Investigation of Copaifera genus as a new source of antimycobaterial agents

Aim: This paper reports on the antimycobacterial activity of the oleoresins and extracts obtained from Copaifera spp. Materials & methods: The minimum inhibitory concentration (MIC) and fractional inhibitory concentration index techniques helped to evaluate the effect of these oleoresins and extracts against six strains of mycobacteria that cause tuberculosis. Results & conclusion: Among the assayed oleoresins and plant extracts, the Copaifera langsdorffii, Copaifera duckei, Copaifera reticulata and Copaifera trapezifolia oleoresins provided the lowest MIC values against some of the tested strains. The combination of Copaifera spp. samples with isoniazid did not evidence any synergistic action. Some Copaifera spp. oleoresins may represent a future source for the discovery of new antimycobacterial drugs due to their low MIC values.

Specifically, Copaifera langsdorffii Desf. (Fabacecae-Ceasalpinoidae), popularly known as 'copaiba', is a native species of tropical regions of Latin America and is widely distributed in the Brazilian Cerrado and other forests. The oleoresin has a strong antioxidant and anti-inflammatory effects and is composed of several sesquiterpenes and unsaturated diterpene acids [34]. The species Copaifera duckei Dwyer belongs to the family Fabaceae, which produces an oleoresin that comes from the exudation of the trunk of the tree. In folk medicine, traditional communities use this oleoresin through oral or topical administration for various medicinal purposes. Several indications have been described, including wound healing, anti-inflammatory action and antigastric and antiulcer effect, among other digestive diseases [35]. Copaifera reticulata is a medium tree known as 'copaibeira' and 'pau d'óleo'. It is native to tropical regions of south America and grows abundantly in several states of Brazil, like Pará, Amazonas and Ceará. The oleoresin of this plant has been used mainly as healing, anti-inflammatory and antiseptic agent. The main chemical constituents of this oleoresin are mono-, sesqui-and diterpenes; many of its biological activities can be attributed to these compounds [36]. In turn, Copaifera trapezifolia Hayne, a species that is easily found in the Brazilian south-eastern region, is noted for its economic and medicinal importance [37,38]. Leandro et al. [20] investigated the antibacterial activity of the hydroalcoholic extract obtained from C. trapezifolia leaves (CTE) against microorganisms that cause dental caries and apical periodontitis, to find that C. trapezifolia leaves exhibits antibacterial activity against some of the tested microorganisms under the employed experimental conditions. The authors emphasized that this plant species can be an important source of biologically active compounds and aid in the search for new effective and safe agents that can act against oral pathogens.
Considering the pharmacological potentials of Brazilian Copaifera species [39], this study aimed to evaluate the antimycobacterial activity of the oleoresins and hydroalcoholic crude extracts obtained from Copaifera spp. leaves against some mycobacterial species and the interaction between compounds from this plant species and the antibiotic isoniazid.

Plant material & chemical characterization
Copaifera leaves and oleoresins (when available) were collected in different states, between August 2012 and May 2014, which was authorized by the Brazilian government through SISBIO (35143-1) and CGEN (010225/2014-5). Table 1 contains information about voucher specimens. To extract the oleoresins, a hole of approximately 1 inch wide and 3 feet above the ground was made into the center of the trunk of the tree with an auger. The oleoresin was drained into an amber bottle by using a pipe connected to a filter and the hole was then properly sealed [40]. The leaf extracts were prepared as described in Furtado et al. [41].

Microorganisms
To evaluate the antimycobacterial activity of the oleoresins and the hydroalcoholic crude extracts from Copaifera spp., the following American Type Culture Collection (ATCC) mycobacteria and clinical isolates

Determination of MIC
To evaluate the antimycobacterial activity of the samples, the MIC was determined by microdilution in a microplate. Resazurin was used to reveal bacterial growth by the resazurin microtiter assay (REMA) method procedure adapted from Palomino et al. [42]. The experiment was conducted in triplicate.
The oleoresins and the hydroalcoholic crude extracts were dissolved in DMSO and serially diluted in Middlebrook 7H9 broth before inoculation. The concentrations of the tested compounds ranged from 31.25 to 2000 μg ml -1 . Isoniazid, dissolved in DMSO, was used as standard drug at concentrations ranging from 0.015 to 1.0 μg ml -1 . The inoculum was prepared by introducing a range of colonies grown in Ogawa-Kudoh (LaborClin) in a tube containing glass beads with 500 μl of sterile water. A 200-μl aliquot was transferred to a tube containing 2 ml of 7H9 broth, incubated at 37 • C for 7 days and compared with McFarland scale 1. After the inoculum was standardized, it was diluted with 7H9 broth at a 1:25 ratio. Growth controls containing no antibiotic and sterility controls without inoculation were also included. The plates were incubated at 37 • C for 7 days. Subsequently, 30 μl of 0.02% resazurin aqueous solution was added to each well. After standing for 18 h, a blue color (coloring resazurin solution) was interpreted as lack of bacterial growth, whereas a pink color indicated the presence of viable micro-organisms. MIC was defined as the lowest drug concentration that was able to inhibit 90% growth of the mycobacterium strain. The assay was conducted in triplicate.

Determination of the fractional inhibitory concentration index
The activities of combinations of the oleoresins and hydroalcoholic crude extracts with isoniazid were evaluated by fractional inhibitory concentration index (FICI) for each of the tested mycobacteria. Checkerboard assays were performed according to the protocol previously described by Bhusal et al. [43] with modifications to investigate the in vitro antimicrobial efficacy of the combination of the compounds with the positive control (isoniazid). The whole experiment was repeated three-times. To determine FICI, the concentrations of oleoresins, hydroalcoholic crude extracts and isoniazid were the same as the concentrations used in the MIC assay. The bacterial inoculum was also employed at the same concentration and the inoculum was prepared in the same way described in the section about the MIC assay.
Serial twofold dilutions of compounds and isoniazid were mixed in each well of a 96-well microtiter plate. Fifty-microliter aliquots of the antimicrobial agent (oleoresins or extracts) at an initial concentration of 8000 μg ml -1 were added in vertical and horizontal orientation, respectively. Next, 100 ml of 1 × 10 8 CFU/ml fresh bacterial suspension was added to each well and incubated as mentioned previously. After 7 days of incubation at 37 • C, resazurin was added to each well to reveal bacterial growth.
FICIs were calculated according to the following formula: FICI = (MIC of antimicrobial agent in combination with isoniazid/MIC of antimicrobial agent alone) + (MIC of isoniazid in combination with antimicrobial agent/MIC of isoniazid alone). Synergism was defined as FICI ≤0.5; additive effect was defined as 0.5 < FICI <1; indifference was defined as 1 ≤ FICI <4 and antagonism was defined as FICI ≥ 4 [44].

Results & discussion
Silva et al. [40] and in Furtado et al. [41] described the chemical characterization of all the oleoresins and leaf extracts. Here, we have investigated the antimycobacterial activity of the oleoresins and hydroalcoholic crude extracts obtained from the leaves of Copaifera spp. by determining the MIC and the FICI. Table 2 summarizes the MIC values determined for the oleoresins and the hydroalcoholic extracts obtained from the leaves of Copaifera spp. against the tested mycobacteria.
MIC ranged from 62.5 μg ml -1 to values above 2000 μg ml -1 . According to Holetz et al. [45], crude extracts with MIC values lower than 100 μg ml -1 , between 100 and 500 μg ml -1 , from above 500 to 1000 μg ml -1 and greater than 1000 μg ml -1 display good antimicrobial activity, moderate antibacterial activity, weak antibacterial activity and absence of antibacterial activity, respectively. Other authors have considered that MICs ≤200 μg ml -1 for extracts indicate good activity against M. tuberculosis [46].  Based on the criteria of Holetz et al. [45], these results corresponded to good antimycobacterial activity and demonstrated that compounds from natural products are an important source of lead drugs for the research and development of new antimycobacterial agents.
The Natural antimicrobial products may lead to different results. The differences observed between the oleoresins and the hydroalcoholic extracts obtained from leaves herein might stem from the distinct composition of the oleoresins and the extracts. From a chemical viewpoint, Copaifera oleoresins comprise a diterpene acid solution in an essential oil consisting mainly of sesquiterpenes [40,41,47]. Currently, over 70 sequiterpenes have been identified in different Copaifera species. From a biological standpoint, the oleoresin consists of a waste product or detoxification plant organism and acts to protect the plant against animals, fungi and bacteria [48]. Additionally, phytochemical studies on these plants have concluded that the hydroalcoholic crude extracts obtained from 'copaiba' leaves are rich in polar compounds, which can be extracted by aqueous alcohol extraction. Thus, Furtado et al. [41] have recently described the presence of phenolic compounds like flavonoid heterosides and galloylquinic acid derivatives in such extracts. The quercetin flavonoids and kaempferol-3-O-α-L-rhamnopyranoside substances were first described in the hydroalcoholic extract of the aerial parts of C. landsgorffii. This class of substances is well known for their different biological activities, especially their anticancer and antioxidant actions [49]. According to Sikkema et al. [50], an important feature accounting for the antimicrobial activity of oils is the presence of hydrophobic lipid components that can partition the bacterial cell membrane, thereby disintegrating structures and making them more permeable.
In most cases herein, the crude hydroalcoholic extracts obtained from the leaves proved to be less effective in fighting bacteria as seen in the MIC results. This is because the oleoresins contain higher concentration of active molecules than the crude hydroalcoholic extracts obtained from the leaves [23].
In this study, the MIC results that were chosen to determine the FICI values were less than or equal to 125 μg ml -1 , these MIC values gave the best results and inhibited mycobacterial growth the most during the tests. The oleoresins and the hydroalcoholic crude extracts that provided MIC values in this range were the C. trapezifolia, C. duckei, C. langsdorffii, C. reticulata, C. oblongifolia and C. multijuga oleoresins and the extracts obtained from C. duckei and C. lucens.
The FICI results obtained after evaluation of the synergistic action between the oleoresins or the hydroalcoholic crude extracts and isoniazid against each of the tested mycobacteria did not evidence any synergism. Indeed, the results were antagonistic or indifferent (data not shown). Even though our data did not support any synergy between the oleoresins or extracts and isoniazid, we investigated the combination of these compounds in an attempt to improve the antimycobacterial activity. Hochfellner et al. [51] evaluated the interaction between quinolone and indoloquinazoline alkaloids with rifampicin or isoniazid against Mycobacterium smegmatis (ATCC 14468). The authors did not find satisfactory interaction between the evaluated molecules and rifampicin or isoniazid.
According to Van et al. [52], antagonistic interactions should not be ignored when investigating interactions in plant-based studies. These authors reported that the increasing number of scientific works exploring interactions among herbal substances is encouraging and relevant in our ongoing quest to understand the mechanisms of action of herbal medicine.
It is worth mentioning that the oils and extracts used in the MIC tests and in combination with isoniazid herein were weighed against a group of bacteria that are difficult to treat because they have cell wall and different proteins. Bearing this in mind, we can consider that the MIC values obtained herein are promising. Not to overlook possible associations of the substances tested herein with different drugs, we shall conduct future studies using a greater variety of drug combinations, which will hopefully result in new treatment alternatives.
Moreover, the literature contains few studies on the use of Copaifera against mycobacteria, another reason this study is important to guide future research in this field.

Conclusion
The oleoresins obtained from C. langsdorffii, C. duckei, C. reticulata and C. trapezifolia inhibited the growth of the mycobacteria M. kansasii (ATCC), M. tuberculosis (ATCC and clinical isolate) and M. avium (ATCC) satisfactorily. The FICI showed that the combination of one of the extracts or oleoresins tested herein with isoniazid exerted antagonistic or indifferent effects on the tested mycobacteria.
In conclusion, the results of this study, especially the MIC values lower than 100 μg ml -1 , suggested that investigation into the properties of the oleoresins and extracts obtained from some Copaifera species could contribute to expanding scientific knowledge about the antimycobacterial activity of this plant genus and aid the discovery of new antimycobacterial drugs.

Future perspective
Although TB has become a treatable disease thanks to the emergence of antibiotics, it is still a difficult disease to treat -treatment lasts long and a large amount of antibiotics is used by the patient. Increased bacterial resistance is a reality in which scientists seek to discover new drugs that can fight infection with minimal damage to the patient. Existing drugs have a large number of side effects and should be replaced with safe molecules. In this context, medicinal plants have been explored as sources of molecules with promising antibacterial activity. Few studies involving antimycobacterial activity and Brazilian medicinal plants have resulted in satisfactory effects. The oleoresins used in this study proved to be safe in the toxicity tests conducted by our research group. The oleoresins evaluated here are promising in the search for new antimycobacterial compounds. It is necessary to understand their mechanism of action and to undertake further studies on molecules obtained from Copaifera spp. oleoresins.
The results suggest that the oleoresins obtained from some Copaifera species can contribute to the discovery of new antimycobacterial drugs, but studies involving its mechanism of action should be undertaken.

Financial & competing interests disclosure
This work was kindly supported by the São Paulo Research Foundation -FAPESP, Processes number # 2011/13630-7 and # 2012/25237-0 and Coordination for the Improvement of Higher Level Personnel (CAPES) and National Council for Scientific and Technological Development (CNPq). The authors have no affiliations or financial involvement with any organization or entity with a financial interest in this work or financial conflict.
No writing assistance was utilized in the production of this manuscript.

Open access
This work is licensed under the Creative Commons Attribution 4.0 License. To view a copy of this license, visit http://creativecomm ons.org/licenses/by/4.0/ Executive summary • The Copaifera langsdorffii, Copaifera duckei, Citrus reticulata, Copaifera multijuga and Copaifera trapezifolia oleoresins were effective against the Mycobacterium spp evaluated in this study. • Among the investigated Copaifera species, C. duckei displayed the best antimycobacterial results.
• Oleoresins in the absence of isoniazid were more effective.