Print version ISSN 0102-695X
Rev. bras. farmacogn. vol.17 no.4 João Pessoa Oct./Dec. 2007
Marcadores morfológicos, anatômicos, macro e micromoleculares para a identificação de Solanum cernuum
Tânia Maria Almeida AlvesI, *; Samanta MarengoI; Cristiane MachadoI; Roberta CaldeiraI; Omar CarvalhoI; Rosy Mary dos Santos IsaiasII; João Renato StehmannII; Carlos ZaniI
IInstituto René Rachou, FIOCRUZ, Av Augusto de Lima 1715, 30190-002, Belo Horizonte, MG, Brazil
IIDepartamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av Antonio Carlos 6627, Campus da Pampulha, 31270-901, Belo Horizonte, MG, Brazil
The plants consumed as remedy by the population may have imprecise taxonomical identification. If these plants are used for the production of phytomedicines such misidentification may affect the quality of the product. Hereby, we describe markers for identification of the entire plant or grounded plant material or the crude extract of Solanum cernuum Vell. (Solanaceae). Specimens from four localities were collected, analyzed and compared. Morphological characters were used to identify the plant when it is not grounded or extracted. However, when the plant material is grounded, the set of trichomes may be used as anatomical marker. The region ITS1, 5.8S and ITS2 of the nuclear ribosomal DNA was cloned and sequenced. The sequence, with length of about 600 base pairs, being 48.1% AT , was deposited in GenBank under the accession number DQ837371. Once this sequence is specific to S. cernuum, it was used as marker for this species. For the crude extract, chromatographic profiles of the leaves extracts were obtained by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Two flavonoids were isolated and identified as quercitrin and afzelin. So, this study presents morphological, anatomical, macro and micromolecular markers to identify S. cernuum.
Keywords: Solanum cernuum, Solanaceae, trichomes, quercitrin, afzelin, ITS rDNA.
Plantas consumidas como remédio nem sempre são identificadas taxonomicamente de maneira correta. Se estas plantas forem utilizadas para obtenção de uma droga vegetal ou um fitoterápico, tal erro pode afetar a qualidade do produto final. Neste trabalho são descritos marcadores para a identificação de Solanum cernuum Vell. (Solanaceae), esteja a planta íntegra, triturada ou como extrato bruto. Indivíduos de quatro localidades de Minas Gerais foram coletados, analisados e comparados. Os caracteres morfológicos foram utilizados para a planta íntegra. Para a planta triturada, o conjunto dos tricomas foi utilizado como marcador anatômico. Um marcador macromolecular também foi determinado. Para tal a região ITS1, 5.8S e ITS2 do DNAr foi clonada e seqüenciada. A seqüência, com cerca de 600 pares de bases dos quais 48,1% são AT, foi depositada no GenBank sob o número de acesso DQ837371. Por ser uma seqüência específica para S. cernuum, ela pode ser usada como marcador desta espécie. Para o extrato bruto foram determinados perfis cromatográficos de extratos das folhas por cromatografia em camada delgada e por cromatografia líquida de alta eficiência. Dois flavonóides foram isolados e identificados como quercitrina e afzelina. Assim, neste trabalho foram determinados marcadores morfológicos, anatômicos, macro e micromoleculares para identificar S. cernuum.
Unitermos: Solanum cernuum, Solanaceae, tricomas, quercitrina, afzelina, ITS rDNA.
The correct botanical identification of plants is one of the steps that will guarantee success of herbal drug technology. Botanical identification is classically based on anatomical and morphological data. However, when the herbal products to be identified are extracts, powder or there are only vegetative plant organs, other tools should be used for identification. Chromatographic techniques to analyze crude extracts, such thin layer chromatography (TLC), high performance liquid chromatography (HPLC) or gas chromatography (GC) hyphenated with detection techniques such as ultra-violet (UV), mass spectrometry (MS) and nuclear magnetic resonance have been successfully employed. For examples see Fan et al. (2006), Borse et al. (2002), Di et al. (2003), Hostettmann and Marston (2002), Yang (2006). Also, DNA-based techniques have been widely used for authentication of medicinal plants especially in the case when those plants are substituted or adulterated with other species morphologically or chemically indistinguishable (Joshi et al., 2004). The internal transcribed spacer (ITS) region of 18S-26S nuclear ribosomal DNA (nrDNA) has proven to be a powerful tool for phylogenetic studies (Baldwin et al., 1995). According to Hsiao et al. (1994), rDNA ITS of Poaceae have enough variability to distinguish between species. Zhao et al. (2001) used characters of rDNA ITS region to distinguish Alpinia galanga (L.) Sw., a plant used as a traditional Chinese medicine, from related species of Zingiberaceae.
The genus Solanum is the biggest one within the Solanaceae with at least 1000-1200 species (Hunziker, 2001). It is rich in steroidal alkaloids as tomatidine (Takagi et al., 1994) and solasodine (Yencho et al., 1998; Barbosa-Filho et al., 1991; Silva et al., 2005).
Solanum cernuum Vell. is a Brazilian shrub or small tree, restricted to Southwest states of the country (Carvalho, 1995, 1996). The leaves are used freshly or powdered as topical for skin diseases or as depurative and diuretic infusion (Carvalho, 1989). The leaves, entire or grounded, are commercialized as " panacea" mostly without any quality control. Although the extract of this species is not commercialized, it would be important to characterize it once there is no information about its chemistry.
Hereby we describe morphological, anatomical, micro and macromolecular markers of S. cernuum with the aim to support the botanical identification of the powdered plant and the crude extract of the leaves.
MATERIAL AND METHODS
As gold standard, four specimens of S. cernuum were collected (Table 1) twice or three times in four different places in the State of Minas Gerais, Brazil, and were identified by Dr JR Stehmann. One voucher was deposited at the herbarium of the Department of Botany, Universidade Federal de Minas Gerais, Brazil under the code BHCB 42855. The voucher from specimen EE-UFMG was deposited at same herbarium under the code BHCB 16886.
For TLC profiles comparisons of Solanum tuberosum L. (BHCB 2193), Solanum americanum Mill., Solanum esculentum Dunal (BHCB 56348), Bauhinia curvula Benth. (PAMG 45207) and Ocimum gratissimum L. (BHZB 2106) were used.
Fertile aerial parts of plants were prepared according to usual techniques for herbarium material conservation (Bridson; Forman, 1992). The measurements were made with a Mitutoyo caliper and the observations were done under stereoscopic microscopic Olympus SZH, coupled to an Olympus C-35AS-4 camera.
To analyze the trichomes distribution, leaves and petioles were embedded in historesin (Reichert Jung) and transversely sectioned in a rotative microtome (Reichert Jung 2035) with a C steal knife (Leica). The transverse sections were stained in 0.05% toluidin blue O in acetate buffer (pH 4.4) (Feder; O'Brien, 1968) for 5 min. Trichomes were isolated, clarified with NaOCl 2% diluted in water 1:8 (v:v), washed in water and stained in safranin 1% in ethanol 50% (Johansen, 1940) and observed at covered glass sheet in an Olympus BH2 microscope coupled to an Olympus C-35AD4 camera.
DNA extraction: DNA was extracted from all specimens but Xavier Chaves (Table 1) using CTAB protocol (Ferreira and Grattapaglia, 1998).
ITS analysis: The ITS region was amplified using the primers ITS4 (5-TCCTCCGCTTATTGATATGC-3), primer designed by Hsiao et al. (1994) and ITSleu1 (5-GTCCACTGAACCTTATCATTTAG-3), primer described by Bohs and Olmstead (2001), anchored, respectively, in the conserved ends of 18S and 26S ribosomal genes. PCR amplification was undertaken in a final volume of 10 µL consisting of: 1-10 ng of DNA, buffer (1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 8.5), 200 µM each dNTP, 0.5 U of Taq DNA polymerase, 5.0 pmol of each primer. The reactions were covered with a drop of mineral oil and subjected to the following cycle program: initial denaturation step for 3 min at 95 °C, and then 32 cycles as follows: annealing at 58 °C for 1 min, extension at 72 °C for 2 min, denaturation at 95 °C for 45 s and a final extension step at 72°C for 5 min.
Cloning and sequencing of PCR products: PCR products of the three specimens cited above were cloned using a pCR-II TOPO kit and transformed into quimiocompetent TOP10 Escherichia coli, according to manufacturers instructions (Invitrogen). Plasmides were isolated using the Quiagen plasmid miniprep kits according to the manufacturers protocol. Sequencing of ITS of rDNA were carried out using a Kit DYEnamic ET dye terminator MegaBace (Amersham). Such products were processed in a DNA automated sequencer MegaBace 500 DNA Analysis System (Amersham Pharmacia Biotech).
Sequence Analysis: Confirmation of the sequences as target regions was undertaken through the search software BLASTn (NCBI). Afterwards, complete sequences were aligned by the software Clustal W (Thompson et al., 1994) using the Bioedit software (http://jwbrown.mbio.ncsu.edu/BioEdit/bioedit.html).
Plant extraction: The plant leaves (Table 1) were oven dried at 40 ºC and powdered at Janke and Kunkel A10 analytical mill and extracted with the mixture of ethyl acetate, methanol and water (10:4:1), at room temperature.
Chromatography: From each extract, 100 mg were applied on silica gel plates Merck 60 F254. The same extraction solvent mixture was used as mobile phase. After elution, the plates were observed under UV254nm and UV366nm and sprayed with Dragendorff and Natural Product reagent (Wagner; Bladt, 1996) for spots detection. Analytical HPLC were run in a Shimadzu chromatographer with LC-10AD pump coupled to a SPD-M10A detector with photodiodo array. Shimpack prep ODS (4.6 X 250 mm, 7 mM) column was eluted with methanol 50% at 1mL/min flow rate. Each injection was run with 20 mL of extract in methanol solution (1mg/mL). Semi-preparative column was run on a Shodex column eluted with 100% methanol.
Structural elucidation: NMR1H and 13C, COSY, DEPT, HMQC and HMBC were run on a Brucker DX-400 spectrometer and samples dissolved in CD3OD. Mass spectra were obtained in a ThermoFinnigan quadrupolo ion trap mass spectrometer model LCQ Advantage with electrospray ion source at positive and negative mode.
RESULTS AND DISCUSSION
Solanum cernuum can be easily identified when entire leaves of the species are commercialized. The leaves are simple, oblong or elliptic, with entire or repand margin, unarmed, discolored, 21.0 to 32.6 cm long, 6.3 to 18.4 cm wide, obtuse or acute at the apex, asymmetric at the base, adaxial surface densely tomentose with prominent venation, abaxial surface sparsely tomentose, petiole 3.2 to 6.9 cm long, slightly winged.
However, when the leaves are not entire, other markers are necessary. In this regarding we also prospect anatomical and macromolecular markers.
Previous work on anatomy established that in Solanum the trichomes are very diverse in shape and size (Edmonds, 1982). Some of these structures are restrict to one species, and can be used to distinguish subgenera, sections (Seithe; Anderson 1982) and groups of Solanum (Mentz et al., 2000), while others are quite common (Siddiqi et al., 1980).
In this research, the trichomes of Solanum cernuum were nominated according to Roe (1971). No different trichomes from those already described for the genus was found. However, the results suggest that the set of trichomes described below are exclusive of S. cernuum and S. castaneum Carvalho (Carvalho, 1996). This set is composed by: glandular trichomes shortly pedicellate, unisseriated with four or more apical cells (found exclusively on leaf surfaces -Fig 1); porrect-stellate (leaf lamina, petiole, and fruit surface -Fig 2); stellate plurisseriated short pedicellate (most are brown, found at leaf lamina, petiole, and fruit surface -Fig 3 and 4); dendroid stelliform (stems, petioles, pedicels, and ovary - Fig 5); paleous (stems, pedicels and the external face of the calyx, and scarce in petioles - Fig 6 and 7). Carvalho (1996) defined this type of trichomes as emergencies, however Siddiqi et al. (1980) classified the paleous dentritic trichomes, not as emergencies, since they are constituted just of epidermal tissues, whereas emergencies are also formed of subepidermal tissues. In fact, a subepidermal layer was not observed in the paleous trichomes of S. cernuum (Fig 8).
In general, the trichomes of Solanum cernuum were found in great quantity in all the observed organs. On leaves, the stellate indument may be constituted of 1 to 3 strata in the abaxial surface. No trichomes were observed on stigmas, styles, filaments and anthers.
The sequencing of nucleotides of DNA has been proposed as a DNA barcode of the organisms (Blaxter, 2003). As macromolecular markers of S. cernuum we found that for DNA extraction the protocol CTAB was the most satisfactory. The nucleotides sequences obtained using PCR technique were confirmed as the target region (ITS1, 5.8S and ITS2) after comparison and alignment with the sequences of genes deposited at GenBank. Within these sequences we highlight the species Solanum exiguum (AY523893), with 36.4% of adenine and timine (AT) and Solanum diversifolium (AY523892), with 35.1% of AT. We found that in S. cernuum this region is constituted by approximately 600 bases pairs, being 48.1% AT. This sequence determined for S. cernuum was deposited at GenBank under the accession number DQ837371.
Besides the morphological, anatomical and macromolecular markers, we also prospected micromolecular markers that could be useful, also, if the plant is found as crude extract.
The crude extracts were obtained as described in the experimental section and yielded 4.4% to 10.5% (x = 7.53 and sx = 1.95) in relation to the plant powder. This result is not related to seasonality.
TLC analysis displayed several spots, after sprayed with Dragendorff reagent, from which four with Rf = 0.30; 0.46; 0.61; 0.66 (Fig 9). The first three were orange and the last one was gray. This profile can be used carefully as marker once all but one extract presented at least three of these spots.
HPLC chromatograms showed peaks at 4.00, 5.42, 6.59 and 9.01 min with variable areas. The chromatograms were different from those obtained from extracts of Solanum esculentum, Bauhinia curvula and Ocimum gratissimum. These species were chosen randomly as control once there is no report on literature of possible adulteration of S. cernuum. Using semi-prep HPLC followed by filtration on Sephadex LH-20 in methanol, the compounds with Rf = 0.61 and 0.66 where isolated (Fig 10). The analysis of their spectral data together with data published in literature (Matthes et al., 1980; Agrawal, 1989; Markham; Ternai, 1976) allowed us to identify them as quercitrin and afzelin, respectively. Flavonol glycosides are frequent in the genus Solanum, mostly derived from kampferol and quercetin (Silva et al., 2003). Quercitrin, a 3-O-a-L-rhamnospyranosil from quercetin was previously isolated from Solanum melongena (Barnabas; Nagarajan, 1989). This is the first report of the occurrence of afzelin in Solanaceae.
The whole leaves of Solanum cernuum can be identified using morphological data. When leaves are not entire, the presence of the set of trichomes described for this species can be used as anatomical markers. However, when the plant material is powdered, it is necessary to investigate the chromatography profile of its crude extract. Afzelin together with quercitrin were isolated as micromolecular markers. The eletrophoretic profile of the entire ITS regions of the rDNA of S. cernuum without a pre-treatment with restriction enzymes cannot be used as marker for the species. The determination of the sequence of nucleotides of the region ITS 1, 5.8S and ITS 2 of S. cernuum published here will support the development of RFLP conditions in order to obtain an eletrophoretic profile useful as macromolecular markers. These macromolecular markers should be used for S. cernuum identification when other markers would not be possible.
Fiocruz, Fapemig, CNPq and Capes for the financial support.
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