Propolis,Bee Honey,and Their Components Protect against Coronavirus Disease 2019 (COVID-19): A Review of In Silico,In Vitro,and Clinical Studies

Despite the virulence and high fatality of coronavirus disease 2019 (COVID-19), no specific antiviral treatment exists until the current moment. Natural agents with immune-promoting potentials such as bee products are being explored as possible treatments. Bee honey and propolis are rich in bioactive compounds that express strong antimicrobial, bactericidal, antiviral, anti-inflammatory, immunomodulatory, and antioxidant activities. This review examined the literature for the anti-COVID-19 effects of bee honey and propolis, with the aim of optimizing the use of these handy products as prophylactic or adjuvant treatments for people infected with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Molecular simulations show that flavonoids in propolis and honey (e.g., rutin, naringin, caffeic acid phenyl ester, luteolin, and artepillin C) may inhibit viral spike fusion in host cells, viral-host interactions that trigger the cytokine storm, and viral replication. Similar to the potent antiviral drug remdesivir, rutin, propolis ethanolic extract, and propolis liposomes inhibited non-structural proteins of SARS-CoV-2 in vitro, and these compounds along with naringin inhibited SARS-CoV-2 infection in Vero E6 cells. Propolis extracts delivered by nanocarriers exhibit better antiviral effects against SARS-CoV-2 than ethanolic extracts. In line, hospitalized COVID-19 patients receiving green Brazilian propolis or a combination of honey and Nigella sativa exhibited earlier viral clearance, symptom recovery, discharge from the hospital as well as less mortality than counterparts receiving standard care alone. Thus, the use of bee products as an adjuvant treatment for COVID-19 may produce beneficial effects. Implications for treatment outcomes and issues to be considered in future studies are discussed.     

Propolis Extract and Its Bioactive Compounds—From Traditional to Modern Extraction Technologies

Propolis is a honeybee product known for its antioxidant, anti-inflammatory, anticancer, and antimicrobial effects. It is rich in bioactive molecules whose content varies depending on the botanical and geographical origin of propolis. These bioactive molecules have been studied individually and as a part of propolis extracts, as they can be used as representative markers for propolis standardization. Here, we compare the pharmacological effects of representative polyphenols and whole propolis extracts. Based on the literature data, polyphenols and extracts act by suppressing similar targets, from pro-inflammatory TNF/NF-κB to the pro-proliferative MAPK/ERK pathway. In addition, they activate similar antioxidant mechanisms of action, like Nrf2-ARE intracellular antioxidant pathway, and they all have antimicrobial activity. These similarities do not imply that we should attribute the action of propolis solely to the most representative compounds. Moreover, its pharmacological effects will depend on the efficacy of these compounds’ extraction. Thus, we also give an overview of different propolis extraction technologies, from traditional to modern ones, which are environmentally friendlier. These technologies belong to an open research area that needs further effective solutions in terms of well-standardized liquid and solid extracts, which would be reliable in their pharmacological effects, environmentally friendly, and sustainable for production.     

Antibacterial effects of Brazilian and Bulgarian propolis and synergistic effects with antibiotics acting on the bacterial DNA and folic acid

Propolis is a honeybee product that has been used since ancient times because of its therapeutic effects. It can be used in the development of alternative therapies for the treatment of many diseases, and because propolis shows antibacterial action, this work was carried out in order to investigate a possible synergism between propolis and antibiotics acting on DNA (ciprofloxacin and norfloxacin) and on the metabolism (cotrimoxazole) against Salmonella typhi. Propolis samples collected in Brazil and Bulgaria were compared in these assays, and the synergism was investigated by using ? and ? of the minimal inhibitory concentration for propolis and antibiotics, evaluating the number of viable cells according to the incubation time. Brazilian and Bulgarian propolis showed antibacterial activity, but no synergistic effects with the three tested antibiotics were seen. Previous works by our laboratory have revealed that propolis has synergistic effects with antibiotics, acting on the bacterial wall and ribosome, but it does not seem to interact with antibiotics acting on DNA or folic acid, and only a bacteriostatic action was seen in these assay conditions.     

Mechanisms underlying the cytotoxic effect of propolis on human laryngeal epidermoid carcinoma cells

Propolis has been used as a traditional remedy for centuries because of its beneficial effects, including anticancer properties. The aim of this study was to compare the cytotoxic mechanism of Cuban red propolis (CP) and Brazilian green propolis (BP) on human laryngeal carcinoma (HEp-2) cells. Cell viability, leakage of lactate dehydrogenase, fluorescence staining, mitochondrial membrane potential (ΔΨm) and the expression of pro/anti-apoptotic genes were assessed. Cell viability and cytotoxic assays suggested a dose-dependent effect of CP and BP extracts with a possible association of intracellular reactive oxygen species production and decreased ΔΨm. Both samples induced apoptosis via activation of TP53, CASP3, BAX, P21 signalling, and downregulation of BCL2 and BCL-XL. CP exerted a higher cytotoxic effect than BP extract. Our findings suggest further investigation of the main components of each propolis sample, what may lead to the development of strategies for the treatment of laryngeal cancer.     

Thermal analysis applied to irradiated propolis

Propolis is a resinous hive product, collected by bees. Raw propolis requires a decontamination procedure and irradiation appears as a promising technique for this purpose. The valuable properties of propolis for food and pharmaceutical industries have led to increasing interest in its technological behavior. Thermal analysis is a chemical analysis that gives information about changes on heating of great importance for technological applications. Ground propolis samples were ⁶⁰Co gamma irradiated with 0 and 10 kGy. Thermogravimetry curves shown a similar multi-stage decomposition pattern for both irradiated and unirradiated samples up to 600°C. Similarly, through differential scanning calorimetry , a coincidence of melting point of irradiated and unirradiated samples was found. The results suggest that the irradiation process do not interfere on the thermal properties of propolis when irradiated up to 10 kGy.     

Calorimetric study on the effect of Co γ-rays on the growth of microorganisms

Using a calorimeter equipped with 24 sample units, the heat evolution from growing Saccharomyces cerevisiae, Escherichia coli and spores of Bacillus pumilus and Bacillus stearothermophilus was detected in the form of growth thermograms. Irradiation with ⁶⁰Co γ-rays affected the growth pattern, which was used for a quantitative analysis of the effect on microorganisms. Irradiation of B. pumilus and B. stearothermophilus spores led to dose-dependent delays in growth, indicating a bactericidal effect. In case of ⁶⁰Co γ-irradiated S. cerevisiae, a dose-dependent reduction of the growth rate constant was observed together with the retardation in growth, indicating a combination of bactericidal and bacteriostatic effects. An equation to determine the number of survivors on the basis of the retardation in growth t and the growth rate constant μ was developed, which proved the opportunity to use the calorimetric technique in predictive microbiology.     

Antioxidant compounds of propolis determined by capillary electrophoresis-mass spectrometry

Propolis is a resinous hive product rich in antioxidant compounds. Capillary electrophoresis coupled to mass spectrometric detection can provide selective information about the analytes present in complex extracts of propolis and has turned out to be an attractive alternative to HPLC methods. Therefore, a CE-ESI-MS method has been developed for the analysis of antioxidant compounds obtained from propolis. For this purpose, different electrophoretic parameters such as the nature, pH, and concentration of the separation buffer, as well as electrospray parameters (dry gas temperature and flow, nebulising gas pressure, and make-up flow) have been carefully optimised. Different phenolic compounds (e.g. pinobanksin 3-acetate, naringenin, pinocembrin, chrysin, daidzein, quercetin 3',7-dimethyl ether, apigenin, and kaempferid) could be detected. To confirm the identity of the phenolic compounds in propolis extracts, accurate mass data of the molecular ions were obtained by TOF MS. Limits of detection ranging from 6 mg/100 g of raw propolis for chrysin to 58 mg/ 100 g of raw propolis for luteolin, were obtained.     

Activity of Cuban propolis extracts on Leishmania amazonensis and Trichomonas vaginalis

In this paper we analyzed the antiprotozoal effects of eighteen Cuban propolis extracts (brown, red and yellow type) collected in different geographic areas, using Leishmania amazonensis (as a model of intracellular protozoa) and Trichomonas vaginalis (as a model of extracellular protozoa). All evaluated propolis extracts caused inhibitory effect on intracellular amastigotes of L. amazonensis. However, cytotoxicity on peritoneal macrophages from BALB/c mice was observed. Only five samples decreased the viability of T. vaginalis trophozoites at concentrations lower than 10 microg/mL. No correlation between the type of propolis and antiprotozoal activity was found. Cuban propolis extracts demonstrated activity against both intracellular and extracellular protozoa model, as well as the potentialities of propolis as a natural source to obtain new antiprotozoal agents.     

Chalcones in bioactive Argentine propolis collected in arid environments

The aim of this study was to assess the chemical and biological profile of propolis samples collected in arid environments of north-western Argentina. The samples were from two phytogeographical regions (Prepuna and Monte de Catamarca Province). Propolis ethanolic extracts (PEE) and chloroform (CHL), hexane (HEX) and aqueous (AQ) sub-extracts of samples from three regions (CAT-I; CAT-II and CAT-III) were obtained. All PEE exhibited antioxidant activity in the DPPH radical scavenging assay (SC50 values between 28 and 43 microg DW/mL). The CHL extract was the most active (SC50 values between 10 and 37 microg DW/mL). The antioxidant activity in the beta-carotene bleaching assays was more effective for PEE and CHL (IC50 values between 2 and 9 microg DW/mL, respectively). A similar pattern was observed for antibacterial activity. The highest inhibitory effect on the growth of human Gram-positive bacteria was observed for CHL-III and CHL-I (Monte region) with minimal inhibitory concentration values (MIC100) of 50 to 100 microg DW/mL. Nine compounds were identified by HPLC-PAD. Two of them (2', 4'- dihydroxychalcone and 2',4'- dihydroxy 3'-methoxychalcone) were found only in propolis samples from the Monte phytogeographical region. We consider that the Argentine arid region is appropriate to place hives in order to obtain propolis of excellent quality because the dominant life forms in that environment are shrubby species that produce resinous exudates with a high content of chalcones, flavones and flavonols.     

超高效液相色谱-高分辨质谱法测定蜂蜜中的3种马桑内酯

建立了采用超高效液相色谱-高分辨质谱测定蜂蜜中3种马桑内酯残留的方法。样品采用0.2 mol/L磷酸盐缓冲溶液(pH=7.5)提取,经Waters HLB小柱净化,以Phenomenex C18色谱柱进行色谱分离,通过高分辨质谱t-MS²负离子扫描模式进行定性和定量分析。结果表明3种目标化合物的检出限(LOD)均为0.05 mg/kg,定量限(LOQ)均为0.1 mg/kg。空白蜂蜜样品在0.1~0.5 mg/kg范围内的3个加标水平的平均回收率为86.3%~95.6%,相对标准偏差为3.0%~8.4%。应用该方法对从新西兰进口的麦卢卡蜂蜜进行检测,检出一份样品含羟基马桑毒素0.3 mg/kg。该方法适用于蜂蜜中马桑内酯残留的检测。     

Analytical methods applied to diverse types of Brazilian propolis

Propolis is a bee product, composed mainly of plant resins and beeswax, therefore its chemical composition varies due to the geographic and plant origins of these resins, as well as the species of bee. Brazil is an important supplier of propolis on the world market and, although green colored propolis from the southeast is the most known and studied, several other types of propolis from Apis mellifera and native stingless bees (also called cerumen) can be found. Propolis is usually consumed as an extract, so the type of solvent and extractive procedures employed further affect its composition. Methods used for the extraction; analysis the percentage of resins, wax and insoluble material in crude propolis; determination of phenolic, flavonoid, amino acid and heavy metal contents are reviewed herein. Different chromatographic methods applied to the separation, identification and quantification of Brazilian propolis components and their relative strengths are discussed; as well as direct insertion mass spectrometry fingerprinting.Propolis has been used as a popular remedy for several centuries for a wide array of ailments. Its antimicrobial properties, present in propolis from different origins, have been extensively studied. But, more recently, anti-parasitic, anti-viral/immune stimulating, healing, anti-tumor, anti-inflammatory, antioxidant and analgesic activities of diverse types of Brazilian propolis have been evaluated. The most common methods employed and overviews of their relative results are presented.     

Analysis of water extracts from airborne dust samples by capillary isotachophoresis

Application of capillary isotachophoresis (CITP) for the analysis of water extracts of the dust samples collected in different periods in air-filtration devices in Prague car traffic tunnels and in Parisian metro station is presented. The extracts were analyzed in cationic mode with a leading electrolyte (LE) of 10 mM KOH, 25 mM acetic acid, pH 4.4, and a terminating electrolyte (TE) of 10 mM β-alanine, adjusted to pH 4.4 with acetic acid, and in anionic mode with LE 10 mM HCl, 20 mM histidine, pH 5.8 and TE 10 mM 2-(N-morpholino)ethanesulphonic acid, pH 3.7. Extracted amounts of UV-absorbing substances, including pollen allergens and organic pollutants, the number of the found components and concentrations of some inorganic ions (e.g. Cl⁻, K⁺, Na⁺, Ca²⁺) in the dust samples were determined. It was found that the extracted amounts of anionic components and their number were much higher than those of cationic components. Significant differences have been found in the analyses of the extracts of different origin. Much more material and more components were present in the extracts of samples from the pollen-rich period than from the pollen-free period, especially in anionic CITP mode.     

亚胺连接的多孔共价有机骨架材料结合固相萃取-液相色谱-串联质谱检测蜂蜜中雌激素

以亚胺连接的多孔共价有机骨架材料(IL-COF-1)作为固相萃取的吸附剂,建立了液相色谱-串联质谱快速检测蜂蜜样品中痕量雌激素的方法。该研究选择雌二醇、己烯雌酚、雌三醇、β-雌二醇和炔雌醇5种雌激素作为目标分析物。在蜂蜜样品中添加雌激素,采用单因素优化法对影响萃取效果的重要因素进行优化,获得最佳条件:IL-COF-1用量为30 mg,样品流速为3 mL/min,样品溶液pH值为7,以5 mL的1%(v/v)氨水-甲醇溶液进行洗脱,流速为0.4 mL/min,萃取过程中不添加NaCl。采用高效液相色谱-三重四极杆质谱联用技术对提取物中的雌激素进行定量分析。以乙腈和5 mmol/L的乙酸铵溶液作为流动相进行梯度洗脱,经C18色谱柱分离,采用电喷雾离子源、质谱多反应监测和负离子扫描模式,实现了蜂蜜样品中5种雌激素的快速定性定量分析。在最佳条件下,方法验证结果中雌三醇、β-雌二醇和炔雌醇的线性范围为1~500 ng/g,雌二醇和己烯雌酚的线性范围为0.1~100 ng/g,相关系数(r)为0.9934~0.9972。检出限(S/N=3)为0.01~0.30 ng/g,定量限(S/N=10)为0.05~0.95 ng/g。添加50 ng/g 5种雌激素进行重复性实验,日内精密度相对标准偏差(RSD)为3.2%~6.6%,日间精密度RSD为4.2%~7.9%。基于IL-COF-1的固相萃取-液相色谱-串联质谱法具有快速准确、灵敏度高等特点,适用于蜂蜜中雌激素的分析和检测。将该方法应用于4个实际蜂蜜样品中雌激素的检测,均未检出目标物;在低中高3个水平下,5种雌激素的加标回收率为80.1%~115.2%,结果令人满意。     

Silica xerogel-hydrogen peroxide composites: their morphology, stability, and antimicrobial activity

Hydrogen peroxide was incorporated into silica xerogel matrix over the concentration range from 3.8 to 68.0 wt% via the sol–gel route. The obtained composites were characterized by scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The release rates of H₂O₂ from the composites into the aqueous phase were examined. In most cases, a 90% release was attained after ca. 10 min, and it was only slightly dependent on H₂O₂ concentration and particle size. The antimicrobial activity of the composite containing 3.59% H₂O₂ was evaluated against Escherichia coli and Micrococcus luteus. A comparative assay was carried out for aqueous solution of H₂O₂ of the same concentration. The results demonstrated a potent microbicidal efficacy of the composite. Furthermore, diffusion range of the hydrogen peroxide from the solid composite into an agar medium matched that of the H₂O₂ in aqueous solution. The stability tests with the xerogels containing 3.8, 26.4, and 68.0% of H₂O₂ showed that after 63 days respective losses of the H₂O₂ at 3 °C were 8.8, 9.7, and 6.2%. Both the DSC results and the stability tests have shown that the molecular water present in the pores stabilizes the composite, probably through improving the binding of the H₂O₂ molecules onto the silica surface.     

Evaluation of the antioxidant power of honey, propolis and royal jelly by amperometric flow injection analysis

In this paper is described the applicability of a flow injection system, operating with an amperometric detector, for measurement in rapid and simple way the antioxidant power of honey, propolis and royal jelly. The proposed method evaluates the reducing power of selected antioxidant compounds and does not require the use of free radicals or oxidants. Twelve honey, 12 propolis and 4 royal jelly samples of different botanical and geographical origin were evaluated by the electrochemical method and the data were compared with those obtained by the DPPH assay. Since a good correlation was found (R² = 0.92) the proposed electrochemical method can be successfully employed for the direct, rapid and simple monitoring of the antioxidant power of honeybee products. Furthermore, the total phenolic content of samples was determined by the Folin-Ciocalteau procedure and the characteristic antioxidant activities showed a good correlation with phenolics (R² = 0.96 for propolis and 0.90 for honey).     

Ultra-high-performance liquid chromatography using a fused-core particle column for fast analysis of propolis phenolic compounds

Propolis is a bee product with a complex chemical composition formed by several species from different geographical origins. The complex propolis composition requires an accurate and reproducible characterization of samples to standardize the quality of the material sold to consumers. This work developed an ultra-high-performance liquid chromatography with a photodiode array detector method to analyze propolis phenolic compounds based on the two key propolis biomarkers, Artepillin C and p-Coumaric acid. This choice was made due to the complexity of the sample with the presence of several compounds. The optimized method was hyphenated with mass spectrometry detection allowing the detection of 23 different compounds. A step-by-step strategy was used to optimize temperature, flow rate, mobile phase composition, and re-equilibration time. Reverse-phase separation was achieved with a C18 fused-core column packed with the commercially available smallest particles (1.3 nm). Using a fused-core column with ultra-high-performance liquid chromatography allows highly efficient, sensitive, accurate, and reproducible determination of compounds extracted from propolis with an outstanding sample throughput and resolution. Optimized conditions permitted the separation of the compounds in 5.50 min with a total analysis time (sample-to-sample) of 6.50 min.     

Determination of Arsenic in Honey,Propolis, Pollen,and Honey Bees by Microwave Digestion and Hydride Generation Flame Atomic Absorption

The toxic properties of arsenic are well known. Honey has been widely used for monitoring this element. The present work reports a novel method for the determination of arsenic in honey, bees, pollen, and propolis, based on the coupling of microwave digestion and hydride generation. Method development included the quantitative reduction of arsenic(V) to arsenic(III), the acid used for dilution, and the complete removal of the gases following digestion. The method performance was satisfactory with recoveries between 83% and 111% and corresponding relative standard deviations between 3.1% and 24%. Among the 32 samples of honey, propolis, pollen, and honey bees analyzed, arsenic was detected in four out of six propolis samples at the method limit of detection (0.4?µg?g^?1). The results indicate that propolis may be an efficient indicator for arsenic.     

高效液相色谱-四极杆飞行时间质谱检测中国杨树型蜂胶、巴西绿蜂胶和杨树胶中的酚类化合物及真伪鉴别

建立了用于检测中国杨树型蜂胶、巴西绿蜂胶和杨树胶中23种酚类化合物的高效液相色谱-四极杆飞行时间质谱（HPLC/Q-TOF MS）法，并进行了差异性分析。蜂胶和树胶样品用甲醇-水（1：1，v/v）溶解后，过0.45 μm有机相滤膜后进样。采用Agilent Eclipse Plus C₁₈色谱柱分离，以乙腈和0.1%（体积分数）甲酸水溶液为流动相进行梯度洗脱，电喷雾正离子模式全扫描方式检测，扫描范围为m/z 100~1000，外标法定量。结果表明，23种酚类化合物在10~200 μg/L范围内线性关系良好，相关系数均大于0.99。桔皮素和刺芒柄花素的检出限和定量限分别为0.2和1 μg/L，其他化合物的检出限和定量限分别为2和10 μg/L。在10、25和50 mg/kg 3个添加水平下，23种酚类化合物在3种样品基质中的提取回收率为70.2%~122.6%，RSD值均小于10%。水杨苷、肉桂酸、咖啡酸和香豆酸可以作为中国杨树型蜂胶掺假鉴别的特征化合物，咖啡酸、阿魏酸、白杨素、咖啡酸苯乙酯、松属素、高良姜素、香豆酸、异鼠李素、山柰素和阿替匹林C可以作为辨别中国杨树型蜂胶和巴西绿蜂胶的特征化合物。该结果对蜂胶产品的质量控制具有一定的参考意义。     

Identification of the plant origin of the botanical biomarkers of Mediterranean type propolis

Propolis is a honeybee product which bees produce by collecting resins from various botanical sources. The chemical composition of propolis is directly dependant on the availability of resinous plant materials in different geographic regions. This study was undertaken to evaluate the resinous plant sources used by bees to produce Mediterranean type propolis. Although this propolis type has already been the subject of numerous studies, its major botanical source had not yet been identified. In this study, using GC-MS analysis, we identify the resin of the common cypress, Cupressus_sempervirens, as the major plant source of the characteristic diterpene fingerprint profile of Mediterranean propolis.     

基于光微热量-荧光光谱联用技术研究光催化热力学和动力学的温度效应

利用光微热量-荧光光谱联用技术,对光催化过程的热谱和光谱信息同步监测,获取了五个温度下,g-C_3N_4@Ag@Ag_3PO_4光催化降解罗丹明B的原位热动力学、光谱动力学信息,探究了温度对相关参数的影响。结果表明,催化降解反应分为三个阶段:(i)污染物和催化剂的光响应过程;(ii)光响应吸热和污染物降解放热的竞争过程;(iii)保持稳定的放热率。吸热和放热的竞争过程符合一级动力学,降解速率随着温度的升高而增大;稳定放热阶段为拟零级反应,在283.15 K、288.15 K、293.15 K、298.15 K、303.15 K下的放热速率分别为0.4668±0.3875μJ·s~(-1)、0.5314±0.3379μJ·s~(-1)、0.5064±0.3234μJ·s~(-1)、0.5328±0.3377μJ·s~(-1)、0.5762±0.3452μJ·s~(-1)。本研究为探究光催化过程的原位热力学、热动力学及光谱信息及机理的推测提供科学依据。