Anti-Trypanosomal Potential of The Red Sea Soft Coral Nephthea Mollis Supported by Metabolomic Pro ling and Molecular Docking Studies


 The total ethanol extract and its derived ethyl acetate fraction of the soft coral Nephthea mollis displayed remarkable in-vitro anti-trypanosomal potential against Trypanosoma brucei with IC50 value of 6.4 and 3.7 (µg/ml, 72 h respectively. Consequently, the total ethanol extract was subjected to LC-HR-ESI-MS metabolomic profiling to discover the constituents that possibly underlie their bioactivities. Therefore, thirty-three secondary metabolites were characterized, among them, sesquiterpenes and diterpenes were found to prevail. In silico modeling was carried out on the dereplicated compounds to provide an insight into their anti-trypanosomal mechanism of action with docking study on ornithine decarboxylase (ORD) which illustrated that five of the dereplicated compounds (‎2-deoxy-12-ethoxy-7-O-methyl lemnacarnol, Nephthenol, ‎4α-O-acetyl-selin-11-en, Eudesma-4,7(11)-diene-8β-ol, and Chabrolidione A) have the highest affinity to the ornithine decarboxylase enzyme. These results highlight the valuable chemical profile of Nephthea mollis as a lead source for anti-trypanosomal natural products.


Introduction
Trypanosomiasis or sleeping sickness is a parasitic infection caused by Trypanosoma brucei which is transmitted generally by an infected y called tsetse, leading to a huge health topic, particularly in the tropical region. The global prevalence of African trypanosomiasis is approximately 600.000 every year [1]. Due to the relatively limited market and the high cost of developing new drugs, there are a few antitrypanosomal drugs available, so there is a great demand for discovering natural products that are effective, safe, and affordable as anti-trypanosomal [2].
The literature survey demonstrated that the genus Nephthea is rich with secondary metabolites including sterols, sesquiterpenes, and diterpenes. Many of these metabolites recently showed a range of biological potentials such as antiviral, cytotoxic, antifouling, antimicrobial, and anti-in ammatory activity [11][12][13][14].
The current approach aims to evaluate the in-vitro anti-trypanosomal potential of the total ethanol extract (TEE) and its derived fractions; petroleum ether and ethyl acetate of Nephthea mollis assisted by LC-HR-ESI-MS metabolomic pro ling to identify the secondary metabolites contributed to this anti-trypanosomal potential of the soft coral. Moreover, in-silico molecular docking simulations within ornithine decarboxylase (ORD; which is the enzyme catalyzing biosynthesis of polyamines in Trypanosoma brucei. ORD is a drug target for the treatment of African sleeping sickness disease and its X-ray was deposited at RCS protein data band as 1NJJ PDB protein) for dereplicated compounds were performed to help better understand the binding mode and their possible anti-trypanosomal mechanism of action [15,16].

Anti-trypanosomal activity
Using the protocol of Huber and Koella, the total ethanol extract and its derived fractions; petroleum ether, and ethyl acetate of Nephthea mollis were tested for their in-vitro anti-trypanosomal results against T. brucei revealed that the total ethanol extract and ethyl acetate fraction of the soft coral Nephthea mollis displayed remarkable in-vitro anti-trypanosomal potential against Trypanosoma brucei with IC 50 value of 6.4 and 3.7 (µg/ml, 72 h) respectively. Unfortunately, the Petroleum ether fraction shows a weak antitrypanosomal activity with IC 50 value of > 50 (µg/ml, 72 h).

Metabolomic analysis
The total ethanol extract of Nephthea mollis was subjected to metabolomic pro ling, for the rst time and the chemical pro ling revealed various classes of secondary metabolites, where sesquiterpenes were the most abundant, in addition to diterpenes and terpenoid-related carboxylic acids ( Figs.1 and 2). The detected compounds were identi ed by using macros, algorithms using MZmine, and online databases (DNP, METLIN, and Marinlit databases) [17]. From these databases, thirty-three compounds were identi ed (Table 1; Fig.3). Fig.1. Total ion chromatogram of total extract of Nephthea mollis (Negative mood).

Furtherly, visual inspection of the resultant docking poses of each compound showed a number of binding interactions (varying from H-bonding to H-pi interactions) between some of the dereplicated
compounds and various amino acids lining ORD active site similar to that found with ORX, (Table 3; Fig.  5). All in all, the obtained molecular docking data showed how good the overlay of the dereplicated compounds of Nephthea mollis within ornithine decarboxylase (ORD) active site, which could explain their anti-trypanosomal activity against Trypanosoma brucei.

Chemicals and reagents
Organic solvents used in this study, including ethanol, ethyl acetate, and petroleum ether (b.p. 60-80 ºC) were of analytical grade and distilled before use. All these solvents were purchased from El-Nasr Company for Pharmaceuticals and Chemicals, Egypt. Solvents of HPLC grade such as acetonitrile and methanol were obtained from SDFCL sd ne-Chem Limited, India.

Extract preparation
The freeze-dried soft coral material (45 g) was extracted with ethanol until exhaustion. The concentrated organic extract (15 g) was suspended in distilled water and extracted successively with different organic solvents, including petroleum ether, and ethyl acetate. The organic phase in each step was separately concentrated under vacuum, yielding the petroleum ether fraction (6 g), the ethyl acetate fraction (1 g), and the aqueous fraction (8 g). The total ethanol extract and its derived fractions; petroleum ether and ethyl acetate were kept at 4°C for anti-trypanosomal assay and other analysis.

In-vitro anti-trypanosomal activity (Huber and Koella assay)
The anti-trypanosomal potential was tested against Trypanosoma brucei following the protocol of Huber

LC-MS Metabolomic pro ling
Metabolomic pro ling of the total ethanol extract of Nephthea mollis was carried out as described by Abdelmohsen et al. [35] using an Acquity Ultra Performance Liquid Chromatography system connected to a Synapt G2 HDMS quadrupole timeof-ight hybrid mass spectrometer (Waters, Milford, USA).
Chromatographic separation was carried out using a BEH C18 column (2.1 × 100 mm, 1.7 µm particle size; Waters, Milford, USA) accompanied with a guard column (2.1 × 5 mm, 1.7 µm particle size). The mobile phase used during the separation consisted of puri ed water (A) and acetonitrile (B) with 0.1% formic acid added to each solvent. A gradient elution started at a ow rate of 300 µL/min with 10% B linearly increased to 100% B within 30 min and remained isocratic for the next 5 min before linearly decreasing back to 10% B for the following 1 min. The volume injected was 2 µL and the column temperature was adjusted to 40°C. The obtained raw data were separated into positive and negative ionization mode using MSConvert software. Accordingly, the les were imported to the data mining software MZmine 2.10 for peak picking followed by deconvolution, deisotoping, alignment, and formula prediction.

Conclusion
The current study highlighted the anti-trypanosomal potential of the total extract and different fractions of the soft coral Nephthea mollis where the total ethanol extract and its derived ethyl acetate fraction displayed remarkable in-vitro anti-trypanosomal potential against Trypanosoma brucei. Such effects are likely underlain by the availability of a range of compounds, mostly sesquiterpenes, and diterpenes that were mined with the help of LC-MS-based metabolomics. Docking studies of the identi ed compounds postulated their mechanism of action, which was further evidenced by in-vitro assays.

Con icts of interest
The authors declare no con ict of interest. Figure 1 Total ion chromatogram of total extract of Nephthea mollis (Negative mood).

Figure 2
Total ion chromatogram of total extract of Nephthea mollis (Positive mood).  Supplementary Files