Copaifera of the Neotropics: A Review of the Phytochemistry and Pharmacology

The oleoresin of Copaifera trees has been widely used as a traditional medicine in Neotropical regions for thousands of years and remains a popular treatment for a variety of ailments. The copaiba resins are generally composed of a volatile oil made up largely of sesquiterpene hydrocarbons, such as β-caryophyllene, α-copaene, β-elemene, α-humulene, and germacrene D. In addition, the oleoresin is also made up of several biologically active diterpene acids, including copalic acid, kaurenoic acid, alepterolic acid, and polyalthic acid. This review presents a summary of the ecology and distribution of Copaifera species, the traditional uses, the biological activities, and the phytochemistry of copaiba oleoresins. In addition, several biomolecular targets relevant to the bioactivities have been implicated by molecular docking methods.


Introduction to the Genus Copaifera
The copaiba trees belong to the genus Copaifera, family Fabaceae, and subfamily Caesalpinoideae. The genus was described the first time by Marcgraf and Piso in 1638, who employed the name "Copaiba" without designating the species [1]. In 1760, Nicolaus Joseph Von Jacquin described the species Copaiva officinalis in the work Enumeratio Systematica Plantarum [2]. Afterwards, in the year 1764, Carl von Linnaeus did a more detailed study of the genus in the work Species Plantarum, in which he described the type species Copaifera officinalis (Jacq.) L. [3]. There are more than 70 Copaifera species distributed throughout the world, with widespread occurrence in Central and South America; there are also four species found in Africa and one species found on the island of Borneo, situated in the Pacific Ocean [4]. Brazil is the country with the greatest biodiversity of Copaifera with 26 species and 8 varieties [5].
The vernacular name copaíba probably originated from the Tupi-Guarani and alludes to the names used by indigenous peoples, copaíva and copahu (kupa'iwa and kupa'u, respectively), which refers to the tree exudate, in reference to the oil stored in its interior [6]. Sixteenth-century records produced by chroniclers during the Brazilian colonization report the widespread use of copaiba oil among the natives as anti-inflammatory and healing agents, and also for esoteric purposes, such as aphrodisiac and contraceptive [4,6,7]. This natural product is known and valued to the present day, mainly in the Amazon region, where the rural population has little access to industrialized pharmaceutical products and public health care [6,8].
The copaiba trees have shrub or arboreal habits, can reach up to 40 m height and 4 m diameter at breast height (dbh), have slow growth, and can live up to 400 years [6]. Their cylindrical trunks contain intercellular secretory channels arranged in bands of marginal axial parenchyma, the lumen from secretory cells is formed schizogenously, and the oleoresin is synthesized in parenchyma cells of the canal. The species have alternate leaves, which are pinnate with 2-12 pairs of leaflets (opposite, alternate, or subopposite), usually glabrous, and may have translucent points and glands at the base of the marginal vein; they have small and interpetiolar stipules and are generally deciduous. The inflorescences are alternate panicles and the flower buds are protected by small bracts; they have small flowers, numerous and sessile, which are monoclamids with a tetramer chalice that forms short tubes and contains internally hirsute sepals. The androecium holds 10 free stamens, glabrous fillets, and oblong and rimose anther; and the gynoecium presents a sessile ovary with two elongate ovules, filiform style, and globular and papillary stigma. The fruits are bivalved, dehiscent, laterally compressed, and monospermic. The seed is a pendulum, oblong-globose, covered by abundant white or yellow aril, and lacking endosperm [1,[9][10][11].
Although the Copaifera genus has been extensively studied taxonomically, there are still difficulties in identifying some species, mainly due to their intricate floral morphology and absence of reproductive structures in the samples studied. With regard to the Amazonian species, the scarcity of field information and illustrations of specimens comprise the main limitations for botanical descriptions of the group. These taxonomic problems have restricted the advance of chemical and pharmacological research, limited the industrial and rational uses of resin oils and wood, and have also hampered the development of projects, plans for sustainable management, and conservation of commercially targeted species [9,12].
The main economic contributions of Copaifera species have been wood and oleoresins. Among Copaifera species that are used in the production of oleoresins, C. reticulata is the most frequent, representing 70% of the production [6]. Copaiba oleoresin is one of the most important renewable natural remedies for the indigenous people from the Amazon region and its use is widely diffused due its various pharmacological properties [13]. The oleoresin is a transparent, colored liquid with variable viscosity, and is constituted by a nonvolatile fraction composed of diterpenes and a volatile fraction composed of sesquiterpenes [14,15]. Its chemical profile may vary according to species, seasonal and climatic characteristics of the environment, soil type and composition, and rainfall index. Biotic pressures, such as insect predation and pathogen infection, also cause differences in oleoresin composition [16,17]. The extraction of copaiba oil is done through the perforation of the trunk with a punch, and the resin is collected with the help of a polyvinyl chloride (PVC) pipe, through which the oil flows and is then stored. This practice is mainly done by plant extraction; therefore, the product of several trees is often mixed, resulting in an additional obstacle to the botanical identity of the copaiba trees. In addition, the lack of parameters to characterize the oil and to perform quality control of the botanical drug also constitutes an obstacle for the registration and exportation of herbal products containing copaiba [18,19].

Ecology and Distribution of Copaifera
The genus Copaifera is native to tropical regions of Latin America, an area of great species diversity [1]. Distributed widely in the Americas, stretching from Mexico to northern Argentina, the genus also occurs in West Africa and Asia [20]. The greatest richness of species occurs in Brazil, where they are distributed from the north to the south of the country. The most common species are C. multijuga Hayne, which is found in the Amazonas, Pará and Rondônia states; C. reticulata Ducke that occurs in Amapá, Pará and Roraima;, and C. langsdorffii Desf., which can occur from the northern to southern regions of Brazil [5]. Other species have more restricted distribution, such as C. guyanensis Desf. (Amazonas), C. majorina Dwyer (Bahia), C. cearensis Huber ex Ducke (Ceará, Bahia, Piauí and Rio de Janeiro), C. elliptica Mart. (Goias and Mato Grosso), C. paupera (Herzog) Dwyer (Acre), and C. lucens Dwyer (Bahia, Espírito Santo, Minas Gerais, Rio de Janeiro, São Paulo) [5]. Although many species of Copaifera have wide occurrence within the Brazilian territory, and may occur in different phytogeographic domains (e.g., C. langsdorffii), some feature endemism, such as C. trapezifolia Hayne, which occurs in an extremely disturbed region of the Atlantic rainforest, of which only 11.6% of the botanical identification of species, floristic inventory of copaiba populations, and ecological studies on its ecosystems are indispensable for the sustainable and rational use of this resource [35,36].

Medicinal Uses
In Pará state (Amazon region, Brazil), people of all ages and social classes consider copaiba one of the most important natural remedies from the Amazon region. Several parts and preparations of the plant are used in folk medicine [24]. The oleoresin or bark decoction is used as an anti-inflammatory and contraceptive by native people from the Brazilian Amazon. The topical application of oil on the skin serves to heal wounds. It is used in massages on the head to cure paralysis, pains, and convulsions. In Amapá state, it is recommended to soak a cotton ball in oil and place on tumors, ulcers, or hives. The daily intake of two drops of oil mixed with one tablespoon of honey is indicated for inflammation, syphilis, bronchitis, and cough [6,37,38]. In Venezuela, the oil is used to prepare a patch that is applied to heal ulcers and wounds, and the decoction of the bark in the form of a bath is used to combat rheumatism, to wash infected wounds such as dog bites, and to use as an anti-tetanus [37,38]. A tea from the seeds is also used as a purgative and for treatment of asthma. In northern Brazil, the practice of "embrocation" (applying oil directly to the throat) is common to treat throat infections [39]. In Belém, the "garrafada"-an infusion of the bark sold in bottles-is currently used as a substitute for the oleoresin due to the difficulty in obtaining the oil in the city [38].
Studies have shown that the ingestion of high doses of copaiba oil can cause adverse side effects, such as gastrointestinal irritation, sialorrhea, and central nervous system depression. A dose of 10 g may cause symptoms of intolerance, nausea, vomiting, colic and diarrhea, and exanthema. Prolonged use may cause kidney damage and topical reactions in susceptible individuals [39,42]. Thus, the advance in pharmacological and quality control studies of copaiba formulations sold at herbal markets is indispensable for the safe use of this plant drug.

Human Nutrition
Copaiba oil was approved in the United States as a food additive and is used in small amounts as a flavoring agent in foods and beverages [43].

Cosmetic Uses
The species of Copaifera are intensively pursued for inclusion in the cosmetics market due to their therapeutic properties and fragrant value of their oils [44]. Copaiba oil is currently used in the cosmetic industry as a fixative for perfumes and perfuming soaps [38]. As an emollient, bactericidal, and anti-inflammatory agent, copaiba oil is used in the production of soaps, lotions, creams and moisturizers, bath foams, shampoos, and hair conditioners [6,24]. In addition, it aids in the treatment of dandruff and acne [38,45]. Despite its fragrant value, little information regarding its odorant potential is available in the literature [44].

Fuel
As a renewable source of hydrocarbons, the use of copaiba oil as an ecologically clean fuel has been evaluated. Experimental plantations were started in the early 1980s near Manaus, Brazil to test its viability as an alternative energy source to fossil fuels [7]. For potential use as fuel, a combination with diesel oil in a ratio of 9:1 (diesel oil to copaiba) has been recommended [6]. Various reports indicate that the liquid can be poured directly into the fuel tank of a diesel-powered car and the vehicle will run normally, with a bluish exhaust smoke being the only noticeable difference [46]. Traditionally, the oil is used in lamps as fuel for lighting [24].

Wood
The copaiba trees are considered hardwoods with high demand due to their properties of strength, as well as insect and xylophagous fungi repellency. The wood is saturated with oil and resin and has been used in both shipbuilding and civil construction, especially in the manufacture of steam caves, pool cues, and decorative and furniture coverings. It is also used in the preparation of lumbers, rafters, door and window frames, and boards in general, including for agricultural implements, general carpentry, flooring furniture, coatings, lamination, plywood sheets. The wood has a high content of lignin and is very good for the production of alcohol and charcoal. C. langsdorffii has traditionally been exploited extensively for charcoal in the Cariri Region, south of Ceará [24,47].

Veterinary Uses
In southern Pará state, farmers have used copaiba oil to prevent foot-and-mouth infection in cattle. The oil is poured on the floor next to the salt lick so that when cattle approach to eat salt, they step in oil soaking their feet [24]. When wounded, some animals lick and rub their bodies in the oil that flows from the trees [24].

Other Uses
Hunters often hunt under the copaiba tree during fruiting because the seeds and oil attract animals [24]. The oleoresin is used in the photographic industry to improve image clarity in areas of low contrast and resolution. The resin has also been used in paper making, as an additive for butadiene in the production of synthetic rubber, as a source of a chiral substrate in the synthesis of biomarkers of sediment and oil residues, and as fixative in the manufacture of varnish, perfume, and paints used in the painting of porcelain, fabrics, and for dying cotton yarn [6,24,38].
A number of parasitic protozoal proteins have been identified as potential targets for antiparasitic chemotherapy [113]. In conjunction with this review, we have examined the potential parasitic targets of Copaifera diterpenoids using molecular docking. It is currently not known what biomolecular targets from Leishmania or Trypanosoma may be responsible for the antiprotozoal activities of copaiba. The Copaifera diterpenoids (Figures 1-3) were screened, in silico, against Leishmania drug targets [114][115][116] and Trypanosoma cruzi protein targets [117] using Molegro Virtual Docker v. 6.0.1 as previously described [114][115][116][117]. The docking energies are summarized in Tables 4 and 5.
Leishmania major methionyl-tRNA synthetase was another Leishmania protein target with good docking energies. Although the docking energies with this protein were excellent (average E dock = −106.9 kJ/mol), they are much poorer than the docking energy of the normal substrate, methionyl adenylate (E dock = −168.1 kJ/mol). Similarly, the T. cruzi target protein with the best docking was UDP-galactose mutase (average E dock = −104.5 kJ/mol), but the normal substrate and co-crystallized ligand, uridine diphosphate (UDP), had a much superior docking energy (E dock = −232.8 kJ/mol). Likewise, L. major UDP-glucose pyrophosphorylase showed an average docking energy of −99.9 kJ/mol, which was much worse than UDP itself (E dock = −145.9 kJ/mol). The diterpenoids showed good docking to T. cruzi spermidine synthase, with an average docking energy of −96.8 kJ/mol; however, these are much worse than the docking energy of the co-crystallized ligand, S-adenosyl methionine, with a docking energy of −133.0 kJ/mol. Thus, although they exhibited good docking properties, it is unlikely that Copaifera diterpenoids can compete with the normal substrate ligands for these proteins.  Copaifera diterpenoids showed excellent docking to L. mexicana pyruvate kinase (average E dock = −103.4 kJ/mol), much better than the normal substrate, phosphoenolpyruvate (E dock = −59.8 kJ/mol). Docking energies with T. cruzi pyruvate kinase were not as impressive (average −80.3 kJ/mol), but still better than phosphoenolpyruvate (E dock = −48.6 kJ/mol) and comparable to the TcPYK inhibitor, ponceau S (E dock = −83.6 kJ/mol). Parasite pyruvate kinases can be expected to be target proteins for Copaifera diterpenoids.
Sterols are the normal substrates for sterol 14α-demethylase (CYP51), and triterpenoids are expected to also target this protein as inhibitors [118]. Nevertheless, Copaifera diterpenoids showed docking energies that may compete with normal sterols for these protein targets. L. infantum CYP51 had an average docking energy with the diterpenoids of −90.2 kJ/mol, which was generally not as good as a normal sterol substrate (obtusifoliol, E dock = −104.4 kJ/mol), but comparable to the known LinfCYP51 inhibitor fluconazole (E dock = −87.5 kJ/mol). Likewise, T. cruzi CYP51 had an average diterpenoid docking energy of −89.5 kJ/mol, but substrate (obtusifoliol) docking of −105.6 kJ/mol, and fluconazole docking energy of −90.9 kJ/mol.
Copaifera diterpenoids generally showed weak docking energies against the parasite cysteine proteases, L. donovani cathepsin B, L. major cathepsin B, or cruzain. This docking behavior of diterpenoids with Leishmania cathepsin B [114] and cruzain [117] was previously observed. Leishmania donovani and T. cruzi cyclophilins also showed weak docking energies.
Although Copaifera diterpenoids showed only weak docking to parasite glyceraldehyde-3-phosphate dehydrogenases, they may still target these proteins. LmexGAPDH had an average E dock of −73.0 kJ/mol and TcGAPDH had an average E dock of −70.3 kJ/mol, but these docking energies are better than the docking energies of the normal substrate, glyceraldehyde-3-phosphate (E dock = −58.9 and −52.6 kJ/mol, respectively).
In order to provide some insight into the mechanisms of activity, a virtual screening of copaiba diterpenoids has been carried out against several bacterial protein targets, including peptide deformylase, DNA gyrase, topoisomerase IV, UDP-galactopyranose mutase, protein tyrosine phosphatase, cytochrome P450 CYP 121, and nicotinamide adenine dinucleotide (NAD + )-dependent DNA ligase [119] (see Table 6). The best bacterial target for copalic acid was Mycobacterium tuberculosis DNA gyrase B (PDB 3ZKD) with a docking energy (E dock ) of −105.7 kJ/mol. The protein with the best docking energy with kaurenoic acid was S. pneumoniae peptide deformylase (PDB 2AIE, E dock = −89.7 kJ/mol). 3α-Alepterolic acid acetate was the best docking ligand to Escherichia coli topoisomerase IV (PDB 1S16) and M. tuberculosis DNAGyrB (PDB 3ZKD) with docking energies of −118.8 and −118.3 kJ/mol, respectively. 3β-Alepterolic acid acetate also showed excellent docking to these two proteins with docking energies of −117.1 and −117.3 kJ/mol, respectively. The best bacterial target for ent-polyalthic acid was M. tuberculosis protein tyrosine phosphatase (PDB 2OZ5, E dock = −107.2 kJ/mol). The copaiba diterpenoid ligand with the best docking properties was 7α-acetoxyhardwickiic acid with S. aureus peptide deformylase (PDB 3U7M, E dock = −120.6 kJ/mol).

Antiproliferative Activity of Copaiba
Copaiba oleoresins have exhibited both in vitro and in vivo antiproliferative activities (Table 3). Copaifera reticulata oleoresin, for example, has shown in vitro cytotoxic activity against GM07492-A human lung fibroblast cells with an IC 50 of 51.85 µg/mL [68]. The oleoresin of C. multijuga has shown in vitro cytotoxic activity against B16F10 murine melanoma cells with an IC 50 of 457 µg/mL [57]. Furthermore, in a mouse model of lung metastasis and tumor growth, oral administration of C. multijuga oleoresin reduced tumor growth, tumor mass, and number of lung nodules after inoculation of B16F10 tumor cells [57]. Likewise, C. multijuga oleoresin, in doses varying between 100 and 200 mg/kg, showed antineoplastic properties against Ehrlich ascetic tumors and solid tumors in an in vivo mouse model [98]. On the other hand, C. officinalis oleoresin actually stimulated growth of Walker 256 carcinoma by 70% in an in vivo rat model [101].

Anti-Inflammatory Activity of Copaiba
Inflammation is the biological response of body tissues to detrimental stimuli, such as pathogenic microorganisms, chemical or physical irritants, or injury. Inflammation is manifested by redness, swelling, heat, and sometimes pain. While acute inflammation is a normal part of the healing process, chronic inflammation often plays a role in chronic diseases such as osteoarthritis, lupus, and inflammatory bowel disease, and can be problematic. Several copaiba oleoresins have shown anti-inflammatory activity, including C. cearensis [13], C. duckei [84], C. langsdorffii [50,91], C. multijuga [13,58,61,100], C. officinalis [62], and C. reticulata [13] (Table 3).
The immune response is a complex cascade of interacting cytokines and reactions, and there are several biomolecular targets important in treating chronic inflammation. We have carried out virtual screening of copaiba diterpenoids against soluble epoxide hydrolase (EPHX2), fibroblast collagenase, phospholipase A2 (PLA2), 5-lipoxygenase, inducible nitric oxide synthase, phosphoinositide 3-kinase, interleukin-1 receptor-associated kinase 4, glutathione S-transferase ω-1, cyclooxygenase-1, cyclooxygenase-2, c-Jun N-terminal kinase, nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), inhibitor of κB kinase β, NF-κB essential modulator, lipid binding protein MD-2, myeloperoxidase, p38 mitogen-activated protein kinase, peroxisome proliferator-activated receptor γ, and cAMP-specific 3 ,5 -cyclic phosphodiesterase 4D (Table 8). The overall best target proteins were murine soluble epoxide hydrolase and murine phospholipase A2, with average docking energies of −108.3 and −100.0 kJ/mol. Secretory phospholipase A2 and cytosolic phospholipase A2 are both targets for anti-inflammatory drug development [129]. Soluble epoxide hydrolase has been identified as a molecular target not only for inflammatory diseases, but also as a target for neurodegenerative diseases and for treatment of pain [130]. Thus, targeting EPHX2 and/or PLA2 by copaiba diterpenoids may explain the anti-inflammatory activities of copaiba oleoresins.

Conclusions
The oleoresins from Copaifera species (copaiba) have been used by native peoples of the Amazon region for thousands of years. These materials have shown remarkable biological activities, including antibacterial, antiparasitic, antineoplastic, and anti-inflammatory activities. Copaiba resins have been distilled to give essential oils that are largely composed of sesquiterpenoids, particularly β-caryophyllene. The resins are also composed of diterpene acids, which are responsible for many of the observed biological activities. Molecular docking of copaiba diterpene acids with documented protein targets has revealed potential mechanisms of activity for these bioactive constituents. Future research to validate the molecular mechanisms of copaiba diterpenoids is encouraged.

Conflicts of Interest:
The authors declare no conflict of interest.