Morphoanatomical study, seasonal variation, and larvicidal activity of volatile oils from the leaves of Campomanesia pubescens (DC.) O. Berg (Myrtaceae)

The aim of this study was to perform from the morpho-anatomical study, physicochemical characterization, chemical composition, seasonal variability and larvicidal activity of the volatile oils of Campomanesia pubescens (DC.) O. Berg leaves. The botanical material was collected in Hidrolândia, Goiás. Morpho-anatomical characterization and phytochemical screening were performed by conventional methods. Leaf 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 larvicidal activity was carried out with third-stage larvae of the Aedes aegypti. The leaf blade, the main vein, and the young stem have a uni-stratified epidermis covered by a cuticle. The mesophyll and cortical parenchyma of the main vein and young stem have secretory cavities and idioblasts with crystals. The main rib contains a bicolateral bundle in an open arch. The young stem has a discontinuous band of sclerenchyma external to the phloem cells. Most volatile oil compounds are spathulenol, caryophyllene oxide, α-macrocarpene, and z-caryophylleneIn phytochemical analysis, tannins, digitalis, flavonoids, and total phenols were detected. The content of volatile compounds was 7.36%, that of total ash was 1.77%, and that of mucilage was 3.52 ml. The volatile oil at the concentration used was inactive against Ae aegypti larvae. The present study contributes to the taxonomic knowledge of the species and provides parameters for quality control of the plant raw material. This work represents the first description of the chemical compounds and seasonal variability of volatile oils from C. pubescens leaves collected in Goiás state.


Introduction
The Myrtaceae family contains 131 genera and 5,900 species. A total of 23 genera and about 1034 species are distributed in Brazilian regions such as Amazon, Caatinga, Cerrado, Atlantic Forest, Pampa, and Pantanal (Sobral, et al., 2015).
Campomanesia pubescens (DC.) O. Berg (Myrtaceae) is widely known as "gabiroba" and is a shrub or sub-shrub with 0.5-1.5 m high. Due to its small size, it can be grown in association with other tree fruits, allowing greater production of food by area. Studies show that in the Cerrado in Goiás, the species blooms from September to November .
The fruits have a sweet taste and can be consumed in nature or processed in the form of pulps, juices, ice cream, soft drinks, sweets, puddings, liqueurs, smoothies or tanned in cachaça . C. pubescens are popularly employed to combat diseases of the urinary tract and diarrhea and have an astringent action (Rodrigues & Carvalho, 2001).
C. pubescens is an aromatic shrub, containing secretory cavities that produce volatile oil. Scientific studies have found ledol and globulol (Cardoso, et al., 2009) to be the major constituents of the volatile oils in the flowers of C. pubecens collected in Mato Grosso do Sul, from the fruits collected in the state of Minas Gerais, limonene, and eucalyptol, in eucalyptus branches, and spatulenol, in the roots bicyclogermacrene, and spatulenol and the leaves, bicyclogermacrene and 1,8 cineol (Chang, et al., 2011).
An in vitro study conducted with extracts and hexane fractions of the fruits of C. pubescens, found antimicrobial activity against Staphylococcus aureus, Pseudomonas auruginosa, Escherichia coli, Salmonella setubal, Saccharomyces cerevisiae and Candida albicans . In another investigation by Guerrero, et al. (2010) demonstrated an increase in the number of monocytes in hematological tests in rats after administration of the hydroethanolic extract of C.
pubescens leaves, suggesting an anti-inflammatory activity. There are no reports in the literature on the seasonal variability of volatile oil compounds or the biological activity of C. pubescens on Aedes aegypti larvae, but studies with other species of the Myrtaceae family, such as Eucalyptus, Pepper, Psidium, and Syzygium, have verified larvicidal action (Dias, et al., 2014).
The mosquito Ae aegypti is the vector of human arboviruses, it breeds in standing water (Consoli & Oliveira, 1994).
The main preventive measure is the use of larvicides associated with the elimination of mosquito breeding sites (Zara, et al., 2016). These chemicals can be toxic to humans and other living organisms causing environmental problems (Amer & Mehlhorn, 2006;Oliveira, et al., 2016). Ecologically sustainable insecticides with lower toxicity and biodegradability are well accepted in this context, volatile oils being an important source (Isman, 2020;Viana, et al., 2018).
Due to the pharmacological potential, this work aimed to carry out the morphoanatomical study of C. pubescens, the quality control of plant raw material (phytochemical screening, content of volatile compounds, total ash, particle size and intumescence index), extract and analyze the chemical constituents of the volatile oils from the leaves, check the monthly variability of volatile oil compounds for a year and their larvicidal activity on third stage larvae of the Aedes aegypti mosquito.

Methodology
Leaves of C. pubescens from Cerrado region were collected monthly for 12 in Hidrolândia, Goiás,Brazil (786 m altitude,16 ° 53 '59" S and 49 ° 13' 29" W). Prof. Dr. José Realino de Paula identified the specimen and a voucher specimen was deposited at the Herbarium of the Federal University of Goiás (n. 67844).
Identification, morphological and phenological descriptions of C. pubescens were made with the naked eye, every month for 12 months, in loco, and using the stereoscopic microscope Olympus SZ-ST and specialized bibliographies (Gentry, 1992, Lorenzi, et al., 2003, Martius, 1897. For the anatomical study, the leaves were fixed in 70% FPA (formaldehyde, propionic acid, and 70% ethanol 1: 1: 18 V / V) for 48 hours and preserved in 70% ethanol. Paradermic sections of the leaf blade (middle third of the inter rib region region) were carried out, cross sections of the leaf blade (leaf margin, middle third of the inter rib region and the main rib); petiole (median region) and young stem branches (second internode internode), stained with Alcian blue/safranin 9: 1 (Kraus & Arduin, 1997) and Steinmetz (Costa, 2001). 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.
For analysis of volatile oils, leaves were collected from ten different individuals monthly, oven-dried with air circulation at 40 °C for 24 hours, triturated immediately before volatile oil extraction. 90g of th poder was submitted to hydrodistillation in a Clevenger-type apparatus for 2 h. After drying with anhydrous Na2SO4, the oils were stored in glass vials Development, v. 10, n.3, e35610313412, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i3.13412 at a temperature of -18 °C until further analysis. Each experiment was performed in triplicate. The composition of the volatile oils from leaves 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).
Principal component analysis (PCA) was used to evaluate the possible interrelationships between the compounds found in volatile leaf oils collected at different months throughout 1 year using Statistica 7 software (Statsoft Inc., Tulsa).
Cluster analysis was applied to study the similarity of the samples based on the distribution of the constituents, and hierarchical grouping was performed according to Ward's minimum variance method (Ward, 1963). Canonical discriminant analysis (DCA) was employed to detect the pattern distribution of samples and to identify the componentes that differed between the groups.
The climatic data in this period were obtained from data from the National Institute of Meteorology (INMET, 2020).
For phytochemical studies, analysis of ash and moisture contents, the leaves were collected, placed in plastic bags, dried in an oven with air circulation at a temperature of 40ºC, and later sprayed in a knife mill.
The determination of the content of volatile compounds was carried out in a moisture analyzer that produces radiation in the infrared region by using a halogen lamp (Ohaus model MB35) (Brazil, 2010). For this, 1 g of the powdered vegetable drug was weighed, the material was evenly distributed on the scale plate and the bowl was heated to constant weight. After starting the process, the moisture content was shown on the device's digital display in approximately 10 minutes. The experiment was carried out in triplicate, the average, standard deviation, and variation coefficient were calculated.
To determine the swelling index, 1 g of the powdered drug was weighed and transferred to a 25 ml becker, the volume of the dry drug in the beaker was measured and 25 ml of water was added. The beaker was capped with a ground cover and stirred every 10 minutes for 1 hour. After stirring, the mixture was left to stand for 3 hours at room temperature. In the end, the volume of plant material plus mucilage was measured. The experiment was carried out in triplicate.
3 crucibles were calcined at 500 ° C for 60 minutes to determine the total ash content. of the pulverized vegetable drug (3 g) was weighed and transferred to the calcined and tared crucible. The sample was incinerated by gradually increasing the temperature: 30 minutes at 200 ºC, 60 minutes at 400 ºC, and 90 minutes at 600ºC. It was cooled in a desiccator and weighed. The experiment was carried out in triplicate.

Morphological study
C. pubescens (Figure 1 A   It is a woody shrub, about 1.5 m tall in clump. Green and medium leaves, discolors (the adaxial face of the leaves has a darker tone compared to the abaxial face), full of trichomes on both sides, simple, crossed, and petiolate opposite. The leaf blade is elliptical, 5-7 cm x 1.5-2 cm with obtuse apex, obtuse base, entire margin, peninervous venation.

Anatomic study
The leaf of C. pubescens, in cross section, shows uni-stratified epidermis with rectangular cells covered by cuticle, The mesophyll, in the transverse section, is dorsiventral, the palisade parenchyma has up to 3 layers of cells, and the lacunous parenchyma of 4 to 6 layers of cells, composed of cells of different shapes ( Figure 2C). In the mesophyll, the presence of idioblasts with polyhedral crystals ( Figure 2C) or druses and secretory cavities ( Figure 2D) was observed. The leaf margin, in the cross-section, is rounded and covered by a cuticle. The midrib, in the cross-section, has a plane-convex to slightly biconvex outline ( Figure 3A). The collenchyma, just below the epidermis, has 2-3 layers of cells, the cortical parenchyma has 5 to 8 layers of cells ( Figure 3B   and secretory cavity ( Figure 5B). A discontinuous sclerenchyma band, separated by parenchymal cells, externally to the phloem ( Figure 5D) are observed. In the medullary parenchyma, there is the presence of scattered sclerenchyma cells the temperature ranged from 8.6 to 37.4 ºC (Table 1).   The results obtained from PCA and the cluster analysis showed the existence of chemical variability among volatile oils obtained from C. pubecens leaves ( Figure 6).  Source: Authors. Development, v. 10, n.3, e35610313412, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i3.13412

Phytochemical Analysis
In phytochemical screening, the presence of tannins, digitalis heterosides, flavonoids (0.84% content), saponins (foam index less than 100) and total phenols (1.42% content) were found. The content of volatile compounds was 7.36% ± 1.03 and a variation coefficient of 14.07%. The total ash content was 1.77% ± 0.0004. The mucilage content was 3.52 ml.
The powder of the leaves of C. pubescens presented, according to the Brazilian Pharmacopoeia

Determination of lethal concentration in Aedes aegypti larves
The volatile oil of C. pubescens does not induce death in the larvae in twenty-four hours in any of the evaluated dilutions (100, 80, 60, 40, and 20μg/mL).

Discussion
Campomanesia pubescens was observed with flowers from September to October and fruiting from October to November, which is in agreement with Silva, et al. (2009) who observed the peak of flowering and fruiting in September and October, respectively, of C. pubescens present in the south of Minas Gerais state. Another study conducted in Mato Grosso do Sul, showed that C. adamantium follows the same flowering and fruiting periods (Nucci & Alves-Junior, 2017). According to Fidalgo and Kleinert (2009), the flowering period of Myrtaceae starts in the transition period to wet weather, justifying the peak of flowering in September, followed by fruiting.
According to Lima, et al. (2011a), C. pubescens occurring in the state of Paraná, is a shrub or sub-shrub, ranging from 0.5m -1.5m, with discolored, petiolate green leaves, densely covered by trichomes, elliptical and lanceolate leaf blade, cuminated apex or acute and acute base, entire margin. The flowers have glabrous petals or with trichomes only on the margins, polystemone (100 to 160 stamens), ovary 5 -8 locular. The fruits are yellowish, globose, and smooth. The present work differs only in the classification of the apex and the base, presenting them as obtuse. According Amaral, et al. (2016) characteristics that proved the identification of the species C. pubescens are associated with the presence of pubescence or puberulence; bracts in the shape of scales or tiny leaves; sepals acute, rounded or intermediate forms; green or yellow-green fruits and ovary with 4 to 7 locules.These being the ones observed in this work. Oliveira, et al. (2018)  C. pubescens is similar to C. adamantium, differentiating itself by conspicuous indument, besides, the corded leaf base does not occur in C. pubescens (Landrum, 1986). Another method of characterizing C. pubescens within the genus Campomanesia is that the leaves are strongly discolored, both fresh and dry in herbaceous materials (Lima, et al., 2011a).
A thick cuticle in the adaxial face, and a thin cuticle in the abaxial face, cover the C. pubescens leaves. The cuticle is an adaptation to the Cerrado environment and acts as a protective layer against the loss of water to the environment and in the protection against excess brightness (Appezzato-da-Glória & Carmello-Guerreiro, 2009). In C. pubescens, the presence of unicellular trichomes was observed, with the absence of a pedal cell, corroborating with Conti, et al. (1997) who described these characteristics as a synapomorphy of Myrtaceae.
The dorsiventral mesophyll presented in the leaves of C. pubescens can be described as another evolutionary aspect, that due to the shape and arrangement of the palisade cells, the chloroplasts can be located parallel to the cell walls, intensifying photosynthesis (Appezzato-da-Glória & Carmello-Guerreiro, 2009). Gomes, et al. (2009) observed that all are hypoestomatic, as in the present study for C. pubescens. However, in C. adamantium they found anomocytic and paracitic stomata, while in C. pubescens they were observed only anomocytic stomata, which can be used as a characteristic for differentiation.
The presence of secretory cavities in different parts of Myrtaceae is widely cited in the literature as a characteristic of the family (Carr & Carr, 1970). The interior of these cavities may consist of oils, phenols, and mucilages for some species (Fahn, 1979). Kuster and Vale (2016) observed that in the leaf secretory cavities are presents among the mesophyll cells.
Histochemical tests demonstraeded voltile oils widely distributed in the secretory cavities of C. adamantium, as presented in this study, observing the presence of secretory cavities in the mesophile, in the cortical parenchyma of the main vein, petiole, and young stem. Idioblasts containing polyhedral crystals or druses were also found in the various organs of C. pubescens, differentiating from C. adamantium which, according to Gomes, et al. (2009) contain only polyhedral crystals. Oliveira, et al. (2018) described secretory cavities distributed on leaves of others species of Campomanesia, parenchymatous cells in the leaf blade endings and the mesophyll, always near the epidermis, forming a space delimited by radially disposed cells. The dorsiventral mesophyll and a uni-stratified epidermis on both leaf blade surfaces was observed. The stomata occupied the same plane as the epidermal cells. Petiole shapes varied among different morphotypes, with the most common types being planarconvex and concave. It was observed unicellular tector trichomes and bicollateral vascular bundles.
Myrtaceae species may present the midrib biconvex, plane-convex or concave-convex, usually a single bundle in the shape of an almost flat or semi-closed arch (Gomes, et al., 2009), in agreement with what was observed in C. pubescens in this work, presenting the main vein plane-convex, single bundle in the shape of a flat arc.
The results of the morphoanatomic study of C. pubescens, corroborate providing basic knowledge, differentiation characteristics and are of great importance to provide standards of control of the vegetable drug if it is used as herbal medicine.
The chemical composition of the volatile oil of C. pubescens showed a variety of components, due to ongoing studies in different regions, with different temperatures, rainfall, altitudes, soil type, and incidence of ultraviolet rays (Gobbo-Neto & Lopes, 2007). According to Sangwan, et al. (2001), the production of volatile oils depends on physiological, biochemical, metabolic and genetic aspects of the plant, and may undergo environmental and molecular modulations that elucidate the chemical variations of volatile oils.
The seasonal analysis of the volatile oil of C. pubescens, during twelve months, it was possible to observe the formation of three groups (Clusters), associated with flowering/fruiting periods (Cluster II) and vegetative period (Clusters I and II). This study was the first to observe the seasonality of volatile compounds in the oils of this species. Since this plant is a shrub, it is much more susceptible to environmental variations, thus reflecting in the variation of secondary metabolites, thus making it difficult to observe a clear correlation pattern.
C. pubescens is widely used in some regions of Brazil as a medicinal plant, it is necessary to have a better knowledge about the secondary metabolites of the plant, which in most cases have observed pharmacological actions. In this work, tannins were detected in the leaves of C. pubescens, confirming the study of Metcalfe and Chalk (1950) when they observed that the species of the Myrtaceae family present tannins that protect the plant against possible attacks by microorganisms and insects (Schmid, 1972). Abe, et al. (2014) when performing phytochemical prospecting of Campomanesia xanthocarpa Mart. ex O. Berg identified the presence of flavonoid, tannins and saponins, which were also found in the present study. However, the flavonoid content is lower. The presence of tannins and main flavonoids confers antioxidant activity in the human body, with possible effects on the prevention of cardiovascular and circulatory diseases (Ness & Powles, 1997;Stoclet, et al., 2004) and cancer (Wang & Mazza, 2002;Katsube, et al., 2003).
Excess water content in vegetable raw materials promotes the development of microorganisms, insects, and unwanted hydrolysis chemical reactions. The humidity index is related to the quality control and preservation of the drug. In the present work, the content of volatile compounds was 7.36% close to the moisture limits for vegetable raw materials established by the Brazilian Pharmacopoeia IV (Brazil, 1988).
Another important method for the quality control of the vegetable raw material is the analysis of the total ash content.
Basically, these are the non-volatile residues coming from the mineral and organo-metallic self constituents (physiological ashes) or associated to foreign materials, especially sand and soil (non-physiological ashes) (Brazil, 1988;Costa, 2001). A content of 1.77% was identified for the leaf powder of C. pubescens, indicating that different values for this, perhaps due to adulteration.
The granulometry in the present study, it was observed that according to the Brazilian Pharmacopoeia IV (Brazil, 1988), leaves powder of C. pubescens is considered coarse because it remains 77% retained in the 710 μm sieve.

Conclusion
It is concluded that the morpho-anatomical study of C. Pubescens is important in the correct taxonomic classification of the species. The phytochemical study, evaluation of volatile compounds content, total ash content, granulometry and intumescence provide quality control parameters for this plant raw material. The majority compounds found in the volatile oils were spatulenol, caryophyllene oxide, α-macrocarpene, and z-caryophyllene. The presence of three clusters was observed, with cluster II corresponding to the reproductive period of the plant (flowering and fruiting) and clusters I and III to the vegetative period. No larvicidal activity of the volatile oil was observed against Ae. aegypti larvae. This work represents the first leaf anatomical description, chemical compounds, and seasonal variability of volatile oils from C. pubecens leaves collected in Goiás.