Leaf anatomy and histochemistry of Oriza ( Pogostemon cablin Beth., LAMIACEAE): Medicinal plant used in community Arari region, Itacoatiara, Amazonas

— Lamiaceae (Labiatae) is one of the largest Angiosperm families (with over 7500 species). Many authors confirm that 25 species from this family are medicinal, both native from Brazil and exotic. This work aimed to characterize the anatomy and histochemistry of the leaves of Oriza (Pogostemon cablin) and identify the key substances of pharmacological importance present in the leaves. The leaves were collected in the Monte Cristo community, Itacoatiara, and were studied via light microscopy and scanning electron microscopy (SEM), following standard anatomy and histochemistry protocols. For electron microscopy, sections were dehydrated in ethanol at different concentrations, undergoing critical point, metallization and then photomicrographed with the SEM. The front view epidermis appeared anfi/hypostomatic (but functionally hypostomatic) with diacytic stomata. The mesophyll is dorsiventral, with palisade parenchyma in a single layer of cells and the spongy parenchyma with 4 or 5 layers, witch two are maiden of parallel cells and the others are irregular. Petiole is non-cylindrical, with a semicircular arrangement of woody fascicles. Histochemically slices reacted to pectin, starch, proteins and phenolic compounds, tannins, acidic lipids, so that oriza showed all the substances assessed in the tests. The presence of the recorded compounds and the anatomical characterization may assist taxonomic pharmacological and allelopathic studies.


I. INTRODUCTION
The use of medicinal plants is a very common practice in many parts of the country, especially in inner cities (Silva 2012). The use of drugs, plants and their derived, directly or indirectly, for the treatment of diseases affecting human beings who seek to achieve physical, mental and social well-being and has been a very common way, especially in developing countries, where the World Health Organization estimates that 80% of the population relies on traditional medicine (Oliveira et al., 2014).
The importance of the species of the genus Pogostemon is related to allelopathy being found by Souza Filho et al., (2009) the constituents of the essential oils of this species synonymous P. heyneanus Benth that identified 15 constituents, with the major compounds alcohol patchouli (21.9%), α-bulneseno (11.8%), α-guaieno (8.6%), seicheleno (6.6%) and α-patchuleno (6.3%) with a repellent action, in addition they are used in folk medicine, have food use and compose incenses in Chinese culture (Ramya et al., 2013).
Pogostemon cablin commonly known as oriza is a plant widely used for medicinal purposes, mainly for the treatment of child sickness, headache and heart problems (CASINO, 2010). The species has its center of origin in India and is grown in Indonesia and Malaysia, intensively, as in South America the plant is grown in Paraguay and Brazil, drawing attention by having an essential oil with characteristic odor, persistent and canforáceo (Maia et al., 2001).
In anatomical studies conducted with Pogostemon cablin, commonly also known by the common name of patchouli in order to determine the types and micromorphological characteristics of trichomes, the results showed that there were eight distinct trichomes types: two non-glandular six glandular trichomes. The nonglandular trichomes are simple, unicellular and multicellular and glandular trichomes are short, with long peduncle, peltate, fingerlike, filiform and fusiform (RUSYDI et al., 2013). In this study, it was not held histolocalização substances in the leaves, and the nature of substances.
For the diagnosis of vegetable drugs are considered important factors such as the nature of the cellular walls and cellular inclusions of organic and inorganic nature can be revealed by histochemical analysis (Oliveira et al., 2005).
In the presence of chemicals in the plants Pogostemon gender and specifically in Pogostemon cablin, it is necessary to analyze and identify compounds through immunohistochemical studies to try to justify the therapeutic capacity (SILVA and FARIA, 2014).
In the Monte Cristo community, Itacoatiara, Amazonas, the Pogostemon cablin is used mainly to treat heart problems, in the form of tea, which encouraged the interest for this study. Thus, this study aims to characterize anatomically Oriza leaves (Pogostemon cablin), identifying the main substances in tissues by immunohistochemistry as a way of providing information for further nafarmacologia research, in addition to the anatomical characterization sheet in general, because previous studies and available literature concentrate efforts only on the trichomes characterization.

Study area and botanical material collection
The botanical material used in this study was collected after an ethnobotanical survey in Arari region, Itacoatiara, Amazonas, in a rural community called Monte Cristo. The criteria used for choosing Pogostemon cablin was the crossing of information between plants mentioned for the treatment of heart and other diseases related to the circulatory system, with the historic municipality of diseases that proved that such diseases have caused the greatest number of visits or death incidences in 2014.
Leaves were chosen because they are used in the preparation of tea to cure possible heart problems. The samples were collected in the morning, in a total of three individuals, withdrawing only mature leaves and in good phytosanitary conditions and replicas, fixed in FAA (formaldehyde, acetic acid, ethyl alcohol 70%) for 24 hours. After this period, they were preserved in 70% ethanol for further analysis.
The plant was herborizada and incorporated into the herbarium collection of the Center for Advanced Itacoatiara in Amazonas State University Studies -CESIT / UEA with number of voucher specimen Hitam 4300-4302. The material was identified by specialist INPA herbarium.

Light microscopy: anatomy and histochemistry
The study of light microscopy was performed in Agroforestry Botany Laboratory of the Federal University of Amazonas -Labaf / UFAM in Manaus, in the period from May to September 2015.
The anatomical analysis was made of the median limb portion of the petiole and leaf set from cross sections made freehand with razor aid in manual microtome. The sections were clarified with sodium hypochlorite solution to 20% safrablau stained and mounted in glycerine.
In the dissociation of the epidermis, sections were taken from the apex, edge of the middle region of the rib base and the leaf blade subjected to the hydrogen peroxide solution and acetic acid in the ratio 1: 1 (Franklin, 1946). After insertion into the solution, the material was kept for 24 hours in an oven at a temperature of 45 ° C. The epidermis was also obtained through paradermal cuts freehand in order to confirm stomata classification and evidence in greater detail trichomes, epidermal cells and epidermal appendages.
Slides were analyzed with the aid of optical microscope and recorded with imaging obtained with the optical microscope Axioskop MC 80 camera.
For histochemical tests of the leaf sections of fresh material was not subjected to reagents and were photographed to document the original color of the analyzed tissue (white).
]With other sections tests for the detection of chemical components of tissue were performed as detailed in Table   Table 1 For all reactions were made photographic records noting the presence or absence of the analyte.

Scanning Electron Microscopy (SEM)
For Scanning Electron Microscopy, cuts the median portion, edge in the middle region and petiole previously dehydrated in ethyl alcohol 90% (10 minutes) and alcohol 95% (15 minutes) and absolute alcohol (twice for 10 minutes) and dried in critical model Bal-Tec CPD 030critical point dryer). The samples were then pasted on metal support with silver-based glue and subjected to plating with gold, Bal-TEC SCD 050 apparatus -Sputter Coater, then being examined and photomicrographed in Electron Microscope JEOL Scanning -JSM -6460 LV -Scanning Electron Microscope. The sample processing stage took place at the Electron Microscopy Laboratory of the National Institute of Amazonian Research and reading in microscopic laboratory of the University of the State of Amazonas -UEA.

III. RESULTS AND DISCUSSION
The epidermis cells presents in front view with different forms on the adaxial and abaxial being more straight walls with light windings in the adaxial side ( Figure 1A) and more sinuous on the abaxial surface ( Figure 1B).
The abaxial epidermis is covered by cuticular ornamentation with random orientation observed only in Scanning Electron Microscopy -SEM ( Figure 1C The species studied were collected in full sun exposure area, which can influence the varied manifestation of anatomical and also external morphology, such as the wall or thickness of the epidermal cells that correspond to the first barrier to filter solar energy. According Taiz and Zeiger (2004), anatomical features are contrasting in leaves of the same plant, depending only on the different light regimes to which it is exposed. Pogostemon cablin in epidermal trichomes still exist on both sides, but higher number on the abaxial and also on the center rib ( Figure 1D). trichomes and glandular were identified. The trichomes are multistage (two to three cells) and uniseriate and are of tapered shape, with the base made by increased epidermal cells adjacent (1G Figures, 1H, 1I) and glandular with multisseriate base and Split head ( The trichomes usually have characteristic shape within species and have taxonomic significance and therefore, is credited also the function is relacionda with the water balance of the plant, one hairy thick surface tends to restrict the flow of air drying (CUTLER, 2011).

International Journal of Advanced Engineering Research and Science (IJAERS
For Ramya et al., (2013), the essential oil is an ingredient used as a "base" material in the perfume industry and the kind Pogostemon cablin studies report that has application not only in perfumes but also therapeutic, medicinal, like incense and food flavorings. These properties justify the concentration of studies only focused on the morphology of trichomes present in the leaf, because, according to Navarro and El Qualidi (2000), the trichomes may still represent an easy tool for Pharmacognostic characterization of plant to be easy observation and analysis.
In cross-section, of Pogostemon cablin leaf mesophyll presents dorsiventral with single-layered epidermis on both sides ( Figure 1D). In the region of the center rib, the cells are of regular shape ( Figure 1D), and the leaf blades, have rectangular shapes. The upper epidermis is papilhosa ( Figure 1E), with stomata slightly above the epidermal cell layer (not shown).
For Lavandula angustifolia species Mill and Lavandula dentata L. leaves have dorsiventral mesophyll, similar to the species under study, consisting of palisade on the adaxial surface, consisting of a layer of cells,
The lower epidermis of Pogostemon cablin still displays intussusception in limbo extension on opposite sides, separated by the central rib ( Figure 1E). SANDES et al.
(2012) observed in both mesophyll faces of Pogostemon cablin trichomes and glandular. The non-glandular trichomes are multicellular and uniseriate, formed by two basal cells, one to three intermediate cells and tapered cell at its end. ://dx.doi.org/10.22161/ijaers.74.3  ISSN: 2349-6495(P) | 2456-1908(O) Just below the upper epidermis, the palisade is presented in a single layer of cells arranged in parallel (Fig 1D and  1E) can be interrupted by idioblasts, secondary beams fiber sheath projections or projections (not shown). In the spongy parenchyma cells are arranged parallel. Are vascular bundles of small arms between the palisade and spongy parenchyma ( Figure 1E). In the region of the midrib, the appearance is biconvex. The adaxial region of the ridge is more prominent and more concave than in abaxial and has one to two layers of collenchyma (fugura 1D). The epidermis of the two faces ://dx.doi.org/10.22161/ijaers.74.3  ISSN: 2349-6495(P) | 2456-1908(O) may be interrupted by larger cells located at the base of the trichomes.

International Journal of Advanced Engineering Research and Science (IJAERS) [Vol-7, Issue-4, Apr-2020] https
The cortex is populated by parenchymal cells constituted of large and intercellular spaces and the vascular bundles are open side ( Figure 1D).
Petiole in cross section does not show a cylindrical shape and rather concave, which has the spherical recessed surface presenting with upper recessed at both ends ( Figure  2A), therefore the liberolenhosos beams are arranged in an open arc ( Figure 2B). Evidence is a uniseriate epidermis, covered with a thin cuticle and trichomes and glandular both adaxial and in abaxial similar to those described for the limbus ( Figure 2C). Stomatal complexes occur above the other epidermal cells ( Figure 2D).
The non-glandular trichomes present aplenty including preventing you from entering the closest to the skin cell structures ( Figure 2G and 2H). In increase, the nonglandular trichomes showed a cuticular ornamentation ( Figure 2G) described, according to Cutler et al. (2011), and between the trichomes also observed glandular trichomes ( Figure 2H). Martins and Martins (2003), in studies of the Mentha species pulegium in electronic photomicrographs, also found the presence of ornamentation in the cuticle of glandular trichomes. Beneath the epidermis was observed up to four layers of angular chollenchyma, interrupted in the regions where the stomata ( Figure 2E). In the middle region of the petiole, there is a concave shape on the adaxial and convex face on the abaxial surface. The vascular system in a horseshoe shape is formed by side beams, showing sclerenchyma tic cap externally to the phloem ( Figure 2F).
The results of several authors for Pogostemon gender focus in the study of trichomes and glands by the interest in the essential oils and other Lamiaceae family species have varied anatomy. For all histochemical tests applied in the courts that species (Table 2) confirmed the reaction of analytes, showing the results with the positive sign (+).
In chlorophyll vegetables, vegetable double membrane and cytoplasmic is cellulosic and its other constituents, such as peptic substances (pectose, pectic acids, metapécticos and callose). Even when the plant is more mature secondary membranes can be of various natures, lignified, suberous, cutinized, cerificadas with hemicellulose and silicified. Still another important factor in diagnosing the plant cell corresponds to the drug inclusions (starch, aleurone, inulin, oil droplets, silica, and calcium oxalate crystals carbonate and other various contents (Oliveira et al., 2005).
The cuticle is thin at both sides of the sheet. lipid droplets were found in the cytoplasm of cells isolated from tissue parenchyma ( Figure 3A). Some cytoplasmic substances in the cells of the adaxial epidermis and palisade reacted to sudan III ethanol, also blushing in glandular trichomes ( Figure 3B). In studies by Guo (2013)  Phenolic compounds are expressed by the full extent of the leaf blade, presenting more concentrated in the abaxial epidermis region, the palisade and spongy parenchyma and was also manifested in the area of the midrib ( Figure 3E). The glandular trichomes of the petiole reacted showing the presence of the compound ( Figure 3F).
Phenolic compounds are substances that are noted for their antioxidant, anti-inflammatory, antitumor and estrogenic, suggesting the action of some phenolic compounds in the prevention of coronary heart disease and cancer (TOMÁS-BARBERAN; ESPÍN, 2001). This information may justify the fact of oriza (Pogostemon cablin) be used by Community Arari in the region for the treatment of diseases of the circulatory system, such as the heart.

International Journal of Advanced Engineering Research and Science (IJAERS)
[ In the area of leaf blade was detected the presence of structural pectins, including cells of trichomes and the epidermis of the leaf and petiole limbo ( Figures 3G and  3H). Unlike what happened with the kind described in issue in Ocimum L. species gratissimum also the Lamiaceae family, ruthenium red unreacted, featuring the absence of pectins in plant tissue cells (SANTOS, 2013). Starch was found mainly in the parenchyma cells of the leaf blade, the petiole showed the formation of a starch sheath ( Figure 3I and 3J). For phloroglucin oriza the flushing cuts reacted only in the area of the conducting vessels ( Figure 3K and 3L). In histochemical tests phoroglucine for Hyptis stricta Benth species, the xylem cells in the midrib region and the petiole showed up lignified (SILVA, 2012), similar to that observed in Pogostemon cablin. For the detection of proteins showed reaction only in the glandular trichomes (3M).
In the case of phenolic compounds, applying the test with potassium dichromate, sections were reacted confirming the presence of these compounds in the blade and also in the petiole (Figures 3N and 3O). For the specific identified the presence of tannins in the glandular trichomes of the lamina and petiole of oriza leaves ( Figure 3P).
Histochemical tests revealed the presence of phenolic compounds, which provide an indication of the presence of some secondary metabolites, such as, for example, tannins.
In the Lamiaceae family, is a kind of tannin that has the ability to precipitate proteins, but it is not suitable for tanning the skin, known as "tannin of Labiatae" (Toledo et al., 2004).
In characterizing ethnobotany and histochemistry of medicinal plants in Goias, Lamiaceae family species were subjected to histochemical tests and obtained as a result of starch, total phenolic compounds, lipophilic compounds and tannins (SILVA and FARIA, 2014) similar to the substances detected in the species that study.
The studies that have been conducted such corroborate the importance of ethnobotanical survey data and ethnopharmacological in the selection of plants with therapeutic potential and bioactivity screening. The results show a significant contribution to the characterization of essential oils activity, confirming the presence of plant areas where there is production as in other species of the genus, but in need of chemical and pharmacological studies to confirm the real action in traditional medicine, addition of secondary compounds and plant extracts flora which can be used in folk medicine.

IV. CONCLUSION
Anatomical nature character of diagnostic values were recorded in this study, such as the presence of glandular trichomes and the presence of primary and secondary compounds confirmed the histochemical tests to complement what exists in the literature for Pogostemon genre. The work that has been carried out focus on to describe the morphology of glandular trichomes to be an area of concentration and production of essential oils characteristics of the species, in addition to mentioning the tectores. Complementing what already exists in the literature was recorded here the anatomical characteristics of the sheet as a whole differs from the other work that can contribute to the correct identification of the species. Thus the description showed stomata diacytic characteristic of the Lamiaceae family, with amphistomatic leaves, dorsiventral mesophyll and epidermis in both unistratified faces. Also in the upper epidermis was observed the buds and stomata slightly above the cell layer. A striking feature was the present intussusception in the cross section separated by the central rib, which seems it is a glandular cavity. The petiole is not cylindrical, with liberolenhosos beams in a semicircle and below the epidermis layer there are cells composing the angular chollenchyma. The species reacted positively to all histochemical tests confirming the presence of various substances such as lipids, phenolic compounds, proteins, starch, tannins and pectins. In electron microscopy demonstrated layer of ornate and random cuticle on the abaxial epidermis and also in granular form in trichomes petiole, results not seen in light microscopy. This information can be considered relevant to aid in the identification of the species, contribute pharmacognostic studies, phytochemicals, industrial, food and other involving the economic value of the species, in addition to demonstrating the quality control of it for medicinal use.