{"paper_id":"8b6f16b9-6b52-4eb0-abde-b54e30cea713","body_text":"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 ].\nThe 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 ].\nThe 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 ].\nAlthough 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 ].\nThe 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 ].\n\nThe 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 natural vegetation cover remains [ 21 ]. Thus, morphological, physiological, and ecological studies are highly relevant for the preservation of species and their natural environment [ 11 ]. A study conducted in the Minas Gerais state on the geographical distribution and environmental characteristics of arboreal species showed that  C. langsdorffii  has wide occurrence throughout the whole state, where latosol type soil predominates, but additionally has a preference for ustic soils (62%) [ 22 ].\nIn relation to the ecological group, copaíba are classified as long-living, late secondary, and climax tree species, demanding of light but tolerant to shade [ 23 ]. They are considered generalists because they are adapted to a wide variety of environments. They can occur in floodplains, riparian forest, and streams of the Amazon basin and the forests of the Cerrado in the center of Brazil [ 24 ].  C. langsdorffii , for example, has great ecological plasticity, occurring in several biomes, such as Cerrado, Atlantic Forest, Caatinga, and Amazon rainforest [ 23 ].  Copaifera  species have great plasticity in relation to edaphic conditions; they occur in areas with fertile soil and well-drained soil and in areas with very poor acidic soils, such as Cerrado fields. They grow well on sandy and clayey soils and generally occupy the forest canopy [ 25 , 26 ].\nPhenological studies on  Copaifera  are important for the rational use of the species and for the preparation of management plans [ 27 ]. The reproduction of copaibas occurs from the fifth year of growth after planting in a climax forest ecosystem [ 6 ].  C. multijuga , commonly found in the Amazon, blooms in the rainy season—between the months of December–April—and fructifies between April and July [ 27 ]. Blooming of  C. reticulata  occurs from January to March, with fruiting from March to August, lasting into October [ 26 , 28 ].  C. langsdorffii , observed in the Tijuca Forest, Rio de Janeiro, blooms between March and April and fructifies between August and September. Another survey carried out near Campinas, São Paulo state, showed that flowering of  C. langsdorffii  occurs in the middle of the rainy season (December–February), with development of fruit during the dry season (April–September) [ 29 , 30 ]. The phenophases of  C. officinalis  were monitored in the municipality of Boa Vista (Roraima state, Brazil), and showed that the flowering of the species occurs between the months of September and November and the fruiting from November to March. Depending on the stage of fruit ripening, the dehiscence can begin in January, in which the seeds enveloped by the aril are exposed, allowing for their dispersal [ 28 ].\nCopaifera  is a hermaphrodite plant of mixed reproduction with a predominance of allogamy. The trees are generally bee pollinated (melittophily), and  Apis mellifera  and  Trigona  spp. are its main pollinating agents [ 25 ].  C. langsdorffii  has high fecundity, producing large quantity of fruits in a short period of time. Its seeds have low nutritional value, mainly composed of carbohydrates, but can attract a wide variety of animals with a general diet [ 30 ]. The dispersion of the copaiba seeds occurs mainly in zoocoric and barocoric forms [ 27 , 28 ]. Some vertebrates, such as birds and mammals, have been observed visiting the fruits of  Copaifera  [ 31 ]. Its seeds have morphological characteristics that fit the ornithocoria syndrome, mainly because they are black with colored, fleshy arils, which, after being swallowed, can be regurgitated intact and remain viable for germination [ 32 ]. A study revealed that 10 species of Passeriformes, such as  Ramphastos toco ,  Cyanocorax cristatellus , and  Turdus rufiventris , visited the fruits of  C. langsdorffii . Likewise, monkeys of the species  Eriodes arachnoides  and  Cebus paella  also eat the fruits of  C. langsdorffii  [ 31 ]. Copaiba seeds may also present hydrocoric dispersion due to their frequent occurrence near waterways [ 25 ]. Copaiba seeds are of conventional behavior and may be conserved in the long term ex situ, with dormancy due to the deposition of coumarin in the tegument, and its germination is of the epigene type [ 25 ]. A tree can produce from 2 to 3 kg of seeds [ 33 ].\nThe population density of copaiba trees in an area is usually very low. It is possible to find only one tree every 5 ha, but they may occur in densities of one to two trees per hectare. The production of oleoresin by species is fairly variable and can be influenced by genetic differences among species, habitat, soil, and intensity of exploitation [ 34 ]. The production of oleoresin per tree ranges from 100 mL to 60 L per year. In addition, not all trees produce oil [ 24 ]. Therefore, detailed investigations regarding extraction methods and equipment that do not harm the plant, correlation of genetic data to 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 ].\n\nIn 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 ].\nCopaiba has a wide range of ethnopharmacological indications, including for the treatment of: cystitis, urinary incontinence, gonorrhea, and syphilis; respiratory ailments, including bronchitis, strep throat, hemoptysis, pneumonia, and sinusitis; infections in the skin and mucosa, such as dermatitis, eczema, psoriasis, and wounds; ulcers and lesions of the uterus; leishmaniasis and leucorrhea; anemia; headaches; and snake bites. It is also used for its aphrodisiac, stimulant, anti-inflammatory, antiseptic, anti-tetanus, antirheumatic, antiherpetic, anthelminthic, anticancer, antitumor (prostate tumors), and antiparalytic properties [ 4 , 6 , 26 , 38 , 40 ].  Copaifera  species are used by people of Igarapé Miri (Pará state) for healing wounds [ 41 ].\nStudies 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.\nCopaiba 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 ].\nThe 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 ].\nAs 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 ].\nThe 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 ].\nIn 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 ].\nHunters 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 ].\n\nThe major components of the essential oils from  Copaifera  species are summarized in  Table 1 . In general, copaiba oils derived from  Copaifera  oleoresins are rich in sesquiterpene hydrocarbons and often dominated by β-caryophyllene [ 15 ]. Some copaiba oils, however, also show significant concentrations of diterpene acids, which are generally analyzed as their methyl esters [ 15 ]. A perusal of internet sources of copaiba oil suggests that the most important commercial sources of copaiba oil are  C. langsdorffii ,  C. officinalis , and  C. reticulata , and the most prized copaiba oils are rich in β-caryophyllene. The oleoresin essential oils from these three  Copaifera  species can have as much as 33% ( C. langsdorffii ), 87% ( C. officinalis ), and 68% ( C. reticulata ) β-caryophyllene (see  Table 1 ).\n\nThe oleoresins of several  Copaifera  species have been shown to be rich sources of clerodane, kaurane, and labdane triterpenoids ( Figure 1 ,  Figure 2  and  Figure 3 ,  Table 2 ). In particular,  C. langsdorffii  resin is composed of biologically active copalic acid [ 70 , 71 ] and kaurenoic acid [ 72 , 73 , 74 ].  C. multijuga  [ 74 ] and  C. paupera  [ 75 ] resins are also good sources of copalic acid.\n\nCopaifera  oleoresins have shown remarkable biological activities, many of which have been attributed to diterpenoid acids (see  Table 3 ). Generally,  Copaifera  oleoresins and their diterpenoid constituents have shown antibacterial, anti-inflammatory, antileishmanial, antiproliferative, antitrypanosomal, and wound-healing activities.\nSeveral  Copaifera  oleoresin oils have shown in vitro antiparasitic activity against  Leishmania amazonensis  promastigotes, including  C. cearensis ,  C. langsdorffii ,  C. lucens ,  C. martii ,  C. multijuga ,  C. officinalis ,  C. paupera , and  C. reticulata  [ 48 ]. The resin oil of  C. martii  showed in vivo antileishmanial activity in a mouse model [ 97 ] and  C. reticulata  resin oil showed activity against  L. amazonensis  axenic amastigotes (IC 50  = 15.0 μg/mL) and intracellular amastigotes (IC 50  = 20 μg/mL) [ 109 ]. Diterpenoids isolated from  C. officinalis —agathic acid, alepterolic acid, kaurenoic acid, methyl copalate, pinifolic acid, and  ent -polyalthic acid—showed antileishmanial activity against  L. amazonensis  promastigotes [ 104 ].\nCopaifera  oleoresins and diterpene acids have also shown antitrypanosomal activities.  C. duckei  and  C. reticulata  resins showed in vitro activity against  T. evansi  trypomastigotes [ 86 ]. The diterpene acids—agathic acid, copalic acid, alepterolic acid, kaurenoic acid, methyl copalate, pinifolic acid, and  ent -polyalthic acid—all showed antitrypanosomal activity against  T. cruzi , including in the epimastigote, trypomastigote, and amastigote forms of the protozoan [ 111 ].\nA 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 ( Figure 1 ,  Figure 2  and  Figure 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  Table 4  and  Table 5 .\nThe  Leishmania  protein target with the best overall docking properties with  Copaifera  diterpenoids was  L. major  dihydroorotate dehydrogenase (average  E dock  = −109.2 kJ/mol). These docking energies were better than the docking energy for the normal substrate, dihydroorotate ( E dock  = −72.1 kJ/mol) and comparable to the co-crystallized ligand for this protein, nitroorotate ( E dock  = −104.2 kJ/mol). Docking energies for  Copaifera  diterpenoids with TcDHODH (average −92.5 kJ/mol) were also better than the normal substrate (dihydroorotate,  E dock  = −64.2 kJ/mol), but worse than the synthetic TcDHODH inhibitor, 5-[2-(5-carboxynaphthalen-2-yl)ethyl]-2,6-dioxo-1,2,3,6-tetrahydro-pyrimidine-4-carboxylic acid (TT2-2-199,  E dock  = −140.7 kJ/mol). Similarly,  Copaifera  diterpenoids docked with  L. donovani  DHODH (average  E dock  = −89.9 kJ/mol) better than dihydroorotate ( E dock  = −60.9 kJ/mol). Based on these docking energies, protozoal dihydroorotate dehydrogenases are likely targets for  Copaifera  diterpenoids.\nLeishmania 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.\nCopaifera  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.\nProtozoal triosephosphate isomerases (LmexTIM and TcTIM) are expected to be targeted by  Copaifera  diterpenoids. The average docking energy with LmexTIM (−90.7 kJ/mol) was much better than either the normal substrate (dihydroxyacetone phosphate,  E dock  = −52.4 kJ/mol) or the co-crystallized ligand, phosphoglycolohydroxamic acid ( E dock  = −61.1 kJ/mol). Likewise, docking energies with TcTIM (average −88.2 kJ/mol) were better than the dihydroxyacetone phosphate ( E dock  = −59.7 kJ/mol) and comparable to the TcTIM inhibitor, 3-(2-benzothiazolylthio)-1-propanesulfonic acid ( E dock  = −85.5 kJ/mol).\nBoth  L. major  pteridine reductase and  T. cruzi  pteridine reductase had docking properties with  Copaifera  diterpenoids with comparable energies (average  E dock  = −93.8 and −96.8 kJ/mol, respectively) with the normal substrate dihydrobiopterin ( E dock  = −96.9 and −100.1 kJ/mol, respectively). Thus,  Copaifera  diterpenoids may compete with dihydrobiopterin for pteridine reductase.\nSterols 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.\nCopaifera  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.\nAlthough  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).\nAdditional  Leishmania  proteins expected to be targeted by  Copaifera  diterpenoids include glycerol-3-phosphate dehydrogenase, which showed excellent docking energies (average −100.4 kJ/mol) to LmexGPDH, better than the normal substrate, glycerol-3-phosphate ( E dock  = −62.5 kJ/mol). Also targeted with a weak docking energy are: glucose-6-phosphate isomerase (Lmex GPI  E dock  average = −73.0 kJ/mol), though better than the docking energy of the normal substrate (glucose-6-phosphate,  E dock  = −62.0 kJ/mol); and phosphomannomutase (LmexPMM  E dock  average = −94.2 kJ/mol), which is better compared to the normal substrate (mannose-6-phosphate,  E dock  = −72.5 kJ/mol).\nAdditional  T. cruzi  protein targets may be dihydrofolate reductase—thymidylate synthase (TcDHFR–TS), which showed an average docking energy with  Copaifera  diterpenoids of −93.2 kJ/mol, comparable to the docking energy of the normal substrate (dihydrofolate, −99.3 kJ/mol), as well as the TcDHFR–TS inhibitor cycloguanil ( E dock  = −83.1 kJ/mol); farnesyl diphosphate synthase (TcFPPS), with docking energies that averaged −96.2 kJ/mol, which is comparable to the docking energy of the normal substrate, isopentenyl diphosphate ( E dock  = −98.9 kJ/mol); and hypoxanthine phosphoribosyltransferase (TcHPRT), having an average  E dock  = −82.1 kJ/mol, compared to the normal substrate, hypoxanthine, with  E dock  of −65.9 kJ/mol.\nCopaiba oleoresin has shown antibacterial activity against several strains, in particular, Gram-positive  Bacillus subtilis  and  Staphylococcus aureus  with minimum inhibitory concentration (MIC) values of 5 μg/mL for both organisms [ 110 ]. Copalic acid, isolated from  C. langsdorffii , showed excellent antibacterial activity against  Bacillus cereus  (MIC 8.0 μg/mL),  B. subtilis  (MIC 5.0 μg/mL),  Kocuria rhizophila  (MIC 5.0 μg/mL),  Streptococcus pyogenes  (MIC 3.0 μg/mL),  S. pneumoniae  (MIC 3.0 μg/mL),  S. agalactiae  (MIC 2.0 μg/mL),  S. dysgalactiae  (MIC 1.0 μg/mL),  S. epidermidis  (MIC 0.5 μg/mL) [ 71 ],  S. salivarius  (MIC 2.0 μg/mL),  S. mutans  (MIC 3.0 μg/mL),  S. mitis  (MIC 5.0 μg/mL),  S. sobrinus  (MIC 3.0 μg/mL), and  S. sanguinis  (MIC 6.0 μg/mL) [ 70 ]. Likewise, kaurenoic acid showed remarkable activity against  S. pyogenes  (MIC 5.0 μg/mL),  S. pneumoniae  (MIC 5.0 μg/mL),  S. dysgalactiae  (MIC 8.0 μg/mL) [ 71 ],  S. epidermidis  (MIC 4–5 μg/mL),  B. subtilis  (MIC 3.1–6.3 μg/mL), and  S. aureus  (MIC 8–10 μg/mL) [ 75 ]. 3α-Alepterolic acid acetate (acetoxycopalic acid) showed moderate antibacterial activity against cariogenic  Streptococcus  bacteria, with MIC values ranging from 12.0 to 60.0 μg/mL [ 70 ].  ent -Polyalthic acid also showed moderate antibacterial activity against  B. subtilis  (MIC 20–30 μg/mL),  S. aureus  (MIC 40–50 μg/mL), and  S. epidermidis  (MIC 40 μg/mL) [ 75 ].\nIn 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).\nCopaiba 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 ].\nDiterpenoids isolated from  Copaifera  species have shown cytotoxic activities ( Table 3 ). Copalic acid, isolated from  C. langsdorffii , showed in vitro cytotoxicity on MO59J human glioblastoma cells and HeLa human cervical adenocarcinoma cells with IC 50  of 68.3 and 44.0 μg/mL, respectively [ 71 ]. Kaurenoic acid has demonstrated cytotoxicity against several human tumor cell lines, including CEM leukemia, MCF-7 breast tumor, HCT-8 colon tumor [ 73 ], AGP01 gastric tumor, and SF-295 glioblastoma [ 74 ]. Growth inhibition of AGP01 and SF-295 cells was also demonstrated by 3β-alepterolic acid and 3β-alepterolic acid acetate [ 73 ]. Methyl copalate showed remarkable cytotoxic activity on P-388 murine lymphoma (IC 50  = 2.5 μg/mL), A-549 human lung carcinoma (IC 50  = 5 μg/mL), HT-29 human colon carcinoma (IC 50  = 5 μg/mL), and MEL-28 human melanoma (IC 50  = 10 μg/mL) cells [ 75 ].\nMolecular docking (Molegro Virtual Docker, Aarhus, Denmark) has been carried out with  Copaifera  diterpenoids on cancer molecular targets, including androgen receptor, aromatase, caseine kinase II, cyclin-dependent kinases 2, 4, and 6, cyclooxygenase 2, DNA (cytosine-5)-methyltransferase-1 and -3A, epidermal growth factor receptor, estrogen receptor α, estrogen receptor β, heat shock protein 90, insulin-like growth factor 1 receptor, 5-lipoxygenase, mitogen-activated protein kinase 1, NF-κB, p90 ribosomal protein S6 kinase, P-glycoprotein, phosphatidylinositol-4,5-bisphosphate 3-kinase, topoisomerase I, topoisomerase IIα, topoisomerase IIβ, tubulin, and vascular endothelial growth factor receptor ( Table 7 ). The best overall cancer targets for the copaiba diterpenoids were human DNA (cytosine-5)-methyltransferase-1 (HsDNMT1), human estrogen receptor β (HsERβ), and human mitogen-activated protein kinase 1 (HsMEK1), with average MolDock docking energies of −102.7, −99.2, and −101.5 kJ/mol, respectively. DNA (cytosine-5)-methyltransferase-1 (DNMT1) is the enzyme responsible for DNA methylation of carbon-5 of cytosine within CpG dinucleotides [ 120 ]. The enzyme is required for embryonic development [ 121 ], but is overexpressed in lung, liver, colorectal, gastric, breast, and lung tumors [ 122 ]. Thus, DNMT1 has emerged as an attractive target for cancer chemotherapy [ 123 , 124 ]. The mitogen-activated protein kinase (MAPK) signaling cascade is one of the most important pathways involved in cellular proliferation and differentiation [ 125 ] and, therefore, inhibition of components of this pathway, such as MEK1, can potentially target tumors that depend on MAPK signaling [ 126 ]. Agonism of estrogen receptor α (ERα) stimulates proliferation of breast, uterus, and prostate tissues, whereas ERβ agonism inhibits proliferation of these tissues [ 127 ]. Thus, compounds that can selectively bind and activate ERβ, but not ERα, could represent effective antitumor agents for treatment of prostate and breast cancer [ 128 ]. Copalic acid and methyl copalate both targeted HsMEK1, with docking energies of −108.2 and −111.0 kJ/mol, respectively, while 3β-alepterolic acid and 3β-alepterolic acid acetate showed excellent docking with HsDNMT1 ( E dock  = −107.2 and −121.7 kJ/mol, respectively). Kaurenoic acid was a relatively weakly docking ligand but did show selective docking to aromatase ( E dock  = −93.7 kJ/mol). The best-docking ligand was patagonic acid, which had a docking energy of −121.8 kJ/mol with HsDNMT1.\nInflammation 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 ).\nThe 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.\n\nMolecular docking analyses were carried out using Molegro Virtual Docker (v. 6.0.1, Molegro ApS, Aarhus, Denmark) against known bacterial [ 119 ],  Leishmania  [ 114 , 115 , 116 ],  Trypanosoma cruzi  [ 117 ], and cancer-relevant and inflammation-relevant protein targets [ 131 ], as previously described [ 114 , 115 , 116 , 117 , 119 , 132 ].\n\nThe 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.","source_license":"CC-BY-4.0","license_restricted":false}