A reliable validated high-performance liquid chromatography-photodiode array detection method for quantification of terpenes in Copaifera pubiflora, Copaifera trapezifolia, and Copaifera langsdorffii oleoresins.

The Copaifera oleoresins are widely used in folk medicine to treat various diseases. The goal of this study was to develop a validated reverse-phase high-performance liquid chromatography method with photodiode array detection (RP-HPLC-PDA) to quantify eight terpenes: ent-hardwickiic acid, ent-copalic acid, ent-7α-acetoxy hardwickiic acid, ent-16-hydroxy-3,13-clerodadiene-15,18-dioic acid, ent-5,13-labdadiene-15-oic acid, junenol, ent-kaurenoic acid, and 13E-ent-labda-7,13-dien-15-oic acid in the oleoresins of Copaifera pubiflora L. (OCP), Copaifera trapezifolia L. (OCT) and Copaifera langsdorffii L. (OCL). The linearity of the method was confirmed in the range of 20.00-500 µg.mL-1 (r2 > 0.999). The limit of quantification was between 1,05 and 16.89 µg.mL-1. Precision and accuracy ranges were found to be %RSD <0.2 and 96% to 110%, respectively. Based on the obtained results, the developed analytical method is rapid, precise, accurate, and sensitive for quantifying these terpenes in Copaifera's oleoresins.

Copaifera multijuga oleoresin and its constituent diterpene (-)-copalic acid: Genotoxicity and chemoprevention study.

Copaiba oleoresins are used in alternative medicine as anti-inflammatory, antitumoral, and antimicrobial treatments. (-)-Copalic acid (CA) is the major diterpene found in exudates from Copaifera species. We have examined the genotoxicity and the chemopreventive potential of Copaifera multijuga oleoresin (CM) and CA. Genotoxicity assessment was examined with the peripheral blood micronucleus test and the comet assay (male Swiss mouse hepatocytes). In the chemoprevention study, we evaluated the effects of CM and CA on the formation of 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) in male Wistar rat colon. Neither agent caused a significant increase in micronucleus frequency relative to controls, but the highest CM dose tested (400mg/kg b.w.) caused DNA damage in the comet assay. Both agents significantly reduced the frequency of DMH-induced ACF. Both CM and CA suppressed ACF formation and may have a protective effect against colon carcinogenesis.

Copaifera duckei Oleoresin and Its Main Nonvolatile Terpenes: In Vitro Schistosomicidal Properties.

In this article, the in vitro schistosomicidal effects of three Brazilian Copaifera oleoresins (C. duckei, C. langsdorffii, and C. reticulata) are reported. From these botanical sources, the oleoresin of C. duckei (OCd) demonstrated to be the most promising, displaying LC50 values of 75.8, 50.6, and 47.2 µg/ml at 24, 48, and 72 h of incubation, respectively, against adult worms of Schistosoma mansoni, with a selectivity index of 10.26. Therefore, the major compounds from OCd were isolated, and the diterpene, (-)-polyalthic acid (PA), showed to be active (LC50 values of 41.7, 36.2, and 33.4 µg/ml, respectively, at 24, 48, and 72 h of incubation). Moreover, OCd and PA affected the production and development of eggs, and OCd modified the functionality of the tegument of S. mansoni. Possible synergistic and/or additive effects of this balsam were also verified when a mixture of the two of its main compounds (PA and ent-labd-8(17)-en-15,18-dioic acid) in the specific proportion of 3:1 (w/w) was tested. The obtained results indicate that PA should be considered for further investigations against S. mansoni, such as, synergistic (combination with praziquantel (PZQ)) and in vivo studies. It also shows that diterpenes are an important class of natural compounds for the investigation of agents capable of fighting the parasite responsible for human schistosomiasis.

RP-HPLC analysis of manool-rich Salvia officinalis extract and its antimicrobial activity against bacteria associated with dental caries

In this paper we screened the dichloromethane extract from the aerial parts of Salvia officinalis L., Lamiaceae, against a representative panel of microorganisms that cause caries, conducted a bioassay-guided fractionation to establish themselves the most active metabolite (manool) and determined the Salvia officinalis fraction with the manool highest concentration to be used to activate an ingredient in oral care products such as toothpastes and mouthwashes. Both manool and S. officinalis extract showed very promising minimal inhibitory concentration values (between 6.24 and 31.36 µg.ml-1) and time kill curves against the primary causative agents of dental caries (Streptococcus mutans) revealed that, at twice its minimal bactericidal concentration (12.48 µg.ml-1), manool required 6 h to completely kill the bacteria. Salvia officinalis extract at twice its minimal bactericidal concentration (31.36 µg.ml-1 ) needed 12 h. The results achieved with Salvia officinalis extract motivated us to develop and validate an analytical RP-HPLC method to detect and determine manool in this extract. The validation parameters were satisfactorily met and evaluated allows us to consider the developed method suitable for use in different labs. In conclusion, our results evidenced that the manool-rich S. officinalis extract can be considered an analytically validated alternative to develop novel and effective antimicrobial agents against the main bacteria responsible for dental caries.

In vitro antimicrobial activity of plant-derived diterpenes against bovine mastitis bacteria.

We evaluated the antibacterial activity of three diterpenes isolated from natural sources against a panel of microorganisms responsible for bovine mastitis. ent-Copalic acid (CA) was the most active metabolite, with promising MIC values (from 1.56 to 6.25 µg mL-1) against Staphylococcus aureus (ATCC and clinical isolate), Staphylococcus epidermidis, Streptococcus agalactiae, and Streptococcus dysgalactiae. We conducted time-kill assays of CA against S. aureus, a commensal organism considered to be a ubiquitous etiological agent of bovine mastitis in dairy farms worldwide. In the first 12 h, CA only inhibited the growth of the inoculums (bacteriostatic effect), but its bactericidal effect was clearly noted thereafter (between 12 and 24 h). In conclusion, CA should be considered for the control of several Gram-positive bacteria related to bovine mastitis.