Anatomical study of the leaves and evaluation of the chemical composition of the volatile oils from Psidium guineense Swartz leaves and fruits

Psidium guineense Swartz is a bush used in urinary tract diseases, diarrhea, and dysentery. The present study aims to perform the anatomical study of the leaves and evaluation of the chemical composition of the volatile oils from Psidium guineense Swartz leaves and fruits. The botanical material was collected in Hidrolândia, Goiás. Anatomical characterization and phytochemical screening of the leaves were performed by conventional methods. Leaf and fruit (green fruits, immature fruits, and ripe fruits) powders were submitted to hydrodistillation in the Clevenger apparatus and the identification of the chemical components of the volatile oils obtained was done by GC-MS. The leaf blade is hypoestomatic with paracytic and anisocytic stomata. Secretory cavities are observed in the central vein, mesophyll, petiole, and young stem. The powder moisture content was 7.4%. The total ash content of the leaf powder was 6.3% and the acid-insoluble ash content was 0.8%. The presence of tannins, flavonoids, and saponins in the leaves were identified. Leaves volatile oil’s majority compounds were 2Z,6E-farnesol (23.1-25.4%), α-copaene (17.7-20.3%), muurola-4,10(1.4) dien-1-β-ol (5.8-6.7%), epi-α-cadinol (5.56.3%), and δ-Cadinene (5.05.9%). Fruits volatile oil’s majority compounds were 2Z,6E-farnesol (31.9-41.4%), α-copaene (13.3-26.6%), δ-cadinene (5.4-9.8%), γhimachalene (3.8-6.1%), and cubenol (2.6-6.1%). This is the first report on anatomical study of the leaves, and chemical composition of volatile oils from leaves and fruits of P. guineense collected in Hidrolândia, Goiás.

Psidium guineense Swartz known as "araçá comum", "araçá-azedo", or "araçá-mirim", is distributed in Brazilian States such as Amazonas, Pará, Goiás, Minas Gerais, São Paulo, Mato Grosso, and Ceará (São Paulo, 1978). P. guineense is a shrub of up to 6 m, with yellowish-brown coriaceous leaves of elliptical shape and flat central rib. The flowers can be solitary or in dichásio with white petals (Silva & Mazine, 2016;Peixoto, et al., 2017). The fruit is a globular berry rich in vitamin C with high dispersion capacity, can be consumed in natura or as an ice cream, beverages, and liquors. The pulp is fleshy, white, mucilaginous, sweet, slightly sour, and aromatic and has numerous small seeds (Manica, 2000).

Plant material
Psidium guineense Swartz leaves, green fruits, immature fruits, and ripe fruits were collected in January and February, during the morning and the first day each month in Hidrolândia -GO (786 m, 16° 53' 59" S and 49° 13' 29" W). Professor Dr.
José Realino de Paula identified the specimen, and a voucher was deposited at the UFG Herbarium, Goiás, Brazil, under code number UFG-67843. The leaves and fruits were dried in an oven with air circulation at 38 ºC by 2 days.

Anatomical study
For the anatomical study, leaves and stems were sectioned and stained with Alcian blue/safranin 9: 1 (Kraus & Arduin, 1997) and histochemical tests Steinmetz and Lugol reagents (Costa, 2001). The photographic recording of the anatomical structures was performed in a photomicroscope (Zeiss-Axiostar plus) with a coupled digital camera (Canon Power Shot G10) using the Axion Vision 4.8 software.

Volatile oils
Healthy leaves, green fruits, immature fruits, and ripe fruits were collected from ten different individuals in January and February, triturated immediately before volatile oil extraction, and 90g of the powder submitted to hydrodistillation in a Clevenger-type apparatus for 2 h. After drying with anhydrous Na2SO4, the oils were stored in glass vials at a temperature of -18 °C until further analysis. Each experiment was performed in triplicate. The composition of the volatile oils was analyzed using a Shimadzu GC/MS-QP5050A fitted with a fused silica SBP-5 (30 m × 0.25 mm I.D.; 0.25 µm film thickness) capillary column (composed of 5% phenylmethyl polysiloxane). The following temperature program was used: the temperature was raised from 60-240 °C at a rate of 3 °C/min and then to 280 °C at a rate of 10 °C/min, ending with 10 min at 280 °C. The carrier gas (helium) had a flow rate of 1 mL/min, and the split mode had a ratio of 1:20. The injection port was set at 225 °C. The operating parameters for the quadrupole mass spectrometer were as follows: the interface temperature was set to 240 °C and the electron impact ionization to 70 eV, with a scan mass range of 40-350 m/z at a sampling rate of 1 scan/s. The components were identified by comparison of the retention indices of the components to those of C9-C28 n-alkanes and comparison of the mass spectra with literature data (Van Den Dool & Kratz, 1963, Adams, 2007.

Anatomic study
The foliar blade of P. guineense is hypoestomatic with paracytic and anisocytic ( Figure 1A) stomata in paradermic section. The epidermis on both sides present cells with straight to slightly curved walls ( Figure 1B). In the transversal section, it has uniestratified epidermis on the adaxial surface and hypodermis with two layers of cells, and uniestratified epidermis covered by thick cuticle on both sides ( Figure 1D) and unicellular trichomes ( Figure 1C). The mesophyll is dorsiventral, present palisade parenchyma with two cell layers, and lacunae parenchyma with four to five cell layers ( Figure 1D). Secretory cavities are present in the lacunous parenchyma ( Figure 1D). A vascular bundle with the extension of the sheath is observe ( Figure 1D). The main rib, in cross-section, has a plane-convex shape ( Figure 2A). The epidermis is uniestratified coated by cuticle ( Figure 2C), observing the presence of simple trichomes ( Figure 2B). After the epidermis, there are about three to four layers of collenchyma cells ( Figure 2D). The cortical parenchyma has from eight to ten layers of cells with secretory cavities ( Figure 2F) and idioblasts containing druse-type crystals ( Figure 2E). The vascular bundle is bicollateral with an arch shape surrounded by a range of sclerenchymatous sheath cells ranging from one to seven layers that emit projections between cells from the external and internal xylem to the phloem ( Figure 2D). In the external phloem, idioblasts cells containing prismatic crystals are presented ( Figure 2E). The petiole, in the transversal section, presents a plane-convex shape ( Figure 3A) with uniestratified epidermis covered by a cuticle (Figure 3B and 3F). It presents a unicellular trichomas tectores. Below the epidermis, a collenchyma with three to four cell layers ( Figure 3B) followed by cortical parenchyma with 10 to 12 cell layers, some presenting amyloplasts ( Figure 3C).
Secretory cavities ( Figure 3B) and cells with points ( Figure 3D) are observed in the cortical parenchyma. The vascular bundle is bicollateral with a revolute arch shape ( Figure 3E). It presents medullary parenchyma with cells of different sizes. Druse-like crystals are observed in the cortical parenchyma and medullary parenchyma and prismatic crystals in the phloem ( Figure 3E). The young stem, in cross-section, has an oval shape ( Figure 4A) and is delimited by a uniestratified epidermis coated with cuticle ( Figure 4B) and numerous unicellular trichomes ( Figure 4C). The cortical parenchyma contains about eight to nine cell layers, presenting secretory cavities Figures 4B and 4C). In the vascular bundle a phloem is observed followed by medullary parenchyma with isodiametric cells of varying sizes. Presence of idioblasts containing druses in the cortical and medullary parenchyma and prismatic crystals in the phloem.

Total ash content, acid-insoluble ash content, and phytochemical screening
The powder moisture content was 7.4%. The total ash content of the leaf powder was 6.3% and the acid-insoluble ash content was 0.8%. The qualitative presence of tannins, flavonoids, and saponins in the leaves were identified.

Volatile Oil
The volatile oil yield from P. guineense leaves varied from 0.05 to 0.06% and 93.5 to 99.7% compounds were identified.

Discussion
In the present study, uniestratified adaxial epidermis with double layer of hypodermis, palisadic parenchyma with 2 layers of cells, numerous unicellular trichomes, paracytic, and anisocytic stomata were observed while Silva, et al. (2007) observed only paracytic stomata in the abaxial epidermis of P. guineense and only one layer of cells in the palisadic parenchyma.
According to Brewer, et al. (1991), the trichomes may be responsible for water retention on the leaf surface by retaining water droplets, improving the photosynthetic process by allowing greater opening of the stomata. Secretory cavities containing volatile oils and crystals in forms of druses described in the leaves and young stem in this study were also described by Oliveira (2015), being a common feature of Myrtaceae species (Metcalfe & Chalk 1979).
The determination of moisture in herbal drugs is important in quality evaluation. Water excess produces unwanted chemical reactions and microbial contamination. Total ash and acid-insoluble ash in high levels indicates impurities like nonorganic materials and silica (Alves, et al., 2010;Brasil, 2019). Official compendia have not established parameters for P.
guineense. However, limit values for moisture are 12.0% and total ash 9.0 % for P. guajava powder (Brasil, 2019). P. guineense powder of the leaves displayed values of 7.4% moisture, 6.3% total ash, and 0.8% acid-insoluble ash. Tannins, flavonoids, and saponins were also identified in the leaves. These compounds were also described in the methanolic extract of P. guineense leaves from India by Sruthi, et al. (2019), alongside coumarins, terpenoids, and quinones.
The chemical composition of the volatile oil of P. guineense showed component variation due to the studies being made in different regions, with different temperatures, pluviometric indexes, altitudes, type of soil, and incidence of ultraviolet rays (Gobbo-Neto & Lopes, 2007). According to Sangwan, et al. (2001) the volatile oils production depends on physiological, biochemical, metabolic and genetic aspects of the plant, and may suffer environmental and molecular modulations elucidating the chemical variations of volatile oils.
Due to the small size of the fruit, the amount of volatile oil obtained was low, a fact that represented a limitation of the study.

Conclusion
Anatomical studies, moisture content and total and insoluble ash in this study contribute to the quality control of plant raw material. Phytochemical screening is important to observe classes of molecules with possible biological activity. In this context qualitative presence of tannins, flavonoids, and saponins in the leaves were observed.
Leaves and fruits volatile oil's majority compounds were 2Z,6E-farnesol and α-copaene. This is the first report on anatomical study of the leaves, and chemical composition of volatile oils from P. guineense leaves and fruits collected in Hidrolândia, Goiás.