Soft Corals Biodiversity in the Egyptian Red Sea: a Comparative MS and NMR Metabolomics Approach of Wild and Aquarium Grown Species † ‡ § ⊥ Mohamed A
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Article pubs.acs.org/jpr Soft Corals Biodiversity in the Egyptian Red Sea: A Comparative MS and NMR Metabolomics Approach of Wild and Aquarium Grown Species † ‡ § ⊥ Mohamed A. Farag,*, Andrea Porzel, Montasser A. Al-Hammady, Mohamed-Elamir F. Hegazy, ¶ ⊥ ¶ ‡ Achim Meyer, Tarik A. Mohamed, Hildegard Westphal, and Ludger A. Wessjohann*, † Pharmacognosy Department, College of Pharmacy, Cairo University, Kasr el Aini st., P.B. 11562, Cairo 12613, Egypt ‡ Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle (Saale), Germany § National Institute of Oceanography and Fisheries, Red Sea Branch, Hurghada 84511, Egypt ⊥ Phytochemistry Department, National Research Centre, 33 El Bohouth St, Dokki, Giza 12622, Egypt ¶ Leibniz Institute of Tropical Marine Ecology, Fahrenheit Str.6, D-28359 Bremen, Germany *S Supporting Information ABSTRACT: Marine life has developed unique metabolic and physiologic capabilities and advanced symbiotic relationships to survive in the varied and complex marine ecosystems. Herein, metabolite composition of the soft coral genus Sarcophyton was profiled with respect to its species and different habitats along the coastal Egyptian Red Sea via 1H NMR and ultra performance liquid chromatography-mass spectrometry (UPLC−MS) large-scale metabolomics analyses. The current study extends the application of comparative secondary metabolite profiling from plants to corals revealing for metabolite compositional differences among its species via a comparative MS and NMR approach. This was applied for the first time to investigate the metabolism of 16 Sarcophyton species in the context of their genetic diversity or growth habitat. Under optimized conditions, we were able to simultaneously identify 120 metabolites including 65 diterpenes, 8 sesquiterpenes, 18 sterols, and 15 oxylipids. Principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS) were used to define both similarities and differences among samples. For a compound based classification of coral species, UPLC−MS was found to be more effective than NMR. The main differentiations emanate from cembranoids and oxylipids. The specific metabolites that contribute to discrimination between soft corals of S. ehrenbergi from the three different growing habitats also belonged to cembrane type diterpenes, with aquarium S. ehrenbergi corals being less enriched in cembranoids compared to sea corals. PCA using either NMR or UPLC−MS data sets was found equally effective in predicting the species origin of unknown Sarcophyton. Cyclopropane containing sterols observed in abundance in corals may act as cellular membrane protectant against the action of coral toxins, that is, cembranoids. KEYWORDS: corals, cembranoids, Sarcophyton, metabolomic fingerprinting, nuclear magnetic resonance (NMR), ultra performance liquid chromatography−mass spectrometry (UPLC−MS), cyclopropyl sterols ■ INTRODUCTION communication in symbiotic relationships.4 Coral reef Seas cover over 70% of the earth. The total global biodiversity ecosystems support enormous biological diversity including is estimated to amount to some 500 × 106 species of structurally and functionally complex benthic communities. prokaryote and eukaryote organisms. Of these, marine The Red Sea is an epicenter for marine biodiversity with a high macrofauna comprise an estimated range of 0.5−30 × 106 percentage of endemic biota including the northern most tropical reefs with stony corals and soft corals. Of the 180 soft species with a broader range of taxonomic diversity than that fi found in the traditional sources of natural products, the coral species identi ed worldwide, approximately 40% are 1 native to the Red Sea. Soft corals are marine invertebrates terrestrial macrofauna. Only a few thousand compounds have 5 been reported from marine origin, and hence seas and oceans possessing a vast range of terpenoid metabolites. These are believed to have an enormous potential of providers for new terpenes, mostly cembranoids, represent the main chemical bioactive metabolites.2 Marine natural products display an defense of corals against natural predators. Soft corals of the 3 genus Sarcophyton are particularly rich in cembrane terpenes.6,7 extraordinary chemical and pharmacological scope. This could 8 be attributed to the necessity of marine invertebrates to release Cembranoids contain a 14-membered macrocyclic skeleton, secondary metabolites as their own chemical defense tools to survive in the specific temperature, salinity, and pressure Received: January 2, 2016 conditions, and to resist their predators or to provide chemical Published: February 19, 2016 © 2016 American Chemical Society 1274 DOI: 10.1021/acs.jproteome.6b00002 J. Proteome Res. 2016, 15, 1274−1287 Journal of Proteome Research Article Figure 1. Photos of soft corals examined and location map of the collection area along the Red Sea, Egypt. Photograph courtesy of Dr. Montasser A. Al-Hammady and Dr. Mohamed A. Farag. Copyright 2016. biosynthesized by macrocyclization of geranylgeranyl- Nevertheless, 1D-NMR on its own cannot always provide diphosphate, and exhibit a wide range of biological properties unambiguous metabolite identifications and suffers from signal including antitumor, neuro-protective, antimicrobial, calcium- overlap. Application of NMR in the field of marine drugs antagonistic, and anti-inflammatory activity.9,10 includes analysis of spatial variation in soft corals from South In addition, marine organisms show intense symbiosis with China23 and profiling of stony reef corals from Hawaii,24 other organisms such as plants or bacteria. Chemical diversity is although in both studies, limited numbers of metabolites were a critical factor in an organism’s adaptation and fitness and a annotated, mostly targeting primary metabolites. In previous primary reason for the large number of natural products found studies, some of the present authors have successfully applied a in marine organisms including their symbionts.11 Metabolomics comparative metabolomic approach combining both NMR and strategies have recently emerged to help us gain a broader MS based technologies with multivariate data analyses for insight into the biochemical composition of living organisms.12 herbal drug analyses.18,25,26 With the recent developments in plant metabolomics The present work is focused on evaluating the capability of techniques,13,14 it is now possible to detect several hundreds 1D and 2D-NMR for metabolomic fingerprinting and profiling of metabolites simultaneously and to compare samples reliably of soft coral extracts, ideally without any preliminary chromato- for differences and similarities in a semiautomated and graphic step, in parallel to more conventional chromatography- untargeted manner. Metabolomics makes use mostly of coupled MS techniques. Such a comparative metabolomics hyphenated techniques, which rely on chromatographic approach is the first time to be applied in a marine-type separation of metabolites using either gas chromatography metabolomics project by exploring the diversity of soft coral (GC) or liquid chromatography (LC) coupled to mass secondary metabolism in the context of its genotype and spectrometry (MS) or, upcoming, nuclear magnetic resonance growing habitat. The effect of the growing habitat on secondary spectroscopy (NMR), to analyze complex mixtures of extracted metabolite accumulation in soft corals was assessed by metabolites.15 While a NMR metabolite profiling approach collecting soft corals from different locations in the Egyptian provides the complete and quantitative metabolite signature of part of the Red Sea and at different sea levels along with soft a complex extract, LC−MS much better resolves individual corals grown in an experimental aquarium facility under chemical components into separate peaks, enhancing the controlled conditions. Five species of the soft coral genus opportunity to uncover novel metabolites of low abundance.16 Sarcophyton, known to have many secondary metabolites The profiling of secondary metabolites in terrestrial plants including cembranoids, which are well represented along the recently has also been utilized, for example, for the quality Red Sea coast, have been comprehensively studied. To verify − controlofherbaldrugs.17 19 Plants,fungi,and(some) the effectiveness of the developed method, extracts from other microorganisms are much richer in (secondary) metabolites soft corals were analyzed for comparison, including Lobophyton than mammals or most other animals, and thus plant studies are and Sinularia sp. from the Red Sea coast. Owing to the more suitable as templates for studies on marine organisms. complexity of coral extracts, statistical multivariate analyses Although the use of metabolomics in plant analyses has been including principal component analysis (PCA) and partial least- extensive over the past 20 years,20 very little has been done in squares-discriminant analysis (OPLS-DA) were performed for terms of applying it to marine organisms. One of the recent samples classification. applications of LC−MS metabolomics was found to be quite effective for marine microbial strain prioritization to support ■ MATERIALS AND METHODS drug discovery of unique natural products.21 With an increasing interest in utilizing NMR for metabolic Soft Coral Material fingerprinting, it is now possible to record NMR spectra from The soft coral Sarcophyton of the family Alcyoniidae is the most crude extracts, providing its valuable metabolite signature.22