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Extraction optimization, structural and thermal characterization of the antimicrobial abietane 7#-acetoxy-6#-hydroxyroyleanone Carlos E. S. Bernardes, Catarina Garcia, Filipe Pereira, Joana Mota, Paula C. Pereira, Maria-João Cebola, Catarina Pinto Reis, Isabel Correia, M. Fátima M. da Piedade, Manuel Eduardo Minas da Piedade, and Patricia Rijo Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00892 • Publication Date (Web): 01 Mar 2018 Downloaded from http://pubs.acs.org on March 2, 2018
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Molecular Pharmaceutics
Extraction optimization, structural and thermal characterization of the antimicrobial abietane 7α-acetoxy-6β-hydroxyroyleanone Carlos E. S. Bernardes a, Catarina Garcia b,c, Filipe Pereira b,c, Joana Motab, P. Pereira b,d, Maria J. Cebola b,e, Catarina P. Reis b, g, Isabel Correia,f M. Fátima M. Piedade a,f, Manuel E. Minas da Piedade a,*, Patrícia Rijo b, g
a
*
Centro de Química e Bioquímica e Departamento de Química e Bioquímica, Faculdade de Ciências,
Universidade de Lisboa, 1749-016 Lisboa, Portugal. b
Center for Research in Biosciences & Health Technologies (CBIOS), Universidade Lusófona de
Humanidades e Tecnologias, 1749-024 Lisboa, Portugal c
Department of Biomedical Sciences, Faculty of Pharmacy, University of Alcalá, Ctra. A2, Km 33.600 –
Campus Universitario, 28871 Alcalá de Henares, Spain d
Center for Natural Resources and Environment (CERENA), Instituto Superior Técnico (IST), Universidade
de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal e
Escola Superior Náutica Infante D. Henrique (ENIDH), Av. Eng. Boneville Franco, 2770-058 Paço de
Arcos, Portugal. f
Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa,1049-001 Lisboa, Portugal. g Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, 1649-003 Lisboa, Portugal
*Corresponding authors Patrícia Rijo, Food Sciences and Phytochemistry, CBIOS, Escola de Ciências e Tecnologias da Saúde, Universidade Lusófona de Humanidades e Tecnologias, Campo Grande, 376, 1749-024 Lisboa, Portugal Tel: +351 317 515 500; fax: +351 217 577 006 E-mail address:
[email protected] Manuel E. Minas da Piedade, Centro de Química e Bioquímica e Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal. Tel: +351-21-7500866; fax: +351-21-7500088 E-mail address:
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Table of contents/ Abstract Graphic Table of contents/ Abstract Graphic ................................................................................................................... 2 Abstract ............................................................................................................................................................... 3 Introduction......................................................................................................................................................... 4 Experimental Section .......................................................................................................................................... 5 Plant Material.................................................................................................................................................. 5 Chemicals ....................................................................................................................................................... 5 Extraction Methods ......................................................................................................................................... 6 Extraction Optimization: Quantification of AHR by HPLC-DAD ................................................................ 6 Preparation and Characterization of the 7α-Acetoxy-6β-hydroxyroyleanone Sample Used in the Structural and Thermal Studies ....................................................................................................................................... 7 Single Crystal X-ray Diffraction .................................................................................................................... 7 Differential Scanning Calorimetry (DSC) ...................................................................................................... 9 Results................................................................................................................................................................. 9 Extraction Optimization of 7α-Acetoxy-6β-hydroxyroyleanone (AHR) by HPLC-DAD ............................. 9 Single Crystal X-ray Diffraction (SCXRD) ................................................................................................. 10 Differential Scanning Calorimetry (DSC) .................................................................................................... 13 Discussion ......................................................................................................................................................... 15 Acknowledgement ............................................................................................................................................ 15 Supporting Information .................................................................................................................................... 16 References......................................................................................................................................................... 16
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Molecular Pharmaceutics
Abstract The abietane 7α-acetoxy-6β-hydroxyroyleanone (AHR), obtained from plant extracts, is an attractive lead for drug development, given its known antimicrobial properties. Two basic requirements to establish any compound as a new drug, are the development of a convenient extraction process and the characterization of its structural and thermal properties. In this work seven different methods were tested to optimize the extraction of AHR from Plectranthus grandidentatus. Supercritical fluid extraction (SFE) proved to be the method of choice, delivering an amount of AHR (57.351 µg⋅mg-1) approximately six times higher than the second best method (maceration in acetone; 9.77 µg⋅mg-1). Single crystal X-ray diffraction analysis of the ARH molecular and crystal structure carried out at 167±2 K and 296±2 K, showed only a single phase, here dubbed form III (orthorhombic space group P21212), at those temperatures. The presence of two other polymorphs above room temperature was, however, evidenced by differential scanning calorimetry (DSC). The three forms are enantiotropically related, with the Form III → Form II and Form II → Form I transitions occurring at 333.5±1.6 K and 352.0±1.6 K, respectively. The fact that the transitions are reversible, suggests that polymorphism is not likely to be an issue in the development pharmaceutical formulations based on ARH. DSC experiments also showed that the compound decomposes on melting at 500.8±0.8 K. Melting should therefore be avoided if, for example, strategies to improve solubility based on the production of glassy materials or solid dispersions are considered.
KEYWORDS: Royleanone, Supercritical Fluid Extraction, Antimicrobial, Single crystal X-ray diffraction, Differential Scanning Calorimetry
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Introduction The growing incidence of infectious diseases caused by bacterial pathogens resistant to multiple classes of antibiotics has developed into a serious health problem 1. This fostered the search for new antibacterial agents from natural resources 2, such as plant extracts, which have traditionally been used to treat human infections 3–5
. The biological activities described for royleanone-related diterpenoids are extensive and include
antimicrobial
properties6.
Previous
in
vitro antimicrobial
studies
showed
that
7α-acetoxy-6β-
hydroxyroyleanone (7α-acetoxy-6β,12-dihydroxy-abieta-8,12-diene-11,14-dione, AHR, Figure 1) obtained from Plectranthus species
7,8
exhibits better activity against specific strains of methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE) resistant Mycobacterium tuberculosis (MDR-TB)
11
9,10
, or multidrug-
, than some of the presently used reference drugs.
Besides antibacterial action, other interesting biological activities were evidenced by various works, such as proven cytotoxicity at low concentrations against different types of human cancer cell lines
12
and
immunomodulatory effects related to antiproliferation of human lymphocytes 13. All these results suggest that AHR can be an interesting lead for future drug development. When attempting to isolate a compound from a plant matrix, the choice of the extraction method is of the utmost importance to attain the highest possible yield. Available methods can be divided into three categories: (i) traditional methods, like aqueous infusions and decoctions; (ii) conventional solvent extraction methods, which make use of abundant quantities of organic solvents; and (iii) non-conventional methods, such as microwave and ultrasound techniques (which use water or an organic solvent as the extracting medium) or supercritical fluid extraction (SFE). The solvent in SFE is a substance that is gaseous at room temperature and pressure, and has enhanced solvent properties above the critical point (supercritical fluid state). A supercritical solvent can, therefore, easily release the extract by a simple pressure-temperature reduction. The supercritical solvent most often used is carbon dioxide which is largely abundant and can be
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Molecular Pharmaceutics
Figure 1. Molecular structure of 7α-acetoxy-6β-hydroxyroyleanone (AHR) obtained with high purity at a relatively low cost
14–16
. Carbon dioxide has several advantages for the
extraction of nutraceutical compounds: it is non-toxic, non-flammable, chemically inert, it has no smell or taste, and it has a low critical point (Tc = 304.13 K; pc = 7.38 MPa) 17.The latter facet is very important for extraction of thermolabile compounds. Furthermore, extracts obtained by SFE have been considered of superior quality in terms of reproducibility and for keeping flavors and aromas true to themselves, when compared to those obtained by conventional methods
18–22
. In this study, all types of methods mentioned
above were tested for the optimization of AHR extraction. Besides optimization of the extraction process from the selected natural resource, two critical aspects that need to be considered as early as possible when the development of a solid form based pharmaceutical product is in view are the structural and thermal characterization of the lead compound. This will allow the identification and control of phenomena such as polymorphism or chemical decomposition, which can have a strong impact in the production, shelf life, and safe use of a medicine. The characterization of the crystal structure and thermal behavior of AHR was, therefore, performed in this work by using single crystal X-ray diffraction and differential scanning calorimetry.
Experimental Section Plant Material P. grandidentatus Gürke was cultivated at the Faculty of Pharmacy Hortum, University of Lisbon, from seeds provided by the National Botanic Garden, Kirstenbosch, Claremont, South Africa. The aerial parts of this species were collected in July–October 2003, and voucher specimens (ref. C. Marques S/N° LISC) were deposited in the Herbarium of the ‘‘Instituto de Investigacão Científica Tropical’’, Lisbon. Previously to the extraction processes, the dried plant material was finely grinded using a grinder mill. The resultant powder was then used in the different extractions processes. Chemicals HPLC grade water, acetone, methanol and trifluoroacetic acid were obtained from Merck (Darmstadt, Germany). CDCl3 from Aldrich (99.80%,
