Structure‐oriented UHPLC‐LTQ Orbitrap‐based approach as a dereplication strategy for the identification of isoflavonoids from Amphimas pterocarpoides crude extract

Hyphenated techniques and especially ultra‐high performance liquid chromatography‐mass spectrometry (UHPLC‐MS) are nowadays widely employed in natural products research. However, the complex nature of plant extracts complicates considerably the analysis and the identification of their constituents. Nevertheless, new MS analyzers with increased resolving power and accuracy such as the orbital trap (Orbitrap) could facilitate drastically this process. The objective of this study is the development of a new structure‐oriented approach based on fast UHPLC‐high‐resolution (HR)MS and HRMS/MS methodologies for the identification of isoflavonoids in crude extracts. In addition, aims to assist dereplication procedures, to decrease the laborious isolation steps and orient the focused isolation of compounds of interest. As a proof of concept, the methanol extract of the stem bark of Amphimas pterocarpoides (Leguminosae) was selected. Based on chromatographic (retention time, polarity) and spectrometric features (ultraviolet spectra, accurate m/z, proposed elemental composition, ring double bond equivalent, and relative isotopic abundance) as well as HRMS/MS spectra, several isoflavonoids were identified. In order to verify the proposed structures, 11 isoflavonoids were selectively isolated and unambiguously identified using 1&2D nuclear magnetic resonance techniques. Moreover, the isolated isoflavonoids were studied in HRMS/MS level, employing electrospray ionization and atmospheric pressure chemical ionization sources, in both modes. Useful information regarding their fragmentation patterns was obtained, and characteristic diagnostic ions were defined for the identification of methoxylated isoflavones, dihydroisoflavones and 5‐hydroxylated isoflavonoids. Based on the current results, the proposed dereplication strategy was verified and could comprise a novel approach for the analysis of crude extracts in the future not only for isoflavonoids but also for other chemical classes of natural products. Copyright © 2013 John Wiley & Sons, Ltd.     

HPLC-coupled spectroscopic techniques (UV, MS, NMR) for the structure elucidation of phthalides in Ligusticum chuanxiong

Ligusticum chuanxiong Hort., a plant that is frequently used in traditional Chinese medicine, has been studied using HPLC-coupled spectroscopic techniques such as HPLC-UV, HPLC-MS as well as HPLC-NMR. With the aid of these modern spectroscopic techniques, the main constituents, namely senkyunolide A, butylphthalide, neocnidilide and Z-ligustilide, have been characterized and identified. Phthalide dimers, present in smaller amounts, have been identified by HPLC-UV and HPLC-MS analysis and compared with reference compounds. Stereochemical features of some phthalide monomers have been determined by detailed spectroscopic studies for the first time.     

Antidiarrhoeic effect and dereplication of the aqueous extract of Annona crassiflora (Annonaceae)

We investigated the antidiarrhoeic effect of the aqueous extract of Annona crassiflora leaves (AEAC). The AEAC decreased the diarrhoeic stools and enteropooling induced by castor oil, without altering total faecal output; moreover, the distance travelled by charcoal meal in the intestine was increased. Twenty-eight compounds were identified by LC-DAD-MS in the AEAC, including flavonoids, alkaloids and proanthocyanidins. In addition, two oligomeric series of condensed tannins of up to nine flavan-3-ol units were characterised by MALDI-MS. These data suggest that the antidiarrhoeic effect of the AEAC is related to its ability to inhibit intestinal secretion and/or to increase intestinal absorption. Moreover, the prokinetic effect of AEAC, together with its inhibitory effect on enteropooling induced by castor oil, explains why this extract decreased diarrhoeic faeces without altering the total faecal output. All these effects are in agreement with the pharmacological activity reported in the literature for many of the secondary metabolites identified.     

Dereplication of known pregnane glycosides and structural characterization of novel pregnanes in Marsdenia tenacissima by high‐performance liquid chromatography and electrospray ionization‐tandem mass spectrometry

In the search for novel natural products in plants, particularly those with potential bioactivity, it is important to efficiently distinguish novel compounds from previously isolated, known compounds, a process known as dereplication. In this study, electrospray ionization‐multiple stage tandem mass spectrometry (ESI‐MSⁿ) was used to study the behaviour of 12 pregnane glycosides and genins previously isolated from Marsdenia tenacissima, a traditional Chinese medicinal plant, as a basis for dereplication of compounds in a plant extract. In addition to [M + Na]⁺ and [M + NH₄]⁺ ions, a characteristic [M‐glycosyl + H]⁺ ion was observed in full‐scan mode with in‐source fragmentation. Sequential in‐trap collision‐induced dissociation of [M + Na]⁺ ions from 11,12‐diesters revealed consistent preferred losses of substituents first from C‐12, then from C‐11, followed by losses of monosaccharide fragments from the C‐3 tri‐ and tetrasaccharide substituents. A crude methanol extract of M. tenacissima stems was analysed using high‐performance liquid chromatography coupled to ESI‐MS. Several previously isolated pregnane glycosides were dereplicated, and the presence of an additional nine novel pregnane glycosides is predicted on the basis of the primary and fragment ions observed, including two with a previously unreported C₄H₇O C‐11/C‐12 substituent of pregnane glycosides. This study is the first report of prediction of the structures of novel pregnane glycosides in a crude plant extract by a combination of in‐source fragmentation and in‐trap collision‐induced dissociation and supports the usefulness of LC‐ESI‐MSⁿ not only for dereplication of active compounds in extracts of medicinal plants but also for detecting the presence of novel related compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

OSMAC Strategy Integrated with Molecular Networking for Accessing Griseofulvin Derivatives from Endophytic Fungi of Moquiniastrum polymorphum (Asteraceae)

Three endophytic fungi isolated from Moquiniastrum polymorphum (Less.) G. Sancho (Asteraceae) were cultivated using the one strain many compounds (OSMAC) strategy to evaluate the production of griseofulvin derivatives. Extracts obtained were analyzed by HPLC–MS/MS and the chromatographic and spectrometric data used to elaborate a feature-based molecular network (FBMN) through the GNPS platform. This approach allowed the observation of differences such as medium-specific and strain-specific production of griseofulvin derivatives and variations of cytotoxic activity in most extracts. To evaluate the efficiency of the OSMAC approach allied with FBMN analysis in the prospection of compounds of biotechnological interest, griseofulvin and 7-dechlorogriseofulvin were isolated, and the relative concentrations were estimated in all culture media using HPLC–UV, allowing for the inference of the best strain–medium combinations to maximize its production. Malt extract-peptone broth and Wickerham broth media produced the highest concentrations of both secondary metabolites.     

Chemical dereplication of marine actinomycetes by liquid chromatography–high resolution mass spectrometry profiling and statistical analysis

Discovery of novel bioactive metabolites from marine bacteria is becoming increasingly challenging, and the development of novel approaches to improve the efficiency of early steps in the microbial drug discovery process is therefore of interest. For example, current protocols for the taxonomic dereplication of microbial strains generally use molecular tools which do not take into consideration the ability of these selected bacteria to produce secondary metabolites. As the identification of novel chemical entities is one of the key elements driving drug discovery programs, this study reports a novel methodology to dereplicate microbial strains by a metabolomics approach using liquid chromatography–high resolution mass spectrometry (LC–HRMS). In order to process large and complex three dimensional LC–HRMS datasets, the reported method uses a bucketing and presence–absence standardization strategy in addition to statistical analysis tools including principal component analysis (PCA) and cluster analysis. From a closely related group of Streptomyces isolated from geographically varied environments, we demonstrated that grouping bacteria according to the chemical diversity of produced metabolites is reproducible and provides greatly improved resolution for the discrimination of microbial strains compared to current molecular dereplication techniques. Importantly, this method provides the ability to identify putative novel chemical entities as natural product discovery leads.     

Dereplication‐guided isolation of a new indole alkaloid triglycoside from the hooks of Uncaria rhynchophylla by LC with ion trap time‐of‐flight MS

Uncaria rhynchophylla (Gou‐Teng) as the monarch herb of many formulae (Fufang), e.g. “Tian‐Ma‐Gou‐Teng‐Yin,” “Ling‐Jiao‐Gou‐Teng‐Yin,” and “Yi‐Gan‐San”, is a famous traditional Chinese medicine documented in the Chinese pharmacopoeia for mental and cardiovascular diseases. In the traditional Chinese medicine system, only the hook‐bearing stems are used as the crude materials for Gou‐Teng, and the hooks are always considered more effective than the stems. Focusing on the mono‐herb and its active constituents from combinatorial formulae is the core idea of reductionism of traditional Chinese medicine theory. Detailed liquid chromatography with mass spectrometry analysis on the hooks of U. rhynchophylla was performed to profile the chemical constituents based on tandem mass spectrometry fragmentation and UV absorption. Under the guidance of liquid chromatography with ion trap/time‐of‐flight mass spectrometry, one new indole alkaloid triglycoside ( 1 ), together with five known compounds 2 – 6 as the main constituents, were isolated from the hooks of U. rhynchophylla by various column chromatography methods. Compound 1 showed moderate activity on MT₁ and MT₂ melatonin receptors with agonistic rates of 79.6 and 46.3% at the concentration of 1 mM. This dereplication strategy can be equally applicable to rapidly disclose the active constituents of other Chinese herbs through targeted purification.     

Dereplication of oligostilbenes in dipterocarpaceous plants using LCMS-ESI-Ion trap-database

A method for compound identification based on mass fragmentation patterns was developed to distinguish known oligostilbenes directly in crude plant extracts. Eleven pure oligostilbenes were used as standard compounds, and a pilot database of MS data was established. Seven extracts from Dipterocarpaceous plants; three from Neobalanocarpus heimii, three from Dryobalanops spp. and one from Dipterocarpus semivestitus were used as test materials. Eleven compounds were positively identified in the heartwood extract, nine in the bark, and six in the leaves of Neobalanocarpus heimii, one compound each in Dryobalanops lanceolata, D. rappa and D. aromatica, as well as two in Dipterocarpus semivestitus extract. Identification of all compounds were performed within a short analysis time.     

Development of Genetic Dereplication Strains in Aspergillus nidulans Results in the Discovery of Aspercryptin

To reduce the secondary metabolite background in Aspergillus nidulans and minimize the rediscovery of compounds and pathway intermediates, we created a “genetic dereplication” strain in which we deleted eight of the most highly expressed secondary metabolite gene clusters (more than 244,000 base pairs deleted in total). This strain allowed us to discover a novel compound that we designate aspercryptin and to propose a biosynthetic pathway for the compound. Interestingly, aspercryptin is formed from compounds produced by two separate gene clusters, one of which makes the well‐known product cichorine. This raises the exciting possibility that fungi use differential regulation of expression of secondary metabolite gene clusters to increase the diversity of metabolites they produce.     

An Integrated LC-ESI-MSn and High Resolution LC-ESI-QTOF Approach for the Identification of Phloroglucinols from Nepalese Hypericum japonicum

Phloroglucinols are characteristic constituents of Hypericum japonicum that are claimed to exert several bioactivities, such as anti-inflammatory, anti-depressant and anti-viral ones. Phloroglucinols are unstable compounds and their synthesis is challenging; thus, isolation from natural sources is still one of the main strategies for obtaining these constituents in purified form. Assessing the presence of phloroglucinols in plant materials can be of interest for compound isolation, and LC-MS approaches afford sensitivity and specificity in this regard. In this work, we combined data from quadrupole-time of flight (QTOF) and ion trap (IT) mass spectrometers in order to assess the presence of the phloroglucinols characteristic of H. japonicum and to elucidate their MS fragmentation pathways. The identified compounds present similar structures bearing the 1,3,5-trihydroxybenzene core with different substitutions, which, in constituents at higher MW, is linked to 3′,3′-dimethyl-6′-oxo-phlorisobutyrophenone by a methylene bridge. Differences in MS² spectra of the considered phloroglucinols are useful for compound identification and differentiation, and to perform dereplication studies. Overall, the proposed approach could be useful for the analysis of phloroglucinols in H. japonicum and other plant species.