Phytochemical Analysis and Antioxidant Activity of Ethanolic Extracts from Different Parts of Dipteryx punctata (S. F. Blake) Amshoff

Abstract

The genus Dipteryx, to which the cumaru tree belongs, contains neotropical species native to Central and South American countries. They are used both in the sale of timber and seeds and for the extraction of the active compound coumarin, used as a flavoring agent. This study evaluated the phytochemical profile and antioxidant activity of extracts of leaves, branches, and fruits (residues and seeds) of the species Dipteryx punctata. The plant material for analysis was collected in five seed-producing areas, in Mojuí dos Campos, Pará, Brazil. The extracts were obtained via Soxhlet extractor using 92.8% distilled ethanol as the solvent and operated till exhaustion (8 h). Chromatographic analyses were performed by thin-layer chromatography (TLC) and gas chromatography coupled to mass spectrometry (GC-MS), followed by phytochemical determination of phenolics and flavonoids and analysis of antioxidant activity (TLC and free radical scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH). The highest extract yields were obtained from D. punctata fruit residues and seeds from all areas, with maximum values of 26.1% and 47.2%, respectively, in Boa Fé (area 3). In the evaluation by TLC, the extracts of leaves, branches, and residues presented the classes of terpenes, condensed and hydrolysable tannins, and flavonoids; coumarin (1,2-benzopyrone) was identified only in residue and seed extracts. The major constituents highlighted in the collection areas were: lupeol in leaves (34.4% in area 5), 4-O-methylmannose in branches and residues (85.5% in area 2 and 90.6% in area 5, respectively), and coumarin in seeds (99.3% in area 3). The best results for the antioxidant action were obtained for extracts from leaves and residues, requiring a concentration of 117.6 µg.mL⁻¹ of the extract from the leaves and 160.4 µg.mL⁻¹ of the extract from the residues to reduce the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical by 50%. This phytochemical study contributes to reducing the scarcity of information on D. punctata. The chemical classes and compounds identified corroborate the antioxidant activity and add value to the species, and the data obtained reinforce the importance of reusing fruit residues, which are chemically rich but discarded in the environment.

1. Introduction

The Amazon has a variety of species that have added economic value to both timber and non-timber forest products, and which generate work and income for various communities in the region, with an emphasis on species of the genus Dipteryx.

This genus, native to Central and South American countries, belongs to the botanical family Fabaceae (Leguminosae) and consists of 11 species [1,2,3,4] that are found in natural forests and highly sought after for monocultures, agroforestry systems, and reforestation [5].

Agroforestry systems (AFSs) integrate forest species, normally of commercial value, with perennial and annual crops, with or without the presence of animals. The most outstanding systems are taungya, silvopastoral, agroforestry, home gardens, and commercial multi-stratified systems and they enable family farmers to obtain income from different species and products throughout the year [6].

Dipteryx odorata (Aubl.) Forsyth f., the best-known species of the genus in the Amazon, is incorporated into plantations and used to obtain wood and extract simple coumarin from the seeds [7,8].

The wood of this species is dense to very dense (0.95 g.cm⁻³ to 1.19 g.cm⁻³), heavy, of good quality, rot-resistant, and resistant to fungi, insects, and marine borers. It is widely used in the shipbuilding and agricultural implement industries. In the construction industry, it is used for the manufacture of beams, rafters, stakes, struts, fence posts, and floorboards, and it can even be used in laminated joinery articles [5,8].

In the almond market (processed seeds), data from 2010 show that Brazil sold 95 tons (t) of cumaru almonds from Pará, generating revenue of BRL 744,000.00. In 2018, there was an increase in sales (170 tons) and production value (BRL 4,105,000.00) [9].

However, other species of the genus, such as Dipteryx punctata (S. F. Blake) Amshoff, can also be exploited to obtain coumarin and other plant products [10].

Dipteryx punctata can grow to a height of 10 m in cultivated formations (Figure 1A). It has a cylindrical trunk (Figure 1B), branches with no exudate and compound, and pinnate leaves, with alternate oval leaflets (Figure 1C). The inflorescence is a panicle with yellow to purple flowers (Figure 1D). The oblong, oval fruits are drupes (Figure 1E), with late dehiscent valves and the endocarp opens only after mesocarp decomposition (Figure 1F).

Many plant species produce substances with cosmeceutical, pharmacological, microbiological, and other properties and have been scientifically researched. In that context, vegetable oils and extracts are particularly important sources of biologically active substances with potential for product development.

The aqueous extract of cumaru fruit residues (epicarp + mesocarp) is used in folk medicine as an antispasmodic and as a tonic to combat coughs, colds, and lung problems. From the cooking of fruits and seeds, a type of tonic is also obtained with anesthetic, diaphoretic, and emmenagogic properties that act as an efficient moderator of heart and respiration rates [5,8].

Plant extracts are concentrated preparations, obtained after harvesting parts of the plant (leaves, branches, fruits, seeds, roots, and rhizomes). They have different consistencies and may be obtained by enzymatic inactivation, drying, and grinding, or by methods involving the use of solvents such as ethyl alcohol or water itself, in which case the compounds are separated by fractionation of the extract and purification of the active principal [11].

Secondary metabolites are chemical compounds found in products of natural origin and the three most important groups are terpenes (giving rise to monoterpenes, sesquiterpenes, diterpenes, triterpenes, and tetraterpenes), phenolic compounds (derived from carbohydrates, such as flavonoids, tannins, and coumarins, among others) and alkaloids (derived from amino acids, the main constituents of proteins) [12].

Among the phenolic compounds, coumarin 1,2-benzopyrone is the simplest representative; it was isolated for the first time from D. odorata fruits by Vogel in 1820 [13]. However, more than 1300 coumarins have been identified from natural sources, especially green plants, and their pharmacological and biochemical properties and their therapeutic applications depend on their substitution pattern [14]. In that sense, products of plant origin emerge as a source of active substances with the potential for various biological activities, including antioxidant, insecticidal, fungicidal, herbicidal, and nematicidal action [15].

Pharmacologically, the antioxidant activity has been extensively investigated with a view to replacing products of synthetic origin with those of natural origin for insertion in human food and use in the prevention of pathologies. Scientifically, it has been demonstrated that the progression and onset of various pathologies may be related to oxidative stress in degenerative, cardiac, inflammatory, immunological, and neurological diseases, among others [16].

The DPPH (2,2-diphenyl-1-picrylhydrazyl) method, for example, is widely used to assess antioxidant capacity [17,18]. The test evaluates the ability of a sample to eliminate or neutralize free radicals, which is monitored using a UV/visible spectrophotometer [19]. Antioxidant activity is usually expressed by the IC50 parameter (inhibitory concentration), which is defined by the antioxidant agent’s ability to sequester 50% of the free radicals present in the solution. The inverse relationship between the amount of sample and the antioxidant activity means that the lower the IC50 value is, the greater the antioxidant activity of the product [20].

In this context, chemically characterizing the extracts from different parts of D. punctata and evaluating their potential in terms of antioxidant activity is an interesting strategy because, in addition to the lack of chemical information about this species, the seeds of Dipteryx spp. are used in the food industry, mainly for the manufacture of sweets and ice cream [7,8]. These facts encourage studies on the capture of free radicals, add economic value to these species, and contribute to the increased demand for them in agroforestry systems. In addition, finding an antioxidant action in fruit residues would open up the possibility of a new destination for this by-product hitherto discarded in the environment, thereby making more efficient use of production. Thus, this study aimed to evaluate the phytochemical profile and antioxidant activity of extracts obtained from leaves, branches, and fruits (residues and seeds) of Dipteryx punctata.

2. Materials and Methods

2.1. Collection Areas

The plant materials were collected in the rural communities of Água Fria, Terra de Areia, and Boa Fé, located in the municipality of Mojuí dos Campos, Eastern Amazon, Pará, Brazil. Collections were carried out in two periods: one in the months of February and March 2018 (average temperature of 25 °C), to obtain leaves, branches, and flowers, and another in September of the same year (average temperature of 30 °C), to obtain fruits (residues and seeds). The five areas with implemented agroforestry systems producing cumaru trees were selected according to the guidance of the local office, in Mojuí dos Campos, of the Technical Assistance and Rural Extension Company of the State of Pará (Emater-PA) [21].

2.2. Description of the Collection Areas

Area 1—located in the Água Fria community (geographical coordinates 2°46′21.69″ S; 54°39′25.13″ W) (Figure 2A). It consists of two hectares, containing 120 cumaru trees, approximately 10 years old, five to nine meters high, in consortium with interspersed rows of orange trees (Citrus sinensis) planted sequentially. The cumaru trees are spaced 16 m between rows and four meters between plants. Area cleaning and tree pruning are carried out regularly.

Area 2—located in the Água Fria community (geographical coordinates 2°46′45.24″ S; 54°38′47.06″ W) (Figure 2B). It consists of 0.5 hectares, containing 70 cumaru trees, approximately 10 years old, five to nine meters high, in consortium with interspersed lines of orange trees planted sequentially. The cumaru trees are spaced eight meters between rows and four meters between plants [22]. Area cleaning and tree pruning are carried out regularly.

Area 3—located in the Boa Fé community (geographical coordinates 2°37′14.33″ S; 54°40′53.22″ W) (Figure 2C). It consists of two hectares, containing 14 cumaru trees, approximately 10 years old and an average of nine meters in height, intercropped with sequentially planted black pepper (Piper nigrum), with no defined spacing. The area is cleaned regularly.

Area 4—located in the Terra de Areia community (geographical coordinates 2°47′53.22″ S; 54°38′28.77″ W) (Figure 2D). It consists of one hectare containing 70 cumaru trees, approximately 10 years old, five to nine meters high, in consortium with separate lines of sequentially planted orange trees. The cumaru trees are spaced eight meters between rows and four meters between plants. This area has not adopted cultivation practices.

Area 5—located in the Boa Fé community (geographical coordinates 2°39′16.95″ S; 54°40′50.08″ W) (Figure 2E). It has two hectares containing 160 cumaru trees, approximately 10 years old and an average of nine meters in height, intercropped in the same row with orange trees and other crops (pineapple and different types of citrus), with the presence of animals (goats, sheep, chickens, and pigs), without defined spacing. Area cleaning and tree pruning are carried out regularly.

2.3. Abiotic Factors in the Collection Areas

The climate is of the humid tropical type, with temperatures ranging from 25 °C to 30 °C, and, in the collection period, the precipitation, according to the Quantis classification, was: rainy season to normal, 85% to 66% (February 2018), normal season dry, 66% to 33% (March 2018), normal dry period, 66% to 33% (September 2018) [23]. The taxonomic classification of the predominant soil in the areas of Mojuí dos Campos is dystrophic yellow latosol, typical, moderate A, very clayey texture, sub-perennial equatorial forest, flat and gently undulating relief [24].

2.4. Collection of Plant Material

Seven D. punctata trees were selected in each of the five areas based on their productivity, that is, plants that bear fruit regularly every year, and with similar morphological and physiological characteristics (height, diameter, and flowering). To obtain extracts from the 35 trees, leaves, branches, and 30 fruits per tree were collected, that is, 210 fruits per area and 1050 fruits altogether [21].

Specimens of the collected materials, botanically identified as Dipteryx punctata, were exsiccated and have been deposited in the Herbarium of the Federal University of Western Pará under registration numbers: HSTM 11897, HSTM 11898, HSTM 11899, HSTM 11900, and HSTM 11901, and in the Herbarium of the Botanical Garden of Rio de Janeiro under registration numbers: RB 772255, RB 772253, RB 772256, RB 772257, and RB 772254. The research was registered in the National System for the Management of Genetic Resources and Associated Traditional Knowledge (SisGen, Brazil) under protocol A1B0150. SisGen is an electronic system created to assist the Genetic Heritage Management Council (CGen), Ministry of Environment and Climate Change, Brazilian Federal Government.

2.5. Obtaining and Plant Extracts and Yields from Leaves, Branches, and Fruits of Dipteryx Punctata

The extracts obtained from all collected parts of the plants and the fruits of D. punctata were separated into residues (epicarp + mesocarp + endocarp) and seeds. The leaves, branches, and fruits were weighed, packed in paper bags, and placed in an oven at 45 °C, with forced air circulation for drying for 20 days. After that, the plant material was weighed again to obtain the dry mass and ground before performing the ethanolic extraction. From each area, 70 g of leaves, 35 g of branches, 70 g of residues, and 35 g of seeds were used for the extraction, which was performed in triplicate [21].

The extraction procedures took place via Soxhlet extractor, in which the previously ground plant materials were transferred to cartridges (sachets) made with filter paper to retain material remnants (largest pore: 44 µm and many pores: 26 µm), and added inside the extractor cup of the device. Then, 92.8% ethyl alcohol (distilled for 4 h with 2.0 g sodium hydroxide for 2 L) was added as a solvent in the glass flask, and extraction was started until exhaustion (8 h) [25]. The ethanolic solutions obtained were evaporated in a rotary evaporator (manufactured by Fisatom) to eliminate the solvent and obtain the ethanolic extracts which were stored in amber glass containers, sterilized, and subjected to drying at room temperature (±27 °C) [25].

To obtain the yields (%), the extracts were weighed and the formula [Mass of oil or extract (g)/Mass of dry plant material (g)]*100 [25] was applied. Analysis of Variance (ANOVA) was performed with the yield data and the treatments means compared by the Tukey test (p ≤ 0.05), using the SISVAR statistical software, version 5.6, Build 90 [26].

2.6. Thin-Layer Chromatography (TLC) for the Evaluation of the Main Classes of Compounds

For the chromatographic analysis of the ethanolic extracts of D. punctata, the study used 10 × 10 cm Alugram Xtra aluminum plates in silica gel 60, (manufactured by Macherey-Nagel GmbH & Co. KG, German Company, Düren, Germany), with a fluorescent indicator and layer thickness of 0.20 mm. The extracts were weighed (10 mg), solubilized in 1 mL of solvent, homogenized, and 10 µL aliquots applied to the plates, 1.5 cm apart from each other.

The system consisted of plates eluted using a mixture of ethyl acetate: hexane (70:30). Subsequently, the plates were dried and revealed to identify the classes of terpenes (1% sulfuric vanillin and thymol standard), condensed tannins (1% hydrochloric vanillin and green tea standard (Camellia sinensis)), hydrolysable tannins (ferric chloride, 1% FeCl₃ and C. sinensis standard), flavonoids (aluminum chloride, 5% AlCl₃ and quercetin standard) and coumarin (potassium hydroxide, 5% KOH and 1,2-benzopyrone standard) [27]. The reagents were from Dinâmica Química Contemporânea LTDA (Brazilian Company located in São Paulo), and the standards were from Sigma-Aldrich (American Company located in Saint Louis, Missouri). Retention factors (Rf) were calculated when possible [25].

2.7. Determination of Total Phenolics and Flavonoids

For the evaluation of the presence of phenolics and flavonoids in the ethanolic extracts of D. punctata, representative samples from all collection areas were weighed and grouped, based on the similarity of the chemical results. Then, the samples of each part of the plant were diluted and aliquots of the solution were added to test tubes to obtain concentrations of 30, 70, 200, and 450 µg.mL⁻¹ for leaves and residues, and 50, 200, 500, and 1000 µg.mL⁻¹ for branches and seeds, up to a volume of 5000 µL in ethyl alcohol P. A.

For the determination of total phenolics with adaptations, triplicates were prepared to contain 500 µL of each evaluated concentration, 2.5 mL of Folin–Ciocalteu at 5% (v/v), and 2.0 mL of sodium carbonate (NaCO₃) to 4% (w/v). The control consisted of 500 µL of ethyl alcohol, 2.5 mL of 5% Folin–Ciocalteu, and 2.0 mL of sodium carbonate (4% NaCO₃). The reading (λ = 740 nm), performed through a UV/Visible spectrophotometer, occurred after keeping the samples in the dark for two hours and the formation of bluish solutions. The calculation of results was based on the seven points analytical curves (10–80 µg.mL⁻¹) of the gallic acid standard (y = 83.532 x − 0.5777 and R² = 0.99), expressed as mg of GAE (gallic acid equivalent) per gram of extract [28].

For total flavonoids with some modifications, triplicates were obtained by adding 2.4 mL of 0.1% aluminum chloride (AlCl₃) solution to 600 µL of each evaluated concentration. The control consisted of 600 µL of ethyl alcohol and 2.4 mL of aluminum chloride (0.1% AlCl₃). The reading (λ = 420 nm) occurred after a period of 30 min and was characterized by the reaction between the flavonoid and aluminum forming a yellowish solution. The calculation of the results was based on the seven points analytical curve (5–80 µg.mL⁻¹) of the rutin standard (y = 83.532 x − 0.5777 and R² = 0.99) and expressed as mg of RE (rutin equivalents) per gram of extract [29].

2.8. Gas Chromatography Coupled to Mass Spectrometry (GC-MS)

The chromatographic analysis of the ethanolic extracts of D. punctata was performed at the Analytical Instrumentation Center of the University of São Paulo (Analytical Center of the Institute of Chemistry, USP), using a QP2020 chromatograph, Shimadzu brand, and linear scanning in the interval of 37 m/z to 660 m/z.

Chromatographic conditions were: injector temperature maintained at 280 °C, the column used was a DB5MS (30 m × 0.25 mm wide × 0.25 mm thick), with initial temperature of 50 °C (3 min), heating of 3 °C/min, final temperature of 280 °C (15 min), detector at 280 °C, carrier gas (He) flow rate of 1.82 mL/min, and volume of injected extracts of 1 µL.

Compounds were identified by comparing their retention indexes (RI), mass spectra, and detected mass fragmentations with those stored in the NIST14 library, and with data from the literature [25,27].

2.9. Antioxidant Activity of Dipteryx Punctata Extracts by TLC and DPPH Free Radical Scavenging

The ethanolic extracts obtained were also evaluated by TLC for their antioxidant potential, according to an adapted methodology [17], using the same elution system for the different classes of compounds, and ABTS (2,2′-azinobis (3-etthylbenzothiazoline-6-sulfonic acid)) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals as developers. Plates developed with ABTS (7 mM) had ascorbic acid as standard (1000 µg.mL⁻¹) and for those developed with methanolic solution DPPH (0.2%), the standard used was gallic acid (100 µg.mL⁻¹).

The antioxidant potential resulting from the reduction in ABTS was evaluated by the presence of white spots on a greenish background after 48 h and that of DPPH by the presence of light-yellow spots against purple staining, after 2 h. All plates were kept in the dark during this period.

After qualitative verification of the antioxidant action on the plaques, the selected quantitative method was DPPH free radical capture with some modifications [30], as only this analysis enables the necessary extract concentrations to be obtained for a 50% reduction in radicals [31]. Representative samples from all collection areas were weighed and grouped; then, the sample from each part of the plate was diluted and aliquots added to test tubes to obtain concentrations of 40, 70, 200, 350, and 500 µg.mL⁻¹ for leaf and fruit residue extracts and 230, 800, 1500, 2500, and 3000 µg.mL⁻¹ for branch and seed extracts, up to a volume of 600 µL in methyl alcohol P. A.

After this process, 2.4 mL of the DPPH radical solution (29 µg.mL⁻¹) diluted in methyl alcohol P.A. were added to the tubes and homogenized in a shaker. For negative control, 600 µL of methyl alcohol and 2.4 mL of DPPH radical were used. All analyses were performed in triplicate. The two readings were performed with a UV/visible spectrophotometer (λ = 516 nm) and monitored at intervals of 40 min (80 min in total) until the absorbance stabilized. The antioxidant action was verified by observing the color change in the sample (purple or violet) to light yellow or pale violet after that period in the dark.

The DPPH index was calculated using the equation: I (%) = [(Abs0 − Abs1)/Abs0] × 100, where Abs0 is the absorbance of the blank and Abs1 is the absorbance of the sample. Regression analysis was performed using the SISVAR statistical software, version 5.6, Build 90 [26]. Based on the relationship between the concentrations tested and the percentage of inhibition calculated, adjusted logarithmic functions were obtained with representation of each part of the plant given via y = a ln (x) ± b (a and b = constants; ln = natural logarithmic function) for the calculation of IC50 values (extract concentration necessary to reduce 50% of the DPPH radical), where IC50 = EXP((50 − b)/a). The logarithmic model was used to plot the graphs in the electronic spreadsheet program, Microsoft Office Excel 365 (Microsoft Corporation, an American Company located in Redmond, Washington), presenting five points plotted for each extract (leaves, branches, residues, and seeds).

3. Results

3.1. Yield of Dipteryx punctata Ethanolic Extracts

There was a significant difference in the yield of ethanolic extracts for both factors (parts of the plant used and collection areas). In all areas, the highest yields were obtained for the seed extracts, except in area 1, in which they did not differ from the residues’ extract (

Table 1. Mean yield of ethanolic extracts from different parts of Dipteryx punctata from agroforestry systems in Mojuí dos Campos, Brazilian Amazon.

Parts of Plant	Mean Yield of Extracts (%)
	Area 1	Area 2	Area 3	Area 4	Area 5
Leaves	17.3 bA	17.9 bA	16.9 cA	14.2 cA	15.2 cA
Branches	7.9 cA	7.1 cA	5.8 dA	9.1 dA	8.6 dA
Residues	24.8 aAB	18.6 bC	26.1 bA	23.3 bAB	20.7 bBC
Seeds	28.4 aC	32.0 aC	47.2 aA	37.1 aB	39.7 aB
CV (%)					8.8

Note: Means followed by the same lowercase letters in the columns and by the same uppercase letters in the rows do not differ from each other by the Tukey test (p ≤ 0.05). CV = coefficient of variation.

).

The extracts from the residues also showed great potential regarding the use of plant biomass, with higher yields than the extracts from the leaves and branches (Table 1).

The highest average yield of seed extracts was observed in area 3 (Table 1) and it was 66.2% higher compared to the extract obtained in area 1 and 47.5%, 27.2%, and 18.9% higher than seed extract yields from areas 2, 4, and 5, respectively. Extracts obtained from fruit residues collected in area 3 also showed higher yields compared to extracts from areas 2 and 5 (Table 1), with increases of 40.3% and 26.1%, respectively.

3.2. Thin-Layer Chromatography (TLC) for the Evaluation of the Main Classes of Compounds

In the phytochemical analysis of ethanolic extracts of D. punctata by TLC, except extracts from seeds, the classes of terpenes, condensed tannins, hydrolysable tannins, and flavonoids were observed in the extracts from leaves, branches, and residues from all areas. Their presence was indicated by the intensity and color of the samples when compared with the standards used (

Table 2. Chemical analysis by thin-layer chromatography (TLC) of ethanolic extracts from different parts of Dipteryx punctata from agroforestry systems in Mojuí dos Campos, Eastern Amazon, Brazil.

Plant Parts from All Areas	TLC
	Terpenes	Condensed Tannins	Hydrolysable Tannins	Flavonoids	Coumarin (1,2-Benzopyrone)
Leaves	+	+	+	+	−
Branches	+	+	+	+	−
Residues	+	+	+	+	+
Seeds	−	−	−	−	+

Note: (+) presence of the compounds class; (−) absence of the class of the compound.

).

As for 1,2-benzopyrone (simple coumarin), the results show its presence in extracts of residues and seeds of D. punctata (Table 2), with a retention factor of 0.78 (Figure 3C,D). For leaves and branches, this compound was not observed (Figure 3A,B), however, for branches, there was the possible presence of other compounds of the coumarin class (Figure 3B).

3.3. Determination of Total Phenolics and Flavonoids

The results obtained in the phenolic and flavonoid determination tests confirmed those found in the TLC. Phenolic compounds were evidenced in all extracts, with leaf and branch extracts showing the highest levels (51.76 ± 0.16 mg GAE g⁻¹ and 48.45 ± 0.50 mg GAE g⁻¹, respectively). The residue and seed extracts showed lower values compared to the other samples, however, these contents are above 24 milligrams of gallic acid equivalents per gram of sample (

Table 3. Analysis of phenolic and flavonoid compounds in ethanolic extracts of Dipteryx punctata samples containing representatives from all collection areas.

Plant Part from All Areas	Total Phenolics	Total Flavonoids
	mg GAE g−1 Sample ± Standard Deviation	mg RE g−1 Sample ± Standard Deviation
Leaves Branches Residues Seeds	51.76 ± 0.16 48.45 ± 0.50 39.97 ± 1.75 25.07 ± 0.20	115.40 ± 3.41 69.71 ± 0.51 71.86 ± 3.79 Not Found

Note: mg GAE g⁻¹ sample = milligrams of gallic acid equivalents per gram of sample; mg RE g⁻¹ sample = milligrams of rutin equivalents per gram of sample.

). About the class of flavonoids, the highest content was obtained in the extract of the leaves, 115.40 ± 3.41 mg RE g⁻¹. For the extracts of branches and residues, the values found were 69.71 ± 0.51 mg RE g⁻¹ and 71.86 ± 3.79 mg RE g⁻¹, respectively; the presence of this class was not detected in seed extracts (Table 3).

3.4. Gas Chromatography Coupled to Mass Spectrometry (GC-MS)

In addition to the major constituent (highest percentage), the extract analyses found compounds present in the plant material obtained from all collection areas with percentages above 5% for at least one of them and those were considered to be markers for the parts of the plant.

In the extracts of the D. punctata leaves, 24 compounds were identified. The markers were: lup-20(29)-en-3-one, with the highest percentage in area 1 (11%) and γ-sitosterol (5.6%), octadecanoic acid ethyl ester (stearic acid ethyl ester) (7.5%), (-)-spathulenol (9.2%), (Z,Z,Z)-9,12,15-octadecatrienoic acid ethyl ester (linolenic acid ethyl ester) (10.6%), and hexadecanoic acid ethyl ester (palmitic acid ethyl ester) (11.8%) area 2, (

Table 4. Chemical compounds identified in the ethanolic extracts of the leaves of Dipteryx punctata, in the five collection areas of the municipality of Mojuí dos Campos, Eastern Amazon, Brazil.

LEAVES EXTRACTS				Area 1	Area 2	Area 3	Area 4	Area 5
Nº	IRT	FRT	COMPOUNDS	RC %	RC %	RC %	RC %	RC %
01	22.47	22.48	2-Methoxy-4-vinylphenol	2.5	-	1.7	-	3.0
02	32.88	33.22	(-)-Spathulenol	4.6	9.2	3.0	3.0	2.4
03	32.95	33.12	Ethyl 3-(2-hydroxyphenyl) propanoate	-	-	1.8	-	-
04	39.15	39.35	(E)-4-(3-Hydroxyprop-1-en-1-yl)-2-methoxyphenol	-	-	-	-	2.1
05	42.66	42.67	Neophytadiene	2.6	4.0	3.3	4.3	3.2
06	43.62	43.65	(3S,3aS,6R,7R,9aS)-1,1,7-Trimethyldecahydro-3a,7-methanecyclopenta[8]anulene-3,6-diol	2.1	-	1.9	2.9	2.1
07	46.77	47.04	n-Hexadecanoic acid (palmitic acid)	4.7	4.0	5.1	4.8	3.5
08	47.71	47.75	Hexadecanoic acid ethyl ester (Palmitic acid ethyl ester)	3.8	11.8	4.6	4.6	3.6
09	49.45	52.71	(Z,Z)-9,12-octadecadienoic acid (linoleic acid)	3.0	5.2	-	-	-
10	51.29	51.38	Phytol	8.0	9.8	11.1	10.2	9.0
11	52.25	52.37	(Z,Z,Z)-9,12,15-octadecatrienoic acid (linolenic acid)	3.5	-	4.9	5.0	3.8
12	52.72	52.74	(Z,Z)-9,12-octadecadienoic acid ethyl ester (Linoleic acid ethyl ester)	2.5	-	2.3	2.4	2.1
13	52.89	52.95	(Z,Z,Z)-9,12,15-octadecatrienoic acid ethyl ester (Linolenic acid ethyl ester)	7.4	10.6	9.2	7.2	7.5
14	53.71	53.75	Octadecanoic acid ethyl ester (Stearic acid ethyl ester)	3.2	7.5	3.3	3.3	2.7
15	54.57	54.82	(E) -3-Methyl-5-((1R, 4aR, 8aR)-5,5,8a-trimethyl-2-methylenedeca-hydronaphthalen-1-yl) pent-2-en-1-ol (copalol)	-	-	2.5	-	-
16	63.08	63.11	Bis(2-ethylhexyl)phthalate	2.2	-	2.4	2.2	1.7
17	67.80	67.99	1,4-Benzenedicarboxylic acid Bis(2-ethylhexyl) ester	-	-	1.4	-	1.6
18	72.36	75.75	δ-Tocopherol	-	-	1.5	-	2.0
19	78.70	78.76	Stigmasterol	3.3	4.5	3.1	3.7	3.4
20	80.05	80.14	γ-Sitosterol	4.9	5.6	4.8	5.5	4.4
21	80.67	80.74	4, 4, 6a, 6b, 8a, 11, 11, 14b-Octamethyl-1, 4, 4a, 5, 6, 6a, 6b, 7, 8, 8a, 9, 10, 11, 12, 12a,14, 14a,14b-octadecahydro-2H-picen-3-one	3.4	4.2	2.7	3.3	2.1
22	81.27	81.42	Lup-20(29)-en-3-one	11.0	7.8	6.4	8.5	5.0
23	81.87	82.15	Lupeol	24.6	15.9	22.8	27.0	34.4
24	83.71	83.72	Stigmast-4-en-3-one	2.6	-	-	2.4	-
Total %				100	100	100	100	100

Note: IRT = initial retention time; FRT = final retention time of the compounds obtained in the collection areas; RC % = relative content in percentage; Appendix A (Figure A1, Figure A2, Figure A3, Figure A4 and Figure A5) = chromatograms of the leaf extracts in the five collection areas.

).

N-hexadecanoic acid (palmitic acid) and phytol were also indicated as markers in D. punctata leaf extracts, with the highest percentages obtained in area 3 (5.1% and 11.1%, respectively) (Table 4).

Lupeol predominated in all areas, and in the material collected in areas 4 and 5, the percentages were 27% and 34.4%, respectively (Table 4). Other substances with percentages below 5% identified in D. punctata leaves in all areas were: neophytadiene, stigmasterol and the 4, 4, 6a, 6b, 8a, 11, 11, 14b-octamethyl-1, 4, 4a, 5, 6, 6a, 6b, 7, 8, 8a, 9, 10, 11, 12, 12a, 14, 14A, 14b-octadecahydro-2H-picen-3-one.

For the ethanolic extracts of D. punctata branches, the analyses identified 16 compounds, among which hydrocoumarin and lupeol are presented as markers. Lupeol was identified as the major compound present in the plant material collected in areas 1, 4, and 5, with percentages above 5% in the other collection areas (

Table 5. Chemical compounds identified in the ethanolic extracts of the branches of Dipteryx punctata, in the five collection areas of the municipality of Mojuí dos Campos, Eastern Amazon, Brazil.

BRANCHES EXTRACTS				Area 1	Area 2	Area 3	Area 4	Area 5
Nº	IRT	FRT	COMPOUNDS	RC %	RC %	RC %	RC %	RC %
01	25.05	25.40	Hydrocoumarin	7.2	1.1	16.8	7.3	9.3
02	33.16	33.19	(-)-Spathulenol	8.3	-	16.9	4.5	3.4
03	39.06	46.19	4-O-Methylmannose	18.1	85.5	25.4	4.2	-
04	46.79	47.04	N-hexadecanoic acid (palmitic acid)	8.9	1.3	-	9.0	9.5
05	47.63	47.87	Hexadecanoic acid ethyl ester (Palmitic acid ethyl ester)	-	-	-	-	4.0
06	51.96	52.11	(Z,Z)-9,12-octadecadienoic acid (linoleic acid)	-	1.0	-	4.5	4.5
07	52.02	52.47	(Z)-9-octadecenoic acid (oleic acid)	6.8	-	-	-	-
08	52.15	52.28	(Z)-9-octadecenal	-	-	-	5.4	5.1
09	52.63	52.87	(Z,Z)-9,12-octadecadienoic acid ethyl ester (Linoleic acid ethyl ester)	-	-	-	-	3.0
10	52.87	52.99	Octadecanoic acid (stearic acid)	-	-	-	5.3	5.2
11	63.08	63.12	Bis(2-ethylhexyl)phthalate	7.4	-	16.9	6.4	6.9
12	67.80	68.00	1,4-Benzenedicarboxylic acid Bis(2-ethylhexyl) ester	-	-	-	-	3.4
13	78.69	78.75	Stigmasterol	7.1	1.4	-	5.4	4.8
14	80.05	80.13	γ-Sitosterol	8.4	1.6	-	6.9	7.1
15	81.29	81.35	Lup-20(29)-en-3-one	8.9	2.3	-	7.3	4.7
16	81.89	82.08	Lupeol	18.9	6.0	23.9	33.9	29.1
Total %				100	100	100	100	100

Note: IRT = initial retention time; FRT = final retention time of the compounds obtained in the collection areas; RC % = relative content in percentage; Appendix A (Figure A6, Figure A7, Figure A8, Figure A9 and Figure A10) = chromatograms of the branches extracts in the five collection areas.

). Only in the plant material collected in area 1 was (Z)-9-octadecenoic acid (oleic acid) detected (6.8%). Other compounds present in the extract of branches collected in this and other collection areas were (-)-spathulenol, 4-O-methylmannose, n-hexadecanoic acid, bis(2-ethylhexyl)phthalate, stigmasterol, γ-sitosterol, and lup-20(29)-en-3-one (Table 5). Except for 4-O-methylmannose, these compounds were also identified in D. punctata leaves extracts.

4-O-methylmannose was the major constituent in the extract of branches collected in area 2 (85.5%) and in area 3 (25.4%) (Table 5). For area 3, all the constituents of the extract of the branches presented percentages above 5%, with the highest values for this area being for hydrocoumarin (16.8%), (-)-spathulenol (16.9%), and bis(2-ethylhexyl)phthalate (16.9%) (Table 5).

In areas 4 and 5, in addition to the common compounds present in the extracts of branches found in all collection areas, the analysis verified the presence of (Z)-9-octadecenal (area 4 with 5.4% and area 5 with 5.1%) and octadecanoic acid (stearic acid) (area 4 with 5.3% and area 5 with 5.2%) (Table 5).

The analyses of the extracts of D. punctata residues by GC-MS identified 31 different chemical substances. Among the 14 compounds analyzed in area 1, for example, were n-hexadecanoic acid (5.1%), hexadecanoic acid 2-hydroxy-1-(hydroxymethyl) ethyl ester (5.5%), (S)-isopropyl lactate (6.5%), 2,3-butanediol (7.3%) and, 9-octadecenoic acid (E,E,E)-1,2,3-propanetriyl ester (15.8%), this last being the outstanding compound of this area, and of area 2, with a percentage of 16%, and area 4, with 10.6% (

Table 6. Chemical compounds identified in the ethanolic extracts of the residues of Dipteryx punctata, in the five collection areas of the municipality of Mojuí dos Campos, Eastern Amazon, Brazil.

RESIDUES EXTRACTS				Area 1	Area 2	Area 3	Area 4	Area 5
N.	IRT	FRT	COMPOUNDS	RC %	RC %	RC %	RC %	RC %
01	3.39	3.68	2-Propenoic acid methyl ester	-	-	0.4	-	0.3
02	3.47	3.79	2,3-Butanediol	7.3	5.0	-	8.0	-
03	3.50	3.72	(S)-Isopropyl lactate	6.5	-	-	-	-
04	3.56	3.83	2,3-Pentanedione	-	-	0.3	-	0.2
05	3.79	4.05	Pyruvic acid methyl ester	-	-	1.3	-	0.8
06	4.13	4.19	Furfural	-	-	0.7	4.8	1.1
07	4.27	4.36	1,2-Diacetylhydrazine	-	-	0.3	-	-
08	4.85	5.14	2-Furanomethanol	3.9	-	0.5	4.8	0.5
09	5.42	5.49	4-Cyclopentene-1,3-dione	-	-	0.4	-	0.3
10	6.82	6.99	1,2-Cyclopentanedione	-	-	0.4	-	0.4
11	7.77	7.83	5-Methyl-2-Furanecarboxaldehyde	-	-	0.3	-	0.2
12	8.35	8.51	2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one	-	-	0.3	-	0.2
13	15.91	15.95	2,3-Dihydro-benzofuran	-	-	0.3	-	0.4
14	20.66	20.76	Butanedioic acid hydroxy diethyl ester (+ /-)	-	3.7	-	4.0	-
15	21.26	22.22	5-Hydroxymethylfurfural	-	-	-	-	0.9
16	25.44	25.74	Hydrocoumarin	4.6	7.7	1.0	8.5	1.8
17	27.70	28.09	Coumarin	8.2	7.2	1.0	9.0	1.7
18	33.09	33.16	Benzenepropanoic acid 2-hydroxyethyl ester	-	4.6	-	4.5	0.2
19	33.25	33.34	(-)-Spathulenol	7.7	8.2	0.3	10.3	-
20	39.08	39.35	1-((1S,3aR,4R,7S,7aS)-4-Hydroxy-7-isopropyl-4-methyloctahydro-1H-inden-1-yl)ethanone	-	-	-	3.4	-
21	43.18	43.22	(-)-Globulol	3.3	4.6	-	4.7	-
22	46.99	47.06	n-Hexadecanoic acid (palmitic acid)	5.1	5.2	-	4.2	-
23	47.74	47.78	Hexadecanoic acid ethyl ester (palmitic acid ethyl ester)	3.9	3.9	-	3.5	-
24	52.23	52.69	(Z)-11-octadecenoic acid (cis-vaccenic acid)	14.6	13.9	2.2	10.0	0.2
25	52.94	53.07	(Z) -9-Octadecenoic acid ethyl ester (ethyl oleate)	9.9	8.5	-	6.4	-
26	54.71	54.72	Copalol	-	3.8	-	3.3	-
27	61.44	63.20	4-O-Methylmannose	-	-	81.7	-	90.6
28	62.09	62.57	Hexadecanoic acid 2-hydroxy-1-(hydroxymethyl) ethyl ester (beta-monoglyceride palmitic acid)	5.5	-	-	-	-
29	66.71	66.91	9-Octadecenoic acid (E,E,E)-1,2,3-propanetriyl ester	15.8	16.0	8.5	10.6	-
30	79.98	80.44	γ-Sitosterol	-	4.1	-	-	-
31	81.98	82.00	Lupeol	3.7	3.7	-	-	-
Total %				100	100	100	100	100

Note: IRT = initial retention time; FRT = final retention time of the compounds obtained in the collection areas; RC % = relative content in percentage; Appendix A (Figure A11, Figure A12, Figure A13, Figure A14 and Figure A15) = chromatograms of the residue extracts in the five collection areas.

). In areas 1, 2, and 4, apart from the common major constituents, (-)-spathulenol was also highlighted.

Fatty acids also showed percentages above 5% in the extracts of D. punctata residues in the collection areas. Ethyl oleate was detected with a percentage of 9.9% in area 1, 8.5% in area 2, and 6.4% in area 4 (Table 6). The constituents found in the residues collected in all study areas were hydrocoumarin, coumarin, and cis-vaccenic acid, making these compounds markers for extracts from D. punctata residues. Hydrocoumarin had the highest percentage in areas 2 (7.7%) and 4 (8.5%) and coumarin and cis-vaccenic acid in areas 1, 2, and 4, with percentages of 8.2%, 7.2%, and 9% for coumarin, and 14.6%, 13.9% and 10% for cis-vaccenic acid, respectively (Table 6).

For the residues collected in areas 3 and 5, most of the constituents identified in the extracts had percentages below 5%, except the ester (E,E,E)-1,2,3-propanetriyl of 9-octadecenoic acid (8.5%) in area 3, and 4-O-methylmannose, which was the major constituent of these areas, with 81.7% (area 3) and 90.6% (area 5) (Table 6). This last compound was also identified in the branches of D. punctata.

In the extracts of D. punctata seeds, 23 compounds were identified, 20 of which were found in area 1. Oleic acid was the major constituent (32.5%) and there were four substances with percentages above 5%, namely, coumarin (5.4%), n-hexadecanoic acid (6.1%), cis-vaccenic acid (8.5%), and (Z)-9-octadecenoic acid 2,3-dihydroxypropyl ester (11%) (

Table 7. Chemical compounds identified in ethanolic extracts of the seeds of Dipteryx punctata, in the five collection areas of the municipality of Mojuí dos Campos, Eastern Amazon, Brazil.

SEEDS EXTRACTS				Area 1	Area 2	Area 3	Area 4	Area 5
Nº	IRT	FRT	COMPOUNDS	RC %	RC %	RC %	RC %	RC %
01	3.78	3.98	O-Methylhydroxylamine	-	1.0	-	-	-
02	6.10	6.11	1-Ethyl-1-methylcyclopropane	-	-	0.7	2.6	-
03	25.30	25.76	Hydrocoumarin	3.3	-	-	-	-
04	27.52	28.04	Coumarin	5.4	98.1	99.3	97.4	93.8
05	28.86	29.09	4-Aminopyrido[3,2-c] pyridazine	-	0.9	-	-	-
06	46.43	46.72	n-Hexadecanoic acid (palmitic acid)	6.1	-	-	-	-
07	47.48	48.33	Hexadecanoic acid ethyl ester (palmitic acid ethyl ester)	4.2	-	-	-	-
08	51.53	52.41	(Z)-11-Octadecenoic acid (cis-vaccenic acid)	8.5	-	-	-	-
09	52.85	53.03	(Z)-9-Octadecenoic acid (oleic acid)	32.5	-	-	-	6.2
10	53.28	53.63	Octadecanoic acid (stearic acid)	4.3	-	-	-	-
11	53.63	54.23	Octadecanoic acid ethyl ester (stearic acid ethyl ester)	3.5	-	-	-	-
12	57.63	58.08	(Z)-13-Eicosenoic acid (cis-13-eicosenoic acid)	1.3	-	-	-	-
13	58.96	59.46	Eicosanoic acid ethyl ester	1.3	-	-	-	-
14	61.93	62.63	Hexadecanoic acid 2-hydroxy-1-(hydroxymethyl) ethyl ester (beta-monoglyceride palmitic acid)	1.4	-	-	-	-
15	62.63	63.26	Bis (2-ethylhexyl)phthalate	1.4	-	-	-	-
16	63.26	64.18	Docosanoic acid	2.7	-	-	-	-
17	64.18	64.63	Docosanoic acid ethyl ester	4.1	-	-	-	-
18	66.03	67.21	(Z)-9-Octadecenoic acid 2,3-dihydroxypropyl ester	11.0	-	-	-	-
19	67.21	67.56	Octadecanoic acid 2,3-dihydroxypropyl ester	1.0	-	-	-	-
20	68.33	68.86	Tetracosanoic acid	1.0	-	-	-	-
21	68.86	69.38	Ethyl tetracosanoate	3.3	-	-	-	-
22	78.48	78.96	Stigmasterol	1.4	-	-	-	-
23	79.81	80.38	γ-Sitosterol	2.1	-	-	-	-
Total %				100	100	100	100	100

Note: IRT = initial retention time; FRT = final retention time of the compounds obtained in the collection areas; RC % = relative content in percentage; Appendix A (Figure A16, Figure A17, Figure A18, Figure A19 and Figure A20) = chromatograms of the seed extracts in the five collection areas.

).

In general, species of the genus Dipteryx have coumarin in their chemical composition. In the seeds obtained from cumaru plants collected in areas 2, 3, 4, and 5, this compound was predominant, with percentages of over 90% (Table 7). Oleic acid was observed in D. punctata seed extracts in areas 1 and 5, and stigmasterol was also identified in D. punctata seed extracts in area 1 (Table 7).

3.5. Antioxidant Activity of Dipteryx punctata Extracts by TLC and DPPH Free Radical Scavenging

From the screening by TLC, it was found that the extracts from the different parts of the plant showed signs of antioxidant activity given the presence of a whitish color at the sample application points on the plates developed with ABTS (2,2′-azinobis(3-etthylbenzothiazoline-6-sulfonic acid)) (Figure 4A1–D1), and the presence of a yellowish color in the plates developed with DPPH (2,2-diphenyl-1-picrylhydrazyl) (Figure 4A2–D2). It was also observed that there were no differences in color or intensity between the extracts from the different collection areas; this characteristic was identified in the chromatographic plates that evaluated the classes of compounds (Figure 4). Quantitative analysis using the DPPH radical scavenging method, performed with extracts of leaves, branches, residues, and seeds, containing representatives from all collection areas, corroborated the TLC results, indicating significant neutralization of the radical by the change in color from purple or violet to light yellow or pale violet with the increasing concentrations tested (Figure 5).

The logarithmic function adequately represents the consumption of DPPH as a function of the increase in concentrations in this study, in the three phases: the first characterized by the gradual increase in the consumption of the DPPH radical, the second by the rapid increase that approaches the logarithmic curve (IC50 value), and the third by the occurrence of a gradual deceleration after the curve, until a constant consumption of this radical and saturation of the extracts is identified (Figure 6).

For the extract of the leaves and residues, the concentration of 500 µg.mL⁻¹ was responsible for the DPPH index of 90% and 91%, respectively; for the branches, the index was 87%, and for the seeds 81%, at the highest concentration tested (3000 µg.mL⁻¹) (Figure 6).

Regarding the consumption of DPPH due to the increase in the tested concentrations, logarithmic functions were obtained with determination coefficients above 90%, enabling the calculation of the IC50 (concentration of the extract necessary to reduce 50% of the DPPH radical). It was verified that a concentration of 117.6 µg.mL⁻¹ is necessary for the extract of the leaves to reduce the DPPH radical by 50% (Figure 6A). For the branches, the increase in radical capture capacity occurred at the concentration of 698.5 µg.mL⁻¹ (Figure 6B), and for the residue and seed extracts, at the concentrations of 160.4 and 1029.5 µg.mL⁻¹, respectively (Figure 6C,D) (

Table 8. Analysis of the antioxidant activity of ethanolic extracts of Dipteryx punctata by the DPPH free radical capture method, representative samples from all collection areas.

DPPH (2,2-diphenyl-1-picrylhydrazyl)
Data	Leaves	Branches	Residues	Seeds
Formula	Y = 32.524 In (x) − 105.05	Y = 27.812 ln (x) − 132.14	Y = 29.355 ln (x) − 99.06	Y = 25.813 ln (x) − 129.06
R2	97.47	98.18	94.64	93.61
CV%	4.43	1.17	3.84	3.70
IC50 (µg.mL−1)	117.6	698.5	160.4	1029.5

Note: R² = coefficient of determination; CV = coefficient of variation; IC50 = extract concentration required to reduce 50% of the DPPH radical.

).

4. Discussion

Regarding the data obtained with ethanolic extractions, many factors can influence the obtaining of higher yields, including the part of the plant studied, the size of the particles, the chosen methodology, the solvent, the concentration used, the extraction time, and the extraction temperature [32].

Soxhlet extractions enable the complete exhaustion of the plant material in a period of approximately eight hours and, when ethyl alcohol is used as a solvent, both apolar and polar substances are extracted as it is an amphiphilic molecule [33]. This explains the higher yield obtained in extracts of cumaru seeds, considering that this part of the plant contains a high oil content imparting an oilier character to this extract.

Extractions of leaves, branches, residues (separated into epicarp + mesocarp, and endocarp) and seeds of Dipteryx odorata and Dipteryx punctata have also been performed in a Soxhlet apparatus and, as in this study, the highest yields were obtained for extracts of fruit residues (epicarp + mesocarp) (44.1% and 54.2%) and for the seeds (48.9% and 44.5%) of the two species, respectively [10]. The yield of Dipteryx odorata seed extractions also resulted in values as high as 46.5% [34].

The yields obtained from fruit residues are very important, as currently, this material serves only as organic matter in the growing areas, being discarded by farmers after collecting and pulping the fruits to extract the seeds [10,21].

Area 3 stands out in terms of the yield obtained for fruit extracts and has a reduced number of cumaru trees. This characteristic can favor the development and production of trees, considering that each species requires specific characteristics of the environment, such as incidence of light, temperature, humidity, gravity, and wind speed [35], and the reproductive phenophases, especially the beginning of flowering, are largely influenced by the temporal variation in abiotic factors [36]. In addition, the greater spacing enhances the trees’ ability to compete and exhibit greater treetop diameters [37]. Thus, designing the best spacing to be adopted in planting, with a view to good development [38], will favor the production of fruits for the commercialization of seeds, an important source of income for producers [21,22].

In this context, studies are important to find out how management systems, spacing, intercropped species, and soil treatments can influence the physiology of these plants, leading to possible improvements in land use and increased production of biologically active natural compounds.

As regards the classes of compounds evaluated by thin-layer chromatography (TLC), this technique helps in the process of choosing the mass/volume of solvent required, as well as the solvent systems that have more affinity with the evaluated samples, because in excess or if insufficient, the amount of extracted compounds can be significantly affected [39].

The class of terpenes was identified in the ethanolic extracts of D. punctata. They have oxygenated functions such as alcohols, ethers, aldehydes, ketones, and lactones [40], and act in growth regulation, as phytoalexins and repellents against the action of herbivorous insects [41]. Among the phenolic compounds, the study identified the classes of tannins, flavonoids, and coumarins. Tannins have the ability to bind to proteins, usually irreversibly, forming precipitates, and are involved in the protection of plants against attacks by invertebrate and vertebrate herbivores as they are characteristically astringent and difficult to digest [42,43].

Flavonoids have different forms and varied functions and the class includes flavones, flavanones, catechins, anthocyanins, proanthocyanidins, and isoflavonoids, among others. These substances have allelopathic effects and can inhibit the growth of plants and fungi [44,45,46]. Coumarins are primarily synthesized in leaves but occur at the highest levels in fruits, followed by the roots and stems. However, seasonal changes and environmental conditions can affect their occurrence in the various parts of the plant [47].

The interest in the quantitative analysis of the total phenolic compounds and flavonoids found in the extracts is due to the ability of these substances to sequester free radicals that are harmful to human health [48,49]. The action of antioxidants, when incorporated into food, not only preserves the quality of the food but also reduces the risk of developing pathologies such as atherosclerosis, brain dysfunction, and cancer [50].

The correlation between the total amount of phenolic compounds and the antioxidant activity has already been reported [51], as well as that of substances with a benzene ring linked to hydroxyl groups in the compounds and double bonds between carbon atoms, which provide greater stability to the molecule that is the donor of electrons or hydrogen atoms [52]. The different classes of compounds and their chemical characteristics also affect the rate of free radical elimination [53].

The results obtained in this work also corroborate this association, since the phenolic compounds were found in greater quantity in the extract of the leaves of D. punctata, and, specifically for total flavonoids, the extracts that stood out most were those of the leaves and the residues of the fruits. Furthermore, by the DPPH free radical capture method, those same extracts were responsible for the lowest IC50 values (117.6 µg.mL⁻¹ for leaves and 160.4 µg.mL⁻¹ for residues), indicating the promising presence of compounds with higher antioxidant activity (the lower the value of extract concentration required to reduce 50% of the DPPH radical, the greater the antioxidant activity).

The TLC analyses also qualitatively demonstrated the presence of antioxidant activity in the extracts of D. punctata, however, the samples used are presented as a complex mixture of compounds, requiring fractionation and tests with isolated substances so that it can be affirmed whether the action is determined by the major constituents of the plant parts or by minor constituents, or whether the effect is due to the synergism of all compounds.

In addition, the analyses carried out in this study were used to select products with potential antioxidant action and direct other tests, mainly in vivo, with a real substrate (specific substrate or specific additive), where several mechanisms are associated, such as absorption cells, metabolic transformations, excretion, presence of competitive enzymes and antioxidants, which can directly affect the activity of these products. In vivo methods are also difficult to implement, as they depend on the application in biological systems, emphasizing the need for multifaceted tests [54], and the forms of molecular interactions and mechanisms of bioactivity of compounds of natural origin on free radicals in the body is still a challenge for scientists [55].

In a study with extracts from the epicarp + mesocarp (residues) of Dipteryx alata by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS), eighteen phenolic compounds were quantified for the first time, predominantly luteolin and trans-cinnamic acid [56]. These results indicate that species of the Dipteryx genus can be explored as a natural source of phenolic compounds with promising properties.

A study with hexanic and ethanolic extracts of D. alata leaves found that the ethanolic fraction had a higher amount of phenolic compounds (112.3 mg GAE g⁻¹) and better antioxidant action, considering that the amounts of extract required to decrease the initial concentration of DPPH by 50% were 52.9 µg.mL⁻¹, 169.1 µg.mL⁻¹, and 181 µg.mL⁻¹ for ethanolic, hexanic, and BHT extracts (Butyl-Hydroxy-Toluene—commercial antioxidant), respectively [57].

It is also interesting to note that the potential presented by the residue extract drives the advancement of new studies and makes it possible to expand the destinations of this raw material discarded in the environment, such as its use for the extraction of bio-oil, coumarin, production of flours and fermented beverages (food and nutritional security), future development of pharmacological products, alternative products for use in controlling plant diseases (organic and agroecological production), and consequent contribution to the appreciation of family farming (production chain).

Analysis of the extracts of the leaves of D. punctata, by gas chromatography coupled to mass spectrometry (GC-MS) verified the presence of fatty acid esters. It is worth mentioning the presence of fatty acids in biodiesel obtained from oilseeds such as soybeans, sunflower, canola, palm, and peanuts, with the commonest being those derived from palmitic acid (hexadecanoic acid) and stearic acid (octadecanoic acid). Examples are the ethyl ester of octadecanoic acid (ethyl stearate, ethyl stearate or stearic acid ester), oleic acid (cis-9-octadecenoic), linoleic acid (cis-9, cis-12-octadecadienoic), and linolenic acid (cis, cis, cis-9,12,15-octadecatrienoic) [58].

Palmitic acid (n-hexadecanoic acid), indicated as a marker due to its presence in the leaf extract of all areas and with percentages above 5% in area 3, is a natural fatty acid found in various oils such as olive oil. It is used in the food industry as a dietary supplement and an agent in dairy products [59]. Phytol, also a marker in the extracts from the leaves of D. punctata, is one of the precursors for the synthesis of different lipids in the chloroplast, and its conversion into phytyl esters of fatty acid (FAPEs), which accumulate in the plastoglobules of the chloroplasts, may occur during stress [60,61].

Among the terpenes, lupane-type triterpenoids such as lup-20(29)-en-3-one (lupenone) and lupeol [21] have also been identified in leaf extracts of plants of the families Asteraceae, Balanophoraceae, Cactaceae, Iridaceae, Musaceae, Urticaceae, Leguminosae, Bombacaceae, etc. Pharmacologically, lupenone stands out for its anti-inflammatory, antiviral, and anticancer activities and as an attenuator of Chagas’ disease [62]. Lupeol, a major constituent in all collection areas, also has anti-inflammatory activity [63], as well as anti-arthritis [64] and anti-leishmania action [65].

For the ethanolic extracts of D. punctata branches, hydrocoumarin, and lupeol (the major constituent in areas 1, 4, and 5) were identified as markers. The class of coumarins to which hydrocoumarin belongs (3,4-dihydro-2H-1-benzopyran-2-one or 3,4-dihydrocoumarin) was detected by TLC in the extracts of the branches, and also as simple coumarin (1,2-benzopyrone). This compound is used in condiments, beverages, gelatins, puddings, perfumes, and cosmetics [66].

4-O-methylmannose was the major constituent in areas 2 and 3; it belongs to the mannan class (polymer of hexoses), which constitutes hemicellulose [67]. Hemicellulose is a heteropolysaccharide common in nature, part of the chemical composition of wood, capable of forming covalent bonds with lignin molecules and hydrogen bonds with cellulose molecules, constituting the lignocellulosic complex and promoting its stability and flexibility [68].

Other compounds such as stigmasterol and γ-sitosterol were identified in the extracts of the branches obtained in all collection areas, except for area 3. Phytosterols are particularly abundant in the plant kingdom, present in fruits, seeds, leaves, and stems [69,70], and, albeit structurally similar to cholesterol, they help to reduce its absorption, thus having anti-inflammatory and antitumor properties [69] in addition to preventing and helping in the treatment of cardiovascular diseases [70,71].

In the extracts of D. punctata fruit residues, in addition to the major constituents, 9-octadecenoic acid (E,E,E)-1,2,3-propanetriyl ester (areas 1, 2, and 4) and 4-O-methylmannose (areas 3 and 5), the study identified the notable presence of 2,3-butanediol and (-)-spathulenol in areas 1, 2, and 4. 2,3-butanediol is a very promising compound in the context of biorefineries, given the use of plant biomass as important substrates for the production of this compound in fermentation broths. It has many applications, being extremely useful in the food, chemical, and pharmaceutical industries due to its physicochemical properties [72].

(-)-Spathulenol is a sesquiterpene that has already been isolated from the hexanic extract of the husks of Dipteryx lacunifera fruits, together with the sesquiterpene β-farnesene and the vinatichoic acid (diterpene furanocassan). Spathulenol and β-farnesene have also been identified in oil extracted from D. lacunifera residues via Clevenger, with percentages of 13.3% and 27.8%, respectively [73]. In addition, spathulenol has been reported in the extract of the epicarp + mesocarp of the fruits of D. punctata [10], and in the essential oil extracted from the flowers of Dipteryx odorata, which are also rich in germacrene D [74].

Hydrocoumarin, coumarin, and cis-vaccenic acid are markers for the residue extracts, the last being a naturally occurring unsaturated fatty acid that has a double bond in the cis configuration. In general, fatty acids and their derivatives are important oleochemical products both for technical applications and for nutrition in the case of natural oils and fats [75]. In the husks of D. odorata fruits, coumarins, lupeol, β-farnesene, betulin, and methyl esters of fatty acids were also identified [76,77,78].

Another fatty acid confirmed in the extracts of the residues was ethyl oleate. This compound, already identified in the extract of the husks of the fruits of D. punctata [10], arises from the esterification of oleic acid with an alcoholic solvent and has applications in the food, cosmetics, perfumery, and pharmaceutical industries [79,80].

In the extracts obtained from the seeds, coumarin in areas 2, 3, 4, and 5, and oleic acid in area 1 were identified as the predominant compounds. Coumarin (1,2-benzopyrone) is very important in the cosmetics and perfumery industry and is extracted from D. odorata seeds in the Amazon region. The seeds of this species are also widely marketed for vascular and lymphatic disorders [81], as the main active ingredient in ethanolic extracts serves to attenuate and/or alleviate diseases [39]. The presence of 1,2-benzopyrone has previously been reported in the seeds and the extracts of the husks and endocarps (integral parts of the fruit residues) of D. punctata. This study confirmed its presence in the extracts of residues and seeds from the different collection areas, which indicates there should be greater reuse of this material as a source for obtaining coumarin.

Macerated Dipteryx sp. seeds were extracted successively with hexane P.A., dichloromethane P.A., and ethyl alcohol P.A. to 96%, obtaining the highest yield and the isolation of 3.4% of coumarin in the hexanic fraction. However, in the dichloromethane and ethanolic fractions, in addition to simple coumarin, hydrocoumarin (3,4-dihydro-2H-1-benzopyran-2-one or 3,4-dihydrocoumarin) was identified [82]. This substance, belonging to the class of coumarins, has also been reported in the extract of D. punctata seeds [10] and this study found it in the extracts of branches, residues, and seeds.

Fatty acids have also been obtained from the oil extracted from D. odorata seeds, such as oleic acid and palmitic acid, with 53% and 13% of the relative total of the mixture, in addition to linoleic, stearic, and vaccenic acid (8%, 7%, and 2%, respectively) [83]. In D. lacunifera, the major component in the fixed oil of the almonds was oleic acid with 75.82 ± 4.310% [80] and 64.71 ± 1.35% [84]; oleic (64.1%) and linoleic (14.1%) acids together contributed to about 99.7% of the total unsaturated fatty acids [85]. When oil was extracted from D. alata seeds by hydraulic pressing, the values were about 50% oleic acid and a significant proportion of linoleic acid (26–28%) [86]. Linoleic acid can promote protection against the development of cancer; it has cardioprotective, anti-atherosclerotic, and nitric oxide potentiating effects [87,88,89].

Bearing in mind the food industry’s use of seeds of the Dipteryx genus, their oil is a possible option for composing healthy diets [86,90], for, albeit not essential, the oleic acid present in this plant product can enhance the levels of the beneficial high-density lipoprotein (HDL) cholesterol fraction and reduce the level of the low-density lipoprotein (LGL) cholesterol fraction, thereby fostering a reduction in cardiovascular diseases [91].

5. Conclusions

The species Dipteryx punctata proved to be an efficient source for obtaining ethanolic extracts in the agroforestry systems implemented in Mojuí dos Campos, Pará, Brazil. The present study of the phytochemical profile contributes to reducing the scarcity of information about this species, and the chemical classes and compounds identified corroborate the antioxidant activity, presenting the potential of the extracts regarding the isolation, identification, and quantification of the substances responsible for that action and calling for more complex biological assays. The data obtained also add value to the species and its cultivation in family agriculture, making it a viable alternative for improving the quality of life and income of the farmer. It also shows the importance of reusing fruit residues, a chemically rich by-product previously discarded in the environment.