Exploring the histolocalization of secondary metabolites in two species of Croton L. (Euphorbiaceae)ABSTRACT
The species Croton heliotropiifolius Kunth and Croton pedicellatus Kunth are used in agriculture and for therapeutic purposes by traditional communities in the city of Campo Maior, Piauí State, Brazil. Both species contain secondary compounds such as flavonoids, triterpenes and phenolic compounds and, according to pharmacological studies, have different biological properties associated with their extracts, such as anti-inflammatory and antioxidant properties. Thus, they are the target of studies that investigate the diversity of secondary metabolites and the site of synthesis and storage of these compounds. In the field, the material was fixed in FAA (formaldehyde-glacial acetic acid, 50% etilic alcohol; 1:1:18 v v-1) for anatomical analyses and in FNT for histochemical analyses. The tests indicate the presence of total proteins, starch, lipids, phenolics and alkaloids in the stem and leaf regions of both species. The laticifers, idioblasts and glandular trichomes are the sites where the compounds were found in both species.
Keywords:
Croton heliotropiifolius; Croton pedicellatus; Crotoneae tribe; histochemistry; Taxonomy
RESUMO
As espécies Croton heliotropiifolius Kunth e Croton pedicellatusKunth são utilizadas na agricultura e para fins terapêuticos por comunidades tradicionais do município de Campo Maior, Estado do Piauí, Brasil. As duas espécies contêm compostos secundários como flavonoides, triterpenos e compostos fenólicos e, segundo estudos farmacológicos, possuem diferentes propriedades biológicas associadas aos seus extratos, como propriedades anti-inflamatórias e antioxidantes. Sendo assim, são alvo de estudos que investigam a diversidade de metabólitos secundários e o local de síntese e armazenamento desses compostos. Em campo, o material foi fixado em FAA (formaldeído-ácido acético glacial, álcool etílico 50%; 1:1:18 v v-1) para análises anatômicas e em FNT para análises histoquímicas. Os testes indicam a presença de proteínas totais, amido, lipídeos, compostos fenólicos e alcaloides nas regiões do caule e da folha das duas espécies. Os laticíferos, idioblastos e tricomas glandulares são os sítios onde os compostos foram encontrados.
Palavras-chave:
Croton heliotropiifolius; Croton pedicellatus; histoquímica; Taxonomia; tribo Crotoneae
Introduction
Popular medicine in Brazil is the result of the union of different cultures, including Europeans, Africans, Asians and native peoples who shared knowledge about local herbs over generations (Lorenzi & Matos 2002). The appropriation of nature by human beings is notorious, with the aim of ensuring better means of survival, making use of natural resources existing in the environment (Brasil 2006).
The economic importance of medicinal plants and phytosanitary preparations is evident in the market that drives a lot of capital in developed countries (Klein et al. 2010). In Brazil, the great diversity of plants found allows access and consumption by the population, without due precautions regarding possible side effects (Ferreira et al. 2019).
Euphorbiaceae is widely used in folk medicine, contributing to the treatment of respiratory, gastrointestinal, inflammatory and skin diseases, in addition to its applications as purgatives and antiparasitics, thus highlighting its therapeutic versatility (Shahidul et al. 2019). In Brazil, approximately 68 genera and 982 species are found, with a predominant presence in the caatinga and cerrado biomes (Flora e Funga do Brasil 2023). Among the genera, Croton L. stands out, which is widely used in folk medicine for its anti-inflammatory, antihypertensive, antifungal, antimicrobial, antioxidant, antidiabetic, antinociceptive and antitumor activities (Junior et al. 2022).
In Campo Maior city, Croton pedicellatus Kunth and Croton heliotropiifolius Kunth stand out, and are used for medicinal purposes (Oliveira 2008). The species Croton heliotropiifolius, popularly known as “velame”, “velaminho” and “velame de cheiro”, is traditionally used in folk medicine against stomach pain, vomiting, diarrhea, and as a muscle relaxant (Randau et al. 2004). In quilombola communities, it is used as an anti-inflammatory, to control body temperature and in the treatment of diabetes, through “teas” made from the fresh leaves of the plant (Magalhães 2020). Phytochemical studies have shown the presence of secondary chemical compounds, such as flavonoids and triterpenes, with active potential in biological activities, such as anti-inflammatory, anti-allergic and antiviral actions (Silva et al. 2020). Croton pedicellatus, popularly known as “velame”, presents secondary compounds, such as phenolic compounds, steroids and terpenoids (Lopes 2012, Lopes et al. 2012). The species has antibacterial activity, helping to combat phytopathogenic infections occurring in agricultural plantations (Silva et al. 2016). The different uses attributed to these Croton species make them promising for diagnostic approaches and correct taxonomic identification, especially when they have similar medicinal uses. This avoids errors in sample quality control.
Histochemistry highlights the main chemical groups, such as acidic polysaccharides, alkaloids, phenolics compounds, terpenoids, among other important groups in plant defense and for therapeutic purposes (Figueiredo et al. 2007). According to Rosa et al. (2021), histological analysis is an essential tool for identifying medicinal plants. Furthermore, it allows the verification and occurrence of irregularities, such as fraud or falsification of these species for medicinal use, since the quality and efficacy of a medicinal species can be compromised by incorrect identification (Santa-Cecília et al. 2014).
The leaves and young stems of Croton species present a wide diversity of secretory structures, such as idioblasts, glandular trichomes and laticifers, which have relevant chemical potential. The histolocalization of these structures can contribute significantly to future bioprospecting studies, especially focused on young leaves and stems (Vitarelli et al. 2015, Gancedo et al. 2022).
Thus, we investigated the anatomy and histochemical profile of the leaves and stems of Croton heliotropiifolius and Croton pedicellatus to measure the diversity of secondary metabolites in the organs and which structures are involved in the synthesis and accumulation of these compounds, in addition to helping in the identification of two species of pharmacological interest.
Material and methods
Sample collection - The samples of Croton heliotropiifolius and Croton pedicellatus were collected in the city of Campo Maior, Piauí, Brasil State, which has the caatinga as its predominant biome, with a semi-arid climate, high temperatures and scarcity of rain. Exsiccates of the two species were produced and deposited in the Graziela Barroso herbarium (TEPB), from the Universidade Federal de Piauí - UFPI (C. heliotropiifolius TEPB 33026 e C. pedicellatus TEPB 33027).
The collected specimens were identified by an expert. In the field, the material was fixed in FAA (formaldehyde-glacial acetic acid, 50% etilic alcohol; 1:1:18 v v-1) for 24 hours (Johansen 1940) for anatomical analyses, and in neutral buffered formalin (NBF) for 48 hours (Lillie, 1965) for histochemical analyses.
Anatomical analysis - The material was sectioned freehand, using a razor blade and Styrofoam, to obtain transverse and longitudinal sections of the stem apex, leaf blade and middle third of the petiole. The sections were clarified in 20% sodium hypochlorite and stained with basic fuchsin-astra blue, mounted in glycerin gelatin and sealed with colorless nail polish (Roeser 1972, Kaiser 1880, Kraus & Arduin, 1997). After the slides were prepared, the material was analyzed to observe the anatomical characteristics.
Histochemistry - After anatomical analyses, the material fixed and stored in NBF (Lillie, 1965) was submitted to histochemical tests. Freehand cuts were made to obtain transverse and longitudinal sections of the leaf blade (apex, middle and base) of the petiole and stem in primary growth of the species Croton heliotropiifolius Kunth and C. pedicellatus Kunth. The material was sent for histochemical tests according to Table 1.
Tests performed on stems and leaves of Croton heliotropiifolius and Croton pedicellatus Chel: Croton heliotropiifolius Kunth. Cped: Croton pedicellatus Kunth.
Microscopic analysis - The analysis and capture of anatomical and histochemical images were obtained using a Coleman optical microscope, with a U-photo system and attached camera, and the anatomical plates were edited in the Arcsoft-photoimpression program and assembled in the CorelDraw program, version 2022. The trichomes were analyzed according to the classification of Pinto-Silva et al. (2023).
Results
Anatomical description - Stellate tector trichomes (Figure 1a) and multiradiate trichomes with ten rays were observed on the stem, leaf blade and petiole of C. heliotropiifolius and C. pedicellatus (Figure 1 b). Glandular trichome, with a rounded shape similar to a circular button, was observed only in C. pedicellatus on the stem, petiole and leaf blade (Figure 1 c).
Details of leaf and stem anatomy of Croton heliotropiifolius Kunth and Croton pedicellatus Kunth. a, b, d, e, f, h, i, j, k, l, m: C. heliotropiifolius Kunth. c, g: C. pedicellatus Kunth. a. Stellate tector trichome. b. Multiradiate trichome. c. Glandular trichome in the form of a bud. d. Stem. e. Cortex. f. Laticifers. g. Idioblast. h. Vascular bundles. Ep: Epidermis. Mp: Parenchymatic medulla. Co: Collenchyma. Id: Idioblasts. La: Laticiferous. D: Druses. P: Phloem. X: Xylem. Ab: Accessory bundles.
The stems of C. heliotropiifolius and C. pedicellatus have similar characteristics, such as a circular outline (Figure 1 d) with a unistratified epidermis with a thin cuticle (Figure 1 e). The cortex is formed by annular collenchyma and fundamental parenchyma (Figure 1 e) and presents a parenchymatic pith (Figure 1 d). There are also the presence of druses and prismatic crystals distributed in the cortex and pith (Figure 1 e). The species are differentiated by the presence of laticifers in C. heliotropiifolius (Figure 1 f), and the presence of idioblasts in C. pedicellatus (Figure 1 g). Regarding the vascular systems, both species present bicollateral bundles with a circular conformation (Figure 1 h).
In the petioles of Croton heliotropiifolius and Croton pedicellatus, it is also possible to highlight similarities such as circular conformation (Figure 1 i) with thin cuticle, unistratified epidermis (Figure 1 j), annular collenchyma (Figure 1 j), presence of druses (Figure 1 k) and collateral vascular bundles (Figure 1 i, l). Regarding the conformation of the vascular bundle, it is an open arch with convoluted ends, with two accessory bundles in C. heliotropiifolius (Figure 1 l), while in C. pedicellatus, it is an open arch (Figure 1 i). Laticifers can be observed in C. heliotropiifolius (Figure 1 m).
The midrib of C. heliotropiifolius has a biconvex conformation (Figure 2 a), while in C. pedicellatus it is flat-convex (Figure 2 b). The epidermis is unistratified, with a thin cuticle in both species (Figure 2 c). The collenchyma is annular, followed by fundamental parenchyma, with druses in both species (Figure 2 c). Another similar characteristic in both species is the presence of a layer of palisade parenchyma crossing the main vein (Figure 2 a-b). Croton pedicellatus presents idioblasts distributed in the epidermal and parenchymal cells and close to the vascular bundles (Figure 2 d). Laticifers are distributed in the midrib of C. heliotropiifolius (Figure 2 e). The vascular system is collateral in both species, with a flat-convex conformation in C. heliotropiifolius (Figure 2 a) and an open arch in C. pedicellatus (Figure 2 b). The mesophyll is dorsiventral in both species (Figure 2 f-g). In C. pedicellatus, there are columnar sclereids starting from the epidermis and crossing the entire mesophyll (Figure 2 g).
Leaf blade details in C. heliotropiifolius Kunth and C. pedicellatus Kunth. a, c, e, f: C. heliotropiifolius Kunth. b, d, g: C. pedicellatus Kunth. a. Midrib of C. heliotropiifolius Kunth. b. Midrib of C. pedicellatus. c. Epidermis and annular collenchyma. d. Idioblast. e. Laticiferous. f. Dorsiventral mesophyll of C. heliotropiifolius. g. Dorsiventral mesophyll of C. pedicellatus. Ep: Epidermis. Co: Collenchyma. D: Druses. La: Laticifers. Pp: Palisade parenchyma. Lp: Lacunose parenchyma. S: Sclereids. Id: Idioblast. P: Phloem. X: Xylem.
Locating the area of secondary metabolites within tissues and cells - Histochemical analyses showed the presence of total proteins (Figure 3 a) in the laticifers and epidermal cells of C. heliotropiifolius and idioblasts of C. pedicellatus. Starch (Figure 3 b) was present in the parenchyma cells of the cortex in all analyzed structures of both species. Total lipids (Figure 3 c-d) were distributed in the parenchyma cells of C. heliotropiifolius and in the glandular trichomes of C. pedicellatus. Phenolic compounds (Figure 3 e) and alkaloids (Figure 3 f) were distributed in the parenchyma cells of the cortex of both species and also in the idioblasts of C. pedicellatus in all analyzed regions. In Croton heliotropiifolius, non-structural phenolic compounds (Figure 3 g) are present in parenchyma cells close to vascular bundles in all regions analyzed. In Croton pedicellatus, this compound is present in parenchyma cells close to the vascular system and idioblasts only in the stem and leaf blade regions.
Histolocalization of secondary metabolites in the stems and leaves of Croton heliotropiifolius Kunth and C. pedicellatus Kunth.: a, b, c C. heliotropiifolius Kunth.: d, e, f, g, h, i C. pedicellatus Kunth. a. Positive reaction for total proteins (arrow) in the petiole. b. Stained starch grains (arrow) in the petiole. c. Lipid inclusions (arrow) evident in the stem. d. Stem trichome with inclusion of neutral lipids (arrow). e. Phenolic idioblasts (arrow) dispersed in the stem. f. Positive reaction for alkaloids (arrow) in the petiole; g) Positive reaction for phenolic compounds (arrow) in the leaf blade. h. Presence of acidic polysaccharides (arrow) in the stem. i. Positive reaction for mucilage (arrow) in the leaf blade.
In Croton pedicellatus, the presence of acidic polysaccharides is observed in the idioblasts of all regions analyzed (Figure 3 h). Mucilages and pectins (Figure 3 i) are also present in the idioblasts and parenchyma cells close to the vascular system of C. pedicellatus. The results of all tests are summarized in Table 1.
Discussion
The Euphorbiaceae family can present a variety of trichomes, including stellate, simple, glandular or non-glandular, lepidote, fascicular, urticating and dendritic (Metcalfe & Chalk 1979). The stellate and multiradiate trichomes found in the two Croton species are described in other studies related to the genus and present a taxonomic contribution, in addition to being widely related to the protection of the plant against herbivory (Lucena & Sales 2006, Liu et al. 2013 Gomes et al. 2018, Pinto-Silva 2023).
Glandular trichomes store secondary compounds, such as phenolics, terpenes, and other substances that aid in the chemical defense of plants (Metcalfe & Chalk 1979). This type of trichome is less frequent in Croton species (Pinto-Silva et al. 2023). Therefore, the presence of this structure in C. pedicellatus is useful in identifying this taxon. In addition, the glandular trichomes found in C. pedicellatus are responsible for synthesizing and storing metabolites such as total lipids that aid in the plant’s defense against microorganisms (Desoti et al. 2017).
The petiole contour of C. pedicellatus and C. heliotropiifolius is circular, a characteristic already described in other studies of the Croton, such as in C. urucurana Baill and C. vulnerarius Baill, according to research on the tribe Crotoneae (Sá-Haiad 2009). However, the two species differ in the conformation of the vascular system. Metcalfe and Chalk (1979) emphasize that the characteristics of the petiole have taxonomic value, as they are not affected by environmental changes. In addition, these species present distinctions in the outline of the main vein and in the conformation of the vascular system. Although there is data suggesting the medicinal use of C. heliotropiifolius, some studies indicate some toxicity of the ethanolic (Carvalho et al. 2018) and methanolic (Silva et al. 2018) extracts of the leaves. These studies demonstrate considerable levels of toxicity in tests with the bioindicator Artemia salina L. Therefore, identifying characteristics that can identify Croton species can influence quality control and avoid the use of potentially toxic species.
Columnar sclereids are associated with the tissue support function, preventing cells from colliding, a characteristic present in xerophytic plants (Barros & Soares 2013), emphasizing an adaptation of C. pedicellatus to the dry environment, in which it is exposed to water scarcity and solar radiation for long periods during the year, a characteristic present in the city of Campo Maior with a semi-arid climate.
Druses are distributed in the three regions analyzed in C. heliotropiifolius and C. pedicellatus. The presence and distribution of druses and crystals may refer to the process of calcium precipitation through transpiration flow (Macnish 2003). Important secretory structures in the species are the presence of laticifers in C. heliotropiifolius and idioblasts in C. pedicellatus. According to Fahn (1979), idioblasts and laticifers are specialized secretory structures that synthesize a variety of secondary compounds such as proteins, phenolic compounds, alkaloids, among others. Therefore, for this study, such structures are responsible for the synthesis and accumulation of compounds that act in the treatment of some diseases.
Phenolic compounds, present in some vegetables, can play an important role in medicinal use, due to their antioxidant properties (Lusa & Bona 2011). The study carried out by Aquino et al. (2017), reinforces the existence of phenolic compounds, such as flavonoids and tannins in Croton heliotropiifolius and Croton blanchetianus, which demonstrate antioxidant potential. Therefore, the metabolites present in the leaves and stems of the species C. heliotropiifolius and C. pedicellatus may confer a natural antioxidant potential.
The presence of lipids in plants helps in microbial activities, such as fighting phytopathogens (Desoti et al. 2017). Species belonging to the Croton exhibit high potential in field activities, such as insecticide and antifungal (Cavalcante et al. 2020). Thus, the presence of lipids in the species C. heliotropiifolius and C. pedicellatus has the potential to repel insects and microorganisms, so the species can contribute to agriculture in fighting fungi and bacteria in agricultural plantations, acting as a repellent for microorganisms.
Plants containing acidic polysaccharides have great therapeutic potential, promoting wound healing and fighting infections (Sousa et al. 2020). Furthermore, they stand out as promising agents with antitumor, immunostimulatory, and anticancer activities (Xei et al. 2015). In this context, C. pedicellatus emerges as a species with potential to be explored in studies focused on wound healing and treatments involving actions on the immune system.
A study related to starch consumption emphasizes that this metabolite can aid in the treatment of hyperglycemic diseases, such as diabetes (Monteiro & Nascimento 2013). The presence of starch in the species C. heliotropiifolius and C. pedicellatus may attribute such therapeutic properties, reinforced by the study by Magalhães (2020) on the use of C. heliotropiifolius in the form of tea for the treatment of diabetes.
The proteins found and analyzed in the stem and leaves of the species C. heliotropiifolius and C. pedicellatus may suggest that these species have antifungal and antioxidant properties, already reported in the study of secretory structures of Croton echinocarpus Müll. Arg. and Croton urucurana Baill, due to the presence of total proteins (Feio et al. 2016).
The presence of mucilages in medicinal plants has a healing potential in the treatment of ulcers, burns, in addition to anti-inflammatory properties (Júnior et al. 2020). And this healing potential has already been reported in Croton lechleri Müll. Arg., as the species helps in the deinflammatory phase of wounds (Rossi et al. 2023). Mucilages are present exclusively in idioblasts of C. pedicellatus, evidencing the therapeutic.
The Croton genus has an abundance of alkaloids distributed in various regions of the plant (Salatino et al. 2007). In plants, alkaloids exert effects against herbivores and pathogens. In medicinal terms, it is used to treat stomach problems, provide pain relief, act as a muscle relaxant, and have antibacterial properties (Roy 2017). The presence of alkaloids reported in the present study reinforces the guidelines of Randau et al. (2004) on the use of Croton heliotropiifolius in the treatment of muscle pain and diarrhea, and the study by Silva et al. (2016) on the use of C. pedicellatus as an antibacterial.
Tests for fatty acids, condensed tannins and carbohydrates were negative in C. heliotropiifolius and C. pedicellatus. These secondary compounds have already been recorded in other species belonging to the genus, such as Croton cajucara Benth and for other genera of Euphorbiaceae, with antimicrobial, antifungal and antioxidant potential (Trindade et al. 2021, Souza et al. 2006, Vieira et al. 2021). However, the absence of these metabolites does not mean that the species do not have such medicinal properties, since other metabolites, such as acid polysaccharides, total lipids and phenolic compounds, can perform these same functions (Lusa & Bona 2011).
Conclusions
The structural anatomical characters contribute to the characterization of C. heliotropiifolius and C. pedicellatus, helping to distinguish the species and, as a result, it contributes to quality control. This can prevent poisoning from the use of similar taxa.
Leaf anatomy can contribute to measuring the diversity of secondary metabolites involved in the numerous biological activities reported in Croton L. species, emphasizing the presence of phenolic compounds, total proteins, alkaloids and lipids in both species. In addition, it can determine which structures are specialized in the synthesis and accumulation of these compounds, such as laticifers, idioblasts and glandular trichomes. These findings may contribute to directing pharmacological and biotechnological studies.
Data availability statement
The dataset for this article is available in the SciELO Dataverse of Hoehnea, at the following link: https://doi.org/10.1590/2236-8906e532025.
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