Improvement of the In Vitro Cytotoxic Effect on HT-29 Colon Cancer Cells by Combining 5-Fluorouacil and Fluphenazine with Green, Red or Brown Propolis.

Cancer is regard as one of the key factors of mortality and morbidity in the world. Treatment is mainly based on chemotherapeutic drugs that, when used in targeted therapies, have serious side effects. 5-fluorouracil (5-FU) is a drug commonly used against colorectal cancer (CRC), despite its side effects. Combination of this compound with natural products is a promising source in cancer treatment research. In recent years, propolis has become the subject of intense pharmacological and chemical studies linked to its diverse biological properties. With a complex composition rich in phenolic compounds, propolis is described as showing positive or synergistic interactions with several chemotherapeutic drugs. The present work evaluated the in vitro cytotoxic activity of the most representative propolis types, such as green, red and brown propolis, in combination with chemotherapeutic or CNS drugs on HT-29 colon cancer cell lines. The phenolic composition of the propolis samples was evaluated by LC-DAD-ESI/MSn analysis. According to the type of propolis, the composition varied; green propolis was rich in terpenic phenolic acids and red propolis in polyprenylated benzophenones and isoflavonoids, while brown propolis was composed mainly of flavonoids and phenylpropanoids. Generally, for all propolis types, the results demonstrated that combing propolis with 5-FU and fluphenazine successfully enhances the in vitro cytotoxic activity. For green propolis, the combination demonstrated an enhancement of the in vitro cytotoxic effect compared to green propolis alone, at all concentrations, while for brown propolis, the combination in the concentration of 100 µg/mL gave a lower number of viable cells, even when compared with 5-FU or fluphenazine alone. The same was observed for the red propolis combination, but with a higher reduction in cell viability. The combination index, calculated based on the Chou-Talalay method, suggested that the combination of 5-FU and propolis extracts had a synergic growth inhibitory effect in HT-29 cells, while with fluphenazine, only green and red propolis, at a concentration of 100 µg/mL, presented synergism.

Differentiating between Monofloral Portuguese Bee Pollens Using Phenolic and Volatile Profiles and Their Impact on Bioactive Properties.

Nowadays, bee products are commended by consumers for their medicinal and dietary properties. This study aimed to differentiate between monofloral bee pollens originating from Portugal using phenolic and volatile profiles and investigate their antioxidant and cytotoxic activity. Total phenolic and flavonoid compounds were recorded between 2.9-35.8 mg GAE/g and 0.7-4.8 mg QE/g, respectively. The LC/DAD/ESI-MSn analytical results allowed us to identify and quantify a total of 72 compounds, including phenolic and phenylamide compounds, whereas GC-MS results revealed the presence of 49 different compounds, mostly ketones, aldehydes, esters, hydrocarbons, and terpenes. The highest DPPH• radical scavenging activity, EC50: 0.07 mg/mL, was recorded in the sample dominated by Castanae sp. pollen, whereas the Rubus sp. (1.59 mM Trolox/mg) and Cistaceae sp. (0.09 mg GAE/g) pollen species exhibited the highest antioxidant activity in ABTS•+ and reducing power assays, respectively. Regarding the anti-carcinogenic activity, only Carduus sp. showed remarkable cytotoxic potential against MCF-7.

Evaluation of Antioxidant and Anticancer Activity of Mono- and Polyfloral Moroccan Bee Pollen by Characterizing Phenolic and Volatile Compounds.

Bee pollen is frequently characterized as a natural source of bioactive components, such as phenolic compounds, which are responsible for its pharmaceutical potential and nutritional properties. In this study, we evaluated the bioactive compound contents of mono- and polyfloral bee pollen samples using spectroscopic and chromatographic methods and established links with their antioxidant and antitumor activity. The findings demonstrated that the botanical origin of bee pollen has a remarkable impact on its phenolic (3-17 mg GAE/g) and flavonoid (0.5-3.2 mg QE/g) contents. Liquid chromatography-mass spectrometry analysis revealed the presence of 35 phenolic and 13 phenylamide compounds in bee pollen, while gas chromatography-mass spectrometry showed its richness in volatiles, such as hydrocarbons, fatty acids, alcohols, ketones, etc. The concentration of bioactive compounds in each sample resulted in a substantial distinction in their antioxidant activity, DPPH (EC50: 0.3-0.7 mg/mL), ABTS (0.8-1.3 mM Trolox/mg), and reducing power (0.03-0.05 mg GAE/g), with the most bioactive pollens being the monofloral samples from Olea europaea and Ononis spinosa. Complementarily, some samples revealed a moderate effect on cervical carcinoma (GI50: 495 µg/mL) and breast adenocarcinoma (GI50: 734 µg/mL) cell lines. This may be associated with compounds such as quercetin-O-diglucoside and kaempferol-3-O-rhamnoside, which are present in pollens from Olea europaea and Coriandrum, respectively. Overall, the results highlighted the potentiality of bee pollen to serve health-promoting formulations in the future.

Development and Characterization of High-Absorption Microencapsulated Organic Propolis EPP-AF® Extract (i-CAPs).

The demand for organic and functional food continues to increase yearly. Among the available functional foods, propolis is a bee product that has various beneficial properties, including antimicrobial, antioxidant, and anti-inflammatory activities. However, it generally is only available in ethanol solution, which has poor bioavailability, as it is relatively insoluble in water. The use of such ethanol extracts is often objectionable because of the alcohol content and because they have a strong and striking taste. Development of alternatives that can efficiently and safely increase solubility in water, and that meet organic production specifications, has been a challenge. To address these concerns, microcapsules were developed using spray-dryer technology from an emulsion based on EPP-AF® propolis and gum arabic (i-CAPS). These propolis-loaded microcapsules were characterized using FT-IR, SEM, TGA, HPLC, and spectrophotometric techniques, along with determination of antimicrobial, antioxidant, antitumor, anti-inflammatory, and antihypercholesterolemic activities, as well as permeability in in vitro models. The production system resulted in microcapsules with a spherical shape and an encapsulation efficiency of 93.7 ± 0.7%. They had IC50s of 2.654 ± 0.062 and 7.342 ± 0.058 µg/mL by FRAP and DPPH antioxidant methods, respectively. The EPP-AF® i-CAPS also had superior antimicrobial activity against Gram-positive bacteria. Antitumor activity was calculated based on the concentration that inhibited 50% of growth of AGS, Caco-2, and MCF-7 cell strains, giving results of 154.0 ± 1.0, 117 ± 1.0, and 271.0 ± 25 µg/mL, respectively. The microcapsule presentation reduced the permeation of cholesterol by 53.7%, demonstrating antihypercholesterolemic activity, and it improved the permeability of p-coumaric acid and artepillin C. The IC50 for NO production in RAW 264.7 cells was 59.0 ± 0.1 µg/mL. These findings demonstrate the potential of this new propolis product as a food and pharmaceutical ingredient, though additional studies are recommended to validate the safety of proposed dosages.

Arbutus Unedo Honey and Propolis Ameliorate Acute Kidney Injury, Acute Liver Injury, and Proteinuria via Hypoglycemic and Antioxidant Activity in Streptozotocin-Treated Rats.

BACKGROUND/AIMS: Honey and propolis have biological and therapeutic effects in various pathological and clinical conditions such as hyperglycemia and diabetes. However, the combined use of honey and propolis has not been reported. The study evaluated the protective effect of Arbutus unedo honey, propolis and their combination in streptozotocin (STR)-induced hyperglycemia, acute kidney injury (AKI), liver injury, dyslipidemia, and proteinuria in male Wistar rats. METHODS: The study identified physicochemical characteristics, mineral and antioxidant content, and antioxidant activity in honey and propolis. Rats were assigned to five groups, with five rats in each group; control, STR-treated, STR-treated + honey (1g/kg/day), STR-treated + propolis (100 mg/day), and STR-treated + honey and propolis. On day 15, blood glucose, insulin, HBA1c, kidney function tests, liver enzymes, lipid profile, hemoglobin, and urine protein, creatinine, glucose, and electrolytes were analyzed. Liver, pancreas, and kidney tissues were studied histologically. The mineral component in honey and propolis was determined by atomic absorption spectrometry. Honey analysis was performed by HPLC. Chemical characterization of propolis was performed by LC/DAD/ESI-MSn . Measurement of blood and urine parameters was carried out with an automated analyzer (Architect c8000) and XT-1800i Automated Hematology Analyzer. Insulin concentration was determined by Elisa and insulin resistance was estimated by using HOMA-IR. RESULTS: Honey and propolis contain a high quantity of antioxidants and exhibit in vitro antioxidant activity. In STR-treated rats, blood glucose, HBA1c, creatinine, blood urea, liver enzymes, and urine protein significantly increased compared to the control group (P<0.05), while insulin, hemoglobin, and body weight significantly decreased. Histological changes were evident in the pancreas, kidney, and liver tissues. These results indicated AKI, liver injury, and pancreatic injury, which was evident with reducing the number of the island of Langerhans and marked hyperglycemia. The use of honey and propolis significantly (P<0.05) attenuated liver and kidney injury, and proteinuria, and improved level of hemoglobin, HBA1c, and insulin toward the normal range. The combination of honey and propolis was more effective than honey or propolis individually (P<0.05). CONCLUSION: the combination of propolis and honey can prevent STR-induced AKI, liver injury, proteinuria, dyslipidemia, anemia, hyperglycemia, and body weight loss, most likely by their hypoglycemic and antioxidant activities.

Volatile Profile of Portuguese Monofloral Honeys: Significance in Botanical Origin Determination.

The volatile profiles of 51 samples from 12 monofloral-labelled Portuguese honey types were assessed. Honeys of bell heather, carob tree, chestnut, eucalyptus, incense, lavender, orange, rape, raspberry, rosemary, sunflower and strawberry tree were collected from several regions from mainland Portugal and from the Azores Islands. When available, the corresponding flower volatiles were comparatively evaluated. Honey volatiles were isolated using two different extraction methods, solid-phase microextraction (SPME) and hydrodistillation (HD), with HD proving to be more effective in the number of volatiles extracted. Agglomerative cluster analysis of honey HD volatiles evidenced two main clusters, one of which had nine sub-clusters. Components grouped by biosynthetic pathway defined alkanes and fatty acids as dominant, namely n-nonadecane, n-heneicosane, n-tricosane and n-pentacosane and palmitic, linoleic and oleic acids. Oxygen-containing monoterpenes, such as cis- and trans-linalool oxide (furanoid), hotrienol and the apocarotenoid α-isophorone, were also present in lower amounts. Aromatic amino acid derivatives were also identified, namely benzene acetaldehyde and 3,4,5-trimethylphenol. Fully grown classification tree analysis allowed the identification of the most relevant volatiles for discriminating the different honey types. Twelve volatile compounds were enough to fully discriminate eleven honey types (92%) according to the botanical origin.

Assessment of the In Vivo and In Vitro Release of Chemical Compounds from Vespa velutina.

Vespa velutina has been rapidly expanding throughout Galicia since 2012. It is causing human health risks and well-known losses in the beekeeping sector. Control methods are scarce, unspecific, and ineffective. Semiochemicals are insect-derived chemicals that play a role in communication and they could be used an integrated pest management tool alternative to conventional pesticides. A previous determination of the organic chemical profile should be the first step in the study of these semiochemicals. HS-SPME in living individuals and the sting apparatus extraction followed by GC-MS spectrometry were combined to extract a possible profile of these compounds in 43 hornets from Galicia. The identified compounds were hydrocarbons, ketones, terpenes, and fatty acid, and fatty acid esters. Nonanal aldehyde appeared in important concentrations in living individuals. While pentadecane, 8-hexyl- and ethyl oleate were mainly extracted from the venom apparatus. Ketones 2-nonanone, 2-undecanone and 7-nonen-2-one, 4,8-dimethyl- were identified by both procedures, as was 1,7-Nonadiene, 4,8-dimethyl-. Some compounds were detected for the first time in V. velutina such as naphthalene, 1,6-dimethyl-4-(1-methylethyl). The chemical profile by caste was also characterized.

Honey Volatiles as a Fingerprint for Botanical Origin-A Review on their Occurrence on Monofloral Honeys.

Honeys have specific organoleptic characteristics, with nutritional and health benefits, being highly appreciated by consumers, not only in food but also in the pharmaceutical and cosmetic industries. Honey composition varies between regions according to the surrounding flora, enabling its characterization by source or type. Monofloral honeys may reach higher market values than multifloral ones. Honey's aroma is very specific, resulting from the combination of volatile compounds present in low concentrations. The authentication of honey's complex matrix, according to its botanical and/or geographical origin, represents a challenge nowadays, due to the different sorts of adulteration that may occur, leading to the search for reliable marker compounds for the different monofloral honeys. The existing information on the volatiles of monofloral honeys is scarce and disperse. In this review, twenty monofloral honeys and honeydews, from acacia, buckwheat, chestnut, clover, cotton, dandelion, eucalyptus, fir tree, heather, lavender, lime tree, orange, pine, rape, raspberry, rhododendron, rosemary, strawberry tree, sunflower and thyme, were selected for volatile comparison purposes. Taking into consideration the country of origin, the technique of isolation and analysis, the five main volatiles from each of the honeys are compared. Whereas some compounds were found in several types of monofloral honey, and thus not considered good volatile markers, some monofloral honeys revealed characteristic volatile compounds independently of their provenance.

Flavonoid Composition and Antitumor Activity of Bee Bread Collected in Northeast Portugal.

Bee bread (BB) is a fermented mixture of plant pollen, honey, and bee saliva that worker bees use as food for larvae, and for young bees to produce royal jelly. In the present study, five BB samples, collected from Apis mellifera iberiensis hives located in different apiaries near Bragança, in the northeast region of Portugal, and one BB commercial sample were characterized by high-performance liquid chromatography coupled to a diode array detector and electrospray mass spectrometry (HPLC-DAD-ESI/MS) in terms of phenolic compounds, such as flavonoid glycoside derivatives. Furthermore, the samples were screened, using in vitro assays, against different human tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), HeLa (cervical carcinoma) and HepG2 (hepatocellular carcinoma), and also against non-tumor liver cells (porcine liver cells, PLP2). The main phenolic compounds found were flavonol derivatives, mainly quercetin, kaempferol, myricetin, isorhamnetin and herbacetrin glycoside derivatives. Thirty-two compounds were identified in the six BB samples, presenting BB1 and BB3 with the highest contents (6802 and 6480 µg/g extract, respectively) and the highest number of identified compounds. Two isorhamnetin glycoside derivatives, isrohamnetin-O-hexosyl-O-rutinoside and isorhamnetin-O-pentosyl-hexoside, were the most abundant compounds present in BB1; on the other hand, quercetin-3-O-rhamnoside was the most abundant flavonol in BB3. However, it was not possible to establish a correlation between the flavonoids and the observed low to moderate cytotoxicity (ranging from >400 to 68 µg/mL), in which HeLa and NCI-H460 cell lines were the most susceptible to the inhibition. To the authors' knowledge, this is the first report characterizing glycosidic flavonoids in BB samples, contributing to the chemical knowledge of this less explored bee product.

Antioxidant activity of aminodiarylamines in the thieno[3,2-b]pyridine series: radical scavenging activity, lipid peroxidation inhibition and redox profile.

The antioxidant activity of the aminodi(hetero)arylamines, prepared by C-N coupling of the methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate with bromonitrobenzenes and further reduction of the obtained nitro compounds, was evaluated by chemical, biochemical and electrochemical assays. The aminodi(hetero)arylamine with the amino group ortho to the NH and a methoxy group in para, was the most efficient in radical scavenging activity (RSA, 63 µM) and reducing power (RP, 33 µM), while the aminodiarylamine with the amino group in para to the NH, gave the best results in ß-carotene-linoleate system (41 µM) and inhibition of formation of thiobarbituric acid reactive substances in porcine brain cells homogenates (7 µM), with EC50 values even lower than those obtained for the standard trolox. This diarylamine also presented the lowest oxidation potential, lower than the one of trolox, and the highest antioxidant power in the electrochemical assays. The para substitution with an amino group enables higher antioxidant potential.

In vitro evaluation of Portuguese propolis and floral sources for antiprotozoal, antibacterial and antifungal activity.

Propolis is a beehive product with a very complex chemical composition, used since ancient times in several therapeutic treatments. As a contribution to the improvement of drugs against several tropical diseases caused by protozoa, we screened Portuguese propolis and its potential floral sources Populus x Canadensis and Cistus ladanifer against Plasmodium falciparum, Leishmania infantum, Trypanosoma brucei and Trypanosoma cruzi. The toxicity against MRC-5 fibroblast cells was evaluated to assess selectivity. The in vitro assays were performed following the recommendations of WHO Special Programme for Research and Training in Tropical Diseases (TDR) and revealed moderate activity, with the propolis extracts presenting the relatively highest inhibitory effect against T. brucei. Additionally, the antimicrobial activity against Staphylococcus aureus, Candida albicans, Trichophyton rubrum and Aspergillus fumigatus was also verified with the better results observed against T. rubrum. The quality of the extracts was controlled by evaluating the phenolic content and antioxidant activity. The observed biological activity variations are associated with the variable chemical composition of the propolis and the potential floral sources under study.

Using Analytic Hierarchy Process to Assess Beekeeping Suitability in Portuguese Controlled Areas: A First Approach.

Beekeeping management is greatly influenced by spatial factors (e.g., land use/land cover, roads, or electrical energy networks), so GIS are a powerful tool to overlap and relate a variety of spatial data levels and, consequently, a very useful tool for beekeeping activity planning. This study was developed within the intervention area of three controlled zones managed by Portuguese Beekeepers Associations. The methodology, based on multi-criteria decision analysis, integrates several criteria, such as hydrographic networks, road networks, soil occupation, solar radiation, and electromagnetic radiation sources. These criteria were proposed and evaluated through online questionnaires carried out with beekeepers. Concerning the selected criteria and the respective geographical data, the most relevant were land use/land cover and water availability, with a significance of 44% and 24%, respectively. The beekeeping suitability map enabled us to evaluate the degree of compliance for the actual location of apiaries, with 60% of the apiaries being installed in high potential areas. In the context of beekeeping planning, the potential of the techniques applied seems to be an important tool for optimizing the location of apiaries and the profitability of beekeeping.

Bee Venom-Loaded Niosomes as Innovative Platforms for Cancer Treatment: Development and Therapeutical Efficacy and Safety Evaluation.

Despite past efforts towards therapeutical innovation, cancer remains a highly incident and lethal disease, with current treatments lacking efficiency and leading to severe side effects. Hence, it is imperative to develop new, more efficient, and safer therapies. Bee venom has proven to have multiple and synergistic bioactivities, including antitumor effects. Nevertheless, some toxic effects have been associated with its administration. To tackle these issues, in this work, bee venom-loaded niosomes were developed, for cancer treatment. The vesicles had a small (150 nm) and homogeneous (polydispersity index of 0.162) particle size, and revealed good therapeutic efficacy in in vitro gastric, colorectal, breast, lung, and cervical cancer models (inhibitory concentrations between 12.37 ng/mL and 14.72 ng/mL). Additionally, they also revealed substantial anti-inflammatory activity (inhibitory concentration of 28.98 ng/mL), effects complementary to direct antitumor activity. Niosome safety was also assessed, both in vitro (skin, liver, and kidney cells) and ex vivo (hen's egg chorioallantoic membrane), and results showed that compound encapsulation increased its safety. Hence, small, and homogeneous bee venom-loaded niosomes were successfully developed, with substantial anticancer and anti-inflammatory effects, making them potentially promising primary or adjuvant cancer therapies. Future research should focus on evaluating the potential of the developed platform in in vivo models.

Phenolic profiling of Portuguese propolis by LC-MS spectrometry: uncommon propolis rich in flavonoid glycosides.

INTRODUCTION: Propolis is a chemically complex resinous substance collected by honeybees (Apis mellifera) from tree buds, comprising plant exudates, secreted substances from bee metabolism, pollen and waxes. Its chemical composition depends strongly on the plant sources available around the beehive, which have a direct impact in the quality and bioactivity of the propolis. Being as Portugal is a country of botanical diversity, the phenolic characterisation of propolis from the different regions is a priority. OBJECTIVE: Extensive characterisation of the phenolic composition of Portuguese propolis from different continental regions and islands. METHOD: Forty propolis ethanolic extracts were analysed extensively by liquid chromatography with diode-array detection coupled to electrospray ionisation tandem mass spectrometry (LC-DAD-ESI-MS(n) ). RESULTS: Seventy-six polyphenols were detected in the samples and two groups of propolis were established: the common temperate propolis, which contained the typical poplar phenolic compounds such as flavonoids and their methylated/esterified forms, phenylpropanoid acids and their esters, and an uncommon propolis type with an unusual composition in quercetin and kaempferol glycosides - some of them never described in propolis. CONCLUSION: The method allowed the establishment of the phenolic profile of Portuguese propolis from different geographical locations, and the possibility to use some phenolic compounds, such as kaempferol-dimethylether, as geographical markers. Data suggest that other botanical species in addition to poplar trees can be important sources of resins for Portuguese propolis.

Potentiating effects of honey on antioxidant properties of lemon-flavoured black tea.

Health benefits including antioxidant potential of black tea (Camellia sinensis), lemon (Citrus limon) and honey bees (Apis mellifera) have been extensively reported. Nevertheless, nothing is reported about the effects of their concomitant use. Herein, those effects were evaluated in infusions of lemon-flavoured black tea with three different kinds of honey (light amber, amber and dark amber) from Lavandula stoechas, Erica sp. pl. and other indigenous floral species from north-east Portugal, a region with high amounts of this food product. Data obtained showed that the use of honey (dark amber>amber>light amber) potentiates the antioxidant activity of lemon-flavoured black tea, increasing the reducing power and lipid peroxidation inhibition properties, as also the antioxidant contents such as phenolics, flavonoids and organic acids including ascorbic acid.

Bee pollen and bee bread nutritional potential: Chemical composition and macronutrient digestibility under in vitro gastrointestinal system.

Bee pollen (BP) and bee bread (BB) have been often investigated as potential functional foods. Both bee products are generally characterized by their high nutritional content, with BB being referred as more digestible than BP, however, there is a lack of scientific studies proving this claim. Here, we present a comparative evaluation of the macronutrient digestibility of BP and BB after applying a simulated in vitro gastrointestinal digestive system, together with the evaluation of its nutritional value and chemical composition. The digestibility scores for protein content were calculated on average as 69% and 76% for BP and BB, respectively, whereas digestibility scores for soluble sugars varied depending on bee product and sugar type. The results demonstrated that the nutritional values of both bee products changed depending on their botanical origin but BB is more accessible in the intestinal lumen, especially regarding protein.

Thermal Stability and Antioxidant Activity of Bioactive Compounds in Bread Enriched with Bee Pollen and Bee Bread.

Bee pollen (BP) and bee bread (BB) are natural food sources containing a wide variety of bioactive compounds, complementing their rich nutritional composition. These bee products are being explored to empower functional foods, with the term functionality being dependent on the bioactive compounds added to the food matrix. However, there is not enough evidence of the effect of heat on these compounds during food processing and production and how it impacts their biological activity. Here, we enriched traditional bread by adding BP and BB at different proportions of 1 to 5% and tested the thermal stability of their bioactive compounds through several spectroscopic and chromatographic analyses. Adding bee pollen and bee bread to bread resulted in a 4 and 5-fold increase in total phenolic content, respectively. While not all the 38 phenolic and phenolamide compounds identified in the raw BP and BB were detected in the processed bread, phenolamides were found to be more resilient to baking and heat treatment than flavonoids. Still, the enriched bread's antioxidant activity improved with the addition of BP and BB. Therefore, incorporating bee products into heat-treated products could enhance the functionality of staple foods and increase the accessibility to these natural products.

Beneficial Effect of Bee Venom and Its Major Components on Facial Nerve Injury Induced in Mice.

Peripheral nerve injury (PNI) is a health problem that affects many people worldwide. This study is the first to evaluate the potential effect of bee venom (BV) and its major components in a model of PNI in the mouse. For that, the BV used in this study was analyzed using UHPLC. All animals underwent a distal section-suture of facial nerve branches, and they were randomly divided into five groups. Group 1: injured facial nerve branches without any treatment. Group 2: the facial nerve branches were injured, and the normal saline was injected similarly as in the BV-treated group. Group 3: injured facial nerve branches with local injections of BV solution. Group 4: injured facial nerve branches with local injections of a mixture of PLA2 and melittin. Group 5: injured facial nerve branches with local injection of betamethasone. The treatment was performed three times a week for 4 weeks. The animals were submitted to functional analysis (observation of whisker movement and quantification of nasal deviation). The vibrissae muscle re-innervation was evaluated by retrograde labeling of facial motoneurons in all experimental groups. UHPLC data showed 76.90 ± 0.13%, 11.73 ± 0.13%, and 2.01 ± 0.01%, respectively, for melittin, phospholipase A2, and apamin in the studied BV sample. The obtained results showed that BV treatment was more potent than the mixture of PLA2 and melittin or betamethasone in behavioral recovery. The whisker movement occurred faster in BV-treated mice than in the other groups, with a complete disappearance of nasal deviation two weeks after surgery. Morphologically, a normal fluorogold labeling of the facial motoneurons was restored 4 weeks after surgery in the BV-treated group, but no such restoration was ever observed in other groups. Our findings indicate the potential of the use of BV injections to enhance appropriate functional and neuronal outcomes after PNI.

Honeybee Venom Synergistically Enhances the Cytotoxic Effect of CNS Drugs in HT-29 Colon and MCF-7 Breast Cancer Cell Lines.

5-fluorouracil (5-FU) and doxorubicin (DOX) are potent anti-tumour agents commonly used for colon and breast cancer therapy, respectively. However, their clinical application is limited by their side effects and the development of drug resistance. Honeybee venom is a complex mixture of substances that has been reported to be effective against different cancer cells. Its active compound is melittin, a positively charged amphipathic peptide that interacts with the phospholipids of the cell membrane, forming pores that enable the internalization of small molecules with cytotoxic activities,. and consequently, causing cell death. Some central nervous system (CNS) drugs have recently demonstrated great anti-cancer potential, both in vitro, in vivo and in clinical trials, being promising candidates for drug repurposing in oncology. The present work evaluated the anti-cancer efficacy of honeybee venom in combination with chemotherapeutic or CNS drugs in HT-29 colon and MCF-7 breast cancer cell lines. The chemical characterization of a Portuguese sample of honeybee venom was done by LC-DAD-ESI/MSn analysis. For single treatments, cells were incubated with increasing concentrations of bee venom. For combination treatments, increasing concentrations of bee venom were first combined with the half-maximal inhibitory concentration (IC50) of 5-FU and DOX, in HT-29 and MCF-7 cells, respectively. Cells were also treated with increasing concentrations of bee venom in combination with the IC50 value of four CNS drugs (fluphenazine, fluoxetine, sertraline and thioridazine). Cytotoxicity was evaluated by MTT and SRB assays. The combination index (CI) value was calculated using CompuSyn software, based on the Chou-Talalay method. Synergy scores of different reference models (HSA, Loewe, ZIP and Bliss) were also calculated using SynergyFinder. The results demonstrate that honeybee venom is active against HT-29 colon and MCF-7 breast cancer cells, having better anti-tumour activity in MCF-7 cells. It was found that bee venom combined with 5-FU and fluphenazine in HT-29 cells resulted in less cytotoxic effects compared to the co-treatment of fluoxetine, sertraline and thioridazine plus bee venom, which resulted in less than 15% of viable cells for the whole range of concentrations. The combination of MCF-7 cells with repurposed drugs plus honeybee venom resulted in better anti-cancer efficacies than with DOX, notably for lower concentrations. A combination of fluoxetine and thioridazine plus honeybee venom resulted in less than 40% of viable cells for all ranges of concentrations. These results support that the combination of honeybee venom with repurposed drugs and chemotherapeutic agents can help improve their anti-cancer activity, especially for lower concentrations, in both cell lines. Overall, the present study corroborates the enormous bioactive potential of honeybee venom for colon and breast cancer treatments, both alone and in combination with chemotherapy or repurposed drugs.

Analytical methods for honeybee venom characterization.

The discovery of new drugs has benefited significantly from the development of research in venomics, increasing our understanding of the envenomation processes. It has been previously reported that honeybee venom (HBV) exhibits several pharmacological activities such as anti-inflammatory, antibacterial, antimutagenic, radioprotective, and anticancer activity and may inclusively act as a complementary treatment for SARS-CoV-2. It composition consists mainly on melittin, phospholipase A2, and apamin but other constituents such as hyaluronidase, mast cell degranulating peptide and secapin are also relevant for its bioactivity. However, and because HBV is not officially recognized as a drug, until now, the international community did not establish quality standards for it. To uncover its exact composition, and boost the discovery of HBV-derived drugs, a significant number of techniques were developed. In this review, a relevant overview of the so far published analytical methods for HBV characterization is organized with the aim to accelerate its future standardization. The literature search was performed within PubMed, Google Scholar, and Science Direct by selecting specific documents and exploring HBV evaluation.