Review 763 Quality Control of Herbal Material and Phytopharmaceuticals with MS and NMR Based Metabolic Fingerprinting Authors Frank van der Kooy, Federica Maltese, Young Hae Choi, Hye Kyong Kim, Robert Verpoorte Affiliation Division of Pharmacognosy, Section of Metabolomics, Institute of Biology, Leiden University, Leiden, The Netherlands Key words Abstract areas of research such as drug discovery from nat- l" metabolic fingerprinting ! ural resources, quality control of herbal material, l" NMR Metabolic fingerprinting techniques have re- and discovering lead compounds. In this review l" MS ceived a lot of attention in recent years and the the current state of the art of metabolic finger- l" medicinal plants annual amount of publications in this field has in- printing, focussing on NMR and MS technologies l" herbal plant preparations creased significantly over the past decade. This in- will be discussed. The application of these two an- crease in publications is due to improvements in alytical tools in the quality control of herbal mate- the analytical performance, most notably in the rial and phytopharmaceuticals forms the major field of NMR and MS analysis, and the increased part of this review. Finally we will look at the fu- awareness of the different applications of this ture developments and perspectives of these two growing field. Metabolomic fingerprinting or technologies in the quality control of herbal ma- profiling is continuously being applied to new terial. Introduction These terms, notably metabolic profiling, meta- ! bolic fingerprinting and metabolomics are some- Plants have been used throughout history as the times used interchangeably. It is not our intention primary source of food, timber, fuel, and medi- to give a formal definition of metabolomics. For a cine, among other purposes. Due to their amazing formal definition and discussion [3,4] should be ability to produce a vast structural array of chem- consulted. A very simplistic view is that metabol- ical components, of which only relatively few omics can be seen in two different ways. Firstly received July 30, 2008 have been identified, plants are one of the most there is the microscopic view (targeted approach) revised January 19, 2009 important sources for the development of new which looks at a specific set of compounds (e.g., accepted February 2, 2009 drugs. Today plants, plant extracts and plant-de- phenolics or terpenoids, etc). This approach is al- Bibliography rived components are being used in a multitude so called metabolic fingerprinting or metabolic DOI 10.1055/s-0029-1185450 of herbal remedies and phytopharmaceuticals. profiling. The second view is the macroscopic Published online March 13, Well-known plants which are used to treat specif- view (untargeted approach) which aims at identi- This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. 2009 ic diseases include Artemisia annua (to treat un- fying and quantifying all the metabolites present Planta Med 2009; 75: 763–775 © Georg Thieme Verlag KG complicated malaria) [1] and the female flower- in a specific organism [3]. Metabolomics is there- Stuttgart · New York · ing tops of Cannabis sativa (to relieve pain and as fore a “systematic study of the unique chemical ISSN 0032‑0943 an appetite enhancer) [2]. A multitude of exam- fingerprints that specific cellular processes leave ”– Correspondence ples exists for the use of plant extracts to treat a behind and, more specifically, the study of their Dr. Frank van der Kooy large array of diseases and the WHO estimates small-molecule metabolite profiles [5]. The me- Division of Pharmacognosy that about 80% of the world population uses me- tabolome represents the collection of all metabo- Section of Metabolomics Institute of Biology dicinal plants as their primary source of medica- lites in a biological organism, which are the end Leiden University tion. Many well-known drugs have also been iso- products of its gene expression. Thus, while Einsteinweg 55 lated from plants including artemisinin from mRNA gene expression data and proteomic anal- PO Box 9502 2300RA Leiden A. annua, paclitaxel from Taxus brevifolia, vinblas- yses do not tell the whole story of what might be Netherlands tine and vincristine from Catharanthus roseus and happening in a cell, metabolic fingerprinting can Phone: + 31715274471 galanthamine from the Amaryllidaceae family. give an instantaneous snapshot of the physiology Fax: + 317 15274511 frank.vanderkooy@ In the literature three terms are used to describe of that cell. One of the challenges of systems biol- chem.leidenuniv.nl the analysis of metabolites produced by plants. ogy is to integrate proteomic, transcriptomic, and van der Kooy F et al. Quality Control of… Planta Med 2009; 75: 763–775 764 Review metabolomic information to give a more complete picture of liv- cules containing NMR-active nuclei, unlike MS where detection ing organisms. of analytes is influenced by selective ionization or ultraviolet The analytical tools used to study the metabolome include spectrometers where only chromophore-bearing compounds mainly chromatography and spectroscopy. Chromatography is are detected. For all proton-bearing molecules, the intensity of used to separate the metabolites which are then being quantified all proton signals is absolutely proportional to the molar concen- and identified with the use of various detectors. The two main tration of the metabolite. Using a proper internal standard, the chromatography systems employed in metabolic fingerprinting real concentration of metabolites can be easily calculated [11]. are gas chromatography (GC) and high-performance liquid chro- Because NMR spectroscopy is based on the physical characteris- matography (HPLC). The detectors used in these chromatography tics of compounds, it has a very high reproducibility. systems depend largely on the type of components to be detected In metabolomics, the detection of metabolites is not the final and quantified. For a targeted approach, mass spectroscopy (MS) step, but all data obtained from the analytical methods should is usually employed as the detector, while nuclear magnetic res- be further analyzed by statistical methods in order to extract all onance (NMR) and MS are used without the chromatography possible information from the data. The accuracy and correctness step as an untargeted analytical tool. In the 1980s the first re- of the data to be further analyzed by the statistical methods are ports on the use of NMR for fingerprinting of plant extracts were inevitably reliant on the robustness of the raw analytical data reported, e.g., Kubeczka and Formácek published a book [6] with set. In this aspect, NMR has a unique advantage, the highest reli- 13C‑NMR fingerprints of essential oils and Schripsema and Ver- ability in metabolomics. Unlike the retention time in chromatog- poorte applied 1H‑NMR for characterizing plant cell cultures and raphy-based techniques, with a few exceptions the chemical quantitative analysis of sugars in the cell culture extracts [7]. But, shift, coupling constant, and integral of each signal in an NMR particularly in the past decade, metabolomics is rapidly develop- spectrum does not change as long as it is measured under the ing for studying plants and quality control of botanicals. The use same conditions: the field strength applied, solvent, pH, and the of these two analytical tools (MS and NMR) will be discussed in temperature. Despite the low intrinsic sensitivity, the robustness more detail below as well as the applications in the quality con- of data and ability to cover a broad range of metabolites, has en- trol of herbal material and the perspectives of these methods. abled NMR to be the favored overall “macroscopic” metabolomics and fingerprinting tool. In addition to the advantages of data ro- bustness, the power of NMR in structure elucidation of metabo- Metabolic Fingerprinting using NMR lites cannot be matched by any other method. NMR has a long ! history in the natural products chemistry field as the tool for Recent advances in analytical chemistry, combined with multi- structural elucidation. With a proper database, it can generate variate data analysis, have brought us closer to the final goal of data which can be kept almost permanently [13]. In this part, metabolomics, comprehensive evaluation of all metabolites, both the general procedure of NMR metabolomic analysis will be dis- quantitatively and qualitatively in living organisms. Among many cussed together with multivariate data analysis. The identifica- different technological platforms, NMR and MS have been suc- tion of metabolites is of high importance and this aspect will be cessfully used for metabolic fingerprinting analysis. These two highlighted. At the end of this section specific applications will be techniques have their respective advantages and limitations, and discussed using NMR as a metabolic fingerprinting technique. are often discussed as being complementary [8,9]. However, as a tool for metabolomics, NMR has some unique advantages over General procedures of NMR-based metabolomics MS-based methods. It can provide a detailed analysis on the bio- NMR-based metabolomics, like other methods using MS or chro- moleculear composition very quickly with relatively simple sam- matography, include sample preparation, extraction, multivari- ple preparation [10–12]. It is a universal detector for all mole- ate data analysis, and identification of metabolites. l" Fig. 1 shows Fig.