Aline Redondo Martins

Development and Characterization of Microsatellite Markers for the Medicinal Plant Smilax brasiliensis (Smilacaceae) and Related Species

Premise of the study: A new set of microsatellite or simple sequence repeat (SSR) markers were developed for Smilax brasiliensis, which is popularly known as sarsaparilla and used in folk medicine as a tonic, antirheumatic, and antisyphilitic. Smilax brasiliensis is sold in Brazilian pharmacies, and its origin and effectiveness are not subject to quality control. Methods and Results: Using a protocol for genomic library enrichment, primer pairs were developed for 26 microsatellite loci and validated in 17 accessions of S. brasiliensis. Thirteen loci were polymorphic and four were monomorphic. The primers successfully amplified alleles in the congeners S. campestris, S. cissoides, S. fluminensis, S. goyazana, S. polyantha, S. quinquenervia, S. rufescens, S. subsessiliflora, and S. syphilitica. Conclusions: The new SSR markers described herein are informative tools for genetic diversity and gene flow studies in S. brasiliensis and several congeners.

Propagation studies in Smilax fluminensis Steud. (Smilacaceae)

Smilax L. species, popularly known in Brazil as salsaparrilha, have been used in folk medicine as tonic against rheumatism and as anti-syphilitic since the sixteenth century; however, until today, their roots have been explored in an extractive way. The aim of this study was to analyze the propagation of Smilax fluminensis aiming to meet the demand and to propose procedures for its sustainable management. It was analyzed the percentage of seed germination, rooting of aerial and subterranean stem cuttings and plant resprouting capacity after removal of the shoots. The monitoring lasted one year. Seeds were subjected to temperatures: 25°C, 30°C, 20-30°C and 20-35°C under a daily photoperiod of eight hours and in the absence of light. For rooting the stem cuttings, aerial and subterranean stems about 20cm long with two nodal regions were subjected to treatment with distilled water (control) and indolbutyric acid (IBA) at 100ppm. The best germination percentages obtained for S. fluminensis were 80% at 20-30°C under light and 85% at 30°C in the dark. Only subterranean stem cuttings showed significant difference concerning fresh and dry matter of roots with higher values in treatments with hormone compared to the control.

The sarsaparilla market in the state of São Paulo (Brazil) and the challenges of cultivation

This study aimed to present information about the sarsaparilla sold in establishments in the state of Sao Paulo, Brazil, assess the genetic diversity of Smilax brasiliensis Spreng., Smilacaceae, and examine the growing conditions and productivity of five species of Smilax. The amount of sarsaparilla sold per month at most pharmacies was 0.4 kg on average. Herbal stores and markets sold averages of 9 kg and 8 kg per month, respectively. The weight of the underground biomass of S. fluminensis (188.3 g) is significantly higher than those of other species (28.3-79.6 g). The study demonstrated that high genetic diversity among the Smilax brasiliensis plants belonging to the CPQBA germplasm bank, which was confirmed by the results of the genotyping study that used a SSR marker on S. brasiliensis. The high consumption of sarsaparilla and the low yield of young plants cultivated from seeds with high genetic variability reinforce the need for further studies on the production of Smilax species.

Aerial stem and leaf morphoanatomy of some species of Smilax

This study aimed to describe the morphoanatomy of the aerial vegetative organs of seven Smilax species, used in Brazilian folk medicine. Samples of leaves and stems were fixed with FAA 50, embedded in historesin, sectioned on a rotary microtome, stained and mounted in synthetic resin. Cuticle ornamentation was analyzed with standard scanning electron microscopy. In the frontal view, the walls of the adaxial epidermis are straight in S. brasiliensis, S. cissoides, S. goyazana and sinuous in the other species. The walls of the epidermis on the abaxial surface are straight in S. brasiliensis, S. goyazana, S. rufescens, sinuous in S. campestris, S. fluminensis, S. oblongifolia, and wavy in S. cissoides. The stomata are paracytic in S. brasiliensis, S. goyazana, S. oblongifolia, and S. rufescens, anomocytic in S. cissoides, S. campestris; anisocytic and paracytic in S. fluminensis. The midrib has three vascular bundles that are individually wrapped by lignified cells in S. brasiliensis, S. cissoides, and S. fluminensis. In the other, the three vascular bundles are surrounded by a single lignified sheath. In the stems the vascular cylinder is surrounded by a sclerenchymatous ring with the exception of Smilax fluminensis, which has a starch sheath and internal layers of thin-walled cells.

Seed ontogeny and endosperm chemical analysis in Smilax polyantha (Smilacaceae)

Smilax polyantha Grisebach is a species native to the Brazilian Cerrado biome and is known as sarsaparilla in folk medicine. Despite its popular use, little is known about the propagation of this species, which is still actively illegally exploited. The present study aims to analyse the seed ontogeny and perform endosperm chemical analyses in S. polyantha to elucidate the structural and chemical factors that could be associated with the low germination rates and structural organisation of the seed. The ovules are orthotropic and bitegmic, have short funicles, single collateral vascular bundles that end in the chalaza, and a hypostasis that is composed of chalazal and nucellar cells. The seed covering is non-multiplicative. In mature seeds, the cellularised endosperm has thick-walled cells, the embryo is small and the tegmen comprises two layers of periclinal elongated cells with a red–orange content, which are covered by a cuticle. Histochemical tests detected the presence of lipids, proteins and polysaccharides in the cellular content of mature seeds. Chemical analyses indicated 46.7% hemicellulose per total weight, 67.3% glucose, 30.7% mannose, 1.9% galactose and an absence of fucose, arabinose and rhamnose. In conclusion, the delayed seed germination in S. polyantha is associated with the seed endosperm cell walls.

Axillary bud and pericycle involved in the thickening process of the rhizophore nodes in Smilax species

The species of the genus Smilax, popularly known as sarsaparilla, are widely used in folk medicine due to the antirheumatic properties of its underground structures. Smilax fluminensis and S. syphilitica occur in forested areas and form thickened stems called rhizophores from which adventitious roots grow. To provide information for more accurate identification of the commercialised product and for elucidating the process of stem thickening, a morphology and anatomy study of the underground organs of the two species was conducted. The adventitious roots differ in colour and diameter depending on the stage of development. They are white and have a larger diameter in the early stages of development, but as they grow, the adventitious roots become brown and have a smaller diameter due to the disintegration of the epidermis and virtually the entire cortex. In brown roots, the covering function is then performed by the lignified endodermis and the remaining walls of the cells from the last parenchyma cortical layer. These results are similar to those found in studies of other Smilax and suggest that the anatomy of the roots can be useful for identifying fraud in commercialised materials. The thickening process of the nodal regions of the rhizophores in both species involves the activity of axillary buds and pericyclic layers.

Karyotype characterization reveals active 45S rDNA sites located on chromosome termini in Smilax rufescens (Smilacaceae).

The genus Smilax (Smilacaceae) includes species of medicinal interest; consequently, their identification is important for the control of raw material used in the manufacture of phytotherapeutic products. We investigated the karyotype of Smilax rufescens in order to look for patterns that would be useful for comparative studies of this genus. To accomplish this, we developed procedures to grow plants and optimize root pretreatment with mitotic fuse inhibitors to obtain metaphase spreads showing clear chromosome morphology. The karyotype, analyzed in Feulgen-stained preparations, was asymmetric, with N = 16 chromosomes gradually decreasing in size; the larger ones were subtelocentric and the smaller chromosomes were submetacentric or metacentric. Nearly terminal secondary constrictions were visualized on the short arm of chromosome pairs 7, 11, and 14, but they were clearly detected only in one of the homologues of each pair. The nucleolus organizer regions (NORs) were mapped by silver staining and fluorescent in situ hybridization of 45S rDNA probes. Silver signals (Ag-NORs) colocalized with rDNA loci were detected at the termini of the short arm of 6 chromosomes. The secondary constriction heteromorphism observed in Feulgen-stained metaphases suggests that differential rRNA gene expression between homologous rDNA loci can occur, resulting in different degrees of chromatin decondensation. In addition, a heteromorphic chromosome pair was identified and was interpreted as being a sex chromosome pair in this dioecious species.

Use of Anatomical, Chemical, and Molecular Genetic Characteristics in the Quality Control of Medicinal Species: A Case Study of Sarsaparilla (Smilax spp.)

Use of Anatomical, Chemical, and Molecular Genetic Characteristics in the Quality Control of Medicinal Species: A Case Study of Sarsaparilla ( Smilax spp.)Species of the genus Smilax, popularly known as sarsaparilla, are used in folk medicine as a tonic, an anti-rheumatic, and an anti-syphilis treatment, and are sold in Brazilian drugstores without any quality control regarding their origin and efficacy. The origin of the material is mainly based on wild extraction. Quality control of herbal drugs should include a more reliable identification of the source involving characterization and definition of their anatomical and chemical characteristics. The current study aimed to verify whether the combined use of anatomical, chemical, and molecular genetic characteristics might be useful in the quality control of medicinal plants, specifically the sarsaparilla sold in compounding drugstores in the state of São Paulo, Brazil. Root samples were subjected to conventional light microscopy and scanning electron microscopy. To determine the chemical profile, thin-layer chromatography (TLC) was applied to ethanol extracts of the roots. The chemical profile of the chemical material sold in stores was compared with the previously determined profiles of medicinal Smilax species (S. goyazana, S. rufescens, S. brasiliensis, S. campestris, S. cissoides, S. fluminensis, S. oblongifolia, and S. polyantha). Although there was considerable similarity between the anatomical structure of the commercial sarsaparilla and the structure reported in the literature for the Smilax species, there were differences in the phloem organization and in the presence of a series of idioblasts containing raphides, phenolic idioblasts, and metaxylem in the center of the plant structure. TLC analysis of the commercial ethanol extracts revealed spots with colors ranging from yellow to green. In addition, the same spots showed components with the same retention factor (Rf), indicating chemical similarity between the different samples. However, the distribution pattern of the spots, as well as the Rf of the commercial samples, differed from those obtained for the eight species of Smilax, which were very similar to each other. Comparing the groups examined in the present study with regard to microsatellite markers and DNA barcoding revealed that commercial sarsaparilla is genetically different from the eight species of Smilax known for their medicinal properties in Brazilian ethnobotanical surveys. This seriously calls into question their effectiveness. This case study of sarsaparilla demonstrates the utility of anatomical, chemical, and molecular genetic characteristics in the quality control of medicinal plants.Utilização de caracteres anatômicos, químicos e genético-moleculares no controle de qualidade de espécies medicinais: O estudo de caso da salsaparrilha ( Smilax spp.)As espécies do gênero Smilax, conhecidas popularmente como salsaparrilha, são empregadas na medicina popular como fortificante, contra o reumatismo e anti-sifilítico e são vendidas em farmácias brasileiras sem que exista um controle de qualidade de sua origem e eficácia. A origem do material comercializado no Brasil é baseada principalmente no extrativismo. O controle de qualidade de plantas medicinais deve basear a identificação da droga vegetal em fontes confiáveis que envolvam sua caracterização anatômica e química. O objetivo do trabalho foi verificar se o uso combinado de características anatômicas, químicas e genético-moleculares poderiam ser úteis no controle de qualidade de plantas medicinais, usando como exemplo, a salsaparrilha vendida em farmácias do Estado de São Paulo, Brazil. Amostras de raízes foram submetidas à técnicas convencionais de microscopia de luz e microscopia eletrônica de varredura. Para determinar o perfil químico dos extratos etanólicos das raízes, foi utilizada cromatografia em camada delgada (TLC). A composição química do material comercializado foi comparada com os perfis químicos de espécies de Smilax previamente identificadas (S. goyazana A. De Candolle, S. rufescens Grisebach, S. brasiliensis Sprengel, S. campestris Grisebach, S. cissoides Martius ex Grisebach, S. fluminensis Steudel, S. oblongifolia Pohl ex Grisebach and S. polyantha Grisebach). Embora haja uma grande semelhança entre a estrutura anatômica da salsaparrilha comercializada e a descrita na literatura para as espécies de Smilax, foram observadas diferenças na organização do floema, na presença de idioblastos em série contendo ráfides, idioblastos fenólicos e presença de elementos de metaxilema no centro da estrutura radicular. As análises em TLC dos extratos etanólicos do material comercializado revelaram manchas com cores que variam de amarelo a verde. Além disso, as mesmas manchas mostraram que seus componentes apresentam o mesmo índice de retenção (Rf), indicando semelhança química entre as diferentes amostras comercializadas. No entanto, o padrão de distribuição das manchas, assim como o Rf das amostras comerciais, diferiram daqueles obtidos das oito espécies de Smilax identificadas, que por sua vez, eram muito semelhantes entre si. Comparando-se os grupos analisados no presente estudo em relação aos marcadores microssatélites e DNA barcoding observou-se que a salsaparrilha comercial é geneticamente diferente das oito espécies de Smilax conhecidas por suas propriedades medicinais em levantamentos etnobotânicos brasileiros, o que torna questionável a eficácia do material comercializado, uma vez que o perfil químico de tais amostras comerciais também é diferente. Sendo assim, através deste estudo de caso envolvendo a salsaparrilha, foi possível verificar que a utilização conjunta de características anatômicas, químicas e genético- moleculares é eficiente no controle de qualidade de plantas medicinais.