2.1. Chemical products and materials

All the chemical reagents and solvents used for extractions, chromatographic and spectroscopic analyses were of analytical grade, from TEDIA^® COMPANY (Fairfield, USA). Column chromatography (CC) was performed using normal phase silica gel (60–200 μm) (SiliCycle Inc., Quebec, Canada). In addition, thin layer chromatography (TLC) was carried out on SiliaPlate TLC chromatography plates with aluminum and glass supports, silica, 200 μm, 20 × 20 cm, F 254 (SiliCycle Inc., Quebec, Canada). The structural elucidation of the isolated substances was carried out using one-dimensional nuclear magnetic resonance (NMR). The NMR spectra were obtained on a VARIAN^® Mercury Plus spectrometer operating at 300 MHz for ¹H and 75 MHz for ¹³C. The chemical shifts were recorded with dimensionless δ values (ppm), with the signal from the deuterated solvent as the internal reference. The solvents were CDCl₃ and C₅D₅N from CAMBRIDGE ISOTOPE LABORATORIES^®.

2.2. Obtaining and processing the agro-industrial residue Carapa guianensis

The extracted cake from the mechanical pressing of C. guianensis seeds was purchased from the local industry in the municipality of Ananindeua, in the state of Pará, in the northern region of Brazil (SisGen registration number A3B8C54). This cake was dehydrated in an oven at a temperature (T) of 45 °C and humidity (U%) of 20% until it reached a constant mass. The dehydrated material was crushed and sieved through a 40 mesh, and the resulting sample was vacuum-packed and stored at 25 °C.

2.3. Preparation of the extracts

The phytochemical analysis of the C. guianensis agro-industrial residue was based on preliminary tests in triplicates, assessing the extraction method (cold and hot) and the extraction efficiency of the organic solvents used (hexane, ethyl acetate, and ethanol). In both extractions, a sample mass/solvent volume ratio of 1:10 m/v was adopted. The cold extractions using a single solvent and successive extractions (increasing polarity gradient) lasted 24 h at room temperature each. Each extraction was carried out in triplicate in a single batch, with the solvents renewed twice. The hot Soxhlet extractions lasted three hours for each solvent. All extract solutions obtained were concentrated to solids in a Syncore rotary evaporator (BÜCHI - Flawil, Switzerland).

2.4. Characterization of the extracts by HPTLC

The chemical profile of the extracts was obtained by HPTLC (High Performance Thin-Layer Chromatography - CAMAG^®). The samples were deposited in spot form, 10 μg/spot for the extracts and 1 μg/spot for the limonoid standard (7-desacetoxy-7-oxogedunin) on silica gel F-254 60 Å chromatoplates with glass support (SILICYCLE^®) in an ATS4 (Automatic TLC Sample4) module. The chromatoplates were then eluted in the AMD2 (Automated Multiple Development) module in gradient mode, following the 12-step adjustment with decreasing polarity and a chromatographic path of 80 mm, using a mixture of organic solvents as the mobile phase: hexane/ethyl acetate/methanol (1% formic acid), ranging linearly from 70/15/15% to 100/0/0%. It was then developed using a selective reagent for terpenes and limonoids (VSA) and scanned using the TLC Visualizer photodocumenter in white light. The data was stored and processed in the WinCats - Planar Chromatography Manager software, version 1.4.6 (CAMAG, Muttenz, Switzerland).

2.5. Fractionation and isolation

The fractionation of 50 g of the extract obtained from the hot ethanolic extraction of the agro-industrial residue of C. guianensis (E9) was performed by column chromatography (CC), using silica Flash G60 (60–200 μm) (SILICYCLE^®) as the stationary phase and, as a mobile phase, a gradient of increasing polarity with seven hexane/ethyl acetate/methanol systems: (1) 90:0.5:0.5, (2) 80:10:10, (3) 70:15:15, (4) 60:20:20, (5) 50:25:25, (6) 00:50:50, and (7) 00:00:100. The fractions obtained were analyzed by HPTLC and combined based on similarities into 4 fractions: F1 (2.0 g), F2 (2.77 g), F3 (1.62 g), and F4 (3.65 g).

Fractions F1 to F3 were subjected to silica gel column chromatography using mixtures of hexane, ethyl acetate, and methanol as eluents, in ascending order of polarity. The fractions were monitored by TLC and revealed with VSA and visible light. Fraction F4 was also subjected to silica gel column chromatography, but using dichloromethane (100%) as the mobile phase. The resulting chromatographic fractions were analyzed by TLC and combined into fractions 55 to 80, which were purified by preparative thin layer chromatography. Two 50 mg injections of samples solubilized in methanol were deposited as a 180 mm × 3 mm band on preparative TLC and eluted in a 15% dichloromethane/methanol system, following a 150 mm chromatographic path. The chromatographic bands were demarcated and removed, each sample was solubilized in methanol and filtered. All the substances were identified by 1D (¹H and ¹³C) and 2D (¹H–¹³C HETCOR, HSQC, and HMBC) NMR spectroscopy , and the data compared with literature:

Gedunin (S1): From F1, fractions 56 to 72, a white amorphous solid (15 mg); ¹H NMR (CDCl₃) and ¹³C NMR (CDCl₃) (see Tables 2 and 3; Section S1, Supplementary Material).

6α-Acetoxygedunin (S2): From F2, fractions 33 to 36 (white crystals) (18 mg); ¹H NMR (CDCl₃) and ¹³C NMR (CDCl₃) (see Tables 2 and 3; Section S2, Supplementary Material).

7-Desacetoxy-7-oxogedunin (S3): From F3, fractions 38 to 52 (white crystals) (15 mg), ¹H NMR (CDCl₃) and ¹³C NMR (CDCl₃) (see Tables 2 and 3; Section S3, Supplementary Material).

Oleanolic acid 3-O-[α-L-ramnopyranosyl-(1→2)-α-L-arabinopyranosyl] (S4): From F4, a white amorphous solid (38 mg); ¹H NMR (C₅D₅N) and ¹³C NMR (C₅D₅N) (see Tables 4 and 5; Section S4, Supplementary Material).

Hederagenin 3-O-[α-L-ramnopyranosyl-(1→2)-α-L-arabinopyranosyl] (Pulsatilla A) (S5): From F4, a white amorphous solid (15 mg); ¹H NMR (C₅D₅N) and ¹³C NMR (C₅D₅N) (see Tables 4 and 5; Section S5, Supplementary Material).

2.6. Fingerprint of hot ethanolic extract (E9) and commercial C. guianensis oil

The fingerprint of E9 and commercial andiroba oil was evaluated according to Section 2.4, this time by injecting the masses of the substances isolated and identified in this work (S1, S2, S3, S4, and S5).

3.1. Extractor efficiency

Regardless of the solvent used, hot extraction gave higher mass percentage yields than cold extraction, with a percentage increase in yield ranging from 38.96 to 53.18%. When comparing the extracting capacity of each solvent, ethanol gave the highest mass yield (26.96%).

When the extraction was carried out with solvents in succession in increasing order of polarity, hexane was the one with the highest mass yield (12.14%). The cold extraction method with successive solvents could therefore be more suitable to extract low polarity components with greater affinity for hexane.

Thus, this work uses a chemical investigation approach to investigate the agro-industrial residue of C. guianensis. The choice of the extracting solvents was according to their selectivity with low, medium, and high polarity molecules, which allowed phytochemical screening. The ethanolic extract was chosen for the other studies due to its higher degradation rate. Above all, the preference for ethanol arises from its prevalence in biological studies using plant extracts, due to its low toxicity and low persistence in Nature. In addition, its low cost and large-scale production from renewable sources make it one of the safest solvents in the recovery of natural assets [19].

At the same time, green chemistry approaches have been developed to mitigate the undesirable effects of traditional extraction protocols, aiming to replace organic solvents with environmentally friendly alternatives. In this scenario, natural deep eutectic solvents (NADESs) have emerged as a pioneering proposal for the recovery of phytochemicals, as they offer advantages such as low toxicity, biodegradability, solute stabilization, sustainability, simple preparation, and tunable viscosity and density [20]. However, it is important to emphasize that for efficient extraction, some parameters must be considered, such as the physical properties of the solvent. For example, NADESs present hydrogen bond networks between the constituents, making them viscous, which compromises the extraction efficiency and, consequently, the affinity capacity of the target compounds with their phase. Therefore, there is a need to adjust this property [21, 22]. This implies additional investigations on the use of NADESs in the activities of isolation, separation, and recovery of bioactive compounds present in plant matrices, in addition to the elaboration and evaluation of extraction protocols using NADESs, the stability of biomolecules, production costs and their effects on human life and health, before their use in the pharmaceutical industry [21].

3.2. Chemical profile by high performance thin layer chromatography (HPTLC)

Chemical profile by HPTLC made it possible to evaluate the metabolic complexity of the compounds present in the extracts of the agro-industrial residue of C. guianensis. Following the methods described by [15], the use of vanillin–sulphuric acid solution (VSA) as a chemical developer allows the detection of fatty acids (blue), as well as terpenes, limonoids (chemotaxonomic marker) and steroids (purple). It was therefore possible to determine the occurrence of substances from these classes in all the extracts, because bands were formed with blue and/or purple tones after the plate was revealed with VSA. The Rf comparison with the standard 7-desacetoxy-7-oxogedunin (track 1 in Figure 1 Rf 0.53) made it possible to observe the presence of the limonoid 7-desacetoxy-7-oxogedunin in some extracts (0.53), based on the proportion of mass and intensity of coloration. It is notably observed in the hot ethanolic extract (tracks 18–19 in Figure 1), in addition to the diversity of compounds displayed through chromatographic bands throughout the chromatoplate, thus proving that this biomass still has a wealth of biomolecules even after extracting andiroba oil.

Table 1 identifies, by means of acronyms, all the extracts obtained from the andiroba agro-industrial residue, in the cold and hot method, with the solvents: hexane, ethyl acetate and ethanol, as well as the standard.

3.3. Structural identification

Tetranortriterpenes identified in the study, gedunin (S1), 6α-acetoxygedunin (S2), and 7-desacetoxy-7-oxogedunin (S3) (Figure 2), are already known in the species, and the study only compared the ¹³C (CDCl₃, 75 MHz) and ¹H (CDCl₃, 300 MHz) NMR signals with data from the literature, in addition to two-dimensional correlation maps 2D ¹H ¹³C (HETCOR) and ¹H ¹³C (HMBC).

Gedunin (S1): characteristic signals of hydrogens and aromatic carbons at δ_H 7.41 (dl, J 1.5 Hz, 2H) and 6.33 (t, J 1.2 Hz, 1H); δ_C 120.4, 143.1, 109.8, and 141.2, respectively, indicating the presence of a furan ring in the structure (Tables 2 and 3; Section S1, Figures S1A–S1C, Supplementary Material).

6α-Acetoxygedunin (S2): typical signals from hydrogens and aromatic carbons at δ_H 7.41 (dl, J 1.0 Hz, 1H) and 6.33 (t, J 1.2 Hz, 1H); at δ_C 120.3, 141.2, 109.8, and 143.1, respectively, indicating the presence of a furan ring in the structure (Tables 2 and 3; Section S2, Figures S2A–S2C, Supplementary Material).

7-Deacetoxy-7-oxogedunin (S3): typical signals from hydrogens and aromatic carbons at δ_H 7.40 (dl, J 1.0 Hz, 1H), 6.35 (dl, J 1.2 Hz, 1H), and 7.38 (t, 1.8 Hz, 1H); at δ_C 120.1, 141.0, 109.7, and 143.1, respectively, indicating the presence of a furan ring in the structure (Tables 1 and 2; Section S3, Figures S3A–S3C, Supplementary Material).

The findings were compared with those of [23, 24] (Tables 2 and 3). These compounds are noted for their diverse biological activities, including insecticidal, antifungal, antimalarial, antibacterial, antiviral, and anticancer properties. Such a variety illustrates their broad applicability, spanning the agricultural and pharmaceutical sectors. For example, their phytotoxic properties make them potential bio-inputs for the formulation of new herbicides in the control of parasitic plants. In clinical trials, the three compounds showed protective effects against liver damage induced by D-GalN/LPS in mice, which shows that they are promising for making hepatoprotective formulations [9, 10].

The research highlights the unprecedented identification of these substances in the Carapa gender, although the triterpene saponins identified in this study have already been reported in the literature (Figure 3). Therefore, the study only compared the ¹³C (C₅D₅N, 75 MHz) and ¹H (C₅D₅N, 300 MHz) NMR signals with the data from [25, 26], (2006) (Tables 4 and 5) in addition to two-dimensional correlation maps 2D ¹H ¹³C (HETCOR and HSQC) and ¹H ¹³C (HMBC).

Oleanolic acid 3-O-[α-L-ramnopyranosyl-(1→2)-α-L-arabinopyranosyl] (S4): typical hydrogen signals from glycidic units such as arabinose were observed at δ_H 4.91 (d, J 5.7 Hz) (anomeric hydrogen H-1′), as well as raminose at δ_H 6.16 (sl) (anomeric hydrogen H-1′′), in addition to the characteristic signals of anomeric carbons for two sugar units of the arabinose type at δ_C 105.1 and raminose at δ_C 101.9. (Tables 4 and 5; Section S4, Figures S4A–S4C, Supplementary Material).

Hederagenin 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl] (Pulsatilla A) (S5): typical hydrogen signals from glycidic units such as arabinose were observed at δ_H 5.13 (d, J 6.3 Hz) (anomeric hydrogen H-1′) and from raminose at δ_H 6.23 (sl) (anomeric hydrogen H-1′′), in addition to signals characteristic of anomeric carbons for two sugar units of the arabinose type at δ_C 104.4 and raminose at δ_C 101.7, and an oxymethylene carbon signal at δ_C 64.0 (Tables 4 and 5; Section S5, Figures S5A–S5C, Supplementary Material).

In summary, this phytochemical study provides new information on C. guianensis and the Carapa gender, revealing greater chemical diversity in the species. Triterpene saponins have previously been detected in C. procera and C. guianensis through preliminary tests [27, 28], but the present study provides the first structural identification of these metabolites in C. guianensis. The glycosides identified in the agro-industrial residues of C. guianensis have important biological characteristics such as: antiviral, antitumor and hepatoprotective [29, 30, 31, 32, 33]. Saponins with the aglycones hederagenin or oleanolic acid in their structures, such as those identified in this study, have antifungal activities due to their ability to bind to sterols in the fungal membrane, causing pore formation and loss of membrane integrity [34]. These promising biomolecules could be of interest to the pharmaceutical and cosmetics industries. The results of this study provide new perspectives for the reuse of C. guianensis industrial waste as a source of bioactive substances of industrial interest.

3.4. HPTLC fingerprint

The fingerprinting of the hot ethanolic extract (E9) and the commercial oil of C. guianensis (OA), using the substances isolated in this research (S1, S2, S3, S4 and S5) in duplicate via HPTLC, enabled a certain similarity to be observed between the chemical composition of OA and E9. This finding is supported by the observation of chromatographic bands suggestive of limonoids when derivatized with VSA, as observed for limonoids S1, S2, and S3 isolated from andiroba agro-industrial residue. This observation can be explained by the fact that the process of obtaining andiroba oil from the seeds of the species does not deplete the limonoid content, since substances of this class are still present in the residue. It was also possible to observe the presence of substances other than limonoids, which are absent in andiroba oil, such as triterpene saponins (S4 and S5), revealing the chemical potential of the agro-industrial residue of Carapa guianensis Aubl. (Figure 4).

The results obtained in this study are relevant and promising, as they show that even after obtaining andiroba oil, the agro-industrial residue of C. guianensis still has a wealth of phytochemicals. This contributes to the phytochemical study of the species, as well as identifying the presence of substances of economic and pharmacological interest. This biomass can be used as an alternative in the search for bioproducts, thus adding value to the residue.