MS/MS Molecular Networking Unveils the Chemical Diversity of Biscembranoid Derivatives, Neutrophilic Inflammatory Mediators from the Cultured Soft Coral Sarcophyton trocheliophorum

Biscembranoids are the distinctive tetraterpenoids owing a 14/6/14 membered tricyclic scaffold that have been mainly discovered in the soft corals, especially the genera Sarcophyton, Lobophytum and Sinularia. Recent findings have demonstrated the great anti-inflammatory potential of biscembranoid analogues in human neutrophils, motivating more chemical and biological explorations targeting these marine-derived natural products. In the current study, the chemical diversity of biscembranoids derived from the cultured-type Sarcophyton trocheliophorum von Marenzeller was illustrated through MS/MS molecular networking (MN) profiling approach. Based on the MN patterns, the prioritization of unknown biscembranoid derivatives was putatively analyzed. As a result, the biscembrane targeting isolation afforded two new metabolites, sarcotrochelides A (1) and B (2), along with six known analogues (3–8). Their structures and relative configurations were determined by spectroscopic methods. In vitro neutrophil inflammatory inhibition was further investigated for all isolates based on reduced superoxide anion (O2•−) generation detections. Compounds 5–8 showed significant dose-dependently inhibitory effects, suggesting the cruciality of 6,7-dihydrooxepin-2(5H)-one moiety and saturated γ-lactone ring in their reactive oxygen species (ROS)-dependent anti-inflammatory properties.


Introduction
Biscembranoids are the distinctive tetraterpenoids owing a 14/6/14 membered tricyclic scaffold that have been mainly discovered in the marine organisms, especially the soft corals belonging to the genus Sarcophyton . These secondary metabolites was first found in 1986 [1] and exhibited various bioactivities, ranging from anti-cancer, anti-inflammatory, neuroprotective, anti-microbial, and immunomodulatory activities. The antiinflammatory effect accounts for the majority of bioactivities of the tetraterpene derivatives. Recent studies have also shown the great anti-inflammatory potential of biscembranoid analogues in human neutrophils, which has attracted more chemical and biological explorations targeting these marine-derived natural products.
Due to the promising pharmacological actions, higher quantity of potent marine-derived compounds is required for subsequent preclinical and clinical trials, but the yields of the secondary metabolites obtained from the wild-type marine organism are usually insufficient for these purposes. Therefore, marine aquaculture has emerged as an effective approach to maintain a sustainable, consistent, and reproducible supply of marine-derived natural products.
In the current study, a MS/MS molecular networking method has been utilized to assist in the discovery of new biscembranoids from the cultured soft coral Sarcophyton trocheliophorum. Based on the MN patterns and NMR spectra, the fraction that was putatively identified to contain biscembranoids was subjected to further purification steps. The biscembrane targeting isolation afforded two new metabolites, sarcotrochelide A (1) and B (2), along with six known analogues (3)(4)(5)(6)(7)(8) (Figure 1). Their structures were determined by spectroscopic methods. In vitro neutrophil inflammatory inhibition was further investigated for all isolates based on ROS generation detections using luminol enhanced chemiluminescence. Compounds 5-8 showed significant dose-dependently inhibitory effects, suggesting the cruciality of 6,7-dihydrooxepin-2(5H)-one moiety in their anti-inflammatory properties.

Characterizing the Distribution of Anti-Inflammatory Biscembranoids Using Multi-informative Molecular Networking (MIMN)
In order to facilitate the operating process for probing anti-inflammatory biscembranoids, a multi-informative molecular networking (MIMN) was applied [29]. In the primary extraction and fractionation part, the organic extract (using dichloromethane:methanol = 1:1) of the cultured-type Sarcophyton trocheliophorum was fractionationed into the EtOAc and the aqueous layers through liquid-liquid partitioning approaches. The chromatographic separation (normal phase) on EtOAc soluble residue further afforded 20 subfractions. Then the chemical and anti-inflammatory MIMN profiles of these fractions were constructed based on the MS/MS analysis and superoxide anion (O 2 •− ) inhibitory assessments in activated neutrophils (Table 1), respectively. The followed-up clustering, classification, and annotation were performed on the GNPS platform (https://gnps.ucsd.edu, accessed on 17 April 2022). Based on the MIMN analyzing results, the clusters of cembrane dimer (m/z 650-750) and monomer (m/z 290-350) exhibited the greatest anti-inflammatory potential with inhibition rate over 70% at the concentration of 10 µg/mL (Figure 2A). The insight MIMN patterns of metabolite distribution ( Figure 2B) revealed that the fraction 12 from the EtOAcsoluble extract contained a variety of anti-inflammatory biscembranoids, resulting the further isolation targeting these characteristic tetraterpenoids from fraction 12.
The relative stereochemistry of 1 was elucidated by correlations in the nuclear Overhauser effect relationships (NOESY) experiment. As shown in Figure 4, the NOESY correlations of H-4 (δH 6.59, br s) with H3-19 (δH 1.89, s), together with the obviously downfieldshifted methyl at C-19 (δC 26  The relative stereochemistry of 1 was elucidated by correlations in the nuclear Overhauser effect relationships (NOESY) experiment. As shown in Figure 4, the NOESY correlations of H-4 (δ H 6.59, br s) with H 3 -19 (δ H 1.89, s), together with the obviously downfieldshifted methyl at C-19 (δ C 26.3, CH 3   Sarcotrochelide B (2) was isolated as a white powder. Its formula was determined as C41H62O9 by the HRESIMS ion at m/z 699.4467 (calcd. for [C41H62O9 + H] + , 699.4472), indicating 11 indices of hydrogen deficiency. The hydroxy-containing structure of 2 was inferred from the IR signal at 3417 cm -1 . 41 carbons, including 9 methyls, 11 methylenes, 10 methines, and 11 quaternary carbons, were revealed by the 13 C NMR spectrum. The NMR signals (Table 3)  H-2 to C-1, C-3, C-14 and C-20; H2-11 to C-10; H-12 to C-13; H2-14 to C-1, C-13 and C-20; and H-33 to C-34. The above-mentioned biscembrane framework put paid to ten of the total eleven unsaturated degrees, implying the existence of an additional ring. It was suggested to be of an ester ring between C-27 and C-30 established via an HMBC from H2-28 to C-27 and C-30. The gross structure of 2 was thereby confirmed, as shown in Figure 2, which possesses a biscembranoid skeleton similar to methyl tortuoate D (4) [18].
The relative configurations of rings A and B of compound 2 were identical to those of co-occurring compound 1 as determined by the similar NOESY and the NMR data. NOESY correlations (Figure 4    H-2 to C-1, C-3, C-14 and C-20; H 2 -11 to C-10; H-12 to C-13; H 2 -14 to C-1, C-13 and C-20; and H-33 to C-34. The above-mentioned biscembrane framework put paid to ten of the total eleven unsaturated degrees, implying the existence of an additional ring. It was suggested to be of an ester ring between C-27 and C-30 established via an HMBC from H 2 -28 to C-27 and C-30. The gross structure of 2 was thereby confirmed, as shown in Figure 2, which possesses a biscembranoid skeleton similar to methyl tortuoate D (4) [18].
The relative configurations of rings A and B of compound 2 were identical to those of co-occurring compound 1 as determined by the similar NOESY and the NMR data. NOESY correlations (Figure 4) 1.54-1.60, m), H-29b/H-33 suggested that H-26 and H-33 was β-oriented. Therefore, the structure of 1 was unambiguously elucidated as shown in Figure 2. On the basis of the above observations and as the relative configurations of 2 have been determined as shown, the structure of compound 2 could be fully established as 1S*, 2S*, 9R*, 12S*, 21S*, 26R*, 27R*, 30R*, 31S*, 33R*.

Bioactivities of the Biscembranoids
The activation effects of N-formyl-methionyl-leucyl-phenylalanine (fMLF) and pathogen-associated molecular patterns (PAMPs) on neutrophils can cause a series of inflammatory responses, such as respiratory burst (O2 •− generation) and degranulation (elastase release) [30]. In order to evaluate the anti-inflammatory activities of the cultured soft coral S. trocheliophorum, the EtOAc, MeOH, and water-soluble extracts, and fraction 12 were assayed in fMLF-induced human neutrophils. All eight pure compounds obtained from fraction 12 were also evaluated for their anti-inflammatory effects in the same in vitro tests. The results showed that fraction 12 exhibited the highest inhibitory effects on superoxide anion generation and elastase release with IC 50 5.45 and 7.48 µg/mL, respectively, among the crude samples (Table 1).
For pure derivatives, compound 6 displayed the strongest activity against superoxide anion generation and elastase release in fMLF/CB-induced human neutrophils, followed by compounds 5, 7, and 8, respectively (Table 4).  The significant difference in the anti-inflammatory effects of the isolates may be caused by some variations in their structures. The common characteristics of the four most active compounds is that they share a 6,7-dihydrooxepin-2(5H)-one moiety and a saturated γlactone ring. In addition, compound 6 possesses a 11Z and 22E double bonds instead of an E geometry of ∆ 11 (12) and ∆ 22 (23) in compound 5 and a Z geometry of ∆ 11 (12) and ∆ 22 (23) in compound 7. Additionally, when compared to compound 6, the 11,12-double bond was replaced by a 10,11-double bond in compound 8, which suggested that the reduced anti-inflammatory effect of compound 8 might be caused by this minor change.

Non-Targeted Fragment Ions Collection Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS)
The acquisition of tandem mass spectral data was carried out based on a Waters SYNAPT G2 LC/Q-TOF (Waters Corporation, Milford, MA, USA) system. The extracts were filtered through a 0.45 µm membrane filter and dissolved in methanol to a final concentration of 5000 ppm for the analysis. For chromatographic part, a C18 column of Waters Acquity UPLC BEH (Waters, 1.7 µm, 2.1 mm × 100 mm) was used for the separation and was maintained in a 40 • C column oven. The analytes were eluated from the column by CH 3 CN (A, containing 0.1% formic acid)/water (W, containing 0.1% formic acid) gradient sequences: 0.01-25 min, 1-100% A; 25.01-30 min, 100% A, with the flow rate of 0.5 mL/min. Automatical injections were executed with injected volumes of 5 µL. The non-targeted MS 1 and MS 2 data were acquired within the range of m/z 100-2000. The automated data-dependent acquisition (DDA) approach was used for the acquisition of MS 2 spectra, and five precursor ions were selected for further fragmentations with ramping of the collision energy from 10-50 eV. Finally, the finalization of MS data were conducted with the assistance of Waters MassFragment software (MassLynx4.1, Waters, MA, USA).

GNPS-Based Molecular Networking Analysis
A GNPS web-based platform (https://gnps.ucsd.edu, accessed on 17 April 2022) [31] was applied to analyze and output the MS/MS molecular networking data (job ID: edcb54569b0042fea7104782189e34af, 17 April 2022). The pre-processing of MS/MS raw data was conducted by converting into mzML file using Proteowizard MSConvert (Ver. 3, GitHub repository, Palo Alto, CA, USA). The conversions were uploaded to GNPS drive using WinSCP software (Ver. 5.21, SourceForge, San Diego, CA, USA) and performed molecule networking analysis. The MS/MS spectra were window-filtered according to the top five strongest ion peaks in the ±50 Da window throughout the spectrum. A molecular network was then created, in which the edges between nodes were kept if the cosine scores were above 0.70 and the separated consensus spectra shared at least four matched peaks. Then the appeared nodes in the network were annotated based on the experimental MS 2 fragmentations of isolates. The data visualization was executed by Cytoscape 3.8.2 (Cytoscape 3.8.2, NRNB, San Diego, CA, USA) [32].

Animal Material, Extraction, and Isolation
Specimens of the wild-type S. trocheliophorum was originally collected by scuba diving from the coast of Pingtung, Taiwan, in 2015 (specimen No. 2015-07-ST). These corals were preserved and aquacultured in National Museum of Marine Biology and Aquarium (Pingtung, Taiwan). The aquaculture condition was mentioned below [33]: The collected wild corals were cut into several sub-strains of 4 to 5 cm, and these sub-strains are naturally placed and attached to porous tiles for domestication and cultivation. These soft corals were kept in cultured tanks (120 tons) with temperature controlled (25-28 • C) coolers and supported by natural light daily. The ecological environment was settled up with live sea rocks, live sea sands, snails, paracanthurus hepatus fishes, sea urchins, sea cucumbers, and other aquaculture soft corals, such as Briareum spp., Paralemnalia sp., Sarcophyton spp., and Sinularia spp. The specimens were then collected by hand in July 2020 and were kept in a −20 • C freezer until extraction. A voucher specimen (specimen no. 202007ST1) was deposited in the Graduate Institute of Pharmacognosy, Taipei Medical University.

Preparation of Human Neutrophils
Venous blood sampling was performed on human donors (aged 20-30 years) according to an approved protocol (IRB No. 202002493A3). The purification of neutrophils was achieved according to a reported procedure [34].

Determination of Superoxide Anion (O 2 •− ) Generation
Under the treatment of compounds 1-8, the O 2 •− generation of human neutrophils was determined by superoxidase dismutase (SOD) inhibitable reduction in ferricytochrome c as previously described [35].

Measurement of Elastase Release
MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide was used as a substrate in an elastase release assay to evaluate the degranulation of azurophilic granules as previously described [36].

Statistics
Statistical analysis was performed using Student's t-test for calculations. p values < 0.05 were considered to be statistically significant.

Conclusions
The chemical investigation of the cultured soft coral Sarcophyton trocheliophorum led to the isolation of two novel metabolites 1-2, along with six known analogues of biscembranoid 3-8. The in vitro tests showed that compound 5-8 exhibited significant inhibitory effects on the superoxide anion generation and elastase release in fMLF/CB-induced human neutrophils. The difference in their bioactivities suggested the importance of the lactone rings and geometry of double bonds in the structures.
The discovery of two novel biscembranoids demonstrated the chemical diversity of this type of metabolite in the aquaculture Sarcophyton trocheliophorum. In addition, the two most bioactive compounds, glaucumolide A (5) and glaucumolide B (6), were obtained with relatively high quantity, measured at 31.59 mg and 53.98 mg, respectively. The results suggested that aquaculture of soft coral could be a prolific and sustainable resource for the drug discovery and development [37].