Anatomical and histochemical analysis of vegetative organs of Vernonia ferruginea Less . ( Asteraceae )

Vernonia ferruginea Less. is a perennial shrub species, present in several regions of Brazil, especially in the savanna. It is popularly used as a phytotherapic. This fact justifies the need to anatomically characterize the plant for its accurate identification and to conduct histochemical studies with the aim of identifying the chemical nature of its cellular constituents. The species-specific data will contribute significantly to pharmaceutical quality control and also provide information about the sites of specific chemical compounds. Samples of V. ferruginea vegetative organs were collected and submitted to the usual plant anatomy and histochemical techniques. The leaves are anfihipoestomática with anomocytic stomata; have tector and glandular trichomes that store essential oils. The stem has collateral-type vascular bundles arranged in a eustele structure; it also has glandular and tector trichomes. The root has brachysclereids, endoderm with various chemical compounds and vascular bundles having axial elements and rays. Few differences were found in the structure of vegetative organs in relation to other species of the genus, confirming the importance of the details shown.

Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License V. ferruginea Less., popularly known as assapeixebranco, assapeixe-do-pará or calção-de-velho, is a perennial shrub or tree; it is erect, highly branched, with a woody stem, rugous at the base and has rusty hairiness at the apex.It measures 2 to 3.5 m high and is native to Brazil.It is one of the most common weeds in the pastures, occurring throughout Brazilian territory except in the Southern Region.V. ferruginea is more common in cerrado and low fertile soils, considered as standard for weak land (Lorenzi, 2000).It has been used over time for treatment, prevention and cure of diseases.Its leaves and roots are considered diuretic, balsamic and antirheumatic and are used in cases of bronchitis and persistent cough.Its roots are diuretic and are used for the treatment of hemoptysis and internal abscesses.It is also used externally for skin affections, muscle aches and rheumatism (Lorenzi and Matos, 2002).
The use of medicinal plants has been described throughout history for centuries; they are as old as the human species (Pereira et al., 2005).The knowledge about these plants is the only therapeutic resource in many communities and ethnic groups (Maciel et al., 2002).In the poorest regions of Brazil and even in large Brazilian cities, medicinal plants are sold in street and popular markets, and found even in residential backyards.Popular observations about the use and effectiveness of these plants contribute significantly to the dissemination of the therapeutic virtues of the plants.They are often prescribed due to their medical effects, even though their chemical constituents are unknown.Thus, users of medicinal plants throughout the world maintain the practice of herbal medicine consumption in vogue.This makes therapeutic information accumulated for centuries valid, indirectly arousing the interest of researchers in studies involving multidisciplinary areas such as botany, pharmacology and phytochemistry that together enrich the knowledge about the inexhaustible natural medicinal source: the world flora (Maciel et al., 2002).
Considering the importance of medicinal plants, the anatomical and histochemical study of the vegetative tissues of medicinal species is essential, and the correct identification and morphoanatomical characterization of these plants is crucial for the quality control of raw material used to produce herbal, ensuring their reliability (Martins and Appezzato-da-Gloria, 2006).
Therefore, this study aimed to identify the disposal of the tissues of V. ferruginea to give an accurate identification of this species, the chemical nature of its cellular constituents and its location through the usual techniques of plant anatomy and histochemistry.Plants collected in the Cerrado in the Municipality of Rio Verde were used for this study.

MATERIALS AND METHODS
The analysis was conducted at the Laboratory of Plant Anatomy, Federal Institute of Goiás -Campus Rio Verde, Goiás.We used vegetative fresh young organs, fixed in FAA70, except for the histochemical tests, collected from V. ferruginea individuals growing in a Cerrado area of the Paturi Farm in the municipality of Rio Verde -GO.The identification of V. ferruginea Less.was performed by specialist and the voucher specimen N o .502 was deposited at the herbarium of Rio Verde in the Goiano Federal Institute.Crosssections with about 10 μm thickness were obtained using a microtome LPC for plant anatomy with a disposable steel blade.Microscopic analyses and photographic records were made in a Leica (DM500) optical microscope with camera (Leica ICC50).For structural analysis, sections were stained with safranin and astra blue (Bukatsch, 1972), and slides were mounted in Canada balsam.

Anatomical characterization
The leaves of V. ferruginea have anomocytic stomata on both surfaces; they are more abundant on the abaxial surface, characterizing the leaf as amphi-hypostomatic.This is also found in Vernonia scorpioides (Toigo et al., 2004) and Vernonia polyanthes (Alves and Neves, 2003).In a frontal view, the epidermal cells of the abaxial surface show slightly sinuous walls; however on the adaxial surface they are sinuous, as seen also in some Asteraceae (Figure 1A to B).
In cross section, the epidermis of the leaves is uniseriated, covered by a thin cuticle.The epidermis shows cubic cells, slightly elongated over the veins.At the midrib, the epidermis consists of elongated cells of different sizes, usually smaller than the mesophyll cells.The cells of the epidermis have little thickening between the anticlinal walls.
On both surfaces of the leaves, numerous multicellular uniseriate tector trichomes are observed, especially on the abaxial surface, all with 3 to 8 cells.Multicellular glandular trichomes are present in less numbers, formed by two basal cells and a multicellular head with about four cells (Figure 1C to F).The presence of trichomes is a factor common to the genus Vernonia, and may vary with type and quantity in each species (Metcalfe and Chalk, 1972).
The mesophyll has two distinct layers of palisade parenchyma closest to the adaxial surface of epidermis; and the size of the cells of the first layer is twice that of the second layer.The spongy parenchyma has approximately five to eight cell layers of irregular shape and varied size, characterizing the mesophyll as dorsiventral (Figure 1D).It is similar with most species of this genus such as V. scorpioides (Toigo et al., 2004), V. polyanthes (Alves and Neves, 2003) and Vernonia brasiliana (Filizola et al., 2003), differing only by the double layer of palisade parenchyma, also observed in Vernonia condensata (Lolis and Milaneze Gutierre, 2003).The mesophyll has slight intercellular spaces, and the cells have a compact arrangement.Around the bundles into the mesophyll, the parenchymal sheaths extend to both epidermis surfaces (Figure 1D).
At the midrib, immediately under the epidermal cells, two to four layers of angular collenchyma cells are observed, a common feature to other members of the family Asteraceae (Metcalfe and Chalk, 1972): Ayapana triplinervia (Nery et al., 2014) and Mikania laevigata (Budel et al., 2009).The fundamental parenchyma shows several layers of cells with different sizes.There are few intercellular spaces in the midrib region.The vascular bundles are collateral with evident procambium layers (Figure 1C).The stem cross-section of V. ferruginea has a uniseriate epidermis showing flattened cubic cells with straight walls or slightly convex external periclinal walls.They are covered by a thin cuticle, with a large number of tector trichomes and few glandular tricomes.Immediately below the epidermis, 2 to 3 layers of angular collenchyma (Figure 2C) and 6 to 9 layers of cortical parenchyma are observed.The stem has various collateral vascular bundles irregularly arranged into the few pith, characterizing a eustele structure (Figure 2A).Numerous secretory ducts of variable diameter with secretory epithelium of seven to eight cells are observed across the cortex among the fundamental parenchyma cells (Figure 2D).Aspects of rosulado and crystal idioblasts of calcium oxalate in form of druse are observed (Figure 2D).
V. ferruginea root has many brachysclereids (stone cells) in the cortical parenchyma, over the vascular bundles; it consists of a sheath over the vascular cylinder.These are sclereids with moderately thick walls and numerous pits, resembling the parenchyma cells in shape (Apezzato-da-Glory and Carmello-Guerreiro, 2006).Between the cortex and vascular cylinder, there is the endoderm, storing various metabolites.This is mainly due to the compact appearance of its cells and the presence of Casparian strips that are little permeable to water and ions (Apezzato-da-Glória and Carmello-Guerreiro, 2006).The vascular cylinder presents a fascicular cambium giving rise to the axial elements and rays, and the inter fascicular cambium originating only rays.The cells of the radial parenchyma present lignified walls, alternating side by side with solitary and multiple radial and racemiform vessels and fibers of average thickness, which provides further rigidity to the root structure (Figure 3).

Histochemical tests
Table 1 shows the metabolites group and the reagent used in the leaf, stem, root and glandular trichomes to identify its presence/absence.The histochemical tests revealed the presence of essential oils in the glandular trichomes and a mixture of essences and resin acids inside the endoderm, vessel elements in the root and parenchyma fill of the leaves.Proteins are present in chlorenchyma, collenchyma, cortical parenchyma and phloem in the leaves and stems.The mesophyll and cortical parenchyma of the stem have a large amount of starch grains.Phenolic compounds are found in the mesophyll and parenchyma of leaves and stems.According to Castro et al. (2004), the phenolic compounds are related to antiulcerogenic properties and  scarring and antiseptic actions.Fibers, vessel elements and root rays showed lignified walls, with the phloroglucinol test.The PAS test was positive in the overall stem parenchyma and radial parenchyma of the root.Lipids were also detected in the root endoderm and the stem ducts, stained by Sudan.In V. polyanthes Less., the phytochemical analysis revealed the presence of alkaloids, glycosides and flavonoids (Lorenzi and Matos, 2002).In a phytochemical screening conducted with V. ferruginea, the presence of flavonoids and terpenoids was detected, but the presence of tannins, saponins and alkaloids was not detected in the extracts studied (Barbastefano, 2007).The presence of chemical constituents such as essential oils, essences, resin acids, lipid compounds and phenolic compounds shows a high variation of primary and secondary metabolites of the species, which might confirm its potential in pharmaceutical production.The identification of the location of the chemical compounds in certain structures of the vegetative organs of the species significantly aids pharmaceutical industries in the extraction of metabolites.This leads to a better utilization of raw materials, to facilitates the process, and enables the use of only those tissues that store the product concerned.

Table 1 .
Histochemical characteristics of organs and glandular trichomes of Vernonia ferruginea Less.