Leaf and Stem Anatomy and Histochemistry of Dalbergia ecastaphyllum

Dalbergia ecastaphyllum (L.) Taub. is a shrubby of Tropical America and Africa. It is recognized as the main botanical source of red-propolis, and also by its uses in folk medicine. This work was performed by light and scanning electron microscopy in order to carry out an anatomical and histochemical study of leaves and stems of this species,to find distinctive characters to support the quality control of its ethnodrugs and derivatives. The leaf epidermis is hypostomatic with straight to curved anticlinal cell walls, papillose on the abaxial surface, with thickened cuticle and coated with epiculticular waxes as rosette. The mesophyll is dorsiventral, with palisade 2-3-layered and the spongy 4-6-layered. The petiole and midrib have vascular system collateral. The stem is cylindrical, with an uniseriate epidermis coated with thickened cuticle and angular collenchyma. The vascular system is a continuous ectofloic siphonostele. Resin idioblasts are concentrated in the inner area of the vascular tissue of the midrib, petiole and stem. The leaf and stem anatomy and histochemistry of D. ecastaphyllum provided distinctive characters for this species that can be used as an additional support for its taxonomy and for the quality control of their ethnodrugs.


INTRODUCTION
Dalbergia L. f., belonging to the Fabaceae family, has about 100 species with pantropical distribution and is considered the second largest genus of the tribe Dalbergieae Bronn ex DC. 1 In Brazil the genus is represented by about 40 species distributed in repre sentative areas of different Brazilian ecosystems and in various types of vegetation, such as Caatinga, Cerrado, Atlantic Forest and Campos Rupestres. 2albergia stands out for having species considered valuable for its decorative and fragrant woods. 3any species of the genus are used in traditional medicine for various purposes: treatment of pain, fever, inflammations, as sexual stimulant, and against worms and larvae. 4Although some species of Dalbergia are referred to by their uses in folk medicine, only some of these uses have been studied biologically or pharmacologically in order to validate its traditional uses. 5In addition, Dalbergia species also have been shown to be important sources of bioactive compo nents, such as is of lavonoids (daidzein, formononetin and biochanin A), neoflavonoids, glycosides, cinnamyl phenols, quinones and furans. 58mong the species of the genus stands out Dalbergia ecastaphyllum (L.) Taub popularly known in Michelline V. Marques Das Neves 1 , Nathalia Diniz Araújo 1 , Eduardo De Jesus Oliveira 2 and Maria De Fátima Agra Brazil as "rabodebugio", "marmelodomangue" and "marmeleirodapraia", a shrubby species, distri buted in tropical America and Africa 9 .In Brazil, it is found mainly in areas of restingas along the Brazilian coast. 1 Propolis is a resinous mixture of substances collected by the bees, Apis mellifera mainly, from several plant sources including Dalbergia ecastaphyllum.Various biological activities have been reported for propolis, including: cytotoxicity, 10 antitumor, 11 antioxidant, 12 and antimicrobial. 13Studies carried out in hives of Apismellifera, in northeastern Brazil, reported the presence of a new type of propolis in areas of the State of Alagoas, which was called "redpropolis", whose chemical composition and botanical origin were studied by Silva et al., 8 Daugsch et al. 7 and Alencar et al., 14 and the results indicate Dalbergia ecastaphyllum as the main species associated with the production of "redprópolis".General information about the anatomy of Dalbergia was referred by Metcalfe and Chalk, 15 that reported the presence of secretory structures producing resins rich in bioactive compounds.Farooqui et al. 16 conducted studies of cuticular characters of three species of Dalbergia: D. emarginata, D. latifolia, and D. sissoides.Moreover, Khan et al. 17 recorded the same information on epidermal markers for Dalbergia sisso.Our work was motivated by the scarcity of anatomical studies for the genus and especially for Dalbergia ecastaphyllum, despite this species being recog nized as the main botanical source of red propolis and many bioactive compounds 5 as well as by its traditional use in Folk medicine. 4his work was carried out in order to perform an anatomical and histochemical study of leaves and stems of Dalbergia ecastaphyllum to find distinctive characters that may constitute parameters to support its taxonomy as well as the quality control of its ethno drugs and derivatives.

Anatomical Characterization
The anatomical analysis was performed on fresh, fixed and dried samples.Dried samples were properly rehydrated and then subjected to the same procedure cited for the fixed material. 20ross sections were performed by free hand with the aid of blades in leaves (lamina and petiole) and stems of Dalbergia ecastophyllum.The cuts were clarified with sodium hypochlorite at 2%, washed in distilled water, neutralized with acetic acid to 1%, washed in distilled water and stained with safra blue, mounted with glycerinated gelatin 50%, and ana lyzed and photographed by optical microscopy.For observation of the epidermis, paradermic sections were performed on both surfaces of the leaf, then clarified in a similar way to the transverse sections, and stained with safranin (1%), and mounted on glycerinated gelatin (50%).Observations and photomicrographs were performed on optical microscope (LeicaDM750, Switzerland) with Qwin system cou pled to a video camera (Leica ICC50 HD) for image capture.The classification of stomata and trichomes was based on Wilkinson 21 and Theobald et al., 22 respectively.

Histochemical Characterization
Cross and longitudinal sections were made by free hand helped by cutting blades, in leaves (midrib and petiole) and stems under the following colorants and reagents: ferric chloride 18 and potassium dichromate 23 for phenolic compounds, 2,4dinitrophenylhydrazine 24 for terpenoids, Wagner reagent 25 for alkaloids, sudan IV and sudan black B 26 for lipids, and acidified phloroglucinol 18 for lignin.The reaction was considered positive when specific, clearly visible and of a different color than the natural color of the substance being examined.Histological slides were also prepared without any processing (in natura), which were called "white".

Scanning Electron Microscopy
The scanning electron microscopy was used for micro morphological analysis of epidermis in dry material, to optimize the observation of the waxes and epidermal appendages, such as stomata, trichomes and papillas.The dried samples were placed on stubs with carbon ribbon, and subsequently coated with gold.The image capture was performed by a scanning electron microscope (Zeiss, model LEO 1430 VP, Cambridge, England).The terminology of epicuticular waxes morphology was done according to Barthlott 27 and Barthlottet al. 28

RESULTS AND DISCUSSION
In front view, the epidermis of Dalbergia ecastaphyllum is hypostomatic, presenting a different pattern from that described by Metcalfe and Chalk 15 for Dalbergia, and by Kahn et al. 17 for D. sisso Roxb.ex DC., that have predominantly amphistomatic leaves (with stomata on both surface).Moreover, Farooqui et al. 16 recorded the amphyhypostomatic type for three additional species of the genus: Dalbergia latifolia Roxb., D. sissoides Graham, and D. sisso, which have predominantly hyposto matic leaves, with rare stomata on the adaxial surface.In frontal view, the anticlinal cell walls of the epidermis are straight to curve on the adaxial surface (Figure .1A), a feature also observed in other species of Dalbergia by Farooqui et al. 16 and Khan et al., 17 and in species of some genera of Fabaceae, like Bauhinia L., according to Lusa and Bona. 29On the other hand, the epidermis on the abaxial surface is predominantly papillose, except on subsidiaries cells of stomata (Figure .1B, 2CF).Similar pattern was referred for Dalbergia latifolia Roxb.by Farooqui et al 16 .The epidermal papillae on the abaxial surface is a com mon character in Dalbergia, and also present in other genera of Fabaceae like Bauhinia, according to Metcalfe and Chalk. 15ector trichomes, with short cells at the base, were observed on both surfaces of the by light and scanning electron microscopy (SEM).On the adaxial surface they are longer and sparse (Figure 2B).than on the abaxial surface, which are numerous and adnate to the surface (Figure . 2CD).This type of trichome corroborate with Metcalfe and Chalk, 15 as well as common characteristic of Dalbergia, and also were reported by Farooqui et al. 16 for Dalbergia sissoo.On the abaxial leaf surface of the studied species a large amount of epicu ticular waxes was observed by SEM, as crystalloid of rosettes type, which were deposited on the epidermal papillae and paracitic stomata (Figure 2DF).This pattern of epicuticular waxes was reported for Bauhinia forficata, another species of Fabaceae, by Lusa and Bona. 29The adaxial surface showed very rough cuticle, however epicuticular waxes were not observed in this area (Figure 2A).In transverse section, the epidermal cells of the adaxial surface were tabular and somewhat rounded (Figure 1CD).followed by a layer of hypodermis that is formed by larger cells (Figure . 1CD).The presence of a hypodermis in Dalbergia is a common character of this genus that was already reported by Metcalfe and Chalk. 15he pattern of mesophyll of Dalbergia ecastaphyllum is of the dorsi ventral type (Figure 1C), with threelayered palisade parenchyma, and four to fivelayered spongy parenchyma, and several collateral vascular bundles distributed throughout the mesophyll.The leaf margin, in cross section, showed to be slightly rounded (Figure 1F) toward the abaxial surface, with palisade and spongy parenchyma more homogeneous, with smaller and compact cells in this area (Figure 1F).According to Metcalfe and Chalk, 15 due to the wide range of morpho logical types, leaves are considered a highly variable structure in Fabaceae that shows great plasticity, but it is usually dorsiventral, and less frequently isobilateral.Moreover, palisade and spongy parenchyma are tissues known to reveal responses related to light, soil and water variations, according to Esau, 30 Levitt 31 and Rozema et al. 32 The midrib, in transverse section, has a planeconvex shape (Figure .3AB, D), with the vascular system constituted by a set of collateral vascular bundle.The distal portion of central vascular bundle exhibits a small semiarch shape with the xylem and phloem polarized in the adaxial and abaxial surfaces, respectively (Figure 3A).The median and proximal portions are constituted by a set of vascular bundles, formed by a larger semiarched shaped central one, and two lateral accessory bundles with circular shape (Figure BD).Throughout the entire midrib, the perivas cular region is marked by the presence of a continuous sclerenchymatic sheath around the vascular system (Figure 3AD).Adjacent to the epidermis, stands 13 layers of collenchyma of lignified cells of the angular type (Figure 3C).Resin idioblasts were observed in the medullar region, adjacent to the xylem (Figure 3A, EF).The petiole, in cross section, has a circular shape, having the cortex with 12 layers of collenchyma of the angular type, underlying the epidermis (Figure . 4A), followed by the fundamental parenchyma.In the distal and the median portions a sclerenchymatous sheath surrounding the collateral vascular system was observed, similar to that found on midrib, which is not evidenced in the proximal portion.The vascular system is formed by 27 vascular bundles, which are arranged in a somewhat irregular arch shape.The medullar portion is formed by the fundamental parenchyma in the central portion (Figure 4AB), in which the presence of resin idioblasts underlying the xylem was observed.
According to Metcalfe and Chalk, 15 the morphology of petiole suffers little influence of the environment and represents a structure of taxo nomic importance.The pattern of the vascular system of petiole in D. ecastaphyllum was also reported to be present in other Fabaceae, such as Caragana arborescens Lam., Erythrina crista-galli L., Galega officinalis L., amongst others.The stem is cylindrical, with an uniseriate epidermis that is coated by a thick cuticle.In cross section, the collenchyma, adjacent to the epidermis, is the angular type with lignified walls.The perivascular region was marked by the presence of a discontinuous sclerenchymatic sheath around the vascular cylinder (Figure 4CD) that is an ect of loic siphonostele.The stem structure of D. ecastaphyllum showed isolated bundles of fibers surrounding the entire vascular system, and also resin idioblasts, similar to that reported for Dalbergia and other genera of  Fabaceae by Metcalfe and Chalk. 15In D. ecastaphyllum resin idioblasts were observed in the internal region of the vascular tissue of leaf and stem.They occur mainly concentrated in areas adjacent to the xylem of the midrib and petiole, and also in the stem.In transverse section they appear as cells with primary walls that contain hydrophilic substances, such as phenolic compounds (Figure 5CF, LO), terpenoids (Figure 5G, P). and alkaloid salts (Figure 5H, Q).Lipidic substances were present only in the cuticle (Figure 5I, RS) and were absent in resin idio blasts.
According to Fahn, 33 secretory tissues occur in most vascular plants, but differing in structure, topographic position and in the materials secreted.The presence of resin idio blasts observed in the midrib and petiole leaf, and in stem (vascular system) of D. ecastophyllum could be the area of formation of the resin used by bees to produce red propolis.Secretory structures are resin producers, which are rich in bioactive compounds, already mentioned for Dalbergia by Metcalfe and Chalk 15 and Farooqui et al. 16 Secretory elements containing tannins, resins and proteins were previously reported by Metcalfe and Chalk 15 for many species of diff erent genera of Fabaceae, including Dalbergia.Secretory elements of D. ecastophyllum are found into the vascular system of the midrib, petiole and stem, which differ from the secretory elements of Myrocarpus, in which the secretory structures are located in the cortex of midrib. 34hytochemical studies have reported the presence of phenolic compounds such as flavonoids, isoflavonoids, as well as other substances in species of Dalbergia referred by Liu et al. 35 , Zhao et al. 36 andSaha et al., 5 amongsto thers, as well as by Matos et al. 37 forDalbergia ecastophyllum.The presence of phenolic compounds was evidenced by histochemical tests performed in leaf and stems of D. ecastaphyllum.

CONCLUSION
The leaf and stem anatomy and histochemistry of D. ecastaphyllum, performed by light and electron microscopy, provided a set of distinctive characters for this species, which can serve as a parameter to be used as an additional support for its taxonomy and the quality control of its drugs.

Figure 2 :
Figure 2: Scanning electron micrograph of the leaf epidermis of Dalbergia ecastophyllum (Neves002). A. General view of the adaxial surface.B. Detail of simple trichome on the adaxial surface.C-F.Indument and papillae of the abaxial surface: C. Detail of the papillae epidermis and trichomes.D. Detail of simple trichome on the abaxial surface.E. Detail of epidermal papillae with epicuticular waxes as rosettes.F. Detail of stomata and papillae covered by epicuticular waxes as rosettes.Legends: (pa) papillae, (st) stomata, (tr) trichome.

Figure 3 :
Figure 3: Leaf midrib in transverse section of Dalbergia ecastophyllum (Neves001).A.General appearance of the midrib at the distal portion.B. General appearance of the midrib at the median portion.C. Detail of vascular system of the midrib at the median portion.D. Detail of angular collenchyma of the midrib at the median portion.E. General appearance plane-convex of the midrib at the proximal portion.F. Detail of vascular system and resin idioblasts of the midrib at the proximal portion.Legends: (co) collenchyma, (ep) epidermis, (hp) hypodermis, (ph) phloem, (pp) palisade parenchyma, (sc) sclerenchyma, (id) idioblast, (sp) spongy parenchyma, (xy) xylem

Figure 5 :
Figure 5: Histochemical tests in leaves and stems of Dalbergia ecastophyllum under different colorants and reagents (Neves 001, 002).A-J.Longitudinal and transverse sections of leaves: A-B.White proof.C-D.Secretory elements colored black by iron chloride III indicating the presence of phenolic compounds.E-F.Secretory elements colored black by potassium dichromate as a positive reaction for phenolic compounds.G. Secretory elements colored red-orange by 2,4-dinitrophenylhydrazine for the presence of terpenoids.H. Secretory elements colored reddish-brown by Wagner reagent as positive reaction for alkaloids.I. Cuticle colored with Sudan IV as a positive reaction for lipids.J. Lignified elements of xylem and sclerenchyma colored by acidified phloroglucinol.K-U.Stems in longitudinal and transverse sections.K. White proof.L-M.Secretory elements colored black by iron chloride III, indicating phenolic compounds.N-O.Secretory elements colored black by potassium dichromate as a positive reaction for phenolic compounds.P. Secretory elements colored red-orange by 2,4-dinitrophenylhydrazine indicating the presence of terpenoids.Q. Secretory elements colored reddish-brown by Wagner reagent, as a positive reaction for alkaloids.R. Cuticle colored with Sudan IV as a positive reaction for lipids.S. Cuticle colored with Sudan black B as a positive reaction for lipids.T-U.Lignified elements of xylem and sclerenchyma colored by acidified phloroglucinol.Legend: (arrow) idioblast.