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Assessment of Anti-Inflammatory Properties Of Talanta 175 (2017) 264–272 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta fl Assessment of anti-in ammatory properties of extracts from Honeysuckle MARK (Lonicera sp. L., Caprifoliaceae) by ATR-FTIR spectroscopy ⁎ R. Nikzad-Langerodia,d, , S. Ortmannb, E.M. Pferschy-Wenzigb, V. Bochkovb, Y.M. Zhaoc, J.H. Miaoc, J. Saukela, A. Ladurnera, E.H. Heissa, V.M. Dirscha, R. Bauerb, A.G. Atanasova,e a Department of Pharmacognosy, University of Vienna, Althanstrasse 9, 1090 Vienna, Austria b Institute of Pharmaceutical Sciences/Pharmacognosy, University of Graz, Graz, Austria c Guangxi Botanical Garden of Medicinal Plants, 189 Changgang Road, Nanning, China d Department of Knowledge-Based Mathematical Systems, Johannes Kepler University Linz, Altenbergerstrasse 69, 4040 Linz, Austria e Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, 05-552 Jastrzebiec, Poland ARTICLE INFO ABSTRACT Keywords: Inflammation is a hallmark of some of today's most life-threatening diseases such as arteriosclerosis, cancer, ATR-FTIR spectroscopy diabetes and Alzheimer's disease. Herbal medicines (HMs) are re-emerging resources in the fight against these Traditional Chinese medicine conditions and for many of them, anti-inflammatory activity has been demonstrated. However, several aspects fl Anti-in ammatory activity of HMs such as their multi-component character, natural variability and pharmacodynamic interactions (e.g. Plant quality control synergism) hamper identification of their bioactive constituents and thus the development of appropriate Chemometrics quality control (QC) workflows. In this study, we investigated the potential use of Attenuated Total Reflectance Cell-based assays Fourier Transform Infrared (ATR-FTIR) spectroscopy as a tool to rapidly and non-destructively assess different anti-inflammatory properties of ethanolic extracts from various species of the Genus Lonicera (Caprifoliaceae). Reference measurements for multivariate calibration comprised in vitro bioactivity of crude extracts towards four key players of inflammation: Nitric oxide (NO), interleukin 8 (IL-8), peroxisome proliferator-activated receptor βδ/ (PPAR βδ/ ), and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB). Multivariate analysis of variance (MANOVA) revealed a statistically significant, quantitative pattern-activity relationship between the extracts' ATR-FTIR spectra and their ability to modulate these targets in the corresponding cell models. Ensemble orthogonal partial least squares (OPLS) discriminant models were established for the identification of extracts exhibiting high and low activity with respect to their potential to suppress NO and IL-8 production. Predictions made on an independent test set revealed good generalizability of the models with overall sensitivity and specificity of 80% and 100%, respectively. Partial least squares (PLS) regression models were successfully established to predict the extracts' ability to suppress NO production and NF-κB activity with root mean squared errors of cross-validation (RMSECV) of 8.7% and 0.05-fold activity, respectively. 1. Introduction which can be isolated, characterized and routinely quantified by an appropriate analytical method [6]. More frequently however, bioactiv- Many of today's most life-threatening diseases such as arterio- ity is brought about by several structurally related molecules with the sclerosis, cancer, diabetes and Alzheimer's disease involve persistent same mode of action or by the simultaneous action of various inflammatory processes [1–3]. Anti-inflammatory activity has been molecules on different cellular targets [7] (which could lead e.g., to shown for a plethora of plant-derived products. Herbal medicines synergism). Furthermore, bioactive molecules might become unstable (HMs) thus represent a valuable resource in the fight against these upon isolation due to oxidative degradation or other chemical mod- conditions [4,5]. However, HMs are complex cocktails that pose unique ifications as matrix integrity is lost, hampering their quantitative challenges to drug discovery, analytical chemistry, quality control (QC), recovery and analysis [8]. Vibrational spectroscopy has been an product standardization and optimization. Ideally, bioactivity is di- attractive analytical tool for non-destructive material testing and is rectly associated with the presence and amount of a single substance particularly well suited for the analysis of HMs and dietary products in ⁎ Corresponding author at: Department of Pharmacognosy, University of Vienna, Althanstrasse 9, 1090 Vienna, Austria. E-mail address: [email protected] (R. Nikzad-Langerodi). http://dx.doi.org/10.1016/j.talanta.2017.07.045 Received 12 April 2017; Received in revised form 11 July 2017; Accepted 14 July 2017 Available online 18 July 2017 0039-9140/ © 2017 Elsevier B.V. All rights reserved. R. Nikzad-Langerodi et al. Talanta 175 (2017) 264–272 the QC context owing to the possibility to comprehensively map their [24], and MrBayes version 3.0b4 [25]. The individual bootstrap (BS) complex chemical makeup to a unique fingerprint [9]. Infrared (IR) consensus trees were visually inspected on a node to node basis to test and Raman spectroscopy have been successfully used in the past to the congruence among the individual DNA datasets by identifying authenticate HMs [10,11], detect adulterants [12,13] and to quantify contradictory nodes with >70% BS support. The collected Lonicera single substances [14] and substance classes [15,16] within plant samples could be assigned to the following 8 species: Lonicera matrices. Due to highly overlapping information present in spectro- japonica Thunberg (3 accessions, 1 comprising flower buds instead scopic data, method development usually involves a pattern recogni- of leaves), Lonicera hypoglauca Miquel (5 accessions), Lonicera tion step that acts as a filter to identify those parts of the spectrum that confusa Candolle (7 accessions), Lonicera macrantha (D.Don) are related to a given property (e.g. quantity of a substance). Sprengel (10 accessions), Lonicera similis Hemsley (4 accession), Establishing a calibration model then amounts to regressing the Lonicera acuminata Wallich (4 accessions), Lonicera reticulata property of interest against these patterns using a set of samples for Champion (2 accessions) and Lonicera bournei Hemsley (1 accession). which the property has been measured by a reference method. Typical One sample could not be identified. Voucher specimens of all plant reference methods that are widely used in QC are liquid- and gas samples are deposited in the Herbarium of the Guangxi Botanical chromatography. Alternatively, methods that directly link chemical Garden of Medicinal Plants. A list of the used plant samples can be composition to (bio-)chemical activity, like for instance anti-oxidant found in the supporting information (Table S-1). For the preparation of capacity, have been developed and proven useful when bioactivity is extracts, dried leaves were used for all samples except sample Nr. 35, mediated by multiple components with similar physicochemical prop- where flower buds of L. japonica were used for extraction. Extracts erties [17] (e.g. flavonoids). Several authors have used radical scaven- were prepared using 96% ethanol as extraction solvent by accelerated ging assays in combination with IR spectroscopy to build calibration solvent extraction (ASE), using an ASE 200 instrument (Dionex, models for direct determination of total anti-oxidant capacity, which is Sunnyvale, CA, USA). 1 g of dried, powdered plant material was mixed an important quality parameter of several dietary products. For a with pelletized diatomaceous earth (ASE Prep DE, Dionex) and filled concise overview on the topic, we refer to Lu et Rasco [17]. Although into the extraction cells. The following extraction parameters were cheap and reliable, such assays can neither capture synergistic effects used: pressure 68 bar; temperature 80°C; cell preheating 1 min; cell nor account for physiological aspects, e.g., related to uptake and heating 5 min; static extraction 5 min; flush 150%; purge 60 s; cycles 3. metabolism in vivo raising questions concerning their biological The extraction solvent was removed in a CentriVap Benchtop relevance [18]. Cell-based assays, on the other hand, continue being Centrifugal Vacuum Concentrator (Labconco, Kansas City, MO, USA) indispensable tools for drug discovery and are more physiologically under vacuum at a temperature of 40 °C. All extractions were relevant models for pharmacological activity studies, but their routine performed in duplicate except for sample Nr. 34 which could be use for monitoring the quality of HMs and dietary products in terms of extracted only once due to scarcity of plant material, resulting in a bioactivity/toxicity has been limited due to high costs [19]. In the total number of 71 extracts. The dried extracts were finally dissolved in present study, we investigated the application of different cell-based DMSO (at 20mgml−1), aliquoted, and kept at −20 °C until further use. assays for anti-inflammatory activity of crude medicinal plant extracts as a reference method for spectroscopic calibration. Four different cell 2.2. Cell-based in vitro assays models were used to quantitatively measure the anti-inflammatory activity of 71 extracts from different, closely related species of the 2.2.1. NF-kB transactivation assay Genus Lonicera L. (Caprifoliaceae) with respect to suppression
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