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.

The effect of Brazilian Green Propolis extract on inflammation in patients with chronic kidney disease on peritoneal dialysis: A randomised double-blind controlled clinical trial.

BACKGROUND: Chronic kidney disease (CKD) patients on dialysis display a low-grade systemic inflammatory burden. Nutritional interventions designed to activate the cytoprotective nuclear factor erythroid-2-related factor 2 (Nrf2) and inhibit nuclear factor-kB (NF-κB) have been proposed to mitigate this burden. Several bioactive compounds have been investigated to achieve this, including propolis, a resin produced by Apis mellifera bees. Considering the safety and efficacy of propolis, it could be a strategy to benefit these patients. Still, there are no studies using propolis in patients with CKD on peritoneal dialysis (DP), and clinical studies to support this application are lacking. HYPOTHESIS/PURPOSE: The objective and novelty of the present study are to evaluate the effects of propolis supplementation on inflammatory markers in patients with CKD on PD. STUDY DESIGN: A longitudinal, double-blind, placebo-controlled trial with CKD patients on PD. METHODS: The patients were randomised into two groups: propolis that received four capsules of 100 mg (400 mg/day), containing concentrated and standardised dry EPP-AF® Brazilian green propolis extract) or placebo, four capsules of 100 mg (400 mg/day), of magnesium stearate, silicon dioxide, and microcrystalline cellulose, for two months. Plasma levels of inflammatory cytokines, including tumour necrosis factor (TNF-α) and interleukin-6 (IL-6), were evaluated by ELISA. Quantitative real-time PCR analyses were performed to evaluate the transcriptional expression levels of Nrf2 and NF-κB in peripheral blood mononuclear cells (PBMCs). Plasma malondialdehyde (MDA) levels, a lipid peroxidation marker, was measured as thiobarbituric acid reactive substances (TBARS). Routine biochemical markers, including C-reactive protein (CRP), were analysed using commercial kits. Carotid Intima-Media Thickness (CIMT) was measured with a doppler ultrasonography device. The study was registered on ClinicalTrials.gov under the number NCT04411758. RESULTS: A total of 19 patients completed the study, ten patients in the propolis group (54 ± 1.0 years, five men, 7.2  (5.1) months on PD) and 9 in the placebo group (47.5 ± 15.2 years, three men, 10.8  (24.3) months on PD). The plasma levels of TNF-α reduced significantly (p = 0.02), and expression of Nrf2 showed a trend to increase (p = 0.07) after propolis supplementation. CONCLUSION: EPP-AF® Green Propolis extract (400 mg/day) supplementation for two months appears as a potential strategy to mitigate inflammation, reducing TNF-α plasma levels in CKD patients on PD.

Effects of propolis on inflammation markers in patients undergoing hemodialysis: A randomized, double-blind controlled clinical trial.

BACKGROUND AND AIMS: Several studies have been performed in vitro and in animals showing that propolis (a resin made by bees) has excellent anti-inflammatory properties, but no study has been performed in patients with chronic kidney disease (CKD) on hemodialysis (HD). The present study aimed to evaluate the effects of propolis supplementation on inflammatory markers in patients with CKD on HD. METHODS: This is a longitudinal, double-blind, placebo-controlled trial with patients randomized into two groups: propolis (4 capsules of 100 mg/day containing concentrated and standardized dry EPP-AF® green propolis extract) or placebo (4 capsules of 100 mg/day containing microcrystalline cellulose, magnesium stearate and colloidal silicon dioxide) for two months. Routine parameters were analyzed using commercial kits. The plasma levels of inflammatory cytokines were evaluated by flow luminometry. RESULTS: Forty-one patients completed the follow-up, 21 patients in the propolis group (45 ± 12 years, 13 women, BMI, 22.8 ± 3.7 kg/m2) and 20 in the placebo group (45.5 ± 14 years, 13 women, BMI, 24.8 ± 6.8 kg/m2). The obtained data revealed that the intervention with propolis significantly reduced the serum levels of tumour necrosis factor α (TNFα) (p = 0.009) as well as had the tendency to reduce the levels of macrophage inflammatory protein-1ß (MIP-1ß) (p = 0.07). There were no significant differences in the placebo group. CONCLUSION: Short-term EPP-AF® propolis dry extract 400 mg/day supplementation seems to mitigate inflammation, reducing the plasma levels of TNFα and MIP-1ß in patients with CKD on HD. This study was registered at clinicaltrials.gov (NCT04411758).

Chemical characterization of Brazilian propolis using automated direct thermal desorption-gas chromatography-mass spectrometry.

BACKGROUND: Propolis, produced by honey bees, is used around the world, displaying several corroborated biological activities. Brazil is one of the leading producers of propolis, with a great diversity of types, each with a characteristically chemical fingerprint influenced by the flora of the local region. The secondary metabolite's composition of propolis strongly impacts its biological properties, and its chemical characterization is of great importance for its quality control. Several chromatographic techniques have been applied to characterize propolis, highlighting the extraction of its volatiles and its analysis through gas chromatography. Fourteen Brazilian propolis samples collected in four states, including brown, green and red propolis types, were chemically characterized using the automated direct thermal desorption-gas chromatography-mass spectrometry (DTD-GC-MS). RESULTS: Red propolis type was characterized by acyclic saturated hydrocarbons, fatty alcohols, terpenes, and phenylpropanoids as nonacosane, α-copaene, ß-amyrin acetate, anethole, and 7-O-methylvestitol. Brown propolis presented hydrocarbons, monoterpenes, and sesquiterpenes, as α-pinene and α-bisabolol. Brazilian green propolis presented polycyclic aromatic hydrocarbons and sesquiterpenes, including 1-methyl-octahydroanthracene, 2,5-dimethyl-γ-oxo-benzenebutanoic acid, nerolidol, and spathulenol. Principal component analysis (PCA) was performed, allowing for clustering brown and red propolis types, indicating a divergence with the chemical composition of the green propolis samples. The hierarchical cluster analysis (HCA) allowed the chemical fingerprint of each propolis type to be differentiated. CONCLUSION: Red propolis was characterized by sesquiterpenes, pterocarpans, and isoflavans; brown propolis was characterized by hydrocarbons, aldehydes, and monoterpenes, while green propolis samples were characterized by the presence of polycyclic aromatic hydrocarbons, sesquiterpenes, and naphthalene derivatives. © 2022 Society of Chemical Industry.

Evaluation of in vitro and in vivo Efficacy of a Novel Amphotericin B-Loaded Nanostructured Lipid Carrier in the Treatment of Leishmania braziliensis Infection.

BACKGROUND: Leishmaniasis is a neglected disease, and the current therapeutic arsenal for its treatment is seriously limited by high cost and toxicity. Nanostructured lipid carriers (NLCs) represent a promising approach due to high drug loading capacity, controlled drug release profiles and superior stability. Here, we explore the efficacy of a unique pH-sensitive amphotericin B-loaded NLC (AmB-NLC) in Leishmania braziliensis infection in vitro and in vivo. METHODS AND RESULTS: AmB-NLC was assessed by dynamic light scattering and atomic force microscopy assays. The carrier showed a spherical shape with a nanometric size of 242.0 ± 18.3 nm. Zeta potential was suggestive of high carrier stability (-42.5 ± 1.5 mV), and the NLC showed ~99% drug encapsulation efficiency (EE%). In biological assays, AmB-NLC presented a similar IC50 as free AmB and conventional AmB deoxycholate (AmB-D) (11.7 ± 1.73; 5.3 ± 0.55 and 13 ± 0.57 ng/mL, respectively), while also presenting higher selectivity index and lower toxicity to host cells, with no observed production of nitric oxide or TNF-α by in vitro assay. Confocal microscopy revealed the rapid uptake of AmB-NLC by infected macrophages after 1h, which, in association with more rapid disruption of AmB-NLC at acidic pH levels, may directly affect intracellular parasites. Leishmanicidal effects were evaluated in vivo in BALB/c mice infected in the ear dermis with L. braziliensis and treated with a pentavalent antimonial (Sb5+), liposomal AmB (AmB-L) or AmB-NLC. After 6 weeks of infection, AmB-NLC treatment resulted in smaller ear lesion size in all treated mice, indicating the efficacy of the novel formulation. CONCLUSION: Here, we preliminarily demonstrate the effectiveness of an innovative and cost-effective AmB-NLC formulation in promoting the killing of intracellular L. braziliensis. This novel carrier system could be a promising alternative for the future treatment of cutaneous leishmaniasis.

Evaluation of potential herbal-drug interactions of a standardized propolis extract (EPP-AF®) using an in vivo cocktail approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Propolis has been employed extensively in many cultures since ancient times as antiseptic, wound healing, anti-pyretic and others due to its biological and pharmacological properties, such as immunomodulatory, antitumor, anti-inflammatory, antioxidant, antibacterial, antiviral, antifungal, antiparasite activities. But despite its broad and traditional use, there is little knowledge about its potential interaction with prescription drugs. AIM OF THE STUDY: The main objective of this work was to study the potential herbal-drug interactions (HDIs) of EPP-AF® using an in vivo assay with a cocktail approach. MATERIALS AND METHODS: Subtherapeutic doses of caffeine, losartan, omeprazole, metoprolol, midazolam and fexofenadine were used. Sixteen healthy adult volunteers were investigated before and after exposure to orally administered 125 mg/8 h (375 mg/day) EPP-AF® for 15 days. Pharmacokinetic parameters were calculated based on plasma concentration versus time (AUC) curves. RESULTS: After exposure to EPP-AF®, it was observed decrease in the AUC0-∞ of fexofenadine, caffeine and losartan of approximately 18% (62.20 × 51.00 h.ng/mL), 8% (1085 × 999 h.ng/mL) and 13% (9.01 × 7.86 h.ng/mL), respectively, with all 90% CIs within the equivalence range of 0.80-1.25. On the other hand, omeprazole and midazolam exhibited an increase in AUC0-∞ of, respectively, approximately 18% (18.90 × 22.30 h.ng/mL) and 14% (1.25 × 1.43 h.ng/mL), with the upper bounds of 90% CIs slightly above 1.25. Changes in pharmacokinetics of metoprolol or its metabolite α-hydroxymetoprolol were not statistically significant and their 90% CIs were within the equivalence range of 0.80-1.25. CONCLUSIONS: In conclusion, our study shows that EPP-AF® does not clinically change CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A activities, once, despite statistical significant, the magnitude of the changes in AUC values after EPP-AF® were all below 20% and therefore may be considered safe regarding potential interactions involving these enzymes. Besides, to the best of our knowledge this is the first study to assess potential HDIs with propolis.

Effect of the Copaifera langsdorffii Desf. Leaf Extract on the Ethylene Glycol-Induced Nephrolithiasis in Rats.

The potential of the Copaifera langsdorffii leaves extract to prevent stone formation was analyzed by means of an ethylene glycol (EG) animal model of nephrolithiasis and an in vitro crystallization assay. Different doses of the C. langsdorffii leaves extract were administered to rats treated with EG. Urine biochemical parameters were quantified. CaOx deposits count and analysis of osteopontin expression were conducted on kidneys fixed in formalin. The in vitro assay was performed by turbidimetry. Phytochemical analyses of the extract were accomplished by HPLC-UV-DAD, and several compounds were isolated. C. langsdorffii leaf extract was able to avoid stone formation. The number of deposits was 50.30 ± 31.29 at the higher extract dose, compared to the value of 179.5 ± 45.96 achieved with the EG control. Significantly lower oxalate levels and OPN expression and increased citrate levels were observed after extract administration. In the in vitro assay, the extract diluted the formed crystals. Phytochemical analyses showed that the extract is rich in phenolic compounds that are capable of preventing stone formation. Thus, on the basis of our results, we suggest that the C. langsdorffii leaf extract has potential application in the prevention of kidney stone formation.

Identification of the cell targets important for propolis-induced cell death in Candida albicans.

Candida albicans is the most common fungal pathogen of humans, forming both commensal and opportunistic pathogenic interactions, causing a variety of skin and soft tissue infections in healthy people. In immunocompromised patients C. albicans can result in invasive, systemic infections that are associated with a high incidence of mortality. Propolis is a complex mixture of several resinous substances which are collected from plants by bees. Here, we demonstrated the fungicidal activity of propolis against all three morphogenetic types of C. albicans and that propolis-induced cell death was mediated via metacaspase and Ras signaling. To identify genes that were involved in propolis tolerance, we screened ~800 C. albicans homozygous deletion mutants for decreased tolerance to propolis. Fifty-one mutant strains were identified as being hypersensitive to propolis including seventeen genes involved in cell adhesion, biofilm formation, filamentous growth, phenotypic switching and pathogenesis (HST7, GIN4, VPS34, HOG1, ISW2, SUV3, MDS3, HDA2, KAR3, YHB1, NUP85, CDC10, MNN9, ACE2, FKH2, and SNF5). We validated these results by showing that propolis inhibited the transition from yeast-like to hyphal growth. Propolis was shown to contain compounds that conferred fluorescent properties to C. albicans cells. Moreover, we have shown that a topical pharmaceutical preparation, based upon propolis, was able to control C. albicans infections in a mouse model for vulvovaginal candidiasis. Our results strongly indicate that propolis could be used as a strategy for controlling candidiasis.

Baccharis dracunculifolia, the main source of green propolis, exhibits potent antioxidant activity and prevents oxidative mitochondrial damage.

Baccharis dracunculifolia DC (Asteraceae) is the main botanical source used by honeybees to produce Brazilian green propolis whose hepatoprotective properties have been already described. In this work we investigated the protective effects of the glycolic extract of B. dracunculifolia (GEBd) against oxidative stress in isolated rat liver mitochondria (RLM). The GEBd was prepared by fractionated percolation using propylene glycol as solvent. The total phenols and flavonoids, which are substances with recognized antioxidant action, were quantified in GEBd and the phytochemical analysis was carried out by HPLC. GEBd exhibited significant scavenger activity towards DPPH radicals and superoxide anions in a concentration-dependent manner, and also a Fe2+ chelating activity. GEBd decreased the basal H2O2 generation and the Fe2+- or t-BuOOH-induced ROS production in isolated mitochondria. Lipid oxidation of mitochondrial membranes, protein thiol groups and GSH oxidation were also prevented by GEBd. This shows that B. dracunculifolia exhibit potent antioxidant activity protecting liver mitochondria against oxidative damage and such action probably contribute to the antioxidant and hepatoprotective effects of green propolis.