Jaqueline Martins Vasconcelos 1, Sebastião Carvalho Vasconcelos Filho 1, Juliana de Fátima Sales 1, Fabiano Guimarães Silva 1*
ABSTRACT: Quina (Strychnos pseudoquina A. St. Hil.). Loganiaceae, a native tree from the Brazilian savannah, is widely used in npopular medicine. In the present study, quina leaves of plants grown in glasshouse or at field conditions were anatomically and histochemically characterized. For the anatomical studies transverse sections taken from the middle third of leaves were stained with 0.05% toluidine blue, pH 4.0. Leaf samples were diaphanized and stained with 1% safranine to study the leaf surface. Phenolic compounds, lipids, starch, and alkaloids were analyzed by several histochemical methods. It was found in the quina leaves the stomata are present only in the abaxial surface and are paracytic. The typical epidermal cells are slightly sinuous and have few trycomas. Cuticle is thin in leaves of plants grown in greenhouse but thick in leaves of plants at field condition. The mesophyll is typically dorsiventral containing collateral vascular bundles. Phenolic compounds were found in the epidermal cells however no lipids, starch or alkaloids were found in any leaf tissue.
Keywords: Quina, anatomy, histochemistry, medicinal plants.
CARACTERIZAÇÃO ANATÔMICA E HISTOQUÍMICA DE FOLHAS DE STRYCHNOS PSEUDOQUINA (A. ST. HILL)
RESUMO: Quina (Strychnos pseudoquina A. St. Hil.) Loganiaceae, uma árvore nativa do cerrado Brasileiro, é amplamente usada na medicina popular. No presente estudo, folhas de quina crescidas em casa de vegetação ou em condições de campo foram caracterizadas anatomicamente e histoquimicamente. Para os estudos anatômicos seções transversais do terço médio das folhas foram coradas com azul de toluidina (0.05%, pH 4.0). Amostras das folhas foram diafanizadas e coradas com safranina (1%) para os estudos da superfície foliar. Compostos fenólicos, lipídios, amido e alcalóides foram analisados por vários métodos histoquímicos. Foi observado que nas folhas de quina, os estômatos estão presentes apenas superfície abaxial e são paracíticos. As células epidérmicas são levemente sinuosas e possuem poucos tricomas. A cutícula é fina em folhas de plantas crescidas em casa de vegetação mas grossa em plantas em condições de campo. As células do mesofilo são tipicamente dorsiventral contendo feixes vasculares colaterais. Compostos fenólicos foram encontrados nas células epidérmicas, contudo nenhum lipídio, amido ou alcalóides foram encontrados nos tecidos foliares.
Palavras-chaves: Quina, anatomia, histoquímica e plantas medicinais.
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1 Departamento de Biotecnologia. Instituto Federal Goiano – Câmpus Rio Verde. Rodovia Sul Goiana, km 01,
Zona Rural, Caixa Postal 66, Rio Verde - GO, CEP 75.901-970. * E-mail: fabiano.rv@ifgoiano.edu.br. Autor para correspondência.
INTRODUTION
Quina (Strychnos pseudoquina A. St. Hil) Loganiaceae, found especially in the savannah, is a plant widely used in folk medicine because of its aphrodisiac, tonic, and anti-fever properties, as well as healing liver ailments, stomach problems and anemia (ALMEIDA et al., 1998).
Quina’s natural occurrence, even in its indigenous habitat, is sparse.
The World Health Organization recommends the use of medicinal plants in poor countries; however, one should consider both, the safety and the degree of efficacy in their use as therapeutics. It is imperative to avoid deleterious side effects that can occur due to intrinsic characteristics of the medicine, many of which are overlooked due to the lack of standardization and product quality control (SILVA et al., 1976; CALIXTO, 2001).
Another consideration is that the indiscriminate use of savannah plants in folk medicine and pharmaceutical industry, and the transformation of savannah areas into grazing and agricultural areas, has caused a considerable reduction in the population density of some plants in their endemic areas (NADEEM et al., 2000).
Studies on the leaf histochemistry and anatomy provide crucial information for the quality control of raw materials used to produce phytotherapics, thus assuring their reliability (MARTINS et al., 2006). Therefore, it is crucial to broaden our knowledge about quina anatomy and hystochemistry. In the present study, quina leaves of plants grown in glasshouse or at field conditions were anatomically and histochemically characterized.
MATERIAL AND METHODS
Quina plants (Strychnos pseudoquina, A. St. Hill) were grown from seeds obtained from fruits harvested at Fazenda Gameleira, in Montes Claros de Goiás– Goiás state, Brazil (latitude 16º06 '20”S, longitude 51º 17' 11' W, 466 m asl) in September 2007. The voucher
specimen is deposited in the Rio Verde herbarium at Instituto Federal de Educação Ciência e Tecnologia Goiano/Campus Rio Verde under the collection number 70.
The fruits were depulped using a steel screen and water. After depulping, the seeds (approximately 100) were germinated in 25-cm diameter pots, containing sand as a substrate, in greenhouse.
Completely expanded leaves of seven month-old plants (collected in the greenhouse of IFGoiano/Campus Rio Verde and in the field at Fazenda Rio Verdinho in the city of Rio Verde) were used for the experiment.
Hand-cut thin sections of the middle third of leaves were stained with 0.05% toluidine blue, pH 4.0 (O’BRIEN et al., 1964), for the anatomical study. Histochemical analyses were performed using potassium dichromate (Johansen, 1940) for the detection of phenolic compounds, Sudan III (BRUNDRETT et al., 1991) for lipids and cuticle, lugol (JOHANSEN, 1940) for starch, and Wagner’s reagent (FURR et al., 1981) for alkaloids. Leaf samples were diaphanized (BERSIER et al., 1960) and stained with 1% safranin to study the leaf surface.
RESULTS AND DISCUSSION
Quina leaves are hypostomatal, with paracytic stomata, and have few trichomes. The common epidermal cells present a slightly sinuous contour and a thin cuticle. Collateral vascular bundles are observed in transverse section with typically dorsiventral mesophyll. The leaves of plants grown in greenhouse and field presented two and three layers palisade parenchyma respectively. These layers consisted of short and large cells and several layers of spongy parenchyma with round, dilated cells, and discrete intercellular spaces (Figure 1).
Pereira et al. (2003) analyzed the leaf morpho-anatomy of Palicourea longepedunculata Gadiner (Rubiaceae) and
found that the leaves of this species were simple, opposite, ovate-hastate, dorsiventral and hypostomatal. Additionally, they
presented a single-stratified epidermis, a thin cuticle, and paracytic stoma, occurring in the same level as the other epidermal cells. The mesophyll consisted of a layer of palisade
parenchyma and several layers of the spongy parenchyma similar to the quina leaves described in this study.
Ocotea puberula (Rich) Ness Lauraceae leaves also presented paracytic stoma and
unicellular tectorial trichomes in the abaxial face, a dorsiventral mesophyll, a biconvex midrib, Fig 1: A-B Anatomy of quina´s leaves. Adaxial (A) and abaxial (B) leaf surface of plants grown in the greenhouse. C-D: Leaf transverse sections of plants grown in the greenhouse. E-F: Leaf transverse sections of plants grown in the field. C-E: Mesophyll, D-F: Midrib. Ed: adaxial
ed pp pp pp pl xi fl E F
and a vascular bundle consisting of a single open-arch collateral bundle (FARAGO et al., 2005). Martins et al. (2006) studied the morpho-anatomy of Smilax polyantha Griseb (Smilacaceae), another savannah species, and found that the leaves of this species are hypostomatal and that the stomata are paracytic. The mesophyll tends to localize dorsiventrally because the adaxial parenchyma consists of cells with pronounced sinuosity.
Cuticle in quina´s upper epidermis is very thick in field-grown plants but is thin in plants grown in the greenhouse thin (Figure 2).
In vivo anatomical studies of Eugenia brasiliensis Lam. (Myrtaceae) leaves
pre-sented a noticeable thickening of the cuticle and epidermal cuticle extracts (DONATO et al., 2007). In contrast, the epidermis of
Policourea longepedunculata Gardiner
(Rubi-ace) (PEREIRA et al., 2003) and Baccharis
anomala DC. (Asteraceae) (Budel et al., 2008)
leaves presented thin cuticle.
The difference in cuticle thickening is likely to be caused by an adaptation that occurred as a function of environment differences in the grown conditions greenhouse or field) and differences in the plants developmental stage Reeve et al. (1993) defined adaptation as a phenotypic variant, resulting in reproductive success within a specific group of variants in a given
environment. According to those authors, the term “adaptation” has been used in plant anatomy to describe anatomical characteristics associated to specific environment conditions. According to Alquini et al. (2006), the cuticle is responsible for some of the epidermal cell functions, among which are the protection against water loss, excessive lighting or sun radiation, and other organisms including fungi, bacteria and insects.
Our histochemical tests showed the presence of phenolic composts in the epidermal cells, but no lipids, starch or alkaloids, in both the greenhouse- and field-grown plant leaves (Figure 3).
G H
Fig. 2: G – H: G: Leaf transverse section of quina (Strychnos pseudoquina A. St. Hill) grown in a greenhouse. H: Leaf transverse section of field-grown quina. Arrow: cuticle stained with Sudan III.
Fig 3: I - J: Leaf transverse section of quinaplants. I: plants grown in the greenhouse. J: Field grown plants. Stained with potassium dichromate (arrow).
Almeida et al. (1998) reported that several flavonoids were isolated from quina’s leaf extracts, among which were quinol and two alkaloids, diaboline and 11- methoxydiaboline. Frank-de-Carvalho et al. (2005) studied the leaf histochemistry of
Gomphrena arborescens L. f.
(Amaranthaceae), a native savannah medicinal plant, and found the presence of phenolic composts in almost all leaf tissues analyzed. Santos et al. (2008) studied the leaf histochemistry of seven Eucalyptus
(Myrtaceae) species, and also found phenolic composts in almost all species analyzed. However, no starch, alkaloids or lipids were found.
CONCLUSIONS
Quina leaves are hypostomatal, with paracytic stomata, presenting few trichomes. The common epidermal cells have a sinuous contour and a thin cuticle in leaves of plants grown in the greenhouse and thick cuticles in those from the field. The leaf has collateral vascular bundles and typically dorsiventral mesophyll. The histochemical tests revealed the presence of phenolic compounds in the epidermal cells, but no lipids, starch or alkaloids were found in quina leaves.
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