Morphology and anatomy of Justicia acuminatissima leaves

Morphology and anatomy of Justicia acuminatissima leaves

Maria Christina S. Verdam^I; Débora T. Ohana^II; Maria Gracimar P. Araújo^III; Fernanda Guilhon-Simplicio^II; Maria Sílvia de Mendonça^IV; Maria M. Pereira^II,*

^IPrograma de Pós-graduação em Ciências Farmacêuticas, Universidade Federal do Paraná, Brazil

^IIFaculdade de Ciências Farmacêuticas, Universidade Federal do Amazonas, Brazil

^IIIInstituto de Ciências Biológicas, Departamento de Biologia, Universidade Federal do Amazonas, Brazil

^IVFaculdade de Ciências Agrárias, Setor Sul, Universidade Federal do Amazonas, Brazil

ABSTRACT

Justicia acuminatissima (Miq.) Bremek., Acanthaceae, is a subshrub found in northern Brazil, where it is widely used by the population of this region as an anti-inflammatory medicine. Despite this popular use, there is no pharmacognostical data to support the correct identification of this species. We therefore performed a morpho-anatomical, histochemical and phytochemical analysis of the leaves of this species, using well-known methods. The leaves are simple, exstipulate, green on the surfaces, and pubescent, with a lanceolate shape, crenate margin, pinnate venation and decussate phyllotaxy. The parenchyma is palisade and spongy, and its vascular system is bilateral. Glandular and non-glandular trichome and cystoliths were also detected. There are diacytic stomata on both the adaxial and abaxial surfaces of epidermis. Histochemical tests revealed the presence of phenolic compounds, amide and protein. Phytochemical tests showed the presence of coumarins, tannins, catechins, saponins and steroids.

Keywords: cystoliths, diacytic stomata, glandular trichoma, non-glandular trichoma

Introduction

The Acanthaceae family comprises about 250 genera and 2500 species widely distributed in tropical regions of the world, with little occurrence in temperate regions (Braz et al., 2002). Brazil has a high diversity of these species, with an estimated occurrence of 40 genera and 542 species. These are mostly distributed in mesophytic forests, with 45% in "Mata Atlantica" and 25% in forests of the "Planalto Central". Another 15% are found in the Amazonian rainforest, and the remainder is distributed in other regions (Silva, 2011; Vilar, 2009).

Acanthaceae species generally grow in the form of plants, subshrubs, shrubs and lianas, and more rarely, trees (Kameyama, 2006). Their leaves are normally opposite, simple and without stipules. These species commonly present cystoliths in epidermal cells of the stem and leaves, and the presence of crystals of almost all shapes and types is an important feature of the family (Kameyama, 2006; Vilar, 2009). Many species are used for ornamental purposes, with mainly economic importance (Metcalfe & Chalk, 1957). In traditional medicine, their use is emphasized for treating fever, pain and stomach disorders (Angonese et al., 1992; Chen et al., 1996). Chemically, Acanthaceae are rich in alkaloids, lignans, flavonoids, terpenoids and biogenic amines. A dichotomist pattern has been observed between the presence of alkaloids and lignans (Angonese et al., 1992).

The Justicia genus has the greatest number of species of the Acanthaceae family, comprising about of 600 members found in tropical and temperate areas of the globe. These species occur in the form of perennial plants or subshrubs, erect or scandent. Their leaves are usually entire and petiolate, with cystoliths in the epidermis (Braz et al., 2002; Kameyama, 2006). In the North and Northeast of Brazil, species of this genus, especially J. pectoralis and J. gendarussa, are widely used by the population for the treatment of inflammatory processes (Oliveira & Andrade, 2000). The genus proved to be rich in amine metabolites, coumarins and lignans (Al-Juaid & Abdel-Mogib, 2004). There is no accurate estimate for the number of the Justicia species in the Brazilian flora (Braz et al., 2002), due to inconsistencies in the circumscription of the genus in the country, which requires taxonomic revisions (Profice, 2010). Therefore, the importance of pharmacobotanical studies with species of this genus is emphasized.

Justicia acuminatissima (Miq.) Bremek. is a native subshrub of the Brazilian Amazon region, used for ornamental and therapeutic purposes (Santos, 2006). In Amazonas (Brazil), where it is popularly known as "saratudo" or "saratudo-de-quintal", its leaves are widely used in the preparation of teas, which are consumed for healing and inflammation relief. However, there is no scientific data providing pharmacognostical identification of this herbal drug, which has morphological similarities with subshrubs and several other plants of the same genus. Therefore, considering that the correct identification of plant material is the first step in producing accurate scientific studies with good quality and reproducibility, we performed morpho-anatomical, histochemical and phytochemical analyses of leaves of J. acuminatissima using gold standard methods, seeking to contribute to pharmacognostical characterization of this species.

Materials and Methods

Plant material

The collection of samples was authorized by the Conselho de Gestão do Patrimônio Genético, chaired by the Ministry of Environment (Brazil), under registration number 034/2008. The plant material was collected at the campus of Instituto Nacional de Pesquisas da Amazônia (INPA), in Manaus-AM, Brazil, on June 24, 2008, in the morning, under intense light. A voucher specimen was deposited in at the Herbarium of INPA, under number 224040.

Leaf morphology

To characterize the leaf morphology, three samples of the species were collected and placed in a bottle containing equal parts of alcohol and water. The phyllotaxy and morphologic characters, such as size, shape, texture, color, coating, margin, venation, midrib and petiole were visually analyzed (Hickey, 1973).

Leaf anatomy

The leaf anatomy was analyzed based on transversal and longitudinal sections of 0.7 mm from the lamina, midrib and petiole of three fresh leaves, obtained by a manual microtome. The material was bleached with 20% sodium hypochlorite and then washed and stained with toluidine blue (O'Brien, 1964). To observe the stomata, as well as the characters from the adaxial and abaxial surfaces, the dissociation of leaf epidermis was performed using commercial sodium hypochlorite (Kraus & Arduin, 1997). The slides were mounted in glycerin and stained with safranin. The structures were analyzed and photomicrographed in an optical microscope with a digital camera attached.

Histochemical analysis

Histochemical tests were carried out using: ferric chloride to detect phenolic compounds; Lugol to detect starch; ruthenium red to detect pectin; and xylidine Ponceau to detect total proteins (Ascenção, 2004; Johansen, 1940).

Phytochemical screening

For the phytochemical screening, the leaves were dried in a oven at 40 ºC and pulverized using a mechanical grinder. Phytochemical screening was performed to verify the presence of cyanogenic glucosides, coumarins, flavonoids, terpenes and steroids (Matos, 1997), saponins (Schenkel et al., 2007) and alkaloids (Costa, 2002).

Results and discussion

Considering that flowers and fruits are only available in certain seasons of the year, the wide variety of anatomical characters of the leaves has been a useful tool for the taxonomic identification of various species (Dipa & Daniel, 2011). On the other hand, it is obvious that knowledge of the main groups of secondary metabolites in medicinal plants is important as a starting point for studies of their biological activities, or even for the isolation and identification of compounds that may be the active principles of plants, especially those used in traditional medicine. In this work, we studied the micromorphological and anatomical characters of Justicia acuminatissima (Miq.) Bremek., Acanthaceae, and investigated the class of secondary metabolites of this herbal drug.

The leaves of J. acuminatissima are simple, lanceolate in shape, with an acuminate apex, cuneate base, crenate margin, and pinnate venation, and are green on both sides. They have a membranous consistency and are pubescent. They are usually between 9 in length and 13 cm and width and between 3 and 5 cm in their central region; they are exstipulate, with decussate phyllotaxy. (Figure 1). The petiole is smooth, flat, and concave-convex in transversal section, and between 1.7 and 2.7 cm in length.

The petiole has a uniseriate epidermis composed of cubic cells with thickened outer periclinal walls and anticlinal walls with some sinuosity. The subepidermal region is composed of an angular collenchyma, followed by a parenchyma with many schizogenous spaces (Figure 2A). As in many other Acanthaceae species, the vascular system is composed of a semicircular central beam and two circular complementary bundles (Patil & Patil, 2012) (Figure 2B). Druses were observed in some cells. Overall, calcium oxalate deposits in druse form are common in Acanthaceae species (Vilar, 2009). Glandular and non-glandular trichomes were found in small numbers (Figure 2C). The mesophyll is dorsiventral, composed of one layer of palisade parenchyma, followed by four to five layers of relatively dense spongy parenchyma in which the cells are almost juxtaposed (Figure 2D). The midrib has a uniseriate epidermis composed of cubic cells. The subepidermal tissue consists of angular collenchyma which is interrupted by sclerenchyma. In the adaxial surface of epidermis, the collenchyma is interrupted by sclerenchyma, which is followed by parenchyma, where the bilateral vascular system is dispersed. In the abaxial surface of the epidermis, the collenchyma is continuous. Some trichomes were seen in the epidermis (Figures 2E and 2F).

 

On both the abaxial and adaxial surfaces, the epidermal cells were elongated and undulate, with U-shaped sinuses, similar to various other Acanthaceae species (Patil & Patil, 2011a). The bases of the trichomes can be seen on both epidermal surfaces (Figures 3A and 3B). Long multicellular and uniseriate non-glandular trichomes, generally five-celled, were found on both sides of the epidermis (Figure 3C) and along the veins (Figure 3D). Glandular trichomes, with one or twocelled heads, were also found on both sides of the epidermis (Figures 3E and 3F). These were generally sessile, but glandular trichomes with very short stalk were also seen in the petiole. Variations in the form and number of cells of the trichomes can be used to identify these plants, including in the powder form (Dipa & Daniel, 2011).

 

The epidermis mainly presents diacytic stomata with elliptical guard cells, as in most Acanthaceae species. The subsidiary cells with U-shaped sinuses, like other epidermal cells, but are not elongated and have very different sizes from each other (Figure 4A). Anisocytic stomata were also viewed in small numbers. However, the amphystomatic pattern shown by J. acuminatissima is an unusual feature in the Justicia genus, and can be used to taxonomically differentiate this species (Patil & Patil, 2011a). Simple and oblong-shaped cystoliths are also found on both sides of the foliar epidermis (Figure 4B).

Species of the Acanthaceae family are known by the presence of various crystal types in their tissues, thus, the presence of cystoliths and druses in J. acuminatissima is easily justified (Vilar, 2009; Watson & Dallwitz, 1992). Cystoliths are calcium carbonate deposits formed in specialized cells called lithocysts. They are mostly located in the epidermis and generally consist of a single stalk, but can take different forms, which are easily identified by microscopy. They have been found only in Moraceae, Urticaceae, Acanthaceae and some other families. The occurrence of cystoliths in the vegetative parts of Acanthaceae species have been repeatedly reported by various researchers. In the leaves and sepals of Justicia, the lithocysts occurred frequently in the epidermis (Lin et al., 2004). The variations in shapes and sizes have been used to create convenient taxonomic distinctions, either exclusively or in conjunction with other endomorphic or exomorphic features of these plants (Patil & Patil, 2011b).

Histochemical tests showed the presence of proteins in the epidermis and amide in the petiole and vascular system. The parenchyma has high phenolic compound content. Phytochemical screening revealed the presence of coumarins and condensed tannins, which confirms the presence of phenolic compounds observed in the histochemical analysis, as well as steroidal saponins. The presence of coumarins seems to be typical in the Justicia genus. In J. pectoralis, one of the better scientifically studied species, these compounds are considered the main chemical constituents (Lino et al., 1997; Oliveira & Andrade, 2000).

Saponins present antiprotozoal, antifungal and antiviral activities, as well as cytostatic effects on various cancer cells, lower serum cholesterol, stimulation of cell-mediated immune system and enhancement of antibody production, as demonstrated in experimental animals (Francis et al., 2002). Tannins have biological activities related to their capacity of protein precipitation and astringent propriety, which has led to their use as antidiarrheal, antiseptic and wound sealant, as well as marked antimicrobial, antifungal and antiviral activities (Monteiro et al. 2005). On the other hand, different coumarins showed antioxidant, anticoagulant, anti-inflammatory and analgesic activities, among many other biological effects (Blanco, 2011; Montagner, 2007). The presence of these compounds therefore suggests good pharmacological potential for J. acuminatissima, which needs to be further investigated.

Overall, J. acuminatissima leaves present morphological, anatomical and chemical features common to most species of the Justicia genus. Moreover, particular characteristics, such as a crenate margin and amphystomatic pattern, and combined characters, such as long multicellular non-glandular trichomes and sessile glandular trichomes on both sides of the epidermis, and the presence of oblong cystoliths in the epidermis and druses in the mesophyll, enables fast and safe identification of this species by leaf analysis.

Received 2012

Accepted 20 Sep 2012

• Al-Juaid SS, Abdel-Mogib M 2004. A novel podophyllotoxin lignan from Justicia heterocarpa. Chem Pharm Bull 52:507-509.
• Angonese MT, Moreira DL, Kaplan MAC 1992. Perfil químico da família Acanthaceae. Bol Mus Biol Mello Leitão 1:3-6.
• Ascenção L 2004. Métodos histoquímicos em vegetais. Viçosa: Editora UFV.
• Blanco NEV 2011. Estudio de la actividad anticonvulsivante de metabolitos secundarios tipo cumarina. Bogotá, 323p. PhD Thesis, Postgraduate Program in Pharmaceutical Sciences, Universidad Nacional de Colombia.
• Braz DM, Carvalho-Okano RM, Kameyama C 2002. Acanthaceae da Reserva Florestal Mata do Paraíso, Viçosa, Minas Gerais. Rev Bras Bot 25:495-504.
• Chen CC, Hsin WC, Ko FN, Huang YL, Ou JC, Teng CM 1996. Antiplatelet aryl naphthalide lignans from Justicia procumbens. J Nat Prod 59:1149-1150.
• Costa AF 2002. Farmacognosia 5 ed. Lisboa: Calouste Gulbenkian.
• Dipa C, Daniel M 2011. Foliar trichomes of some members of family Acanthaceae and their taxonomic utility. Int J Pharm Bio Sci 2:231-235.
• Francis G, Kerem Z, Makkar HPS, Becker K 2002. The biological action of saponins in animal systems: a review. Br J Nutr 88:587-605.
• Hickey LJ 1973. Classification of the architecture of dicotyledons leaves. Am J Bot 60:17-33.
• Johansen DA 1940. Plant Microtechnique New York: Mcgraw Hill.
• Kameyama C 2006. Flora da Reserva Ducke, Amazonas, Brasil: Acanthaceae. Rodriguesia 57:149-154.
• Kraus JE, Arduim M 1997. Manual básico em morfologia vegetal Rio de Janeiro: Seropédica.
• Lin ML, Yen TB, Kuo-Hang LL 2004. Formation of calcium carbonate deposition in the cotyledons during the germination of Justicia procumbens L. (Acantaceae) seeds. Taiwania 49:250-262.
• Lino CS, Taveira ML, Vianna GSB, Matos FJA 1997. Analgesic and anti-inflammatory activities of Justicia pectoralis Jacq. and its main constituents: coumarin and umbelliferone. Phytother Res 11:211-215.
• Matos FJA 1997. Introdução à Fitoquímica Experimental, 4. ed. Fortaleza: Edições UFC.
• Metcalfe CR, Chalk L 1957. Anatomy of dicotyledones, vol II. Oxford: Clarendon Press.
• Montagner C 2007. Atividades antifúngica, citotóxicas (células tumorais humanas) e hemolítica de cumarinas natuarais e semi-sintéticas. Florianópolis, 125p. Dissertation of the Master's degree, Postgraduate Program in Biothecnology, Universidade Federal de Santa Catarina.
• Monteiro JM, Albuquerque UP, Araújo EL, Amorim ELC 2005. Taninos: uma abordagem da química à ecologia. Quim Nova 28:892-896.
• O'Brien TP, Feder N, McCully ME 1964. Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma 59:368-37.
• Oliveira AFM, Andrade LHC 2000. Caracterização morfológica de Justicia pectoralis Jacq. E Justicia gendarussa Burm. F. (Acanthaceae). Acta Amazonica 30:569-578.
• Patil AM, Patil DA 2011a. Investigations on foliar epidermal characteristics in some Acanthaceae. Current Botany 2:1-8.
• Patil AM, Patil DA 2011b. Occurrence and significance of cystoliths in Acanthaceae. Current Botany 2:1-5.
• Patil AM, Patil DA 2012. Petiolar anatomy of some hitherto unstudied Acanthaceae. J Exp Sci 3:5-10.
• Profice SR 2010. Novas combinações para espécies brasileiras de Acanthaceae. Rodriguésia 61:85-88.
• Santos JL 2006. Uso e diversidade de espécies vegetais cultivadas na reserva de desenvolvimento sustentável do Tupé. Manaus, 86p. Dissertation of the Master's degree, Postgraduate Program in Agriculture in the Humid Tropics, Instituto Nacional de Pesquisas da Amazônia.
• Schenkel EP, Gosmann G. Athayde ML 2007. Saponinas. In: Simões CMO, Schenckel EP, Gosman G, Mello JCP, Mentz LA, Petrovick PR (org.) Farmacognosia: da planta ao medicamento. Porto Alegre: Editora da UFRG, p. 711-740.
• Silva MG 2011. Estudo taxonômico da subtribo Ruellinae Nees (Acanthaceae) no Estado do Paraná, Brasil. Curitiba, 66p. Dissertation of the Master's degree, Postgraduate Program in Botany, Universidade Federal do Paraná
• Vilar TS 2009.Acanthaceae Juss. no Distrito Federal, Brasil. Brasília, 105p. Dissertation of the Master's degree, Postgraduate Program in Botany, Universidade de Brasília.
• Watson L, Dallwitz MJ 1992. The families of flowering plants: descriptions, illustrations, identification, and information retrieval. http://delta-intkey.com/angio/www/acanthac.htm, accessed Aug 2012.

Publication Dates

• Publication in this collection
  16 Nov 2012
• Date of issue
  Dec 2012

History

• Received
  2012
• Accepted
  20 Sept 2012