Non-Targeted Electrospray Mass Spectrometry-Based Metabolic Fingerprinting and PLS-DA-Based Classification of Brazilian “Malvas”

http://dx.doi.org/10.5935/0103-5053.20140288 J. Braz. Chem. Soc., Vol. 26, No. 2, 365-372, 2015. Printed in Brazil - ©2015 Sociedade Brasileira de Química 0103 - 5053 $6.00+0.00 Article Non-Targeted Electrospray Mass Spectrometry-Based Metabolic Fingerprinting and PLS-DA-Based Classification of Brazilian “Malvas” Cleverson A. F. Martins,a Mário S. Piantavini,a Rômulo P. Ribeiro,a Erika Amano,b Bruna V. Dal Práb and Roberto Pontarolo*,a Departamento de Farmácia, Universidade Federal do Paraná, 81531-990 Curitiba-PR, Brazil a Departamento de Botânica, Universidade Federal do Paraná, 80210-170 Curitiba-PR, Brazil b Malva sylvestris, popularly known as “malva” in Brazil, is a medicinal plant used for its antiinflammatory effects. However, multiple plants are known as “malvas” in Brazil; these species include Sida cordifolia (“malva-branca”) and Pelargonium graveolens (“malva-cheirosa”). In this work, electrospray mass spectrometry metabolic fingerprints from these species were obtained by direct infusion of extracts of commercial samples, purchased as crushed dried leaves. The spectral data were used to classify these species using a partial least squares discriminant analysis method. The identities of all samples were confirmed by morphological analyses, emphasizing the trichomes morphology. While over 80% of the samples were labeled as M. sylvestris or “malva”, in the present study only 37% of these samples were classified as M. sylvestris species. Approximately 50% of these samples were classified as S. cordifolia, whose consumption may be dangerous for patients with cardiovascular diseases. Thus, these analytical and chemometric procedures worked as a fast and simple method for classifying species of “malvas”. Keywords: Malva sylvestris, Sida cordifolia, Pelargonium graveolens, ESIMS, PLS-DA Introduction Traditionally, several medicinal plants have been used for the treatment and management of various inflammatory conditions. Among these plant species, Malva sylvestris L. Malvaceae has been used medicinally throughout the world since 3000 B.C.1 M. sylvestris is effective against mouth and throat diseases and can reduce and relieve swelling, toothaches, thrush, gum inflammation and periodontitis.2,3 Although it is a non-native plant, the Brazilian Public Health System promotes the use of M. sylvestris (popularly known as “malva”) by encouraging the planting, marketing, dispensing and consumption of this species.4 Furthermore, both the macro and microscopic characteristics of this plant are described in the Brazilian Pharmacopoeia.5 However, according to the local name, approximately 21 additional medicinal plants are also known as “malvas”. In addition to M. sylvestris, the species Sida cordifolia L. Malvaceae (“malva-branca”) and Pelargonium graveolens L’Hér Geraniaceae (“malva-cheirosa”) are widely used and possess antiinflammatory properties. Thus, due to similarities and ambiguities in nomenclature, it is necessary *e-mail: to develop accurate identification methods to prevent multiple distinct species from being miscategorized as M. sylvestris. Additionally, the commercially available forms of these plants are limited, consisting of either crushed leaves or powders, which complicates the use of morphological evaluation as a means of distinguishing among species. S. cordifolia, a plant common to several regions of the country,6,7 is a species rich in sympathomimetic amines such as ephedrine and pseudoephedrine, and alkaloids with cardiovascular effects such as vasicine and vasocinone. Thus, the consumption of this plant can be dangerous in patients with central nervous system diseases, including anxiety and behavioral disorders6,8 and can lead to serious cardiovascular problems, like heart attacks and strokes.7,8 P. graveolens is also well acclimatized throughout Brazil, but despite its antibacterial, antifungal, antioxidant and anti-diabetic effects are well established, it is mainly used to treat stomach and bladder inflammatory disorders.9 Despite being a laborious and time-consuming operation, the morphological identification of raw materials is one of the first steps in establishing quality control of medicinal plants. However, it is important to use complementary methods to ensure the correct identification of each species. A fast, versatile, sensitive, low solvent-consuming and high A 366 Non-Targeted Electrospray Mass Spectrometry-Based Metabolic Fingerprinting J. Braz. Chem. Soc. throughput alternative to morphological identification is electrospray mass spectrometry (ESIMS) with direct sample injection. By analyzing the unique mass fingerprint of each sample, this technique provides rapid characterization of complex phytochemical matrices without the requirement of time-consuming sample preparation steps or prior chromatographic separation. When used in combination with chemometric analyses, ESIMS-based fingerprinting can be a useful method for species recognition.10-12 Among existing supervised pattern recognition techniques, partial least squares-discriminant analysis (PLS-DA) has been successfully employed to classify samples. The PLS-DA classification is achieved by correlating matrices containing instrumental variable dataset and class affiliations of each sample, and decomposing the two matrices using an algorithm to obtain maximum separation between classes.11 PLS-DA classification coupled with different analytical methods can be useful for quality control purposes of herbal products, such as storage duration and stability prediction,13 distiction of herbs collected from different geographical regions14 and identification of genuine and fake products.15 The aim of this study was to use an ESIMS-based metabolic fingerprinting method alongside a PLS‑DA‑based supervised pattern recognition method to classify commercial samples of “malvas” and to distinguish canonical M. sylvestris from other similar species. was collected in Curitiba, Paraná (25°18’45.87”S; 49°0’29.48”W) in April 2013. Commercial samples (dried leaves) of “malvas” (n = 60) were purchased in 2012-2013 from different suppliers (herb stores and public markets) in bulk and industrialized bags. These samples were originally labeled as M. sylvestris or “malva” (n = 49), S. cordifolia or “malva-branca” (n = 7) and P. graveolens or “malva-cheirosa” (n = 4). All samples were analyzed prior to their expiration dates. Once purchased, no additional drying was performed to preserve the original characteristics and chemical composition. All samples were maintained at a controlled temperature (20.0 ± 0.2 °C) and humidity (45-55%). Experimental Mass spectrometry experiments were performed on an Applied Biosystems MDS Sciex API 3200 Triple Quadrupole Mass Spectrometer (Toronto, Canada) equipped with an ESI source. A Harvard 22 Dual Model syringe pump (Harvard Apparatus, South Natick, USA) with a flow rate of 10 μL min-1 was used for direct infusion of samples into the mass spectromet (...truncated)