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CHEMICAL SCIENCES • An. Acad. Bras. Ciênc. 97 (1) • 2025 • https://doi.org/10.1590/0001-3765202520240635 linkcopy

Open-access Systematic investigation of phenolic compounds distribution in Libidibia ferrea (ironwood) seedling, using metabolomics and histochemical techniques

AuthorshipSCIMAGO INSTITUTIONS RANKINGS

Abstract

Ironwood (Libidibia ferrea) is cultivated and widely used due to its medicinal properties attributed especially to phenolic compounds. In the seedling stage, different environmental conditions influence the synthesis, composition, and accumulation of bioactive compounds in different ways, mainly under water stress. Thus, given the wide distribution, this study aimed to analyze the occurrence and distribution of phenolic compounds in L. ferrea seedling for three different states/climate typologies: Pará/Af, Amazonas/Am, and Maranhão/Aw. Gas chromatography-mass spectrometry (GC-MS) and plantlet histochemistry were applied to determine the phenolic compounds distribution present in the extracts of leaflets and roots, respectively. Three different metabolic profiles were described, of which Pará/Af and Maranhão/Aw were similar, while Amazonas/Am showed quantitative differences in the composition. Among the main phenolic compounds were found gallic acid, ferulic acid, and caffeic acid, and presented different concentrations according to origin. The only phenolic compound found for all origins was gallic acid. Therefore, it was demonstrated that the chemical profile of phenolic compounds is conserved in L. ferrea, and significant variations were not observed, which ensures the use of this species regardless of origin.

Key words
chromatography; gallic acid; histochemistry; metabolites; natural products

INTRODUCTION

In the Amazon, Libidibia ferrea (Max. Ex Tul.) L. P. Queiroz, has as relevant synonyms Caesalpinia ferrea Mart. ex Tul. (basionym) and Libidibia ferrea (Mart. ex Tul.) L.P.Queiroz var. ferrea (Oliveira & Fernando 2024), is cultivated and widely used due to its antifungal, antibacterial, antiulcerogenic, anti-inflammatory and healing properties attributed to certain groups of metabolites, especially phenolic compounds (Port’s et al. 2013, Ferreira & Soares 2015, Ferreira et al. 2019, Prazeres et al. 2019, Luna et al. 2020, Almeida et al. 2021). These compounds play several important roles in plants and represent a striking example of metabolic adaptation that enables plants to adapt to biotic and abiotic changes, such as growth, reproduction, plant tolerance, and allelopathic activity (Boudet 2007, Wink 2008).

The ability to synthesize phenolic compounds was acquired throughout evolution in different plant strains when these compounds started to meet specific needs (Verma & Shukla 2015). The diversity of these secondary metabolites and their natural distribution, until now was not fully known, and this work is an effort to map these substances, especially those in tropical forest areas. The type and concentration of compounds produced by a plant are determined by the species, genotype, physiology, developmental stage, and environmental factors (Isah 2019).

Kubitzki & Gottlieb (1984) reinforce the importance of knowing these metabolites and highlight their importance as markers of phylogenetic evolution and/or ecogeographic diversity of species. Additionally, the distribution and abundance of plant species are determined during the early growth stages, when seedlings and young plants are more susceptible to environmental conditions (Harper 1977, Kitajima & Fenner 2000).

Changes in the growth pattern have already been recorded for L. ferrea at different stages, also demonstrating that this species has high adaptability under the different abiotic conditions that it is subjected to due to its wide distribution (Lenhard et al. 2010, Ferreira et al. 2015). However, according to Ambrosio et al. (2017), variability in the chemical composition of plants is the result of the genetic evolution of the species that, in its center of origin, has undergone processes of diversification and selection, in which specific environmental factors promote variations at a lesser amplitude than those of genetic origin.

Currently, information is still needed on the constitution of the metabolome of economically important groups, mainly medicine and civil construction, in which the target molecules have bioactive potential, but can also serve as a basis for studies related to the responses of plants to different factors (Cheynier et al. 2013). This approach, when aligned with efficient methods of analysis and detection, provides strategic information that can result in a new understanding and be used in other studies.

Due to the growing interest in studying the relationship between secondary metabolites and abiotic factors using powerful tools and techniques, we aimed to identify the occurrence of phenolic compounds in the root and leaflet tissues of seedlings from three regions with different climatic typologies, especially to investigate the spatial distribution of these compounds and their possible functions and storage locations.

MATERIALS AND METHODS

Botanical material collection

Ripe fruits were collected during July and August of 2014 and 2015 in the cities in the North of Brazil: Belém, state of Pará (1°44’2.4’’S, 48°31’30’’W), Manaus, state of Amazonas (3°23’45.6’’S, 60°02’16.8’’W), and Pinheiro, state of Maranhão (2°48’3.6’’S, 45°06’14.4’’W), located in the Northeast region encompassing the Mid-North sub-region of the country. The ripe fruits were collected from the matrices at the Research Campus of the Museu Paraense Emílio Goeldi in Belém-Pará (1°44’2.4’’S, 48°31’30’’W), under collection permit no. 13989-2/SISBIO. The taxonomic determination was performed by D.Sc. Flávia Lucas, Curator of the Herbarium (MFS) of the Universidade do Estado do Pará, using samples of flowering branches. The voucher for the specimen is deposited under the number 002780/MFS.

To characterize the collection sites, a map of the climatic typology was generated (Figure 1) using Köppen classification groups adapted by Martorano et al. (1993). This resulted in 10 climatic divisions that were classified as Af (Af1, Af2, Af3), Am (Am1, Am2, Am3, Am4), and Aw (Aw3, Aw4, and Aw5), where each unit has specific precipitation conditions and dry seasons (Table I). The area where data were collected from Pará State is classified as type Af (subtype Af1), which is characterized by not having a dry season and having precipitation in the least rainy month equal to or greater than 60 mm, and an annual precipitation greater than 3.000 mm. The region where the material was collected in Amazonas State is classified as Am (subtype Am3), which has a moderate dry season (3 months) with monthly precipitation below 60 mm, and annual precipitation that ranges from 2.000 to 2.500 mm. In Maranhão State, the collection region is classified as Aw, which has a well-defined dry winter (6 months), monthly rainfall of less than 60 mm, and annual rainfall that ranges from 1.000 to 1.500 mm.

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Figure 1
Map of the climatic typology of the Brazilian Amazon.
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Table I
Chemical profile of the hydroalcoholic extract of leaflets of L. ferrea from Amazonas, Pará, and Maranhão.

Obtaining seedlings and extracts

The fruits were processed at the Forest Seed Laboratory (LASF) of Embrapa Amazônia Oriental, in Belém, Pará. One lot from each source was selected at random for seedling production. The asepsis of the seeds and the pre-germination treatment to break the seed coat dormancy. They were scarified and then immersed in water for 24 hours, following the recommendations in the instructions for analyzing seeds of forest species (Brasil 2013).

Subsequently, the seeds were distributed in trays with a substrate consisting of a mixture of sand and sawdust sterilized at the proportion of 1:1. For each origin site, 100 seeds were used, which consisted of four repetitions of 25 seeds. The trays were kept in a semi-shaded place and irrigated twice a day. The number of seedlings was counted each day. The seedlings were collected after 25 days of cultivation. The individuals considered normal, or seedlings that had their essential structures (root system and aerial part), were collected. Subsequently, the samples were dried in an oven with forced air circulation at 45°C for 48 hours. After complete dehydration, the material was ground with a knife mill and stored away from light and moisture.

To obtain the crude extracts, extractions were made with solvents of different polarities (hexane, chloroform, ethyl acetate, and 70% v/v hydroalcoholic solution), with the aid of ultrasound at a temperature of 45°C for 30 minutes, following the methodologies used in the Laboratory of Systematic Research in Biotechnology and Molecular Biodiversity (LABSISBIO) at Universidade Federal do Pará (UFPA).

Obtaining the chemical profile of the hydroalcoholic extract of seedling leaflets and roots

A sample of each of the dry extracts of the leaflets and roots, from each source, was weighed, transferred to conical tubes with a volume of 2 mL, and subjected to the processes of alkaline hydrolysis for lignin phenolic compounds, acid hydrolysis for ether and ester bonds, and derivatization by silylation (dos Santos et al. 2021). The processes described below refer to the methodologies for analyzing phenolic compounds developed at the Laboratory of Systematic Research in Biotechnology and Molecular Biodiversity (LABSISBIO) at Universidade Federal do Pará (UFPA).

Hydrolysis and extraction of phenolic compounds

For the extraction process of the phenolic compounds, 50 mg of the hydroalcoholic extract was subjected to alkaline hydrolysis, with agitation assisted by ultrasound, for 30 minutes at 60°C, by adding 400 µL of 4M NaOH in a 2 mL conical bottle with a bottle cap. The homogenization of the system was carried out by vortexing for 2 minutes. Afterward, 500 µL of 6M HCl was added, vortexed for 2 minutes, and measured at pH 2. Subsequently, the system was subjected to ultrasonic-assisted stirring for 30 minutes at 60°C. For the extraction of phenolic compounds, three extractions were made with 500 µL of AcOEt, followed by centrifugation at 10.000 RPM for 2 min. The supernatant was separated and dried with N2 under inert conditions.

Derivatization method

100 µL of N, O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA) + 1% of trimethylchlorosilane (TMCS) was added at 35°C during a period of 1 h. After derivatization, 400 µL of ethyl acetate was added and transferred to a glass vial with a cap and 2 mL septum.

Metabolomics analysis of phenolic compounds by gas chromatography-mass spectrometry (GC-MS)

The gas chromatography analyses were performed with a ThermoScientific Trace 1300 Gas Chromatograph (GC) coupled to a ThermoScientific MS-ISQ Single Quadrupole mass spectrometer with an AI 1310 autosampler equipped with a ZB-5HT capillary column (30 m × 0.25 mm × 0.1 µm). Helium gas was used as a carrier at a flow rate of 1 mL min-1. 1.0 µL sample injection in Splitless mode. The injector operated at 220°C and the oven temperature programming went from 50°C to 200°C (8°C min-1); it was maintained for 1 min., then raised to 300°C (15°C min-1) and maintained for 5 min; finally, it was raised to 350°C (15°C min-1) and maintained for another 9 minutes. The MS-ISQ operated with an interface at 280°C, ionization source at 280°C, mass range (40-1.000 Da), and electronic ionization at 70 eV. The identification of the substances was carried out by comparing the mass spectra with those of the commercial mass spectra libraries NIST2011, WILEY2009, FAMES2011; these are the largest libraries of electron ionization (EI) mass spectra in the world.

Histochemical characterization

Samples of leaflets and roots from the three sources were fixed in FAA (formaldehyde-glacial acetic acid, 50% etilic alcohol; 1:1:18, v/v/v) for 24 hours (Johansen 1940) to preserve hydrophilic substances and perform the structural characterization, in neutral buffered formalin (NBF) for 48 hours (Lillie 1965) to preserve lipophilic substances, and in ferrous sulfate in formalin (FSF) for 48 hours (Johansen 1940) to show total phenolic compounds from their precipitation. Subsequently, the material was dehydrated to absolute ethanol, embedded in hydroxyethyl methacrylate (Historesin Leica®; solutions prepared according to the manufacturer), and sectioned with a rotary microtome with automatic advancement (RM 2245, Leica®, Nussloch, Germany), using tungsten razors (Leica®, Biosystems, Nussloch GmbH), to obtain transversal and longitudinal cuts (5-7 μm) of the median portion of the leaflet blade (midrib and margin) and root. The sections were stained with 0.05% toluidine blue in acetate buffer, at pH 4.6 (O’Brien et al. 1964), and mounted with synthetic resin (Permount®, New Jersey, USA). Toluidine blue is a metachromatic dye that exhibits different colors based on the substrate it reacts to: mucilages and walls rich in pectin stain purple, cellulosic walls stain blue, and lignified walls and non-structural phenolic content stain green.

More specific histochemical tests for phenolic compounds were also made: hydrochloric vanillin (Mace & Howell 1974) for condensed tannins, and aluminum chloride (Charrière-Ladreix 1976) for flavonoids. The test controls were made according to their respective techniques. The photographic documentation was made using a digital camera (AxioCam HRc; ©Carl Zeiss, Göttingen, Germany) coupled to an optical microscope equipped with a mercury lamp (HBO 50W) and the blocking filters BA420 and BA515 (excitation filters BP330-385, BP450- 480 and dichromatic mirror DM400 and DM500, respectively) from the Laboratory of Morphophysiology Applied to Health at the Universidade Estadual do Pará (UEPA).

RESULTS

Chemical profile of the hydroalcoholic extract of seedling leaflets and roots using gas chromatography and mass spectrometry (GC-MS)

The collection locations were determined because they are regions with different climates, which are represented on the map in Figure 1: Pará presents high incidence of rainfall (Af typology), Amazonas has an intermediate incidence of rainfall (Am typology), and Maranhão presents low incidence of rainfall (Aw typology). These aspects are relevant because in the Amazon different climates can be found. Germination was carried out in order to assess the distribution of the compounds in the seedling and to see if these compounds were already present in the vegetative organs, such as leaves, stems and roots. In general, it is already known that these compounds are present in various organs of the adult plant, especially in the leaves (Soares de Melo Junior & Silva 2020), stem and fruit peels (Grisi et al. 2020) which are traditionally used, but it was necessary to know whether they would be present in the seedling stage, since monitoring seedlings and young plants is crucial due to their sensitivity to environmental conditions (Harper 1977, Kitajima & Fenner 2000). These metabolites may be related to the adaptability and resistance of the seedling, so it is important to investigate whether there is a relationship between the establishment of these plants with the environment. The chemical profile of the seedling leaflets and roots of L. ferrea (jucá) was determined for the first time, since most works about this subject have been conducted with mature tissues and structures, such as seeds, fruits, and branches. Monitoring the phenolic compounds in seedlings by GC-MS made it possible to draw inferences about the action and importance of these compounds at this stage of development and associate them with the origins of the samples (Pará, Amazonas, and Maranhão). It is important to note that so far there have been no commercial plantings of this species, with only backyard cultivations in rural domestic areas. This species which is considered important as a medicinal plant, was introduced to the Amazon by Northeasterners who brought it during the period of exploitation of the rubber plantations in the Amazon.

The chromatogram (Figure 2) was registered for the sample of the derivatized leaflet extract from Amazonas State. The peak corresponding to gallic acid, with a retention time of 21.02 min, does not represent a majority compound but can be associated with a chemical marker.

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Figure 2
GC-MS chromatographic profile of the hydroalcoholic extract of leaflets of L. ferrea from Amazonas.

In the chromatograms (Figure 3, Figure 4) it is possible to identify the peaks referring to the silylated phenolic acids that were detected in the samples. The peaks with retention times at 21.03 and 21.02 min correspond to gallic acid in its silylated form (Table I). The second and third structures, observed in the two chromatograms, correspond, from left to right, respectively, to ferulic acid and caffeine that are also in their silylated forms.

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Figure 3
GC-MS chromatographic profile of the hydroalcoholic extract of leaflets of L. ferrea from Maranhão.
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Figure 4
GC-MS chromatographic profile of the hydroalcoholic extract of leaflets of L. ferrea from Pará.

There is a similarity in composition between the extracts from the states of Pará (typology Af1) and Maranhão (typology Aw4) (Table I), which can be better visualized in the profile of their chromatograms. The similarity between the composition of the samples of these two locations is evident; however, there was a difference in the composition of the extract from the state of Amazonas (Am3 typology), which is the region with no dry season and greater annual precipitation. The latter has a composition rich in fatty acids and some esters, such as α - linolenic, myristic, stearic, lauric, and palmitic acids that together correspond to approximately 27.00% of the total relative area, of which more than one-third corresponds to α-linolenic acid (an omega 3 fatty acid).

In extracts from Pará and Maranhão, the amount of fatty acid is twice as high as the amount found in the extract from Amazonas; 22.14% of the relative area in the chromatogram of the sample comes from Maranhão and 18.76% of the sample comes from Pará. Phenolic acids are found in greater quantities in the samples from Pará and Maranhão (Table I), among which there is the presence of caffeic acid and ferulic acid that are not present or are in quantities undetectable by GC-MS in the sample from Amazonas. In general, for the column type used here, the amount detected is above 100 µg per liter. Gallic acid is a metabolite in the phenolic class and present in greatest quantity in samples from the sites of origin (Pará: Af1, Amazonas: Am3, and Maranhão: Aw4): 3.28% (Amazonas State), 4.96% (Maranhão State), and 4.34% (Pará State). The only phenolic acid detected in all extracts, although in low concentrations, was gallic acid, which refers to peak 13 (Table II).

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Table II
Chemical profile of the hydroalcoholic extract of roots of L. ferrea from locations in Amazonas, Pará, and Maranhão, identified by GC-MS after the derivatization process.

Histochemistry and identification of phenolic compounds

The histochemical tests applied to the cross sections of the leaflets and roots of L. ferrea showed no differences between the three sources. It was also observed that total phenolic compounds and flavonoids are present in pluricellular glandular trichomes located on the abaxial surface of the leaflet blade (Figure 5a-c). Simultaneously, in the roots, in addition to total phenolic compounds and flavonoids, tannins were also present in idioblasts, especially in regions with greater meristematic activity, such as in the vicinity of the endoderm and pericycle, and in the regions where secondary roots are emitted (Figure 5d-g, Table III).

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Figure 5
Positive results of the tests applied to L. ferrea. a-c: foliar glandular trichomes; d-g: roots. a, e (inset): toluidine blue, non-structural phenolics in trichomes (arrowhead) and roots (arrows); b, f: ferrous sulfate in formalin, total phenolics in trichomes (arrowhead) and roots (arrows); c, g: aluminum chloride, flavonoids in trichomes (arrowhead) and roots (arrows); d: hydrochloric vanillin, condensed tannins in roots (arrows). Bars: a-c and inset: 25µm; d-g: 75µm.
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Table III
Results of the histochemical tests for phenolic compounds applied to the secretion present in L. ferrea seedlings. Notes: positive result (+); negative result (-); LL: leaflet; R: root.

Phenolic compounds were detected in the leaflet parenchyma, close to the epidermis, and especially in secretory structures. Perhaps they are tannins since the GC-MS analysis detected gallic acid derivatives in leaflets. The fresh material tested for flavonoids showed a positive response to the reagents Natural A (N-diphenyl boroyl oxyethylamine 1% in methanol) and developer of aluminum chloride. Material fixed in NBF and tested with Natural A reagent also showed a positive response, although a little weaker than the tests with fresh material. Perhaps the positive response for flavonoids was observed in the fixed material due to the neutral pH of the stock solution.

DISCUSSION

The analysis of the seedling root extracts revealed chemical profiles that are very similar for the three places of origin studied (Pará, Amazonas, and Maranhão) and rich in fatty acids, which are primary metabolites essential to the maintenance of the plant metabolism.

According to Ambrosio et al. (2017), the variation in the chemical composition of plants results from the genetic evolution of the species that, in its center of origin, has undergone processes of diversification and selection. Specific environmental influences promote variations at a lesser amplitude than those of genetic origin. The relationship between the chemical composition of a given species and climatic data may reveal changes in the metabolic profile or establish patterns of responses to environmental variations.

The total phenolic content is found to be higher in the fruiting stage, while this amount is reduced in the flowering and vegetative stages (Cirak et al. 2013). This phenomenon is different from what was expected; in general the contrary occurs, leading to higher phenolic content in the flowering stage (Chepel et al. 2020). Phenolic concentrations also differ based on the ontogenetic stage (Cirak et al. 2013). In the evolutionary history of plants, the participation of secondary metabolites in the success of adaptation to the most diverse terrestrial environments is clear, allowing some classes of metabolites to be used as chemical markers for certain groups of plants (Kubitzki & Gottlieb 1984). The APG IV (2016) shows the appearance of gallic acid and ellagic acid in the clade that includes the order Fabales, which is also called the nitrogen-fixing clade. This may lead to the hypothesis that these two compounds represent chemical markers for this group.

Phenolic acids are an important and abundant subgroup of phenolic compounds with the basic chemical structure of C6-C1 (related to hydroxybenzoic acids) or C6-C3 (related to hydroxycinnamic acids) and consisting of a phenolic ring and a carboxyl group. In some cases, phenolic acids are the precursor of other important compounds (xantons, tannins, coumarins, and quinones). The profile observed here does not reflect the presence of ellagic acid as a main compound, even though it is associated with gallic acid. On the contrary, caffeic acid, ferulic acid, and gallic acids were the predominant phenolics observed.

The concentration of phenolic acids can be correlated to climatic typology; in northeastern Brazil, Leguminosae produces high amounts of gallic and ellagic acids in the arid and dry zone due to the stress from water shortage and high temperatures. These phenolic compounds possibly act as providers of regulatory ecosystem services, by promoting the ability to adapt in adverse climatic conditions to control competitiveness in the area of ​​occurrence of the plants, which ensures their maintenance and permanence (Kabtni et al. 2020).

For the secondary metabolism, the investigation of the metabolic profile of seedlings can assist in the selection and conservation of promising chemotypes that can be used in biotechnological research. França (2017) noted that seedlings grown in vitro can be used as a source of standard compounds for metabolome studies and as a potential model for elicitation tests or in the stimulation of secondary metabolite synthesis. At the same time, Gripenberg et al. (2017) state that, although polyphenols are one of the most common groups of secondary metabolites of plants, they play a fundamental role in the establishment of plants and constitute one of the first chemical barriers, even though the mortality of seedlings due to attacks by herbivores, microorganisms, competition, and environmental factors can act as an important regulator in the population structure.

Phenolic compounds are a class of metabolites that are of great interest due to their considerable biological activity, and higher plants synthesize several thousand known phenolic compounds and the number of these that are fully characterized is continually increasing (Boudet 2007). Despite that they are safe to consume, the limited bioavailability of these compounds continues to be highlighted as a main concern (Arnoso et al. 2019). For these aspects, the studied plant presents a set of phenolics that can be a source of beneficial metabolites.

According to Wink (2008), the biosynthesis of secondary metabolites is very complex, since it involves specific enzymes for each pathway and is highly regulated in terms of compartmentalization, time, and space. Such complexity is also related to the processes of synthesis and storage of these metabolites. In general, the tissues and organs that are important for the survival and reproduction of plants, such as those of the epidermis and bark, flowers, fruits and seeds, have different metabolic profiles and more of these metabolites.

Phenolic compounds are often produced from stressful conditions, such as attacks by herbivores, infections, and UV radiation (Naczk & Shahidi 2004). According to Dickson (2000), they are components of defensive adaptations and, thus, are present from the early stages of the plant and in external structures, such as trichomes. It is believed that the hypothesis of defensive adaptation is the most coherent for the studied species.

CONCLUSIONS

Gas chromatography coupled with mass spectrometry (GC-MS) was an efficient method for detecting gallic acid as a common structure in the leaflets and roots from the three climatic typologies (Af/Pará, Am/Amazonas, and Aw/Maranhão). The main phenolic compounds found in the leaflets and roots of L. ferrea using histochemical techniques were total phenolic compounds, including flavonoids and tannins.

We conclude that the presence of gallic acid in extracts of leaflets of L. ferrea seedlings shows that this species produces this phenolic acid in the initial stage of development and that the concentration of this acid is greater in mature tissues, such as leaves and fruits (data not quantified but observed by color intensification in the qualitative assays). The presence of phenolic compounds acts in the protection and selectivity of this species, which helps it become established. The presence of total phenolic compounds and flavonoids in glandular trichomes is associated with defense activities against herbivory, microorganisms, and solar radiation. In the roots, total phenolic compounds, flavonoids, and tannins occur in specialized structures.

Seedlings from the Af/Pará and Aw/Maranhão typologies have higher amounts of phenolic acids and possibly represent the L. ferrea chemotype found in its diversification (radiation) and origin center, which are seasonally dry forests. However, it was demonstrated that the chemical profile of phenolic compounds is conserved in this species, and drastic variations due to high plasticity were not observed, which ensures its use regardless of origin.

ACKNOWLEDGMENTS

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. The authors would like to thank the Museu Paraense Emílio Goeldi, and Propes/Universidade Federal do Pará for the scholarship and technical support. ACF thanks to senior postdoctoral grant (FAPERJ/E-26/203.422/2023).

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Publication Dates

  • Publication in this collection
    24 Mar 2025
  • Date of issue
    2025

History

  • Received
    12 June 2024
  • Accepted
    12 Dec 2024
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