Tanja Gödecke

Quantitative 1H NMR. Development and potential of an analytical method: an update.

Covering the literature from mid-2004 until the end of 2011, this review continues a previous literature overview on quantitative (1)H NMR (qHNMR) methodology and its applications in the analysis of natural products. Among the foremost advantages of qHNMR is its accurate function with external calibration, the lack of any requirement for identical reference materials, a high precision and accuracy when properly validated, and an ability to quantitate multiple analytes simultaneously. As a result of the inclusion of over 170 new references, this updated review summarizes a wealth of detailed experiential evidence and newly developed methodology that supports qHNMR as a valuable and unbiased analytical tool for natural product and other areas of research.

Importance of Purity Evaluation and the Potential of Quantitative 1H NMR as a Purity Assay

In any biomedical and chemical context, a truthful description of chemical constitution requires coverage of both structure and purity. This qualification affects all drug molecules, regardless of development stage (early discovery to approved drug) and source (natural product or synthetic). Purity assessment is particularly critical in discovery programs and whenever chemistry is linked with biological and/or therapeutic outcome. Compared with chromatography and elemental analysis, quantitative NMR (qNMR) uses nearly universal detection and provides a versatile and orthogonal means of purity evaluation. Absolute qNMR with flexible calibration captures analytes that frequently escape detection (water, sorbents). Widely accepted structural NMR workflows require minimal or no adjustments to become practical 1H qNMR (qHNMR) procedures with simultaneous qualitative and (absolute) quantitative capability. This study reviews underlying concepts, provides a framework for standard qHNMR purity assays, and shows how adequate accuracy and precision are achieved for the intended use of the material.

In vitro Serotonergic Activity of Black Cohosh and Identification of Nω-Methylserotonin as a Potential Active Constituent

Cimicifuga racemosa (L.) Nutt. (syn. Actaea racemosa L., black cohosh) is used to relieve menopausal hot flashes, although clinical studies have provided conflicting data, and the active constituent(s) and mechanism(s) of action remain unknown. Because serotonergic receptors and transporters are involved with thermoregulation, black cohosh and its phytoconstituents were evaluated for serotonergic activity using 5-HT7 receptor binding, cAMP induction, and serotonin selective re-uptake inhibitor (SSRI) assays. Crude extracts displayed 5-HT7 receptor binding activity and induced cAMP production. Fractionation of the methanol extract led to isolation of phenolic acids and identification of N(omega)-methylserotonin by LC-MS/MS. Cimicifuga triterpenoids and phenolic acids bound weakly to the 5-HT7 receptor with no cAMP or SSRI activity. In contrast, N(omega)-methylserotonin showed 5-HT7 receptor binding (IC50 = 23 pM), induced cAMP (EC50 = 22 nM), and blocked serotonin re-uptake (IC50 = 490 nM). These data suggest N(omega)-methylserotonin may be responsible for the serotonergic activity of black cohosh.

The Tandem of Full Spin Analysis and qHNMR for the Quality Control of Botanicals Exemplified with Ginkgo biloba

Botanical dietary supplements and herbal remedies are widely used for health promotion and disease prevention. Due to the high chemical complexity of these natural products, it is essential to develop new analytical strategies to guarantee their quality and consistency. In particular, the precise characterization of multiple botanical markers remains a challenge. This study demonstrates how a combination of computer-aided spectral analysis and 1D quantitative ¹H NMR spectroscopy (qHNMR) generates the analytical foundation for innovative means of simultaneously identifying and quantifying botanical markers in complex mixtures. First, comprehensive ¹H NMR profiles (fingerprints) of selected botanical markers were generated via ¹H iterative full spin analysis (HiFSA) with PERCH. Next, the ¹H fingerprints were used to assign specific ¹H resonances in the NMR spectra of reference materials, enriched fractions, and crude extracts of Ginkgo biloba leaves. These ¹H fingerprints were then used to verify the assignments by 2D NMR. Subsequently, a complete purity and composition assessment by means of 1D qHNMR was conducted. As its major strengths, this tandem approach enables the simultaneous quantification of multiple constituents without the need for identical reference materials, the semiquantitative determination of particular subclasses of components, and the detection of impurities and adulterants.

Essential Parameters for Structural Analysis and Dereplication by 1H NMR Spectroscopy

The present study demonstrates the importance of adequate precision when reporting the δ and J parameters of frequency domain 1H NMR (HNMR) data. Using a variety of structural classes (terpenoids, phenolics, alkaloids) from different taxa (plants, cyanobacteria), this study develops rationales that explain the importance of enhanced precision in NMR spectroscopic analysis and rationalizes the need for reporting Δδ and ΔJ values at the 0.1–1 ppb and 10 mHz level, respectively. Spectral simulations paired with iteration are shown to be essential tools for complete spectral interpretation, adequate precision, and unambiguous HNMR-driven dereplication and metabolomic analysis. The broader applicability of the recommendation relates to the physicochemical properties of hydrogen (1H) and its ubiquity in organic molecules, making HNMR spectra an integral component of structure elucidation and verification. Regardless of origin or molecular weight, the HNMR spectrum of a compound can be very complex and encode a wealth of structural information that is often obscured by limited spectral dispersion and the occurrence of higher order effects. This altogether limits spectral interpretation, confines decoding of the underlying spin parameters, and explains the major challenge associated with the translation of HNMR spectra into tabulated information. On the other hand, the reproducibility of the spectral data set of any (new) chemical entity is essential for its structure elucidation and subsequent dereplication. Handling and documenting HNMR data with adequate precision is critical for establishing unequivocal links between chemical structure, analytical data, metabolomes, and biological activity. Using the full potential of HNMR spectra will facilitate the general reproducibility for future studies of bioactive chemicals, especially of compounds obtained from the diversity of terrestrial and marine organisms.

Mass spectrometric dereplication of nitrogen-containing constituents of black cohosh (Cimicifuga racemosa L.)

Black cohosh preparations are popular dietary supplements among women seeking alternative treatments for menopausal complaints. For decades, triterpene glycosides and phenolic acids have dominated the phytochemical and biomedical research on this plant. In this study, we provide evidence that black cohosh contains an unexpected and highly diverse group of secondary nitrogenous metabolites previously unknown to exist in this plant. Using a dereplication approach that combines accurate mass measurements, database searches and general knowledge of biosynthetic pathways of natural products, we identified or tentatively identified 73 nitrogen-containing metabolites, many of which are new natural products. The identified compounds belong to several structural groups including alkaloids, amides or esters of hydroxycinnamic acids and betains. Among the alkaloids, several classes such as guanidino alkaloids, isoquinolines and β-carbolines were identified. Fragmentation patterns for major compound classes are discussed, which provides a framework for the discovery of these compounds from other sources. Identification of alkaloids as a well-known group of bioactive natural products represents an important advance in better understanding of the pharmacological profile of black cohosh.

Occurrence of Progesterone and Related Animal Steroids in Two Higher Plants

Previously, the presence of a wide variety of chemically diverse steroids has been identified in both flora and fauna. Despite the relatively small differences in chemical structures and large differences in physiological function of steroids, new discoveries indicate that plants and animals are more closely related than previously thought. In this regard, the present study gathers supporting evidence for shared phylogenetic roots of structurally similar steroids produced by these two eukaryotic taxa. Definitive proof for the presence of progesterone (3) in a vascular plant, Juglans regia, is provided. Additional evidence is gleaned from the characterization of five new plant steroids from Adonis aleppica: three 3-O-sulfated pregnenolones (6a/ b, 7), a sulfated H-5beta cardenolide, strophanthidin-3-O-sulfate (8), and spirophanthigenin (10), a novel C-18 oxygenated spirocyclic derivative of strophanthidin. The ab initio isolation and structure elucidation (NMR, MS) of these genuine minor plant steroids offers information on preparative metabolomic profiling at the ppm level and provides striking evidence for the conserved structural space of pregnanes and its congeners across the phylogenetic tree.

In vitro metabolic interactions between black cohosh (Cimicifuga racemosa) and tamoxifen via inhibition of cytochromes P450 2D6 and 3A4

Women who experience hot flashes as a side effect of tamoxifen (TAM) therapy often try botanical remedies such as black cohosh to alleviate these symptoms. Since pharmacological activity of TAM is dependent on the metabolic conversion into active metabolites by the action of cytochromes P450 2D6 (CYP2D6) and 3A4, the objective of this study was to evaluate whether black cohosh extracts can inhibit formation of active TAM metabolites and possibly reduce its clinical efficacy. At 50 μg/mL, a 75% ethanolic extract of black cohosh inhibited formation of 4-hydroxy- TAM by 66.3%, N-desmethyl TAM by 74.6% and α-hydroxy TAM by 80.3%. In addition, using midazolam and dextromethorphan as probe substrates, this extract inhibited CYP3A4 and CYP2D6 with IC50 values of 16.5 and 50.1 μg/mL, respectively. Eight triterpene glycosides were identified as competitive CYP3A4 inhibitors with IC50 values ranging from 2.3–5.1 µM, while the alkaloids protopine and allocryptopine were identified as competitive CYP2D6 inhibitors with Ki values of 78 and 122 nM, respectively. The results of this study suggests that co-administration of black cohosh with TAM might interfere with the clinical efficacy of this drug. However, additional clinical studies are needed to determine the clinical significance of these in vitro results.

Integrated standardization concept for Angelica botanicals using quantitative NMR.

Despite numerous in vitro/vivo and phytochemical studies, the active constituents of Angelica sinensis (AS) have not been conclusively identified for the standardization to bioactive markers. Phytochemical analyses of AS extracts and fractions that demonstrate activity in a panel of in vitro bioassays, have repeatedly pointed to ligustilide as being (associated with) the active principle(s). Due to the chemical instability of ligustilide and related issues in GC/LC analyses, new methods capable of quantifying ligustilide in mixtures that do not rely on an identical reference standard are in high demand. This study demonstrates how NMR can satisfy the requirement for simultaneous, multi-target quantification and qualitative identification. First, the AS activity was concentrated into a single fraction by RP-solid-phase extraction, as confirmed by an alkaline phosphatase, (anti-)estrogenicity and cytotoxicity assay. Next, a quantitative (1)H NMR (qHNMR) method was established and validated using standard compounds and comparing processing methods. Subsequent 1D/2D NMR and qHNMR analysis led to the identification and quantification of ligustilide and other minor components in the active fraction, and to the development of quality criteria for authentic AS preparations. The absolute and relative quantities of ligustilide, six minor alkyl phthalides, and groups of phenylpropanoids, polyynes, and poly-unsaturated fatty acids were measured by a combination of qHNMR and 2D COSY. The qNMR approach enables multi-target quality control of the bioactive fraction, and enables the integrated biological and chemical standardization of AS botanicals. This methodology can potentially be transferred to other botanicals with active principles that act synergistically, or that contain closely related and/or constituents, which have not been conclusively identified as the active principles.

Protostane and Fusidane Triterpenes: A Mini-Review

Protostane triterpenes belong to a group of tetracyclic triterpene that exhibit unique structural characteristics. Their natural distribution is primarily limited to the genus Alisma of the Alismataceae family, but they have also been occasionally found in other plant genera such as Lobelia, Garcinia, and Leucas. To date, there are 59 known protostane structures. Many of them have been reported to possess biological properties such as improving lipotropism, hepatoprotection, anti-viral activity against hepatitis B and HIV-I virus, anti-cancer activity, as well as reversal of multidrug resistance in cancer cells. On the other hand, fusidanes are fungal products characterized by 29-nor protostane structures. They possess antibiotic properties against staphylococci, including the methicillin-resistant Staphylococcus aureus (MRSA). Fusidic acid is a representative member which has found clinical applications. This review covers plant sources of the protostanes, their structure elucidation, characteristic structural and spectral properties, as well as biological activities. The fungal sources, structural features, biological activities of fusidanes are also covered in this review. Additionally, the biogenesis of these two types of triterpenes is discussed and a refined pathway is proposed.