Análise quimiométrica usando RMN de 13C como ferramenta para diferenciar duas espécies de Maytenus

Artigo de Pesquisa

http://dx.doi.org/10.5935/2446-4775.20170003

Análise quimiométrica usando RMN de 13C como ferramenta para diferenciar duas espécies de Maytenus

Chemometric analysis using 13C NMR as a tool to differentiate two species of Maytenus

Autores:
1SOUSA, Grasiely F. de*;
2SILVA, Fernando C.;
3ALEME, Helga G.;
4MESSIAS, Maria Cristina T. B.;
4VIEIRA-FILHO, Sidney A.;
1DUARTE, Lucienir P.
Instituições
1Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
2Universidade do Estado de Minas Gerais, Belo Horizonte, MG, Brazil.
3Universidade Federal de São Paulo, São Paulo, SP, Brazil.
4Universidade Federal de Ouro Preto, Outo Preto, MG, Brazil.
*Correspondência:
grasiely_sousa@yahoo.com.br

Abstract

Maytenus robusta foi recentemente sinonimizada com Maytenus gonoclada devido a sobreposição de caracteres morfológicos utilizados na classificação botânica. No presente trabalho foram investigadas as diferenças químicas entre Maytenus gonoclada e Maytenus robusta usando análise quimiométrica. Os dados de RMN de 13C de triterpenos pentacíclicos obtidos a partir dessas espécies foram avaliados por meio da Análise de Componentes Principais (PCA). Os friedelanos foram os principais metabólitos que contribuíram para o reconhecimento de diferenças entre os táxons. Análises quimiométricas demonstram ser uma importante ferramenta em taxonomia de Celastraceae.

Palavras-chave:
Maytenus gonoclada.
Maytenus robusta.
Metabolomics.
Pentacyclic Triterpenes.
Chemotaxonomy.

Resumo

Varronia curassavica Jacq (=Cordia verbenacea DC), Cordiaceae, is an American species widely spread from Argentina to Mexico. The species is tradicionally used for medicinal purposes in inflamatory diseases and is raw material for the first Brazilian phytotherapic (Acheflan®, Aché). Here, we aimed to describe the morphoanatomy of V. curassavica, searching useful features to its identification. The species has craspedodromous venation and wavy to dentate leaf margins. The epidermis has one layer of cells, with two types of glandular stalked trichomes, one possessing a globular head and other a kidney-shaped head, and two types of non-glandular trichomes, one of them possessing cystolith. Leaf blade is hypostomatic. Mesophyll is dorsiventral. Glandular trichomes are possibly the best feature to identify the species, since we can use it to distinguish V. curassavica from two other species of Varronia.

Palavras-chave:
Maytenus gonoclada.
Maytenus robusta.
Metabolômica.
Triterpenos Pentacíclicos.
Quimiotaxonomia.

Introduction

Maytenus Molina sl. is the largest genus in the family Celastraceae, comprising about 300 species, widely distributed in the tropics and subtropics with 49 of them occurring in Brazil (MCKENNA et al., 2011). In several countries Maytenus species are routinely used in traditional medicine to treat a variety of illnesses, such as gastric disorders, rheumatism and diarrhoea (LEITE et al., 2001; SOSA et al., 2007). Phytochemical studies show that many of these species present bioactive metabolites, especially pentacyclic triterpenes (NIERO, ANDRADE and CECHINEL FILHO, 2011). The Maytenus genus has been chemically characterized by the production of terpenoids, especially dihydroagarofuran sesquiterpenes, triterpenes and some unusual constituents, such as dimers and trimers of sesquiterpenes, diterpenes and triterpenes (NIERO, ANDRADE and CECHINEL FILHO, 2011). Pentacyclic triterpenes such as friedelan-3-one and friedelan-3β-ol are considered taxonomic markers of Maytenus (OLIVEIRA et al., 2007).

Maytenus is a polyphyletic genus and additional research is important to better understand its taxonomy (JOFFILY and VIEIRA, 2005). For example, some species were synonymized with M. gonoclada, such as M. robusta Reissek(GROPPO and ERBERT, 2015) and M. salicifolia Reissek (GROPPO, 2009) because of their morphological diagnostic characteristics were overlapping, mainly about stem apex form, fruit shape and size. Biral and Lombardi (2012) considered M. gonoclada and M. robusta as synonymous because of the taxonomic distinction problems, but highlighted the need for more precise characteristics to establish this definition. On the other hand, Niero, Andrade and Cechinel Filho (2011) together with Carvalho-Okano (1992) pointed out that these species present relevant differences. M. gonoclada fruits are orbicular, smaller and its branches have quadrangular shape whereas M. robusta fruits are elliptical, bigger and its branches have a flattened-cylindrical shape. In addition, M. gonoclada is typical from highlands, occurring in campos rupestres or mountain forests, while M. robusta occurs in the Atlantic Lowland Rainforest (CARVALHO-OKANO, 1992; CARVALHO-OKANO and LEITÃO-FILHO, 2004).

Metabolomics represent an important tool to taxonomic classification. This technique is based on a qualitative and quantitative evaluation of a number of compounds in plants or other organisms. This tool utilizes chemical data from NMR, IR, and/or MS spectrometry in combination with multivariate analysis, to study the metabolome of live beings (MAULIDIANI et al., 2012). The Principal Component Analysis (PCA) is one of the chemometric methods routinely used. Its purpose is the data reduction from linear combinations of the original variables, what provides a better visualization of the results (MASSART, 1998).

PCA has been recognized as an important statistical tool and has been used to differentiate samples according to their chemical composition (MAULIDIANI et al., 2012). Cruz and coworkers (2008) used PCA to investigate the relationships between the families Celastraceae and Hippocrateaceae, using a large number of compounds together with botanical information. The results contributed to the inclusion of Hippocrateaceae in Celastraceae family. Maulidiani and coworkers (2012) studied the metabolites of three species of Apiaceae utilizing NMR spectral data and PCA. They differentiated the species Centella asiatica, Hydrocotyle bonariensis and H. ibthorpioides, showing the potential of multivariate data analysis.  In this context, the combination of NMR spectroscopy and PCA can provide more information to clarify the taxonomy of Maytenus species.

This study investigated the chemical differences between M. gonoclada Martius and M. robusta Reissek (synonymized with M. gonoclada Martius) (GROPPO, 2009) using combination of 13C NMR spectral data with PCA analysis.

Materials and Methods

A literature review was performed in order to obtain the 13C NMR chemical shifts of the triterpenes from leaves and branches of M. gonoclada and M. robusta (OLIVEIRA et al., 2007; SILVA et al., 2011a; SILVA et al., 2011b; SILVA et al., 2013, NIERO et al., 2006; SOUSA et al., 2012a; SOUSA et al., 2012b). The compounds (13 triterpenes for each species) were analyzed based on the chemical shifts of the carbon atoms C1 to C30, generating a matrix. These data were subjected to PCA using Matlab R2009b program (Math Works, Natick, MA, USA) together with PLS Toolbox 6.2 (Eigenvector Research, Wenatchee, WA, USA) for the chemometric treatment.

In order to obtain more accurate results, the matrix was re-evaluated and only the chemical shifts of triterpenes with the friedelane skeleton (9 of M. gonoclada and 10 of M. robusta) were selected. This new analysis was performed because the friedelanes provided more consistent data since their occurrence is more reported in both species than triterpenes with other skeletons. Then, the matrix was re-subjected to PCA.

Results and Discussion

To provide more information to support differences or similarities among the two species of Maytenus, a study was performed applying PCA to the 13C NMR data of triterpenes from leaves and branches of M. gonoclada and M. robusta. A number of 26 triterpenes were selected, 13 of each species (TABLE 1).  

TABLE 1: Triterpenes isolated from M. gonoclada and M. robusta, grouped in accordance with skeleton.
Triterpenes Pentacyclic Skeleton Species
MG MR
friedelan-3-one Friedelane + +
friedelan-3β-ol + +
friedelane-3,16-dione + +
29-hydroxyfriedelan-3-one + +
friedelane-3,11-dione + -
12α-hydroxyfriedelane-3,16-dione + -
friedelane-3,12-dione + -
12α-hydroxyfriedelan-3-one + -
12α,29-dihydroxyfriedelan-3-one + -
11β-hydroxyfriedelan-3-one - +
friedelane-3β,11β-diol - +
21α-hydroxyfriedelane-3,15-dione - +
friedelane-3,15-dione - +
3,4-seco-friedelan-3,11β-olide - +
3,4-seco-friedelan-3-oic acid - +
21β-H-hop-22(29)-en-3β-ol Hopane - +
3,4-seco-21β-H-hop-22(29)-en-3-oic acid - +
21β-H-hop-22(29)-en-3-one - +
Lupeol Lupane + -
α-amirine Ursane + -
β-amirine Oleanane + -
Taraxerol Taraxerane + -
MG = M. gonoclada; MR = M. robusta; (+) Isolated; (-) Not yet found in this species of Maytenus.

The triterpenes 13C NMR spectral data of both Maytenus species were obtained from the literature. Although the collected spectra were obtained with various solvents and NMR equipments, the differences did not invalidate the study because the chemical shift assignments of the analyzed triterpenes are well known. Firstly, the PCA analysis considered the data of the 26 triterpenes using the 13C NMR chemical shifts as variables. However, this analysis was unproductive due to the high number of scores that demonstrated inconsistencies in the results. This may be justified by the high skeleton variety of the compounds, as well as the low number of examples for each triterpene class except the friedelanes. Therefore, the PCA analysis was repeated employing the 13C NMR chemical shifts of the 19 friedelanes. Each carbon atom was considered a variable and projected in smaller dimensions through linear combinations. According to the results, only three major components comprised 92.0% of the total variance of the model after analysis of the 30 original variables. In other words, three components are sufficient to explain the difference between M. gonoclada and M. robusta. The principal components PC1, PC2 and PC3 showed a variance of 83.48%, 5.19% and 3.29%, respectively (FIGURE 1).

FIGURE 1: Principal Component Analysis (PCA) highlighting PC1, PC2 and PC3 to differentiate Maytenus gonoclada (A) and Maytenus robusta (B) according to the friedelanes 13C NMR data.
Figura 1
FIGURE 2: Loading plot of PC1, PC2 and PC3 of friedelanes from Maytenus gonoclada and Maytenus robusta.
Figura 2

The most important variables in the construction of PC1, PC2 and PC3 are connected to the carbon atoms 3, 16 and 12, respectively. Indeed, the friedelanes from M. gonoclada tend to present carbonyl groups at C3, C12 and C16 and hydroxyl groups at C3 and C16. On the other hand, M. robusta friedelanes tend to have predominantly carbonyl group at C15, hydroxyl groups at C11 and carbonyl, carboxyl and lactone groups at C3 (FIGURE 3).

Despite the morphological similarities and difficulties for the taxonomic distinction between M. gonoclada and M. robusta, differences in their chemical profile corroborate the separation of both species. Besides the friedelanes, the hopanes mark a distinction since this class of compounds is uncommon for Maytenus genus. Several Maytenus species are commonly used in folk medicine especially to treat gastric disorders and the imparity in their chemical constitutions should affect their therapeutic properties. The differences in Maytenus species chemical compounds can be exploited in the quality control of vegetable raw material intended for medicinal use.

FIGURE 3: Chemical structures of friedelanes from Maytenus gonoclada (MG) and Maytenus robusta (MR).
Figura 3

Conclusion

This study indicated chemical differences between M. gonoclada and M. robusta based on 13C NMR chemical shifts of friedelanes through PCA analysis. The obtained results corroborate the classification of M. gonoclada and M. robusta as being different species as proposed by Niero, Andrade and Cechinel Filho (2011) and Carvalho-Okano (1992).

Acknowledgments

The authors thank to Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support.

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Histórico do artigo

Submissão:
10/10/2016
Aceite:
12/07/2017
Publicação:
25/09/2017

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