Extraction of Alkaloids for NMR-Based Profiling: Exploratory Analysis of an Archaic Cinchona Bark Collection

Abstract

Introduction !The crude drug cinchona (Cinchonae cortex) used against malaria is known since the colonization of South America in the beginning of the 17th century. The antimalarial effect is mainly due to the content of quinine (1), which interferes with the development of the Plasmodium parasites in the red blood cells [1]. Cinchona also contains the likewise active analogue alkaloids quinidine (2), cinchonine (3), and cinchonidine (4) (l " Fig. 1) [1][2][3]. The most abundant alkaloid is quinine that accounts for 70-90 % of the total alkaloid content which can be as high as 25 % of the dry weight [4]. Even though there are effective artemisinin-based combination therapies against malaria, there is still an interest in using the bark of Cinchona species (Rubiaceae) for reasons of availability and evolved malaria drug resistance [5,6]. As a result of the economical values of cinchona during the 19th century, plantations were spread to different tropical regions such as Indonesia and India. Different species and hybrids were tested for production of the drug in order to maximize the yield of quinine. During this period, samples of Cinchonae cortex were collected from different parts of the world and eventually ended up in a collection hosted by the Museum of Natural Medicine at the University of Copenhagen. A profiling study of this archaic collection, focused on alkaloids, could possibly indicate species that were not selected due to the quinine content but to other types of antimalarial alkaloids. Since the samples are collected over a long time period, from many different sites, and for many different reasons, we expect a large variation of types and amounts of alkaloids. Profiling of extracted alkaloids might indicate odd samples that contain other types of alkaloids than the expected and so allows finding out about the possible variations and combinations of the known and major alkaloids. NMR is a rational choice of analytical platform for this study. It is rapid, robust and gives reproducible results [7,8]. NMR also gives different spectra of diastereomers, and this makes it possible to differentiate between the main alkaloids. However, the traditional liquid-liquid extraction to separate alkaloids based on their basic properties is not practical for a profiling study in terms of sample throughput and potential problems with robustness. In this study, we present the use of a direct Abstract ! A museum collection of Cinchonae cortex samples (n = 117), from the period 1850-1950, was extracted with a mixture of chloroform-d 1 , methanol-d 4 , water-d 2 , and perchloric acid in the ratios 5 : 5 : 1 : 1. The extracts were directly analyzed using 1 H NMR spectroscopy (600 MHz) and the spectra evaluated using principal component analysis (PCA) and total statistical correlation spectroscopy (STOCSY). A new method called STOCSY-CA, where CA stands for component analysis, is described, and an analysis using this method is presented. It was found that the samples had a rather homogenous content of the well-known cinc...

NMR-Based Metabolic Profiling of Edible Olives—Determination of Quality Parameters

“…To our knowledge, STOCSY has been, up till now, used only in the study of some medicinal plants like Paeonia mascula [ 44 ], Cinchona sp. [ 45 ], Isatis tinctorial [ 46 ] and Keetia sp. [ 47 ], and a couple of other cases [ 48 , 49 , 50 ], while there is a recent study by Kew et al involving Scotch whisky [ 51 ].…”

Hundred Fifty Years of Herbarium Collections Provide a Reliable Resource of Volatile Terpenoid Profiles Showing Strong Species Effect in Four Medicinal Species of Salvia Across the Mediterranean

“…Likewise, herbarium materials could be a resource of chemical data potentially relevant for chemotaxonomy (Cook et al, 2009; Yilmaz et al, 2012), chemical ecology (Zangerl and Berenbaum, 2005), environmental bioindicators (Foan et al, 2010; Monforte et al, 2015) as well as drug discovery and authentication (Saslis-Lagoudakis et al, 2015; Rønsted et al, 2017).…”

“…However, the exploration of herbarium material for plant metabolite data is comparably understudied and the reliability of chemical data from herbarium samples is uncertain due to the expectation that plant specialized metabolites may not be well-preserved over longer time scales. Whereas alkaloids for example are generally considered highly stable (Cook et al, 2009; Dewick, 2009; Yilmaz et al, 2012), low molecular weight terpenoids tend to be both volatile and thermolabile and may be easily oxidized or hydrolyzed altering the chemical composition of the plant material dependent on the conditions during processing and storage of the plant material (Turek and Stinzing, 2013; Tasca et al, 2018).…”

“…Using proton nuclear magnetic resonance ( 1H NMR) spectroscopic metabolite screening, Jafari et al (2018) found no significant difference in metabolite profiles of recently dried and up to 25 year old fungi specimens. Cook et al (2009) found no age effect on toxic diterpenoid alkaloid composition of Delphinium occidentale S. Watson from up to 100 year old herbarium specimens and Yilmaz et al (2012) found no significant degradation of quinine alkaloids in 50–150 years old historical Cinchonae bark samples. Likewise, a study by Zangerl and Berenbaum (2005) found no sampling age effect on furanocoumarins in the phototoxic invasive Pastinaca sativa L. over 150 years.…”

An Optimised Method for Routine Separation and Quantification of Major Alkaloids in Cortex Cinchona by HPLC Coupled with UV and Fluorescence Detection

“…In most of these studies focus has been on the separation of the four major alkaloids but as more than 30 different alkaloids have been identified from Cinchona species an efficient separation also including gradient elution is necessary in order to obtain the full pattern of constituents in the plant samples (Kaufman and Rúveda, ; Kacprzak, ). In addition to the use for authentication of commercial barks, efficient separation and quantification of quinine alkaloids in Cinchona barks can also aid the study of archaic museum materials (Yilmaz et al , ) or the study of chemical diversity of modern samples (Kacprzak, ; Okunade et al , ; Ruiz‐Mesia et al , ).…”