Phytochemical, Antiplasmodial, Cytotoxic and Antimicrobial Evaluation of a Southeast Brazilian Brown Propolis Produced by Apis mellifera Bees.

Seven phenolic compounds (ferulic acid, caffeic acid, 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, 3-hydroxy-4-methoxybenzaldehyde, 3-methoxy-4-hydroxypropiophenone and 1-O,2-O-digalloyl-6-O-trans-p-coumaroyl-ß-D-glucopyranoside), a flavanonol (7-O-methylaromadendrin), two lignans (pinoresinol and matairesinol) and six diterpenic acids/alcohol (19-acetoxy-13-hydroxyabda-8(17),14-diene, totarol, 7-oxodehydroabietic acid, dehydroabietic acid, communic acid and isopimaric acid) were isolated from the hydroalcoholic extract of a Brazilian Brown Propolis and characterized by NMR spectral data analysis. The volatile fraction of brown propolis was characterized by CG-MS, composed mainly of monoterpenes and sesquiterpenes, being the major α-pinene (18.4 %) and ß-pinene (10.3 %). This propolis chemical profile indicates that Pinus spp., Eucalyptus spp. and Araucaria angustifolia might be its primary plants source. The brown propolis displayed significant activity against Plasmodium falciparum D6 and W2 strains with IC50 of 5.3 and 9.7 µg/mL, respectively. The volatile fraction was also active with IC50 of 22.5 and 41.8 µg/mL, respectively. Among the compounds, 1-O,2-O-digalloyl-6-O-trans-p-coumaroyl-ß-D-glucopyranoside showed IC50 of 3.1 and 1.0 µg/mL against D6 and W2 strains, respectively, while communic acid showed an IC50 of 4.0 µg/mL against W2 strain. Cytotoxicity was determined on four tumor cell lines (SK-MEL, KB, BT-549, and SK-OV-3) and two normal renal cell lines (LLC-PK1 and VERO). Matairesinol, 7-O-methylaromadendrin, and isopimaric acid showed an IC50 range of 1.8-0.78 µg/mL, 7.3-100 µg/mL, and 17-18 µg/mL, respectively, against the tumor cell lines but they were not cytotoxic against normal cell lines. The crude extract of brown propolis displayed antimicrobial activity against C. neoformans, methicillin-resistant Staphylococcus aureus, and P. aeruginosa at 29.9 µg/mL, 178.9 µg/mL, and 160.7 µg/mL, respectively. The volatile fraction inhibited the growth of C. neoformans at 53.0 µg/mL. The compounds 3-hydroxy-4-methoxybenzaldehyde, 3-methoxy-4-hydroxypropiophenone and 7-oxodehydroabietic acid were active against C. neoformans, and caffeic and communic acids were active against methicillin-resistant Staphylococcus aureus.

Isolation of diterpenes from Araucaria sp Brazilian brown propolis and development of a validated high-performance liquid chromatography method for its analysis.

Propolis comprises a complex resinous product composed of plant's parts or exudates, pollen, bee wax, and enzymes. Brazilian brown propolis from Araucaria sp displays several biological activities. Considering the lack of validated analytical methods for its analysis, we are reporting the development of a validated high-performance liquid chromatography with photodiode array detector method to analyze Araucaria brown propolis. The crude propolis were extracted and chromatographed, furnishing six main diterpenes. The isolated standards were used to draw the analytical curves, allowing the studies of selectivity, precision, accuracy, recovery, robustness, the determination of limits of detection and limits of quantification. The mobile phase consisted of 0.1% acetic acid in water and acetonitrile, using an octadecylsilane column, 1 mL/min flow rate and detection at 200 or 241 nm. Relative standard deviation values obtained for intra-day and inter-day precision were lower than 4% for all diterpenes. From the five parameters for robustness, wavelength detection and flow rate were the critical ones. Limits of detection and quantification ranged from 0.808 to 10.359 µg/mL and from 2.448 to 31.392 µg/mL, respectively. The recoveries were between 105.03 and 108.13%, with relative standard deviation values around 5.0%. The developed method is precise, sensitive, and reliable for analyzing Araucaria brown propolis.

Antinociceptive and anti-inflammatory activities of Copaifera pubiflora Benth oleoresin and its major metabolite ent-hardwickiic acid.

ETHNOPHARMACOLOGICAL RELEVANCE: Copaifera species folkloric names are "copaíbas, copaibeiras, copaívas or oil stick", which are widely used in Brazilian folk medicine. Among all ethnopharmacological applications described for Copaifera spp oleoresins, their anti-inflammatory effect stands out. However, the knowledge of anti-inflammatory and antinociceptive properties of Copaifera pubiflora Benth is scarce. AIM OF THE STUDY: To investigate the cytotoxic, anti-inflammatory, and antinociceptive activities of C. pubiflora oleoresin (CPO), and its major compound ent-hardwickiic acid (HA). MATERIAL AND METHODS: The phosphatase assay was used to evaluate the cytotoxicity of CPO and HA in three different cell lines. CPO and HA doses of 1, 3, and 10 mg/kg were employed in the biological assays. The assessment of motor activity was performed using open-field and rotarod tests. Anti-inflammatory activity of CPO and HA was assessed through luciferase assay, measurement of INF-γ, IL-1ß, IL-6, IL-10, and TNF-α in a multi-spot system with the immortalized cell line THP-1, zymosan-induced arthritis, and carrageenan-induced paw edema. Acetic acid-induced abdominal writhing and formalin tests were undertaken to evaluate the antinociceptive potential of CPO and HA. In addition, the evaluation using carrageenan was performed to investigate the effect of CPO in pain intensity to a mechanical stimulus (mechanical hyperalgesia), using the von Frey filaments. A tail-flick test was used to evaluate possible central CPO and HA actions. RESULTS: In the cytotoxicity evaluation, CPO and HA were not cytotoxic to the cell lines tested. CPO and HA (10 mg/kg) did not affect animals' locomotor capacity in both open-field and rotarod tests. In the luciferase assay, CPO and HA significantly reduced luciferase activity (p < 0.05). This reduction indicates a decrease in NF-κB activity. HA and CPO decreased INF-γ, IL-1ß, IL-6, IL-10, and TNF-α at 24 and 72 h in the multi-spot system. In zymosan-induced arthritis, CPO and HA decreased the number of neutrophils in the joint of arthritic mice and the number of total leukocytes (p < 0.05). In experimental arthritis HA significantly decreased joint swelling (p < 0.05). CPO and HA also increased the mechanical threshold during experimental arthritis. HA and CPO significantly inhibited the carrageenan-induced paw edema, being the doses of 10 mg/kg the most effective, registering maximum inhibitions of 58 ± 8% and 76 ± 6% respectively, p < 0.05. CPO and HA reduced the nociceptive behavior in both phases of formalin at all tested doses. The highest doses tested displayed inhibitions of 87 ± 1% and 72 ± 4%, respectively, p < 0.001, in the first phase, and 87 ± 1% and 81 ± 2%, respectively, p < 0.001, in the second phase. Oral treatment of CPO and HA (1, 3, 10 mg/kg) significantly reduced the nociceptive response in acetic acid-induced abdominal writhings, and the 10 mg/kg dose was the most effective with maximum inhibitions of 86 ± 2% and 82 ± 1%, respectively, p < 0.001. Both HA and CPO significantly decreased the intensity of mechanical inflammatory hyper-nociception on carrageenan-induced hyperalgesia at all tested doses, and 10 mg/kg was the most effective dose with maximum inhibitions of 73 ± 5% and 74 ± 7%, respectively, p < 0.05.CPO increased the tail-flick latencies in mice, and concomitant administration of naloxone partially reduced its effect. CONCLUSIONS: CPO and HA may inhibit the production of inflammatory cytokines by suppressing the NF-κB signaling pathway, resulting in anti-inflammatory and antinociceptive activities.

Brazilian southeast brown propolis: gas chromatography method development for its volatile oil analysis, its antimicrobial and leishmanicidal activities evaluation.

INTRODUCTION: Propolis is widely used in folk medicine, and many factors can affect its chemical composition, including abiotic factors that can influence plants and bees. Therefore, analytical methods are powerful techniques in the quality control of such products. OBJECTIVE: Develop and validate an analytical method for quantifying volatile compounds in Brazilian brown propolis, and evaluate its biological activities. METHODS: A gas chromatography flame ionisation detector (GC-FID) analytical method was validated, attending the parameters of international validation guidelines as ANVISA 2017 and ICH 2005, for quantification of compounds present in volatile oils from propolis. Evaluation of cytotoxic, antimicrobial, and leishmanicidal activities of the oil. RESULTS: The compounds 1,8-cineole, terpinen-4-ol, α-copaene, ß-caryophyllene, γ-muurolene, nerolidol, spathulenol, and γ-palmitolactone were isolated from the volatile fraction of a Brazilian brown propolis and used in the method validation. All the validation parameters of the method were satisfactory. The volatile fraction displayed a significant leishmanicidal activity, with half maximal inhibition concentration (IC50 ) = 21.3 µg/mL against amastigote forms and IC50 = 25.1 µg/mL against promastigote forms of Leishmania amazonensis. The oil also displayed an antibacterial effect by inhibiting the growth of Streptococcus mutans and Staphylococcus aureus at 25 µg/mL and 50 µg/mL, respectively, but it was not cytotoxic against AGP-01, He-La and CHO-K1cell lines, with IC50 > 100 µg/mL. CONCLUSION: The GC-FID method can be a useful tool in the quality control of propolis material. The southeast brown propolis showed a high chemical complexity in its volatile fraction, which displayed leishmanicidal activity and bactericidal activity.

Artepillin C as an outstanding phenolic compound of Brazilian green propolis for disease treatment: A review on pharmacological aspects.

Propolis is a viscous resin consisting of plant material (shoots, flowers, and plant exudates), salivary secretions and waxes produced by Apis mellifera bees. Its popular use aroused the interests of scientific research, which proved to be a potential source of various bioactive substances. The chemical composition of propolis depends on several factors, such as the different types of plant sources collected by bees, geographic origin, and the time of year in which they are produced, but it is known that phenolic represent the main bioactive constituents of propolis. Baccharis dracunculifolia DC (Asteraceae) is the most important botanical source of propolis and a native to southeastern Brazil. It is widely known as the green propolis because of its deep green color. One of its major phenolic acids is artepillin C (Art-C), a diprenyl-p-hydroxycinnamic acid derivative. This review aims to provide a comprehensive summary of the pharmacological effects of Art-C. The limited number of publications on this topic over the past two decades have been collected from databases and summarized. Numerous biological activities have been described for the Art-C, such as gastroprotective, anti-inflammatory, antimicrobial, antioxidant, antitumor. This article describes aspects of occurrence, synthesis, biological activities and pharmacokinetic approaches.

Use of spinning band distillation equipment for fractionation of volatile compounds of Copaifera oleoresins for developing a validated gas chromatographic method and evaluating antimicrobial activity.

Copaifera is a tree that produces an oleoresin that has great historical and economic importance. These oleoresins display several pharmacological properties, such as anti-inflammatory and antimicrobial, among others. The commercialization of Copaifera oleoresin occurs, in many cases, without any quality control, which facilitates its adulteration. Validated analytical methods can provide a safe quality control. In this work, the 800 Automatic Spinning Band Distillation equipment was used to perform the fractionation of the volatile oils obtained by hydrodistillation of Copaifera multijuga, C. paupera, C. Publifora and C. langsdorffii, aiming to isolate and purify the major compounds present in these oils. For purification, classical column chromatography was used, furnishing six isolated sesquiterpenes. The sesquiterpenes were used as standards in the development and validation of the method by GC-FID. The evaluated parameters were selectivity, linearity, precision, accuracy and robustness and they are all in accordance with ANVISA and International Conference on Harmonization guidelines. The developed method is reliable for the quantification of sesquiterpenes in Copaifera oleoresins. Both volatile oils and isolated sesquiterpenes had their minimum inhibitory concentration determined against strains of Gram-negative and Gram-positive bacteria and yeasts. Copaifera langsdorffi oleoresin was the only one active against all of the evaluated microorganisms, displaying good antimicrobial potential.