Development and Validation of a Rapid and Reliable RP-HPLC-PDA Method for the Quantification of Six Diterpenes in Copaifera duckei, Copaifera reticulata and Copaifera multijuga Oleoresins

Abstract

Oleoresins from Copaifera species are extensively used in folk medicine in Brazil, which are employed mainly in the production of cosmetic formulations in Brazil, North America and Europe. Considering the lack of validated analytical methods for the analyses of diterpenes in Copaifera oleoresins, it was developed a validated and reliable reversed-phase high-performance liquid chromatography with photodiode array detection (RP-HPLC-PDA) method for the analysis of six diterpenes, including: (-)-polyalthic acid; (13E)-ent-labda-7, 13-dien-15-oic acid; ent-8(17)-labden-15,18-dioic acid; (-)-copalic acid; (-)-3β-acetoxycopalic acid and (-)-3β-hydroxycopalic acid. These compounds were isolated from C. duckei, C. reticulata and C. multijuga oleoresins by chromatographic means. The analytical curves were linear with regression coefficients (r 2 ) between 0.9903 and 0.9999. The limits of detection (LOD) and quantification (LOQ) values were 0.35 to 3.09 µg mL -1 and 1.05 to 9.36 µg mL -1, respectively. The method also displayed good precision and accuracy. The developed analytical method is reliable and a useful tool for the analysis of Copaifera oleoresin and its products.Keywords: Copaifera spp., oleoresins, diterpenes, RP-HPLC-PDA IntroductionThe Copaifera genus (Leguminoseae) consists of approximately 72 species, from which 16 of them occur only in Brazil, mainly in Northern region in the states of Amazonas, Pará and Ceará.1,2 These trees are popularly known as "copaiba", "copaibeiras" or "pau d'óleo", and the oleoresins obtained from their trunks are extensively used in folk medicine due to their antiinflammatory, analgesic, wound healing, antimicrobial, antileishmanial and antitumoral properties.2,3 Such ethnopharmacological relevance has stimulated several researchers to investigate their biological activities, which corroborated their pharmacological potential. [4][5][6][7][8] In addition to their pharmaceutical properties, these balsams are also largely used as dietary supplement, employed in the production of flavoring agents, and extensively commercialized as crude oil in Brazil. Moreover, these natural products are exported to Europe and North America, to be used mainly in cosmetic formulations. 2,9 Despite the great relevance of these oleoresins for pharmaceutical and cosmetic industries, most of these commercial balsams have not been authenticated, and their chemical profile has not been well established, thus hindering their industrial and biomedical applications, as well as impairing the quality and economic value of such products. 9,10 Chemically, the oleoresins of copaiba are predominantly composed by a mixture of volatile and non-volatile compounds comprising mainly sesquiterpenes and acid diterpenes. Acid diterpenes stands out for a wide range of the biological properties reported for these oleoresins, such as: antimicrobial, antitumoral, antinociceptive and anti-inflammatory, among others. 3,4,6,[11][12][13][14][15] Despite this fact, most of the studies reporting the chemical identification and quan...

An Overview of Biotransformation and Toxicity of Diterpenes

“…Most of the studies with biotransformation of diterpenes aim to obtain new active derivatives. Although diterpenes are common and widespread in natural medicines for oral use in folk medicine [ 47 , 48 ], a lower number of publications focus on the use of biotransformation to investigate their metabolism for safety assessment.…”

Quantification of diterpene acids in Copaiba oleoresin by UHPLC-ELSD and heteronuclear two-dimensional qNMR

“…Copaiba oleoresin was obtained through perforation of the trunk of a C. reticulata tree NMR spectra were recorded using a Bruker Avance III 300 NMR spectrometer operating at 300 MHz for the proton channel and 75.5 MHz for the 13 C channel with a 5 mm PABBO broad band probe with a z gradient unit. Measurements were performed at 298 K, the temperature was calibrated with a methanol-d4 solution.…”

“…The second study by Bardají et al, which investigated only C. reticulata, accepts these findings, whereas the third study introduces ent-kaurenoic (5) acid as another major diterpene acid, but still does not address the unidentified main compound. The compound was finally identified as (13E)-ent-labda-7,13-dien-15-oic (8) acid by Carneiro et al in a study on the quantification of six diterpene acids by HPLC-PDA [13]. Unfortunately, the presented method shows deficits in separation, as ent-copalic acid (6) and (13E)-ent-labda-7,13-dien-15oic (8) are co-eluting, and as it does not take ent-kaurenoic acid (5) into account, which was before declared as one of the most abundant diterpenes.…”

Biological Activities of Two Major Copaiba Diterpenoids and Their Semi-synthetic Derivatives

“…Thus, from then onwards, two forms of polyalthic acid existed in the literature. Except for the last 3 years, where (+)-and (−)-or ent-prefixes were used to differentiate between the two enantiomers (Bardají et al 2016;Borges et al 2016;Carneiro et al 2018;Çiçek et al 2018;Pfeifer Barbosa et al 2019;Senedese et al 2019), only one publication in almost four decades adopted such prefixes (Miyazawa et al 1995). The missing stereochemistry did not represent a problem as long as the studies were dealing with Copaifera, because the ent-configuration is abundant in this genus (Leandro et al 2012).…”