In vivo study of anti-inflammatory and antinociceptive activities of Copaifera pubiflora Benth oleoresin.

Copaifera pubiflora Benth oleoresin (CPO) is used as an anti-inflammatory, wound healing, and antimicrobial. This paper reports the cytotoxic, anti-inflammatory, and antinociceptive activities of CPO. CPO (10 mg/kg) did not affect locomotor capacity in the open-field and rotarod tests and was not cytotoxic to CHO-k1, THP-1, and L929 cell lines. It was active in the formalin test at 3 mg/kg by 86 ± 3% and 96 ± 3%, respectively, for the first and second phases. At 10 mg/kg, CPO inhibited 90 ± 7%, the pain in the mechanical hyperalgesia test. In the tail-flick test, CPO at 3 mg/kg affected the tail-flick latencies in mice by 77 ± 20%, which in combination with naloxone was only partially reduced. At 3 mg/kg CPO inhibited 80 ± 12% the carrageenan-induced paw edema, and at 3 mg/kg it reduced by 91 ± 5% the nociception on acetic acid-induced abdominal writhing. Therefore, CPO possesses anti-inflammatory and antinociceptive activities.

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.

Development of a validated ultra-high-performance liquid chromatography tandem mass spectrometry method for determination of acid diterpenes in Copaifera oleoresins.

Species of Copaifera genus (Fabaceae - Caesalpinoiodidaeae) produces an important commercial oleoresin that displays many medicinal properties. Copaifera oleoresins (COR) are composed mainly of a mixture of diterpenes and sequiterpenes, and the main reported acid diterpenes for this genus are kaurenoic, copalic, hardwickiic and polyaltic acids. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for identification and quantification of nine acid diterpenes. The developed method was applied in the analyses of 10 authentic COR samples collected in the North and Southeast of Brazil and six commercial COR samples. Samples preparation consisted of simple dilution of oleoresins in methanol followed by filtration. Validation parameters of the method for nine acid diterpenes were satisfactory: selectivity/specificity was defined by retention time and MS/MS analyses for each analyte; generally all analytical curves presented r2>0.99, Lack-of-fit test not significant and RSD<20% for all concentration levels; limit of detection and limit of quantification were on the scale of nanogram per milliliter; inter- and intra-day precision and accuracy were adequate. Regarding the robustness, the method was sensible to small deliberate variations of temperature and additives to the mobile phase, such as formic acid and ammonium hydroxide. Results of 16 analyzed samples of COR showed qualitative and quantitative differences of acid diterpenes among all samples. The diterpenes ent-kaurenoic acid 1, ent-polyalthic acid 3, ent-copalic acid 5 and, ent-3-ß-acetoxy copalic acid 9 were found with more frequency in COR analyzed samples. Additionally, the content of the acid diterpenes found in 16 Copaifera oleoresin samples was analyzed by Principal Component Analysis (PCA), suggesting a botanical origin for the commercial samples. The developed UPLC method was shown to be reliable for the analysis of acid diterpenes in commercial Copaifera oleoresins.