Secretory structures in Cochlospermum regium ( Schrank ) Pilg . ( Bixaceae ) : Distribution and histochemistry

Instituto Federal Goiano (IFGoiano)Campus Rio Verde Rod. Sul Goiana Km 01, Zona Rural, CEP 75.901-970, Caixa Postal 66, Rio Verde – GO, Brasil. Universidade de Rio Verde (UniRV), Fazenda Fontes do Saber, CEP: 75.901-970, Caixa Postal 104, Rio Verde – GO, Brasil. Universidade de Brasília (UnB), Campus Darcy Ribeiro, Departamento de Botânica, CEP 70919-970, Caixa Postal 04457, Brasília-DF, Brasil.


INTRODUCTION
The use of medicinal plants for the treatment and prevention of diseases is one of the oldest practices in human history.Because of studies that have demonstrated their safety and efficacy, at present, medicinal plants and their products are sold in pharmacies and health stores, bearing commercial labels (Veiga Junior et al., 2005).Due to the growing demand for herbal medicines, the number of companies in this field has increased in Brazil, resulting in the issuing of the Presidential Decree No. 5813, of June 22, 2006, which approved the National Policy on Medicinal Plants and Herbal Medicines.This decree regulates the production, distribution and rational use of medicinal plants in a sustainable manner, thus ensuring the safety, efficacy and quality of these products, in addition to expanding the therapeutic options and user access to this therapeutic modality (Brazil, 2006).
Cochlospermum regium (Schrank) Pilg., known as the yellow cotton tree, is a small shrub that displays plasticity in flowering time, blooming several times over the year (Camillo et al., 2009;Inácio, 2010).This plant is known to have several pharmacological actions of medicinal interest, such as the treatment of gastritis, ulcers, gynecological infections, (Oliveira et al., 2007;Camillo et al., 2009), intestinal infections, skin disorders, arthritis (Camillo et al., 2009), urogenital disorders, and as a purgative (Vila Verde et al., 2003;Guarim-Neto, 2006).Examples of the many uses of C. regium include tea made from roots and bark, topical use of the decoction in sitz baths, white wine garrafada (single or mixed plant species macerated in white wine and sold in bottles) called enóleo, and aqueous infusion for the treatment of various diseases (Nunes et al., 2003;Vila Verde et al., 2003;Guarim Neto, 2006;Sólon et al., 2009).However, if the genus Cochlospermum, as a whole, is considered, not only the species, pharmacological properties including hepatoprotective, antimalarial, antibacterial, antitumor, antiviral, detoxifying, antileishmanial, antihypertensive, antidiabetic, anti-inflammatory, antischistosomiasis, and antidiarrheal activities, among others, have also been reported (Antunes, 2009).
A number of chemical compounds produced by the plant may be associated with secretory cells and tissues, such as idioblasts, trichomes, emergences, cavities, and ducts.This study aimed to identify the secretory structures and the major classes of substances in vegetative organs of C. regium.

Plant
Leaves, stems and roots of C. regium, Bixaceae, were collected in the "Cerrado" (Brazilian savannah) of the Environmental Protection area of the University of Rio Verde (UniRV), more specifically at the "Fazenda Fontes do Saber" in the city of Rio Verde, Goiás,Brazil (17° 47' 53'' S;51° 55' 53'' W).The identification of C. regium was performed by a specialist (first author) and the voucher specimen number HRV 362 was deposited at the Herbarium of Rio Verde in the Goiano Federal Institute (IFGoiano) Campus Rio Verde.

Assays
Samples for anatomical study were fixed in formalin, acetic acid and 70% alcohol (FAA 70 ).Transverse and longitudinal sections of the fixed plant organs were then cut using a microtome (model LPC, Rolemberg and Bhering, Belo Horizonte, Brazil) with disposable steel blades and stained with toluidine blue (O'Brien et al., 1965) or astra-blue/safranin stain (Bukatsch, 1972).For analysis of the leaf surface the blade sections were cleared (Kraus and Arduin, 1997), stained with safranin and mounted in 50% glycerol.Fresh samples of vegetative organs of C. regium were used for histochemical analysis.Sections of the vegetative organs of the plant were also cut with the microtome.The sections were stained with Sudan IV (red) for lipids (Pearse, 1980); Xylidine Ponceau for proteins (O'Brien and McCully, 1981); periodic acid-Schiff reagent (PAS) for total carbohydrates (Maia, 1979); ferric chloride (Johansen, 1940) and potassium dichromate (Gabe, 1968) for phenolic compounds; Nadi reagent for essential oils and oleoresins (David and Carde, 1964); vanillin-HCl for tannins (Mace and Howell, 1974); Wagner and Dittmar reagents for alkaloids (Furr and Mahlberg, 1981); and phloroglucinol for lignins (Johansen, 1940).The images were captured using a digital camera (Leica DC 50, Leica Microsystems, Germany) attached to a Leica DM500 microscope.

RESULTS
The leaf of the C. regium showed a single-layered epidermis with anomocytic stomata confined to the abaxial surface, characterizing the leaf as hypostomatic (Figure 1a  and b).In cross-section view, the epidermis exhibited upright cells on the adaxial side, and cells with rectangular shape on the abaxial side.In frontal view, the epidermal cells had a polyhedral shape with straight walls (Figure 1c  and d).The midrib of the C. regium leaf contained a collateral vascular bundle.Immediately below the epidermis, a thin layer of angular collenchyma was observed and fundamental parenchyma delimited numerous ducts distributed in the cortical region of the midrib (Figure 1c).A large number of cells just underneath the phloem, intensely stained with safranin, were identified as idioblasts, which together with ducts, constitute the secretory structures of C. regium (Figure 1c).The mesophyll was dorsiventral with 1 or 2 layers of palisade parenchyma cells followed by 3 to 4 layers of spongy parenchyma cells delimiting small intercellular spaces (Figure 1d).The stem of C. regium had a singlelayered epidermis and a phellogen layer throughout its length (Figure 2a), indicating a secondary growth of the organ.The vascular cambium can be observed between the secondary xylem and phloem (Figure 2b).External to the phloem, there were several layers of fibers with walls of thin to medium thickness.Ducts, such as those present in the leaf, were observed in the medullary region (Figure 2b).
Secondary growth was also observed in the root of C. regium.The root surface had a thick periderm.The phloem layer was visible underneath the periderm as well as numerous layers of vascular cambium cells.Ducts were distributed in the root cortex and near the phellogen differentiation region, and delimited by fundamental parenchyma (Figure 3a and b).In the xylem, there was a predominance of elements of solitary vessels of simple perforation plates with a helical secondary wall deposition pattern (Figure 3c  parenchyma were present in association with vessels (Figure 3e).Storage parenchyma (radial and axial) rich in starch grains and a small amount of thin-walled fibers were observed below the cambium (Figure 3e).Histochemistry revealed phenolic compounds (including tannins and lignin), starch, lipid compounds, alkaloids and essential oils in the different plant organs.
The leaves of C. regium had many cells distributed in the fundamental parenchyma containing phenolic compounds, which were detected using potassium dichromate and ferric chloride.The test with vanillin-HCl confirmed that these cells were tannin idioblasts.The same phenolic content was found in chlorenchyma cells.The phloroglucinol reaction indicated the presence of lignin only in the vessel elements, confirming the absence of fibers, sclereids or any other cells with lignified walls.
Essential oils were found in some idioblasts.Proteins were widely distributed in the phloem and fundamental parenchyma.Total carbohydrate was observed in some idioblasts, ducts and parenchyma.Reserve lipids were detected in idioblasts and inside the ducts.Alkaloids were not found in the leaves.Likewise, several tannin idioblats were observed in the cortex and pith of the C. regium stem.A large number of fibers with lignified walls were present outside the phloem and xylem cells (vessel elements) and had a positive reaction to phloroglucinol.Essential oils occurred in isolated form in a few idioblasts.Proteins and carbohydrates were widely distributed in the parenchyma, cortex, and pith.In addition to lipid reserves, a thin cuticle layer was revealed with Sudan IV.
The C. regium root had a large number of cells containing phenolic compounds.A large number of tannin idioblasts were found in the fundamental parenchyma of the cortex and radial parenchyma cells were observed in the secondary xylem.Starch grains were abundant in both the axial and radial parenchyma in the roots of C. regium.Essential oils were detected with Nadi reagent in single cells (idioblasts) dispersed among fundamental parenchyma cells; many of these cells had globules of essential oils and resin acids.The analysis of C. regium performed in the present study revealed that this species has a rather complex structural organization, and a rich and varied chemical composition, confirming the great potential for research in this understudied plant.The leaves, roots and stems of C. regium have a complex tissue organization and are rich in secretory structures, such as ducts and idioblasts.A varied composition of secondary metabolites, especially phenolic compounds and a mixture of lipophilic compounds are found in these organs.The wide variety of compounds present in C. regium is evidence of its pharmacological potential, as well as of the possible applications of this plant in various industrial sectors.This perspective points to C. regium as a promising medicinal plant and should be the basis for continuing research on this so important and yet so little known species.

DISCUSSION
Various idioblastic cells associated with secretory structures were observed on C. regium leaves.Categorically, idioblasts are individual cells with a different chemical composition from that of the surrounding cells; they vary in shape and are classified according to the substances that they synthesize (Castro and Demarco, 2008) and part of the bioactive compound precursor synthesis and storage may exist within the idioblast vacuoles (Mahroug et al., 2006).Several classes of phenolic compounds are synthesized and stored in a large central vacuole or multiple cells of varying sizes present in the idioblastic cytoplasm.These cells are involved in the secretion of mucilage, gum and phenolic compounds, and epithelial cells that delimit ducts can secrete heterogeneous material with mixed composition (Appezzato-da-Glória and Carmello-Guerrero, 2006).The immunohistochemical analysis revealed phenolic compounds (including tannins and lignin), starch, lipid compounds, alkaloids and essential oils in different organs of the plant.Firstly, essential oils were detected only in leaves, but later found in plant roots by Solon et al. (2009) in a study on the antimicrobial properties of these compounds.Inácio (2010) reported the presence of essential oils, mainly terpenes and oxygenated derivatives in leaves and roots of C. regium.The same author also highlighted the high potential of C. regium for use in perfumes, the aromatherapy industry, and pharmaceutical applications.Unlike other vegetative organs, the C. regium root had several conduits that contain alkaloids, especially in the cortical region.Solon et al. (2009) reported that the roots of C. regium contain a variety of chemical compounds such as tannins and other phenolic compounds, mucilage and terpenes.
According to Appezzato-da-Gloria and Carmello-Guerreiro ( 2006  (terpenes, free fatty acids, phenolic compounds and waxes), mucilage, proteins, amino acids and essential oils, among others.Other studies have reported that the roots of C. regium contain saponins, triterpenoid compounds, flavonoids, tannins and other phenolic compounds (Andrade et al., 2008).The analysis of essential oils from the C. regium roots by gas chromatography and mass spectrometry led to the identification of various substances (Solon et al., 2009).
The ducts are secreting structures composed of an epithelium terminating in an elongated lumen in which the secreted material has a varying chemical nature (Fahn, 1990).Aguiar-Dias and Cardoso-Gustavson (2011) reported the presence of secretory ducts in the angles of origin of the development of Polygala angulata rods under primary growth.In North American species, the resin ducts secret, while in P. angulata, a species found in the Brazilian "cerrado", the mucilage ducts secret, which is probably a xeromorphic adaptation to an arid environment.The C. regium root parenchymal tissue is rich in carbohydrates, with a lower content of proteins and lipids.