Print version ISSN 0102-695X
Rev. bras. farmacogn. vol.16 no.1 João Pessoa Jan./Mar. 2006
Observações de sementes de algumas espécies herbáceas de Phyllanthus L. (Phyllanthaceae) em MEV
Clarice A. MachadoI, *; Paulo L. de OliveiraII; Lilian A. MentzII
IPontifícia Universidade Católica do Rio Grande do Sul, Faculdade de Farmácia, Laboratório de Farmacognosia, Av. Ipiranga 6681, Prédio 12, 90619-900, Porto Alegre, RS, Brasil
IIPrograma de Pós-Graduação em Botânica - PPG-BOT, UFRGS, Av. Bento Gonçalves 9500, 91570-950, Porto Alegre, RS
Six medicinal herbaceous Phyllanthus L. (Phyllanthaceae) species, P. tenellus Roxb., P. niruri L., P. stipulatus (Raf.) Webster, P. urinaria L., P. caroliniensis Walt., and P. amarus Schum. & Thonn. are difficult to distinguish. In order to solve this problem, the ornamentation of the seed surfaces was analyzed by SEM (scanning electron microscopy). Differences between lateral, dorsal and ventral surfaces were observed. The seeds were cut into sections at different angles, exposing testa sculptures. Three types of surface ornamentation patterns were observed and they showed to be avaluable diagnostic.
Keywords: Phyllanthus, Phyllanthaceae, seed ornamentation, scanning electron microscopy (SEM).
Seis espécies herbáceas de Phyllanthus L. (P. tenellus Roxb., P. niruri L., P. stipulatus (Raf.) Webster, P. urinaria L., P. caroliniensis Walt. e P. amarus Schum. & Thonn.) mencionadas como medicinais apresentam dificuldades na identificação. Com o objetivo de auxiliar neste problema e determinar diferenças entre as espécies, o padrão de ornamentação das sementes foi estudado utilizando técnicas de microscopia eletrônica de varredura (MEV). As sementes apresentaram ornamentação distinta, sendo observadas as superfícies laterais, dorsais e ventrais. As espécies puderam ser separadas em três tipos de ornamentação, que demonstraram importância como caractere diagnóstico para a sua separação.
Unitermos: Phyllanthus, Phyllanthaceae, ornamentação da superfície da semente, microscopia eletrônica de varredura (MEV).
The genus Phyllanthus L. has a pan-tropical distribution, with over 800 described species of trees, shrubs, and herbs (Webster; Carpenter, 2002). According to Judd et al. (2002), APG II (2003), and Samuel et al. (2005) Phyllanthus genus should be now reported as belonging to the Phyllanthaceae family, instead to the former subfamily Phyllanthoideae of the Euphorbiaceae. Phyllanthus species are largely used in Brazilian traditional or folk medicine as a diuretic and are also indicated in the treatment of jaundice, hepatitis, kidney stones and diabetes (Oliveira; Saito, 1987/1989; Calixto et al., 1997; Calixto et al., 1998).
This complex genus is considered to have at least ten subgenera (Webster, 1967). It is difficult to identify the species correctly, such as the case of P. niruri and P. amarus (Unander et al., 1990; 1991; 1992), and to establish which of the species has useful medicinal properties with the available ethno-botanical and ethno-pharmacological data. In herbaceous Phyllanthus, floral characters have limited utility for the identification of species and supra-specific taxa, due to the small size of the flowers and the relative narrow range of variation in number and shape of parts, but it is unquestionable that the mean size and the ornamentation pattern of seeds tend to be the best diagnostic (Webster, 1970; Silva; Sales, 2004). Based on a literature revision, the present work focuses on some medicinal herbaceous Phyllanthus species occurring in Brazil known as quebra-pedra (Smith; Downs, 1959; Smith et al., 1988; Falcão et al., 2005; Barbosa-Filho et al., 2005) in order to study the surface ornamentation patterns of P. amarus Schum. & Thonn., P. caroliniensis Walt., P. niruri L., P. stipulatus (Raf.) Webster, P. tenellus Roxb., and P. urinaria L. seeds. The infra-generic classification of the six Phyllanthus species listed below (Table 1) is based on Webster (1967; 1970; 2002).
Chen and Wu (1997) compared SEM photographs of pollen grains and seed surface ornamentation of herbaceous Phyllanthus species in Taiwan. They studied P. amarus Schum. & Thonn., P. tenellus Roxb., P. urinaria L.subsp. nudicarpus Rossign. & Haic., reviewed in this paper, and other five species. Also Silva and Sales (2004) studied some species with a taxonomic approach, with short seed descriptions and illustrations.
MATERIAL AND METHODS
Seeds of Phyllanthus amarus, P. caroliniensis, P. niruri, P. tenellus, and P. urinaria were kindly provided by the experimental station CPQBA-UNICAMP (courtesy of Prof. Dr. Pedro Melillo de Magalhães, Campinas-SP, Brazil), and were also collected from the field at different locations in Brazil. The seeds of P. stipulatus were collected from the field. The vouchers are deposited at ICN herbarium, UFRGS (Brazil) (Table 2). Five mature seeds of each species were air dried and selected for the exposure of ventral, lateral, and dorsal views, and divided into sections to verify ornamentation pattern on longitudinal or transversal view (Chen; Wu, 1997). They were mounted on stubs with double-sided carbon adhesive tape. The specimens were gold coated (20-25 nm), post-coated with carbon on ion sputter coater (Bal-Tec SCD 005), and observed under XL-30 Philips Scanning Electron Microscope at 15 kV accelerating voltage. The terminology used for surface patterns was based on Barthlott (1990) and Barthlott et al. (1998) and adapted when needed. In order to determine the terminology of ornamentation pattern of P. amarus and P. stipulatus,some sculptures on transversal sections mounted on stubs were broken. The SEM images showed a massive core. AccordingBarthlott et al. (1998), the best word for this structure is rodlet.
RESULTS AND DISCUSSION
The six studied species of Brazilian herbaceous Phyllanthus, all seeds were wedge-shaped (trigonous) (Burger; Huft, 1995), and two seeds were present in each locus of the capsular fruits. Through micromorphologic examination of each seed coat, three types of rib disposition on dorsal and ventral face were recognized. The seed descriptions and illustrations of Silva and Sales (2004) for P. amarus, P. caroliniensis, P. niruri, P. stipulatus and P tenellus can not be compared with the SEM micromorphology described in Table 2 because of the lower magnification used.
The seed coat on dorsal face of the first type group presented longitudinal ribs with transversal finger-shaped rodlets. In P. amarus (Table 2; Figs 1-6), the seed coat had 6-9 dorsal regular longitudinal ribs, where the rodlet end forms the ribs. The same pattern was recognized in P. stipulatus (Table 2, Figs 7-13), but the finger-shaped rodlets were irregularly arranged in different sizes, forming 11-15 irregular dorsal longitudinal ribs, with spaces between the fingers-shaped rodlets. The lateral and ventral faces results are on Table 2, and the dates (with Figs. 1-6) showed that the seed ornamentation is consistent with Chen and Wu (1997) studies to P. amarus.
In the second type group, the seed coat on dorsal face presented longitudinal ribs formed by stellate verrucose ornamentation in longitudinal arrangement. In P. caroliniensis (Table 2, Figs 14-20), the seed coat presented a regular pattern of about 10 longitudinal ribs, and the verrucose coat was lost. The verrucose coat and the crust were easily detachable on dorsal and lateral faces. P. niruri (Table 2) presented regular longitudinal and concentric ribs on dorsal and lateral faces (Figs 23-26). However, in some seeds, these ribs were occasionally irregular (Figs 21, 22). P. tenellus (Table 2, Figs 27-32) usually had irregular longitudinal and concentric alignment ribs, with less prominent verrucose ornamentation, which look like a fried egg (Figs 29 and 30), particularly on the lateral face.
The third type was represented by P. urinaria (Table 2, Figs 33-38). The seed coat showed transversal ribs on the dorsal face, and longitudinal ribs on the lateral face. This is a typical shape, which was already mentioned by Chen and Wu (1997), bearing symmetrical or asymmetrical ornamentation.
Webster (1956) mentioned a curious and unique kind of seed ornamentation in P. urinaria, as well as the presence of deep pits on the sides of transverse ridges on radial walls. Dot-shaped microverrucose ornamentation was found on the ribs, and crust waxes on dorsal and ventral faces (Figs 34-36). They appear as a deep pit due of their black color. Crusts were mainly found in valleys. The transversal section of all seeds shows pits on the seed coat, and in P. stipulatus (Fig 39), it is easy to see several pits inside the cell wall.
A partial relationship between Websters (1956; 1967; 1970) infra-generic classification and the three types of ornamentation observed can be implied. The first type of seed ornamentation was found in three distinct subgenera (Isocladus, Kirganelia and Phyllanthus). However, in the last subgenus (Phyllanthus), three types of ornamentation for three species studied were observed, showing the importance and the need of studies on more species of the genus.
There is considerable micromorphological diversity among seeds of herbaceous Phyllanthus species. Our observations reinforce the results of previous studies. A new image on seed of the pits in the seed coat, never published before, was taken. These results, as well as those of Chen and Wu (1997), emphasize the importance of SEM in seeds studies, and underscore the need of more extensive use of this technique in taxonomic classifications in Phyllanthaceae.
Phyllanthus niruri and P. tenellus were published in the Brazilian Pharmacopoeia (F. Bras., 2004). The data found in this study allows the differentiation of these herbaceous species from others, and can be used as a diagnostic trait in the assessment of the authenticity of plant raw materials.
We wish to thank Faculdade de Farmácia PUCRS, Dr. Sérgio de Meda Lamb (in memorian), and CEEM PUCRS (Centro de Microscopia e Microanálises, Pontifícia Universidade Católica do Rio Grande do Sul), Dr. Pedro Melillo de Magalhães from Experimental Station CPQBA-UNICAMP (Campinas, SP- Brasil), Prof. Dr. Nelson Ivo Matzenbacher for collecting some species, Dr. Giovanna Machado and Dr. Carmem Isse Gomes for the images acquisition at XL30 Phillips SEM on CEEM; Dr. Inês Cordeiro (Instituto Botânico de São Paulo-SP) and Dr. Grady L. Webster (UC Davis Califórnia, in memoriam) for sending some articles and identifications, Dr. Gilsane Lino von Poser from Programa de Pós-Graduação em Ciências Farmacêuticas (UFRGS), Dr. Eliane Heuser from Faculdade de Biociências (PUCRS) and Dr. Jorge Ernesto de A. Mariath from Programa de Pós-Graduação em Botânica (UFRGS) for their valuable comments and considerations on the manuscript. This work is part of Doctoral Thesis of the first author.
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