Anatomy and histochemistry of the leaf blade of Syzygium cumini (L.) Skeels Anatomia e histoquímica da lâmina foliar de Syzygium cumini (L.) Skeels

Diabetes is a noncommunicable disease responsible for about 1.6 million deaths in 2016 worldwide. The use of medicinal plants is commonly reported in the literature as an adjuvant in the treatment of diabetes. Syzygium cumini (L.) Skeels, popularly known as azeitona preta or jambolão, is an important medicinal species of the Myrtaceae family used for diabetes. This work aimed to conduct an anatomical and histochemical study of the leaf blade of S. cumini. Semipermanent histological slides were prepared for analysis of the leaf blade in optical microscopy, polarization and scanning electron microscopy. Histochemical tests were performed in cross-sections of fresh leaf slides, using specific reagents for each group of metabolites. Microscopic analysis allowed the identification of important elements in the diagnosis of the species. The histochemical tests evidenced the presence of phenolic compounds, tannins, triterpenes and steroids, alkaloids, essential oils, lipophilic compounds, starch, lignin and calcium oxalate crystals. The results presented contribute to the pharmacobotanical standardization of the species.


Anatomical characterization
The anatomical study was performed using leaf blades obtained between the third and fifth nodes, fixed in FAA50 (JOHANSEN, 1940). Various cross-sections were obtained by hand, using a common razor blade, at the middle region of the leaf blades.
Paradermal sections were also performed on the adaxial and abaxial faces. All sections were clarified in 50% sodium hypochlorite solution (KRAUS; ARDUIN, 1997).

Histochemical characterization
Histochemical tests were made on cross-sections of fresh leaf blades obtained by the same method used in anatomical study (JOHANSEN, 1940). The specific reagents

RESULTS AND DISCUSSION
In frontal view, under optical microscopy, the leaf blade of S. cumini presents epidermal cells with sinuous walls in the adaxial face (Fig. 1a) and cells with straight or slightly wavy walls on the abaxial face (Fig. 1a). It is also verified the presence of secretory cavities, which occur on both sides (Fig. 1a-b). Under SEM, it is seen that the epidermal cells are covered by smooth cuticle (Fig. 1c-d).
The leaf blade is hypoestomatic (Fig. 1a-b), corroborating other studies with the There is a divergence in the literature regarding the types of stomata found in the leaf blade of the species. In the present study were identified anomocytic and anisocytic stomata (Fig. 1b). According to Metcalfe and Chalk (1950), stomata are usually anomocytic in the Myrtaceae family. This was the only type described by  In cross-section, the midrib is slightly concave on the adaxial face and slightly convex on the abaxial face (Fig. 2a). Ruggiero (2004) also described the shape of the midrib of the species as concave-convex, while Siqueira-Nunes and Martins (2010) described as plane-convex. The epidermis is uniseriate, formed by rounded cells, covered by thick cuticle (Fig. 2a).
In the central parenchyma region of the midrib is located a bicollateral vascular bundle, which is continuously surrounded by a sclerenchyma (Fig. 2a) (Fig. 2b-c). The druses of the parenchyma have a mean diameter of 19.82 µm ± 1.86 and the druses of the phloem have a mean diameter of 10.22 µm ± 1.33.
Secretory cavities are observed in the midrib and mesophyll (Fig. d-e). According to Metcalfe and Chalk (1950), the secretory cavities are generally present in the Myrtaceae family and secrete oily substances.
The mesophyll is isobilateral (Fig. 2e). This is another predominant feature in

Página | 626
palisade parenchyma is located below both sides of the epidermis, consisting of two layers on the adaxial face and one layer on the abaxial face. The spongy parenchyma consists of seven to nine layers of irregular cells (Fig. 2e). In this last tissue occur druses, which have a mean diameter of 22.82 µm ± 4.02 (Fig. 2e-f). Soh and Parnell (2011) studied 81 species of Syzygium from Southeast Asia and the South West Pacific area and described, with respect to crystals, that in some species druses predominate and prismatic crystals are rare; in others, prismatic crystals predominate and druses are rare; and also that there are species where the occurrence of both types of crystals is the same. The Figure 3a shows the cross-section of the leaf blade of S. cumini without addition of any reagent. Phenolic compounds were evidenced in the epidermis (Fig. 3b), in the parenchyma of the midrib (Fig. 3b), in the palisade parenchyma and spongy parenchyma (Fig. 3c). Tannins were found in the same places where the phenolic compounds were observed, besides being also visualized in the phloem and in the secretory cavities (Fig. 3d). Triterpenes and steroids were identified in the secretory cavities (Fig. 3e), in the collenchyma (Fig. 3f), in the parenchyma of the midrib (Fig. 3f) and in the palisade parenchyma (Fig. 3g). Alkaloids were visualized in the parenchyma of the midrib (Fig. 3h) and in the spongy parenchyma (Fig. 3i). Essential oils were found in the secretory cavities (Fig. 3j).
Lipophilic compounds were evidenced in the cuticle (Fig. 3k)

Página | 628
in xylem and sclerenchyma (Fig. 3l). The presence of starch was demonstrated in the parenchyma of the midrib (Fig. 3m). The Figure 3n shows the presence of druses in the leaf blade of S. cumini and the Figure 3o shows the dissolution of the druses with the test of hydrochloric acid 10%, indicating that they are of calcium oxalate. BANDIOLA, 2017). Ruggiero (2004) analyzed the essential oil of the leaves of the species collected in São Paulo and found α-pinene (36.54%), α-terpineol (12.85%) and βpinene (12.74%) as major constituents.

CONCLUSION
The present study provided anatomical information useful for the correct identification of S. cumini. The histochemical tests evidenced the presence of phenolic compounds, tannins, triterpenes and steroids, alkaloids, essential oils, lipophilic compounds, starch and lignin. Besides that, the study showed the chemical nature of the crystals and contributes with the taxonomy of the genus.