Chemical Profiling and Biological Activity of Copaiba Oil‐Resin Samples and Development of HPLC‐DAD Method for the Analysis of β‐Caryophyllene

Abstract

Figures

The sesquiterpenes found in copaiba oil‐resin samples.

The colour variation between the seven samples of copaiba oil‐resin.

TLC of soybean oil, β‐ caryophyllene and samples of copaiba oil‐resin. Samples: (OS) soybean oil; (CAR) β‐caryophyllene; (1) CO1; (2) CO2; (3) CO3; (4) CO4; (5) CO5; (6) CO6; and (7) CO7. Eluent: Hexane:ethyl acetate (9:1). (a) Spray reagent anisaldehyde sulphuric acid reagent; (b) Spray reagent sulphuric acid 20%.

Chromatographic profiles by GC–MS for soybean oil, β‐caryophyllene and samples of copaiba oil‐resin. Samples: 3A (OS) soybean oil; 3B (CAR) β‐caryophyllene; (3C) CO1; (3D) CO2; (3E) CO3; (3F) CO4; (3G) CO5; (3H) CO6; and (3I) CO7. Analysis conditions: Capillary column (HP‐5MS, 25 μm thickness and 30 m) using an injection volume of 1 μL in split mode (1:10) with nitrogen as the carrier gas at a flow rate of 12.7 mL/min. Temperature gradient: 50 min at 100°C, 100°C–200°C with a variation of 1.5°C/min, 200°C–300°C with a variation of 2.5°C/min and 5 min at 300°C. Injector temperature at 220°C, mass source temperature 290°C and acquisition range 20–500 m/z. The identified compounds (1–26) are presented in Table 4.

The most abundant sesquiterpenes in copaiba oil‐resin samples.

Chromatographic profiles in HPLC‐RP of samples of copaiba oil‐resin. HPLC (Agilent 1200‐UV), column XDB‐C18 (4.6 × 250 mm × 5 μm), flow rate of 0.8 mL/min. Detection of ƛ 218 nm. Injection volume: 10 μL. Mobile phase: Ultra‐purified water (Phase A) and acetonitrile HPLC grade (Phase B), both with 0.1% formic acid in a linear gradient elution: 0–40 min 90% of B and 40–45 min 90%–95% of B.

Analytical curve β‐caryophyllene concentration (μg/mL) versus peak area.

Tables

TABLE 1. Samples of copaiba oil‐resin.

Sample/species	Form of acquisition/location	Packaging/documents sent by the suppliers
CO1	Compounding pharmacy/Belo Horizonte/MG	Dark glass bottle/not sent
CO2	Central market/Belo Horizonte/MG	Clear plastic bottle/not sent
CO3	Central market/Belo Horizonte/MG	Clear plastic bottle/not sent
CO4	Website store	Dark glass bottle/microbiological analysis
CO5	Website store	Dark glass bottle/CG analysis report
CO6	Central market/Belém/PA	Clear plastic bottle/not sent
CO7	Central market/Belém/PA	Clear plastic bottle/not sent

TABLE 2. Physicochemical analyses of seven commercial samples of copaiba oil‐resin.

Sample	Colour	Refractive index (ƞD20)	Density (mg mL−1)	Acidity index** (mg KOH/g)	Ester index (mg KOH/g)	Solubility 1.0 g of samples (volume of ethanol)
OS	Light yellow	1.4743	0.9072	3.22 ± 0.10	5.14 ± 0.82	3.95
CO1	Light yellow	1.4782	0.9319	4.86 ± 0.13	21.19 ± 9.11	5.32
CO2	Yellow	1.5051	0.9368	44.03 ± 3.22	9.21 ± 0.73	42.54
CO3	Yellow	1.5061	0.9368	31.37 ± 0.04	7.42 ± 0.77	43.73
CO4	Light yellow	1.5085	0.9356	53.22 ± 0.19	4.21 ± 1.42	64.84
CO5	Dark yellow	1.5093	0.9299	37.01 ± 0.20	11.67 ± 1.61	64.11
CO6	Dark yellow	1.5089	0.8798	37.42 ± 0.78	10.53 ± 0.9	63.47
CO7	Brown	1.5142	0.9536	58.12 ± 0.69	11.21 ± 1.41	45.89

TABLE 3. Compounds identification and relative composition of copaiba oil‐resin samples.

Compounds a	RRICal b	RRILit c	Retention time (min)Relative peak area (%)
			CO2	CO3	CO4	CO5	CO6	CO7
α‐Bergamotene	1400	1411	21.134 8.22	21.155 8.77	21.155 14.22	21.155 7.4	21.120 4.9	21.045 4.94
α‐Cadinol	1600	1652	—	—	—	34.975 1.09	34.675 2.1	35.575 1.03
α‐Copaene	1301	1374	17.291 4.54	17.335 6.61	17.315 6.31	17.400 9.10	17.375 7.87	17.260 3.75
α‐Cubebene	1300	1345	—	—	15.720 1.59	15.735 2.02	15.730 1.57	15.700 0.84
α‐Guaiene	1401	1437	—	—	—	22.530 1.74	—	—
α‐Humulene	1401	1436	24.812 4.15	—	22.065 3.59	—	—	—
α‐Muurolene	1401	1500	—	—	—	25.935 5.63	25.185 0.82	25.140 0.62
β‐Bisabolene	1500	1505	25.857 4.17	25.865 3.97	25.940 8.70	26.155 2.22	25.910 4.04	25.875 5.00
β‐Cadinene	1400	1491	18.679 1.43	—	—	26.875 5.06	26.845 5.05	—
β‐Cedrene	1400	1419	—	—	21.455 0.75	—	—	—
β‐Copaene	1401	1430	23.937 6.14	23.870 2.50	—	24.025 5.22	25.050 5.29	23.935 7.33
β‐Elemene	1301	1389	18.281 3.70	18.290 4.17	18.230 2.96	18.255 1.70	18.265 2.51	—
β‐Eudesmene	1400	1489	24.262 6.70	—	—	—	24.285 2.84	—
β‐Farnesene	1400	1440	—	—	—	20.385 4.43	—	—
β‐Humulene	1401	1452	22.203 9.34	22.195 7.7	—	22.210 7.11	22.205 7.25	22.140 7.79
Caryophyllene	1400	1417	20.325 37.90	20.330 32.65	20.045 12.58	20.325 28.34	20.325 28.37	20.240 30.17
Caryophyllene oxide	1501	1582	—	30.845 17.96	30.880 22.71	—	30.545 0.99	—
Caryophyllenyl alcohol	1501	1570	—	—	—	—	—	29.650 1.58
δ‐Cadinene	1500	1522	26.758 3.13	26.735 1.8	—	—	—	26.790 5.52
δ‐Cadinol	1601	1644	—	—	—	35.530 1.35	34.980 1.20	35.020 1.31
δ‐Elemene	1300	1335	15.047 0.77	15.45 0.88	15.035 0.31	15.055 1.44	15.050 1.47	—
γ‐Cadinene	1500	1513	—	—	—	26.290 0.57	—	—
γ‐Elemene	1400	1434	—	20.875 1.41	—	—	28.850 3.53	—
γ‐Muurolene	1401	1478	—	23.620 2.45	23.555 1.38	—	26.130 0.97	23.630 2.51
Gemacrene B	1501	1559	28.833 4.31	28.870 4.69	—	—	—	24.850 2.73
Isocaryophyllene	1400	1408	20.390 6.01	24.145 2.03	24.135 4.64	24.810 2.82	20.405 5.71	—
Longifolene	1401	1427	—	24.754 0.62	24.715 1.00	25.185 0.89	24.825 3.45	—

TABLE 4. Content of β‐caryophyllene (% ± s) in different samples of copaiba oil‐resin.

Sample	CO1	CO2	CO3	CO4	CO5	CO6	CO7
% β‐caryophyllene	3.36 ± 0.30	31.41 ± 0.40	21.84 ± 0.46	33.05 ± 0.12	37.12 ± 0.16	27.14 ± 0.12	17.63 ± 0.88

TABLE 5. The antibacterial (% inhibition/MIC) and cytotoxicity of samples of copaiba oil‐resin.

Samples	Antibacterial activity % inhibition at maximum concentration (MIC)		Cytotoxicity activity IC50 RAW 264.7(μg/mL)
	S. epidermidis a ATCC 12228	S. aureus b ATCC 25923
CO1	41.32 ± 3.87	41.32 ± 8.65	1119.75 ± 154.98
CO2	72.82 ± 0.56 (0.29)	99.29 ± 2.81 (1.17)	14.93 ± 1.88
CO3	82.30 ± 7.72 (2.34)	98.80 ± 1.34 (2.34)	10.06 ± 0.73
CO4	66.84 ± 0.46	90.8 ± 0.3 (1.16)	12.43 ± 1.84
CO5	75.08 ± 0.38 (4.64)	86.77 ± 1.24 (1.16)	10.70 ± 1.54
CO6	71.40 ± 0.3 (4.39)	100.33 ± 0.96 (2.19)	7.83 ± 0.93
CO7	66.4 ± 3.6	91.7 ± 0.1 (0.59)	13.51 ± 1.63
Soy oil	Inactive	21.0 ± 0.4	Not evaluated
β‐Caryophyllene	Inactive	40.37 ± 1.11	26.7 ± 3.46
Chloramphenicol (50 μg/mL).	97.59 ± 3.61	99.9 ± 0.70	Not evaluated

Full text

Introduction

1

The Copaifera genus belongs to the Fabaceae family, and the species occur mainly in Central and South America (Trindade et al. 2018; Frazão et al. 2024). In Brazil, the species of copaiba trees can be found in all the regions (Flora e Funga do Brasil 2025). Although many research groups have studied the taxonomy of the Copaifera genus, its botanical identification is difficult. According to Trindade et al. (2018), it has been attributed to their intricate floral morphology and absence of reproductive structures in the samples.

The Copaifera species are also known as ‘Copaiba balsam’, ‘Copaiba’, ‘Angelim copaiba’, ‘Copaiba branca’, ‘Iguapo copaiba’, ‘Jutaí copaiba’, ‘Copaiba vermelha’, ‘True copaiba’, ‘Capaibarana’, ‘Copauba’, ‘Mari‐Mari’ and ‘Óleo vermelho’ (Tappin et al. 2004; Arruda et al. 2019; Frazão et al. 2024). The different species are used traditionally for their anti‐inflammatory, antiseptic and healing properties. In addition, its oil‐resin has been used to treat a variety of other problems, such as urinary, skin, rheumatic and respiratory diseases (Tappin et al. 2004; Pieri et al. 2009; Arruda et al. 2019; Lee et al. 2023; Leandro et al. 2024). Several other uses of copaiba oil‐resin have been described, including as a raw material for biodiesel production, as a fuel, in the wood, photographic and paper industries and for the manufacture of paints (Trindade et al. 2018; Frazão et al. 2024). According to Silva‐Junior and co‐workers (2023), the copaiba oil‐resin is one of the main non‐timber forest products (NTFP) commercialized in the popular market in the Amazon State.

However, only one herbal medicine is registered with the Brazilian Health Regulatory Agency (ANVISA, consulted on 31/07/2025) for the commercial use of copaiba oil as an anti‐inflammatory. In contrast, a variety of products, including crude oil, dietary supplements (capsules) and a wide range of cosmetics containing copaiba oil‐resin in combination with other plants, are currently available for online sales and at public markets.

Supplies of this exuded oil‐resin come from natural populations of copaiba, which are slow‐growing plants and, therefore, limited in availability. The oil‐resin is obtained through artisanal procedures and may exhibit variability in its phytochemical profile because of biotic and abiotic factors. The phytochemical profile of copaiba oil‐resin can vary depending on the species, season and harvesting location. Moreover, its chemical composition may be affected by epigenetic and genetic responses to abiotic stress. Sesquiterpenes and diterpenes are the main classes of compounds detected in oil‐resin (Veiga Junior. et al. 1997; Veiga Junior and Pinto 2002; Lee et al. 2023; Frazão et al. 2024).

The botanical identity of the product cannot be guaranteed, as it is sustainably harvested from different species of copaiba. However, copaiba oil‐resin extraction from native forests has increased from 165 tons in 2020 to 223 tons in 2023 (Ministerio do Meio Ambiente e Mudança do Clima 2024).

Despite the widespread use of copaiba oil‐resin for its medicinal properties and its economic importance, the species has not yet been described in the Brazilian Pharmacopoeia. To overcome this situation, further research is needed to confirm its efficacy, validate its use and ensure its safety for official medicinal use of the copaiba oil. Given the lack of parameters in official compendia, the inherent variability of copaiba oil‐resin and the increasing market demand for this product, it is important to develop procedures to standardize the raw material.

Adulteration of oil‐resin has been reported by adding low‐value mineral or vegetable oil. In Brazil, copaiba oil‐resin is adulterated mainly by the addition of low‐value vegetable oils (soybean or corn) (Veiga Junior and Pinto 2002). Different parameters can be used to evaluate the quality of plant material, including physicochemical properties, chromatographic fingerprinting and chemical marker analysis. According to Rasheed et al. (2012), markers are chemically defined constituents of an herbal drug that are of interest for quality control purposes, regardless of whether they have therapeutic activity. The compounds β‐caryophyllene, humulene and methyl copalate have been used as chemical markers in analytical methods for the quality control of copaiba oil‐resin (Arruda et al. 2019; De Oliveira et al. 2022; Almeida Borges et al. 2013).

The objective of this study was to compare the chromatographic profiles of seven samples of copaiba oil‐resin with respect to chromatographic and physicochemical properties, thereby contributing to the standardization of general parameters used in official compendia and to the evaluation of biological activities related to the medicinal use of copaiba oil‐resin by the population. In addition, an in silico prediction approach was used to assess the toxicity of several sesquiterpenes identified in the samples.

Experimental Section

2

Samples

2.1

A total of seven samples of copaiba oil‐resin (CO) and one of soybean oil were acquired. The samples were identified by letters and numbers (Table 1) and refrigerated during the work. This study received authorization for access to and remittance of genetic material for scientific research from SISGEN (Registration 010801/2015‐4). The details about the samples, acquisition method and packaging are given in Table 1.

Standards/Reagents and Solvents

2.2

The β‐caryophyllene standard (purity ≥ 80%) was acquired from Sigma‐Aldrich. The commercial soybean was purchased at a local market in Belo Horizonte (Brazil). All solvents and reagents for analyses were of analytical or chromatographic grade and were purchased from Anidrol, Vetec and Sigma‐Aldrich.

Physicochemical Analyses

2.3

Acidity and Ester Index

2.3.1

The methods described previously by Vasconcelos and Godinho (2022) were followed, with some adaptations. Briefly, for the acidity index (AI), 2.0 g of oil‐resin was weighed and added to 25 mL of an ethyl ether:ethanol (2:1) mixture. Three drops of phenolphthalein alcohol solution (1%) were added and then titrated with NaOH (0.1 mol/L). The equation obtained the AI value:

$$[eqn]$$

where V is NaOH volume (mL) used for titration, M is the molarity of NaOH solution and m is the mass of each sample (g). The result is expressed in milligrams of NaOH per gram of oil‐resin.

For the determination of the ester index (EI), 2.0 g of oil‐resin was weighed, 5.0 mL of ethanol and three drops of phenolphthalein alcohol solution (1%) were added, and the mixture was titrated with NaOH (0.1 mol/L). In the resulting solution, 20.0 mL of KOH solution (4%) was added. The mixture was heated under reflux for 30 min. After cooling, two more drops of alcoholic phenolphthalein solution (1%) were added to the mixture followed by a new titration using a hydrochloric acid (0.5 mol/L) solution. A titration without the oil sample was performed as a comparison (blank). The EI values were calculated using the equation:

$$[eqn]$$

where V (mL) is the difference between the volume of hydrochloric acid solution spent in the titration of the sample and the blank, M is the molarity of the hydrochloric acid (0.5 mol/L) solution and m is the weight of each sample (g). The result was expressed in mg KOH/g of oil‐resin.

Solubility

2.3.2

The solubility in ethanol was determined using 1.0 mL of each oil sample, to which ethanol of analytical grade was added under agitation for 5 s until the sample was fully dissolved. The result was expressed as the amount of ethanol required to solubilize 1.0 g of oil‐resin completely at room temperature.

Refractive Index

2.3.3

The refractive index was determined using a refractometer (Mettler Toledo, Model R5), with duplicate readings recorded at 20°C.

Density

2.3.4

The density of the liquid was determined using a pycnometer (5.0 mL) at 20 ± 1°C. The density of the oil‐resin was measured in triplicate, with the average density value and its respective standard deviation (σ) (Zenebon and Pascuet 2008).

Chromatographic Analyses

2.4

Thin‐Layer Chromatography Profile

2.4.1

The chromatographic profile was performed on thin‐layer chromatography (TLC) using aluminium plates coated with silica gel 60 F254 (Merck, Germany) containing a fluorescent indicator for the seven commercial samples of copaiba oil‐resin, along with the respective standards. Elution was performed with a hexane:ethyl acetate (9:1) mixture, and the plate was sprayed with anisaldehyde‐sulphuric acid reagent, then heated at 120°C. The same eluent was used on the second plate, which was revealed with sulphuric acid reagent (20%). The retention factors (RFs) of the main spots were calculated. The colour aspects of spots and their RFs were measured to compare with those of soybean oil, a standard oil used in the adulteration of copaiba oil‐resin (CO), and β‐caryophyllene (CAR) (Vasconcelos and Godinho 2022; Barbosa et al. 2009). β‐caryophyllene was used as a positive control.

Gas Chromatography/Mass Spectrometry (GC/MS) Analysis

2.4.2

An aliquot (50 μL) of each sample was diluted in ethyl acetate (950 μL), homogenized by vortexing for 1 min and analysed by gas chromatography (Shimadzu QP2010 Ultra). The samples were eluted on a nonpolar stationary phase capillary column (HP‐5MS, 25 μm thickness and 30 m) using an injection volume of 1 μL in split mode (1:10) with nitrogen as the carrier gas at a flow rate of 12.7 mL/min. Temperature gradient was as follows: 5 min at 100°C, 100°C–200°C with a variation of 1.5°C/min, 200°C–300°C with a variation of 2.5°C/min and 5 min at 300°C. Injector temperature was 220°C, mass source temperature was 290°C, and acquisition range was 20–500 m/z. The chemical composition of the oils was determined by comparing spectral data with data deposited in the National Institute of Standards and Technology (NIST11) library and by comparing the relative retention index calculated from the analysis of hydrocarbon standards with those reported in the literature. The equation for determining the linear retention index (RRI) is described below:

$$[eqn]$$

where RRI is the linear retention index, T _ x _ is the retention time of the compound of interest, T _ n _ is the retention time of the previous alkane, T _ n + 1_ is the retention time of the subsequent alkene and C is the number of carbons from the previous alkane.

Quantification of β‐Caryophyllene by HPLC and Validation of Methods

2.4.3

Analytical Conditions

2.4.3.1

The analyses were performed on an Agilent Technologies 1200 series HPLC system equipped with a diode‐array detector and an Agilent data handling system (Agilent Technologies, USA). The separation was achieved on a reversed‐phase XDB‐C18 column (4.6 × 250 mm, 5 μm), with a flow rate of 0.8 mL/min, detection at ƛ 218 nm and an injection volume of 20 μL. The elution was carried out with acetonitrile HPLC grade (Phase A) and ultra‐purified water (Phase B), both containing 0.1% formic acid, in a linear gradient elution: 0–40 min 90% B and 40–45 min 90%–95% B. The samples were solubilized in HPLC‐grade acetonitrile, centrifuged at 10,000 rpm for 10 min (Eppendorf, Model 5415D) and filtered through 0.45 μm PTFE syringe filters (Unifil).

Method Validation

2.4.3.2

The validation of the analytical method used to quantify the compound β‐caryophyllene in copaiba oil‐resin samples was performed according to the parameters recommended by the official guides (INMETRO  2011; FDA 2015; Agencia Nacional de Vigilancia Sanitaria 2017).

System Suitability

2.4.3.2.1

The system suitability parameters were obtained using the Agilent ChemStation software, as follows: retention factor (K), number of theoretical plates (N), tailing factor (Tf) and resolution (Rs). β‐caryophyllene solution in acetonitrile (60 μg/mL) was utilized to evaluate the parameters.

Selectivity

2.4.3.2.2

The spectral purity of β‐caryophyllene in all samples was evaluated using spectral data and peak purity metrics.

Linearity

2.4.3.2.3

The analytical curve using β‐caryophyllene was obtained using the method developed with the chromatographic conditions previously described. The calibration curve was obtained using β‐caryophyllene solution (20, 40, 60, 80 and 100 μg/mL) diluted in acetonitrile. All the samples were injected in triplicate. The area values were plotted against the corresponding concentrations, and statistical analysis was performed.

Matrix Curve

2.4.3.2.4

The matrix calibration curve was obtained using a β‐caryophyllene solution (20, 40, 60, 80 and 100 μg/mL), and the addition of 100 μL of a sample (1.0014 g/mL) was diluted in acetonitrile. All the samples were injected in triplicate. The area values were plotted against the corresponding concentrations, and statistical analysis was performed.

Accuracy (Recovery)

2.4.3.2.5

Accuracy was verified by adding 100 μL of copaiba Oil‐Resin (Sample CO6, 1.12 mg/mL) to β‐caryophyllene (μg/mL) at three concentration levels (Table S2), diluted in acetonitrile. All the samples were injected in triplicate. The area values were plotted against the corresponding concentrations, and statistical analysis was performed. Recovery was determined using the equation below:

$$[eqn]$$

Limit of Detection and Quantification

2.4.3.2.6

The limits of detection (LOD) and quantitation (LOQ) were determined using the standard deviation of the response (SD) and the slope of the calibration curve (S) in the equations.

$$[eqn]$$ $$[eqn]$$

Precision

2.4.3.2.7

The precision was evaluated by determining repeatability (repeatability precision) and intermediate precision. The samples were assessed in triplicate on two consecutive days. The RSD value was calculated to determine the repeatability (repeatability precision) and intermediate precision.

Robustness

2.4.3.2.8

The robustness of the method was investigated by varying the following parameters: wavelengths (λ 210.0, 218.0 and 220.0 nm), flow rate (0.7, 0.8 and 0.9 mL/min) and oven temperature (20°C, 21°C and 22°C). The analyses were performed using three replicate samples of the CAR standard solution (40, 60 and 80 μg/mL). The concentrations of β‐caryophyllene were calculated, along with the relative standard deviation. The mean, SD and RSD were calculated. It was considered acceptable when the CV was less than or equal to 5% (Moura‐Silva et al. 2023).

Analysis of β‐Caryophyllene in Copaiba Oil‐Resin Samples

2.4.3.2.9

The developed method was applied to detect the concentration of β‐caryophyllene (μg/mL) in copaiba oil‐resin. A solution of copaiba oil‐resin sample was prepared in acetonitrile (200 μg/mL). All the samples were analysed in triplicate. The concentration of β‐caryophyllene was determined using the analytical curve. The analysis of CAR in the CO samples was performed using the previously described chromatographic conditions (Section 2.4.3.1 ).

Biological Analysis

2.5

Antimicrobial Activity

2.5.1

Susceptibility tests against Staphylococcus epidermidis (ATCC 12228) and Staphylococcus aureus (ATCC 25923) were performed using a modified version of the Clinical and Laboratory Standards Institute (2018) methods for bacteria. The oil‐resin samples were diluted in Mueller–Hinton Broth (MHB) to 8.8–9.5 μg/mL, based on the oil‐resin sample density. The bacterial isolates were grown in Mueller–Hinton Agar plates at 37°C, for 18–24 h. Then, the inoculum was adjusted in saline solution using a spectrophotometer at 625 nm to a concentration of 1–5 × 10^8^ CFU/mL and diluted in MHB to 1–5 × 10^5^ CFU/mL. The inoculum (100 μL) was added to wells containing 100 μL of the oil‐resin samples at concentrations of 8.8–9.5 μg/mL, in triplicate (one well intended for the sample control), resulting in a final concentration of 4.39–4.76 μg/mL of the oil‐resin in the wells. Chloramphenicol at 50 μg/mL and 0.5% DMSO were used as positive and negative controls, respectively. Evaluation of microbial growth was carried out by adding the inoculum to a well containing only MHB. Sterility of the culture medium was confirmed by incubation in the assay plate. Assays were performed in 96‐well microplates, in duplicate. The microplates were incubated at 37°C for 24 h.

As a microbial growth indicator, 20 μL of 2,3,5‐triphenyltetrazolium chloride (TTC) at 5 mg/mL was added to each well. The plates were incubated at 37°C for 3 h. The TTC was solubilized with 100 μL of 7 μg/mL sodium lauryl sulfate in isopropanol and measured in a microplate reader at 485 nm. The result was expressed as the percentage inhibition of oil‐resin samples relative to the microbial growth control, as described by Fukuda et al. (2006). In the present study, samples with inhibition ≥ 70% were considered promising, and the minimal inhibitory concentration (MIC) of the oil‐resin samples was determined by microdilution assay in MHB (Clinical and Laboratory Standards Institute 2018). The promising oil‐resin samples, solubilized in DMSO at 4.39–4.76 μg/mL, were serially diluted in MHB to seven concentrations. The inoculum, controls, plate assembly, incubation and measurement of results were performed as described above. The MIC was defined as the lowest concentration of each oil‐resin sample that inhibited ≥ 70% of the microbial growth.

Cytotoxic Evaluation

2.5.2

Cultivation of Macrophages

2.5.2.1

The RAW 264.7.1 (murine macrophage cells) were maintained at 37°C and 5% CO_2_ in DMEM medium supplemented with 10% heat‐inactivated fetal bovine serum, 3.7 g/L NaHCO_3_ and pH 7.

Sample Dilution

2.5.2.2

The oil‐resin copaiba samples were diluted to 50 mg/mL in DMSO, and a serial dilution was performed in DMEM medium supplemented with 1% heat‐inactivated fetal bovine serum, 3.7 g/L NaHCO_3_, and adjusted to pH 7.

Cytotoxicity Assay

2.5.2.3

Macrophages RAW 264.7 were plated in 96‐well plates at a density of 0.6 × 10^5^ cells per well in 200 μL of DMEM medium supplemented with 1% heat‐inactivated fetal bovine serum, 3.7 g/L NaHCO3, pH 7 and incubated for 24 h at 37°C and 5% CO_2_. After that, the crude extract or essential oil was added to the wells in serial concentrations (500–0.9 μg/mL for the CO2 to CO7 samples and 2000–3.9 μg/mL for the CO1) and incubated for 72 h, at 37°C and 5% CO_2_. A resazurin reduction assay measured cytotoxicity; 20 μL resazurin solution (1 mM) was added to each well, and the plate was incubated for 2–3 h at 37°C and 5% CO_2_. Optical densities at 570 and 600 nm were measured using an ELISA plate reader (Thermo Fisher Scientific Multiskan GO). Each experimental condition was performed in quadruplicate. The data were evaluated to calculate the percentage of cell death and the cytotoxic concentration for 50% of the cells (CC_50%_) determined by the DrFit software (2015).

In Silico Prediction of the Toxicity of Sesquiterpenes

2.6

The compounds β‐caryophyllene (1), α‐copaene (2), β‐copaene (3), α‐humulene (4) and bergamotene (5) (Figure 1) found in the samples of copaiba oil‐resin were drawn, and their SMILES codes were obtained using the ACD/ChemSketch programme (ACD/ChemSketch 2015). Their SMILES codes were used as input variables to screen for potential targets in the ADMETlab 3.0 programme (Fu et al. 2024). The toxicity parameters were calculated: hERG blockers, DILI, AMES toxicity, rat oral acute toxicity, skin sensitization, carcinogenicity, eye corrosion, eye irritation, respiratory, human hepatotoxicity, drug‐induced nephrotoxicity, drug‐induced neurotoxicity, ototoxicity, hematotoxicity, genotoxicity, RPMI‐8226 immunotoxicity and cytotoxicity against Hek293 and A549.

The sesquiterpenes found in copaiba oil‐resin samples.

Statistical Analysis

2.7

Statistical analysis was performed using Microsoft Excel. The results were expressed as mean ± standard deviation (SD) from triplicate experiments. Outliers were diagnosed using the standard Jackknife residue test and applied successively until no new extreme values were detected, or up to a maximum exclusion of 22% of the original number of outcomes. The assumptions regarding the regression analysis were verified using the Ryan–Joiner test for residual normality, the Durbin–Watson test for residual independence and the Brown–Forsythe test (or the modified Levene test) for residual homoscedasticity. F tests were used to assess the adequacy of the linear model fit, based on regression significance, and the linearity deviation was evaluated against the pure error. Linearity was also statistically evaluated by analysis of variance (ANOVA) to assess linearity deviation and regression significance.

Results and Discussion

3

Given the economic importance of copaiba oil‐resin and its wide range of pharmaceutical applications, various studies are underway to standardize it (Barbosa et al. 2009). The samples commercialized in Brazil are a mixture of different Copaifera species (Veiga Junior and Pinto 2002; Barbosa et al. 2009), which is not considered a counterfeit.

Initially, the physicochemical properties of seven samples of copaiba oil‐resin (Table 3) were investigated using simple, low‐cost and accessible methods previously described (Vasconcelos and Godinho 2022; Barbosa et al. 2009; Zenebon and Pascuet 2008). The samples appeared as transparent liquids with colouration ranging from light yellow to dark brown (Figure 2 and Table 2). According to Veiga Junior and Pinto (2002), the colour of copaiba oil‐resin varies widely depending on the species.

The colour variation between the seven samples of copaiba oil‐resin.

The refractive index (Table 2) of commercial oil‐resin samples reported in the literature (Souza et al. 2023; Gottlieb and Iachan 1945) ranged from 1.503 ± 0.001 to 1.507 ± 0.001. In the present study, the refractive index of the evaluated samples ranged from 1.50514 to 1.51421, except for Sample CO1. The latter presented a refractive index similar to that of soybean oil (1.4743). Barbosa et al. (2009) proposed that an association between refractive index results and thin‐layer chromatography can be used as a detector of copaiba oil adulteration.

The density values determined for Samples CO1 to CO7 ranged from 0.9299 to 0.9536 g/mL and were similar to those obtained from the literature (Souza et al. 2023; Gottlieb and Iachan 1945; Silva et al. 2012) for commercial samples of Copaifera sp. (0.92 ± 0.01 to 0.94 ± 0.002) and C. reticulata (0.97 ± 0.05) except CO6, whose density value was 0.8798 g/mL.

To determine the acidity and ester index (EI) of the samples, an analytical method by titration was used according to the methodology described by Vasconcelos and Godinho (2022). The acidity index (AI) values obtained for the majority of the analysed samples ranged from 31.37 ± 0.04 mg KOH/g to 58.12 ± 0.69 mg KOH/g. In contrast, CO1 showed a low AI value (4.86 ± 0.13 mg KOH/g), similar to that observed for soy oil (3.22 ± 0.10 mg KOH/g). The EI for the majority of the evaluated samples ranged from 4.21 ± 1.42 mg KOH/g to 11.67 ± 1.61 mg KOH/g, except for CO1, which had an EI of 21.19 ± 9.11 mg KOH/g. These analytical methods for determining acidity and EI are simple and accessible. However, they should not be used to evaluate sample purity, which is contrary to Vasconcelos and Godinho (2022). For these authors, the methods mentioned earlier were suitable for assessing the authenticity of the copaiba oil‐resin. The values of acidity and ester index depend on the species and the commercial source (Silva et al. 2012; Souza 2010; Barbosa et al. 2009; Souza et al. 2023; Gottlieb and Iachan 1945).

The solubility of the sample is an important parameter for the pharmaceutical industry, even though it cannot be used to assess adulteration. However, when combined with other physicochemical features, it may indicate a possible contamination (Vasconcelos and Godinho 2022). In this study, the solubility of 1.0 g of the majority of samples in absolute ethanol ranged from 42.54 to 64.48 mL, except Sample CO1. The latter shows a solubility value similar to that of the soil oil, as specified in Table 2.

The chemical composition of copaiba oil‐resin was analysed using three different chromatographic techniques. The thin‐layer chromatography (TLC) plate was developed with the anisaldehyde‐sulphuric acid reagent (Figure 3a) and 20% sulphuric acid (Figure 3b) for seven samples of copaiba oil‐resin from different suppliers, soybean oil (OS) and the β‐caryophyllene standard (CAR). In Figure 3a, OS showed a dark lilac spot (RF = 0.58), and CAR showed a lilac spot (RF = 1.0). Samples CO1 and CO2 showed a lilac spot (RF = 0.58) similar to OS, indicating that both samples may have been adulterated with OS. All the samples, except CO1, showed a spot (RF = 1.0), indicating the presence of β‐caryophyllene. As expected, the samples exhibited a characteristic chromatographic profile, with additional dark lilac spots indicative of terpenes. In Figure 3b, the soybean oil (OS) and Samples CO1 and CO2 showed an orange spot at RF = 0.58, indicating possible adulteration of both samples with OS.

TLC of soybean oil, β‐ caryophyllene and samples of copaiba oil‐resin. Samples: (OS) soybean oil; (CAR) β‐caryophyllene; (1) CO1; (2) CO2; (3) CO3; (4) CO4; (5) CO5; (6) CO6; and (7) CO7. Eluent: Hexane:ethyl acetate (9:1). (a) Spray reagent anisaldehyde sulphuric acid reagent; (b) Spray reagent sulphuric acid 20%.

Gas chromatography coupled to mass spectrometry is one of the most used techniques in the analysis of copaiba oil‐resin. The terpenes are the main class of compounds found in copaiba oil‐resin, and the presence of sesquiterpenes and diterpenes has been described. According to Leandro et al. (2012), the sesquiterpenes can comprise more than 90% of the oil‐resin. The diterpenes are present in minor amounts.

The GC/MS total ion chromatograms (Figure 4) illustrate the chemical profile of the copaiba oil samples. Table 4 describes the volatile compounds identified in the samples by GC/MS as well as the relative amount of each compound. As can be seen, the compositional patterns of the oils are quite similar, with differences observed in both composition and compound levels. These differences are expected, as the chemical composition and concentrations of compounds can vary across Copaifera species (Lee et al. 2023). Additionally, variations in compound concentration can be observed within the same species growing in different locations and at varying collection times.

Chromatographic profiles by GC–MS for soybean oil, β‐caryophyllene and samples of copaiba oil‐resin. Samples: 3A (OS) soybean oil; 3B (CAR) β‐caryophyllene; (3C) CO1; (3D) CO2; (3E) CO3; (3F) CO4; (3G) CO5; (3H) CO6; and (3I) CO7. Analysis conditions: Capillary column (HP‐5MS, 25 μm thickness and 30 m) using an injection volume of 1 μL in split mode (1:10) with nitrogen as the carrier gas at a flow rate of 12.7 mL/min. Temperature gradient: 50 min at 100°C, 100°C–200°C with a variation of 1.5°C/min, 200°C–300°C with a variation of 2.5°C/min and 5 min at 300°C. Injector temperature at 220°C, mass source temperature 290°C and acquisition range 20–500 m/z. The identified compounds (1–26) are presented in Table 4.

The terpenes cannot be detected in Sample CO1 in the analysed concentration. Its profile was similar to that of soybean oil, indicating contamination of the CO1 sample with this vegetable oil. This result is consistent with TLC, which showed contamination of CO1 with soybean oil (Figure 3).

Only the β‐caryophyllene was identified in all six copaiba oil‐resin samples (Figures 5 and 6). β‐caryophyllene was the dominant compound in most of the samples, except for Sample CO4, in which caryophyllene oxide was the most abundant compound, suggesting that this sample may have undergone oxidation (Table 3).

The most abundant sesquiterpenes in copaiba oil‐resin samples.

Chromatographic profiles in HPLC‐RP of samples of copaiba oil‐resin. HPLC (Agilent 1200‐UV), column XDB‐C18 (4.6 × 250 mm × 5 μm), flow rate of 0.8 mL/min. Detection of ƛ 218 nm. Injection volume: 10 μL. Mobile phase: Ultra‐purified water (Phase A) and acetonitrile HPLC grade (Phase B), both with 0.1% formic acid in a linear gradient elution: 0–40 min 90% of B and 40–45 min 90%–95% of B.

By displaying the five most prominent components of each sample, Figure 5 clearly highlights the compositional differences among them. The results agree with data reported in the literature (Veiga Junior and Pinto 2002; Leandro et al. 2012).

An analytical method using HPLC‐DAD to analyse CAR in copaiba oil‐resin samples was proposed, as it is the main constituent of the CO samples and is commercially obtainable. The process was based on previous studies described by Oliveira et al. (2022).

The seven samples of copaiba oil‐resin showed a similar HPLC chromatographic profile, except for CO1 (Figure 4). The presence of β‐caryophyllene (RT = 15.559 min) aligns with the results obtained with the other chromatographic techniques used in this work.

The method demonstrated the ability to separate and identify the component CAR, even in samples with varying structures and compositions. Moreover, to confirm the retention time and selectivity of the method, the CAR standard was added to the CO samples, resulting in an increase in the CAR peak area relative to the CO samples without CAR addition.

Both samples, β‐caryophyllene and CO6, were used to validate the proposed method and to evaluate the system's adequacy (Table S1). The system suitability parameters were determined in accordance with the FDA criteria, except for resolution (Rs) (US Food and Drug Administration  2015). Although the observed resolution was lower than the US Food and Drug Administration recommendation, the resolution was considered good (Ravisankar et al. 2021).

The analytical curve (Figure 7) was expressed as the area of the CAR peak versus concentration of CAR (μg/mL): ** Y = 18.148X − 39.106** was obtained with the correlation coefficient, (R ^2^) 0.9996, in accordance with the criteria of RDC n° 166/2017 (Brasil 2017), which establishes as a minimum acceptable criterion that the analytical curve presents a correlation coefficient greater than or equal to 0.990.

Analytical curve β‐caryophyllene concentration (μg/mL) versus peak area.

The limit of detection (LOD) represents the lowest concentration of β‐caryophyllene that can be reliably detected. In contrast, the limit of quantification (LOQ) is the lowest concentration that can be accurately measured. Based on the response standard deviation and slope, the LOD was 1.26 μg/mL, and the LOQ was 3.83 μg/mL.

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The matrix effect was evaluated according to RDC 166/17 (Brasil 2017). The matrix effect was not significant (Figure S1 and Table S2), with a p‐value greater than 0.05, so the hypothesis that the slopes are equal is not rejected. In this case, the lines are parallel.

In an ideal situation for the accuracy (recovery) evaluation, the matrix will be free of the target analyte. However, in phytochemical analysis, the latter occurs naturally in the matrix. Therefore, the most common technique for determining accuracy in natural product studies is the standard addition method. In terms of accuracy (Table S3), the method provided recoveries ranging from 101.0 ± 6.9% to 108.1 ± 2.8% for β‐caryophyllene. The results are in accordance with the acceptable ranges for tests on complex matrices (80%–120%), such as natural products (Betz et al. 2011).

Intra‐day and inter‐day precision were determined. The obtained concentrations and their respective relative standard deviations (RSDs) are displayed in Table S4. All the RSD values for CA concentrations in intra‐ and inter‐day analyses were less than 5%, indicating that the results were adequate for the analytical purposes of the developed method (Agencia Nacional de Vigilancia Sanitaria 2003).

Considering robustness, the method was not robust (Table S5) when the wavelength was modified. It can be explained by the fact that many substances absorb at 210 nm. After changing the flow and then the temperature of the method developed, slight differences in the standard deviation were observed in the results. The HPLC flow rate is a significant factor in separation during analysis. A wider peak can occur at lower flow rates because of incomplete separation at higher flow rates. This way, the analytical methodology has robustness in terms of flow. The method developed was robust to oven temperature, with no significant standard deviation observed in the results. This parameter is important for the CAR and other CO compounds because they are volatile. The validation parameters showed satisfactory results. The method can be used to quantify the concentration of β‐caryophyllene in copaiba oil‐resin (Tables 4 and S6).

Copaiba oil‐resin exhibits various biological activities (Arruda et al. 2019; Frazão et al. 2023), with antimicrobial activity among the most frequently cited. The activity of the samples evaluated against S. epidermitis and S. aureus is shown in Table 5.

In the antibacterial susceptibility testing, four (4) samples were considered active against S. epidermidis , with growth inhibition values ranging from 71.40 ± 0.3 (CO5) to 82.30 ± 7.72 (CO3). In contrast, almost all samples were active against S. aureus , with inhibition of microbial growth exceeding that observed for S. epidermidis , ranging from 86.77 ± 1.24 (CO5) to 100.33 ± 0.96 (CO6). The most active copaiba oil‐resin sample was CO3, which demonstrated activity against both bacterial species evaluated, particularly against S. aureus . The antibacterial activity of copaiba oil‐resin has been vastly documented (Santos et al. 2008; Abrão et al. 2015; Tobouti et al. 2016; Frazão et al. 2024). This antibacterial effect seems to be associated with the mixture of sesquiterpenes and diterpenes, which affects the integrity of the bacterial cell wall (Tobouti et al. 2016).

Most species of the Copaifera genus have already been reported to exhibit antibacterial activity. Santos et al. (2008) demonstrated bactericidal action of Copaifera martii, C. officinalis L. and C. reticulata oils against * S. aureus , methicillin‐resistant S. aureus (MRSA), S. epidermidis *, Bacillus subtilis and Enterococcus faecalis (Santos et al. 2008). These authors also reported the lysis of the bacteria by the oil‐resin from C. martii , resulting in cellular agglomerates visible at scanning electron microscopy.

The copalic acid isolated and purified from C. langsdorffii was evaluated against a range of bacterial species. It showed antibacterial activity against S. aureus (surgical wound), Staphylococcus haemolyticus , S. capitis , E. faecalis and Streptococcus pneumoniae (Abrão et al. 2015).

Interestingly, the documented antibacterial activity of Copaifera species is marked against Gram‐positive bacteria. One reason for this fact is the differences in bacteria's cell walls: The stratified wall of Gram‐negatives would hinder the permeability of the antibacterial, suggesting that this resistance is due to the structural constitution of the bacteria themselves (Abrão et al. 2015).

According to Zielińska et al. (2023), the microbiota can affect wound‐healing dynamics. Therefore, the development of a product with healing properties and antibacterial activity would be highly significant, as it would address the need for effective wound treatment while simultaneously preventing infections and promoting tissue regeneration. Indeed, S. epidermidis promotes keratinocyte proliferation, whereas S. aureus colonization impedes healing. S. epidermidis upregulates Toll‐like receptors (TLRs) and modulates the downstream pathway of TNF‐α, which, through skin CD8+ T cells, accelerates the progression of keratinocytes. S. epidermidis produces lipoteichoic acid, which reduces inflammation via TLR2 signaling. S. aureus colonization may negatively affect wound healing because of elevated levels of keratinocyte cytokines and chemokine ligands, IL‐1β, IL‐6, CXCL‐1 and TNF‐α (Zielińska et al. 2023). In addition, S. aureus is among the pathogens associated with slow wound healing (Uberoi et al. 2024).

The in vitro cytotoxicity was evaluated using the RAW 264.7 cell line. These cells are a murine macrophage cell line commonly used in research to study inflammation and immune responses. In order to evaluate the statistical difference between the cytotoxic activity of the samples (Table 5), the Kruskal–Wallis test was performed using the R program (https://www.r‐project.org). The results were not significant (p > 0.05), indicating that the cytotoxic activity of samples is not statistically different. Sample CO1, which is clearly adulterated, exhibits cytotoxic activity that is very different from the others and was not evaluated in this analysis. To assess the chemical profile of the samples through the study of sesquiterpenes obtained in the GC analysis, HCA and PCA analyses were performed using the sesquiterpenes identified in the GC/MS analysis with the aid of the R program (https://www.r‐project.org). Two groups were clearly observed (Figures S2 and S3). Group 1 included Samples CO2, CO3, CO4 and CO7. Group 2 comprised Samples CO6 and CO5.

HCA was used to determine similarities between the samples based on the sesquiterpene content detected by GC/MS. The HCA results were represented in the dendrogram (Figure S2), which showed clustering of the samples into two main groups, Clusters I and II. The group was supported by the PCA, which accounted for 63.6% of the total information. Dim1 (PC1) and Dim2 (PC2) accounted for 35.9% and 27.7% of the total variance, respectively (Figure S3). The results of the chemometric analyses performed on sesquiterpenes detected by GC–MS in copaiba oleoresin indicate that the cytotoxic activity against the RAW 264.7 cell line is not exclusively related to them. Although the sesquiterpene profiles differ, two groups can be observed, and their biological activities are similar. β‐caryophyllene was cytotoxic to the RAW 264.7 cell line; however, the cytotoxicity of copaiba oil‐resin did not correlate with β‐caryophyllene concentration. This indicates that the cytotoxicity evaluated in this work is not solely due to β‐caryophyllene. In studies by Barbosa et al. (2019), the anticancer potential of copaiba oleoresin was attributed mainly to its diterpene content. Furthermore, Frota et al. (2025) demonstrated cytotoxicity in different cell lines for both terpene classes (diterpenes and sesquiterpenes). According to Vargas et al. (2015), the anti‐inflammatory, antimicrobial, antileishmanial, analgesic and antitumor activities are attributed to the intact oil, which results from the synergistic activity of the substances present in the copaiba oil‐resin.

To evaluate the toxicity properties of compounds, both in vitro and in vivo methods have been used. However, these experimental approaches are time‐consuming and expensive and raise ethical concerns about animal use in research. In silico methods have been employed to predict toxicity, offering a cost‐effective and time‐efficient alternative to traditional approaches. Examples of in silico tools for toxicity prediction include admetSAR (Yang et al. 2019), ProTox‐II (Banerjee et al. 2018) and ADMETlab 3.0 (Fu et al. 2024). Although in silico toxicity prediction methods are well established for isolated compounds, assessing the toxicity of plant extracts computationally remains difficult because of their complex mixtures and varying metabolite levels. Our previous work (Pereira Rocha et al. 2018; Rocha et al. 2018; Sudan et al. 2021) focused on predicting activity for extracts based on the main compounds identified using the Active‐IT program (Almeida et al. 2024). In this study, we examined the toxicity of β‐caryophyllene (1), α‐copaene (2), β‐copaene (3), β‐humulene (4) and α‐bergamotene (5) (Figure 1) using ADMETlab 3.0. We selected this tool because it is a free, fast, reliable, easy‐to‐use and straightforward online platform. According to the prediction results (Table S7), all evaluated sesquiterpenes have a low likelihood of being hERG blockers, causing DILI (drug‐induced liver injury), mutagenic effects (AMES toxicity), genotoxicity or adverse effects from a single exposure (Rat oral acute toxicity). They also show cytotoxic activity against RPMI‐8226 (Multiple Myeloma cell line), A549 (human non‐small cell lung cancer cell line) and Hek293 (human embryonic kidney cells).

In the reviewed literature, limited data are available on the toxicity of sesquiterpenes (Table S8). The most studied compound is β‐caryophyllene. The results for this compound align with existing literature, except regarding cytotoxicity. All the evaluated sesquiterpenes have been shown to be cytotoxic against various cell lines (Türkez, Toğar, et al. 2014; Türkez, Celik, and Toğar 2014; Fidyt et al. 2016; Dalavaye et al. 2024). Additionally, these sesquiterpenes demonstrated a high likelihood of causing skin sensitization. The findings for β‐caryophyllene are consistent with those reported by Inan et al. (2023) The tested sesquiterpenes have the potential to cause ocular irritation or corrosion and induce respiratory toxicity. In silico predictions suggest that β‐caryophyllene (1), β‐copaene (3) and humulene (4) are likely neurotoxic; however, β‐caryophyllene (1) has been described as non‐neurotoxic (Oliveira et al. 2023). No data are available for β‐copaene (3) and humulene (4). Regarding the human hepatotoxicity, most evaluated sesquiterpenes are likely hepatotoxic except for bergamotene. According to the literature, β‐caryophyllene (1), β‐copaene (3) and humulene (4) are not hepatotoxic (Oliveira et al. 2023; Lacerda Leite et al. 2021). All tested sesquiterpenes have a low probability of being nephrotoxic, except α‐copaene (2), which has a medium probability. Furthermore, β‐caryophyllene (1), α‐copaene (2) and β‐copaene are highly likely to cause hematotoxicity.

The evaluated sesquiterpenes showed a low likelihood of being genotoxic. Previous research (Di Sotto 2008; Gonçalves et al. 2011) demonstrated that β‐caryophyllene (1) has no cytotoxic, genotoxic or mutagenic effects. Studies by Almeida et al. (2012) with commercial copaiba oil‐resin also found no genotoxic or mutagenic effects.

Conclusions

4

The physicochemical characteristics evaluated, such as colour, acid value, ester value and solubility, varied among the samples of copaiba oil‐resin. However, physicochemical analysis alone is not sufficient to confirm the material's quality or detect adulteration. The TLC analysis indicated a possible adulteration in two samples (CO1 and CO2) with soybean oil (OS). According to GC/MS data, the CO1 sample was probably contaminated, as the terpenes were not detected at the concentration used. Of the seven copaiba oil‐resin samples analysed, only CO1 showed a distinct chromatographic profile (TLC, HPLC and GC). The HPLC‐DAD method for the quantification of β‐caryophyllene in copaiba oil‐resin was efficient, and acceptable values were obtained for the validation parameters. The β‐caryophyllene content in the samples was very distinct. These results can be interpreted as effects of sample collection or adulteration. All the samples studied, except CO1, showed antibacterial activity and were cytotoxic against the RAW 264.7 cell line. The combined analytical techniques with biological analyses may be used to assess the quality of copaiba oleoresin, to establish a protocol for its quality control.

Author Contributions

G.A.P. Graça and J.R. Vasconcelos carried out the physicochemical TLC and HPLC analysis as well as the original draft writing. D.O. Scoaris, P.P. Marcellini and I.R.M. Silva carried out the microbiological analysis. A.F. Silva and V.C.F. Mosqueira contributed for funding acquisition and conceptualization. L.S. Salomon and J.S.C. Santos carried out the cytotoxicity evaluation. C.G. Silva and J.C.D. Lopes designed this study, conceptualized, wrote, reviewed and edited. V.L. de Almeida designed this study and provided the resources and project management, writing, review and editing. The authors wrote and approved the final manuscript.

Funding

This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (APQ‐00373‐21 and BIP‐00213‐24).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Analytical curve β‐caryophyllene concentration (μg/mL) versus peak area (blue) and analytical curve with β‐caryophyllene concentration and fortified extract (100 μg) in orange. Figure S2: Dendrogram obtained by hierarchical cluster analysis of samples of copaiba oil‐resin. Figure S3: Classification by principal component analysis (PCA) of samples of copaiba oil‐resin. Table S1: System suitability of β‐caryophyllene (60 μg/mL) and CO2 sample (166.5 μg/mL). Table S2: Evaluation of matrix effect: Concentration of β‐caryophyllene (μg/mL) and fortified sample (100 μg) and area observed. Table S3: Accuracy data of the HPLC analytical method for quantification of β‐caryophyllene (μg/mL). Table S4: Results of repeatability (intra‐day) and intermediate precision (inter‐day) assays for β‐caryophyllene. Table S5: Robustness of the method considering the concentration of β‐caryophyllene (μg/mL)* and the variation in column temperature, flow and wavelengths. Table S6: Determination of β‐caryophyllene in copaiba oil‐resin samples. Table S7: Toxicity of β‐caryophyllene, β‐humulene, β‐copaene, α‐copaene and α‐bergamotene using ADMETlab 3.0. Table S8: Toxicity data found in the literature of β‐caryophyllene, β‐humulene, β‐copaene and α‐copaene.