, however, believed that pesticides and microorganisms play a huge role in the mass extinction of bee colonies. Insecticides are chemicals that are dangerous to both humans and the environment. They can cause enormous damage to bees’ nervous system and permanently weaken their immune system, making them vulnerable to other factors. Some of the insecticides that negatively affect bees are, for example, neonicotinoids, coumaphos, and chlorpyrifos. Microorganisms can cause various diseases in bees, weakening the health of the colony and often resulting in its extinction. Infection with microorganisms may result in the need to dispose of the entire hive to prevent the spread of pathogens to other hives. Many aspects of the impact of pesticides and microorganisms on bees are still unclear. The need to deepen knowledge in this matter is crucial, bearing in mind how important these animals are for human life.     

Optimized determination method for trans-10-hydroxy-2-decenoic acid content in royal jelly by high-performance liquid chromatography with an internal standard

An optimized reversed-phase high-performance liquid chromatography method was developed to detect the trans-10-hydroxy-2-decenoic acid (10-HDA) content in royal jelly cream and lyophilized powder. The sample was extracted using absolute ethanol. Chromatographic separation of 10-HDA and methyl 4-hydroxybenzoate as the internal standard was performed on a Nova-pak C18 column. The average recoveries were 95.0-99.2% (n = 5) with relative standard deviation (RSD) values of 1.3-2.1% for royal jelly cream and 98.0-100.0% (n = 5) with RSD values of 1.6-3.0% for lyophilized powder, respectively. The limits of detection and quantitation were 0.5 and 1.5 mg/kg, respectively, for both royal jelly cream and lyophilized powder. The method was validated for the determination of practical royal jelly products. The concentration of 10-HDA ranged from 1.26 to 2.21% for pure royal jelly cream samples and 3.01 to 6.19% for royal jelly lyophilized powder samples. For 30 royal jelly products, the 10-HDA content varied from not detectable to 0.98%.     

固相萃取-超高效液相色谱-串联质谱法测定蜂产品中10种头孢类药物

建立了蜂产品中10种头孢类药物（头孢喹肟、头孢噻肟、头孢洛宁、头孢哌酮、头孢匹林、头孢氨苄、头孢乙腈、头孢拉定、去乙酰基头孢匹林、头孢唑林）含量的液相色谱-串联质谱测定方法。蜂产品样品中头孢类药物用乙腈-水（80：20,v/v）溶液提取,离心,上清液经Oasis PRIME HLB固相萃取柱净化,氮吹后复溶,进行液相色谱-串联质谱分析。采用Acquity BEH C18色谱柱,以0.1%（v/v）甲酸水溶液-甲醇体系作为流动相进行梯度洗脱,ESI源正离子模式电离,多反应离子监测模式（MRM）检测,基质校准外标法定量。结果表明,10种头孢类药物在一定浓度范围内峰面积与质量浓度的相关系数（r²）大于0.999,线性关系良好;检出限为0.15~1.5 μg/kg,定量限为0.50~5.0 μg/kg;在阴性蜂产品样品中的加标回收率为75.0%~89.8%,相对标准偏差（RSD）为1.4%~4.6%（n=5）。该方法检测周期短,准确度和精密度高,能满足多种蜂产品样品中头孢类药物的检测需要。     

蜂王浆产品中5种大环内酯类抗生素残留量的高效液相色谱-质谱/质谱检测方法

建立了高效液相色谱-质谱/质谱测定蜂王浆中5种大环内酯类抗生素(螺旋霉素、竹桃霉素、泰乐菌素、罗红霉素、交沙霉素)残留的方法。先用三氯乙酸沉淀蜂王浆中的蛋白质,上层清液再用乙腈提取、C18小柱净化。每种抗生素选择一个母离子和两个子离子进行监测。5种大环内酯类抗生素在0.002～0.05 mg/L 范围内与其峰面积具有良好的线性关系,检测低限均为20 μg/kg,3个加标水平（每种抗生素的添加水平均为20, 100 和 200 μg/kg）下的加标回收率为73.0%～90.2%,相对标准偏差为5.6%～10.5%。     

Sambucus Nigra Extracts–Natural Antioxidants and Antimicrobial Compounds

Due to the health-promoting properties of elderberry fruits, which result from their rich chemical composition, this raw material is widely used in herbal medicine and the food industry. The aim of the study was to demonstrate the antibacterial activity of the elderberry fruit extracts. The research showed that the content of phenolic acids and flavonoids in the extracts determined their antibacterial activity. The research showed that the content of phenolic acids and flavonoids in the extracts determined their antibacterial activity. The following phenolic acids were predominant: chlorogenic acid, sinapic acid, and t-cinnamic acid. Their average content was, respectively, 139.09, 72.84, 51.29 mg/g extract. Rutin and quercetin (their average content was 1105.39 and 306.6 mg/g extract, respectively) were the dominant flavonoids. The research showed that the elderberry polyphenol extracts exhibited activity against selected strains of bacteria within the concentration range of 0.5–0.05%. The following bacteria were the most sensitive to the extracts: Micrococcus luteus, Proteus mirabilis, Pseudomonas fragii, and Escherichia coli. Of the compounds under analysis, apigenin, kaempferol and ferulic, protocatechuic, and p-coumarin acids had the greatest influence on the high antibacterial activity of elderberry extracts. The results of the microbiological and chemical analyses of the composition of the extracts were analyzed statistically to indicate the bioactive compounds of the greatest antimicrobial significance.     

Natural‐Product‐Inspired Aminoepoxybenzoquinones Kill Members of the Gram‐Negative Pathogen Salmonella by Attenuating Cellular Stress Response

Gram‐negative bacteria represent a challenging task for antibacterial drug discovery owing to their impermeable cell membrane and restricted uptake of small molecules. We herein describe the synthesis of natural‐product‐derived epoxycyclohexenones and explore their antibiotic activity against several pathogenic bacteria. A compound with activity against Salmonella Typhimurium was identified, and the target enzymes were unraveled by quantitative chemical proteomics. Importantly, two protein hits were linked to bacterial stress response, and corresponding assays revealed an elevated susceptibility to reactive oxygen species upon compound treatment. The consolidated inhibition of these targets provides a rationale for antibacterial activity and highlights epoxycyclohexenones as natural product scaffolds with suitable properties for killing Gram‐negative Salmonella.     

Screening of Plant Pollen Sources,Polyphenolic Compounds,Fatty Acids and Antioxidant/Antimicrobial Activity from Bee Pollen

In this study, the botanical origin, total flavonoid and phenolic content, antioxidant activity, phenolic profile and fatty acid composition of mixed bee pollen loads collected in Bayburt, Turkey, were determined. In addition to these assays, antibacterial activity of bee-collected pollen extract (BCPE) against a variety of food-borne pathogenic bacteria was determined in vitro. Pollen loads were classified into five botanical families based on their color: Asteraceae, Fabaceae, Campanulaceae, Cistaceae and Rosaceae. Total flavonoid, total phenolic, CUPRAC and CERAC concentrations were 173.52 mg GAE/g, 79.21 mg QE/g, 85.59 mg Trolox/g and 118.13 mg Trolox/g, respectively. Twenty-three phenolic compounds were scanned in bee pollen extract by LC-MS/MS, with rutin being the most abundant. Cis-4,7,10,13,16,19 docosahexaenoic acid was the predominant fatty acid, followed by cis-11-eicosenoic acid, palmitic acid, and alfa linolenic acid. In addition, the agar well diffusion (AWD) and micro-broth dilution methods were used to determine of the antibacterial activity of the BCPE sample. MIC values were observed to vary between 2.5–5 mg/mL for Gram-positive bacteria and 5–10 mg/mL for Gram-negative bacteria. These findings indicate that bee pollen could be a potential source of antioxidants and antimicrobials.     

Green Synthesis and Characterization of Silver Nanoparticles Using Spondias mombin Extract and Their Antimicrobial Activity against Biofilm-Producing Bacteria

Multidrug resistant bacteria create a challenging situation for society to treat infections. Multidrug resistance (MDR) is the reason for biofilm bacteria to cause chronic infection. Plant-based nanoparticles could be an alternative solution as potential drug candidates against these MDR bacteria, as many plants are well known for their antimicrobial activity against pathogenic microorganisms. Spondias mombin is a traditional plant which has already been used for medicinal purposes as every part of this plant has been proven to have its own medicinal values. In this research, the S. mombin extract was used to synthesise AgNPs. The synthesized AgNPs were characterized and further tested for their antibacterial, reactive oxygen species and cytotoxicity properties. The characterization results showed the synthesized AgNPs to be between 8 to 50 nm with -11.52 of zeta potential value. The existence of the silver element in the AgNPs was confirmed with the peaks obtained in the EDX spectrometry. Significant antibacterial activity was observed against selected biofilm-forming pathogenic bacteria. The cytotoxicity study with A. salina revealed the LC50 of synthesized AgNPs was at 0.81 mg/mL. Based on the ROS quantification, it was suggested that the ROS production, due to the interaction of AgNP with different bacterial cells, causes structural changes of the cell. This proves that the synthesized AgNPs could be an effective drug against multidrug resistant bacteria.