Litoarbolide A: an undescribed sesquiterpenoid from the Red Sea soft coral Litophyton arboreum with an in vitro anti-malarial activity evaluation

Abstract Soft corals distributed across the Red Sea coasts are a rich source of diverse and bioactive natural products. Chemical probing of the Red Sea soft coral Litophyton arboreum led to isolation and structural characterization of an undescribed sesquiterpenoid, litoarbolide A (1), along with 14 previously reported metabolites (2-15). The chemical structures of the isolates were assigned based on NMR as well as high resolution electrospray ionization mass spectrometry (HR-ESI-MS) data. Litoarbolide A is supposed to be the biosynthetic precursor to other sesquiterpenoids, which formed via further post-translational modifications. Furthermore, these metabolites were evaluated for anti-malarial activity, where only the acyclic sesquiterpenoid of a sec-germacrane nucleus (7) showed an activity against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum with IC50 at 3.7 and 2.2 mg/mL, respectively. Moreover, the isolated metabolites were all non-toxic to the Vero cell line. These findings support the consideration of L. arboreum in further natural anti-malarial studies. Graphical Abstract


Introduction
The Red Sea has long been recognized as one of the most biodiverse and high endemic marine environments harboring several categories of marine organisms. Among them are algae, coral, invertebrates, mangrove, and seagrass (Ellis et al. 2017). Particularly, the Red Sea harbors more than 200 soft and 300 hard coral species, which have not been explored appropriately yet in comparison with other ecosystems (Tammam et al. 2020). Soft corals have received a great attention, since their species are considered promising sources of novel marine bioactive compounds with unique structural diversity to include sesquiterpenoids, diterpenoids, and steroids and exhibition of a broad spectrum of bioactivities such as anti-microbial, anti-cancer, antiinflammatory, anti-viral, and anti-fouling actions (Sang et al. 2019).
Specifically, the soft corals of the genus Litophyton (Nephthedae) are abundant in the region of the Gulf of Aqaba of the Red Sea with accepted 13 species (van Ofwegen 2016). Litophyton species have been shown to contain various chemical constituents, including sesquiterpenes, steroids, ceramides, c-lactones, and cembrane-type diterpenes (Duh et al. 1998, Ellithey et al. 2013Ghandourah et al. 2015;Grote et al. 2008;Li et al. 1994; Rezanka and Dembitsky 2001;Zhang et al. 2003;Zovko Kon ci c et al. 2016). The diversity of the secondary metabolites from Litophyton led to a wide range of biological activities including anti-HIV, cytotoxic, antiproliferative, and antituberculosis (El Sayed et al. 2000;Ellithey et al. 2013;Grote et al. 2008). Previous work performed on Litophyton arboretum which is distributed across the Red Sea showing that this species is rich in metabolites with promising biological activities such as antibacterial and anti-cancer (Abou El-Kassem et al. 2018;Ellithey et al. 2013;Grote et al. 2008;Li et al. 1994;Rezanka and Dembitsky 2001;Shaker et al. 2010). Moreover, malaria is one of the life-threatening diseases that transmitted through the bite of an infected Anopheles female mosquito carrying one of several protozoans belonged to the genus Plasmodium (P. falciparum, P. ovale, P. vivax, P. knowlesi, and P. malariae) (Fattorusso and Taglialatela-Scafati 2009;Sutherland et al. 2010). Soft corals have been reported to provide diterpenes such as laevigatol A isolated from soft corals living in Vietnamese seas. It showed moderate anti-plasmodial activity with an IC 50 <5.0 lM (Thao et al. 2015;Thao et al. 2014). In addition, series of diterpene glycosides were obtained from the Caribbean soft coral Pseudopterogorgia elisabethea, among them pseudopterosin V, possessing an anti-malarial activity at IC 50 ¼ 2.2 lM against chloroquine-resistant P. falciparum colonies (Rodr ıguez et al. 2004).
The present work focuses on the isolation of sesquiterpenes and steroids and evaluating their anti-malarial activity. Chemical investigation of L. arboreum has led to the isolation and structure elucidation of an undescribed sesquiterpene; litoarbolides A (1), in addition to fourteen known metabolites (2-15) that are mainly identified in this organism for the first time. In addition, the anti-malarial activity of the isolated metabolites was evaluated via in vitro anti-malarial assay against D6, CQ-sensitive and W2, CQ-resistant strains of P. falciparum. All the compounds were also screened for their in vitro cytotoxicity against the mammalian vero cell lines.

Fractionation of the crude extract
The frozen L. arboreum sample was chopped into small pieces using a blender and extracted exhaustively with a mixture of CH 2 Cl 2 and MeOH (50:50). The extract, obtained after evaporation of solvents, was subjected to a VLC silica gel column and fractionated into different major fractions using hexane and EtOAc with gradient increase in the polarity. The fractions were subjected to a series of different chromatographic techniques to purify the metabolites. Using RP-HPLC for our final purification step, we were able to isolate these metabolites with a very close structural features (see Figure 1).

Structural characterization of isolated compounds
Litoarbolide A (1) was isolated as a colorless amorphous solid with a molecular formula of C 15 H 22. It showed a pseudo molecular ion at m/z 203.1797 for [M þ H] þ in the HRESIMS for C 15 H 23 þ (calcd m/z 203.1792) indicating five degrees of unsaturation. The 1 H-NMR and 13 C-NMR data (Table S1)  2) and C-9 (d C 125.8) ( Figure  S9). In addition, the 1 H-1 H-COSY of 1 ( Figure S9)  ). These evidences indicated that 1 was a sesquiterpenoid of guaiane-type (Bowden et al. 1980). The relative configurations of 1 were deduced from the NOESY data in which H-1 and H-5 were supposed to be a-oriented due to the observed cross peaks between H-1 (d H 5.03)/Ha-8 (d H 3.37) and H-5 (d H 5.15)/Ha-8 (d H 3.37) ( Figure S10). Furthermore, the relative configuration of 1 was established by proposed biogenesis in which H-1 retained in the a-position. On the basis of the above data, the structure of 1 was assigned to be 1a,5a-H-guaia-4(14),6,9-trien ( Figure 1).
The chemistry of isolated compounds has proved the diverse structural chemotypes of sesquiterpenes and steroids from marine invertebrates. All these compounds were identified from their NMR and mass spectral data analysis, as well as on comparison with literature values. The various NMR spectra of the isolated compounds, 1 H-and 13 C-NMR can be found in Supplementary materials, i.e. Figures S1-S38.

Anti-malarial and cytotoxic activities
Sesquiterpenoids are well-known for their anti-malarial and selective cytotoxic activity, i.e. artemisinin. Additionally, discovery of marine-derived new drugs has gained much interest, especially for malaria which is the most challenging vector born disease worldwide (Ezzat et al. 2021). Representatives of the isolated compounds were evaluated for their anti-malarial and cytotoxic activity. In contrast with the highly potent anti-malarial sesquiterpene lactone artemisinin isolated the aerial parts of Artemisia annua L. plants (Abdin et al. 2003), the results shown in Table S2 indicated that compound 3 and 4 displayed weak anti-malarial activity against CQ-resistant (W2) strains of P. falciparum and only compound 7 exhibited activity for both D6 and W2 strains with IC 50 values at 3.7 and 2.1 mg/mL, respectively. The weak activity of isolated terpenoids was consistent with previous publications reported for terpenoids isolated from soft corals (Rodr ıguez et al. 2004;Thao et al. 2015;Thao et al. 2014). The results confirmed the deduced structure activity relationships and valued the importance of the endoperoxide bridge for the anti-malarial activity. In addition, cyclization of the sesquiterpenoid structure may weaken the anti-malarial activity. Moreover, the cytotoxic activity investigated the kidney fibroblast (Vero) cell line demonstrated that the isolated compounds were highly safe in the range of tested concentrations.

General experimental procedures
Optical rotations were measured in CHCl 3 and MeOH, using AUTOPOL II Automatic Polari-meter (Rudolph, Hackettstown, NJ, USA). IR spectra were recorded on an Agilent Technologies Carry 630 FTIR. 1 D-and 2 D-NMR experiments were used TMS as internal standard and carried out on Bruker Avance III-500 or Bruker Avance III 400 MHz spectrometer using CDCl 3 or CD 3 OD as solvents. An Agilent Technologies 6200 series mass spectrometer was employed for MS. High-performance liquid chromatography was performed on Waters 2795 HPLC System/Waters 996 PDA detector at 220 nm equipped with a semi-preparative reversed-phase column (Merck, HibarPurospher RP-18e, 5 mm, 250 Â 10 mm) and a preparative reversed-phase (WatersXBridge-OBD, 5 mm, 19 Â 100 mm) and a flash chromatography using Isolera Four, Biotage, USA. Column chromatography (CC) was performed over flash silica gel (SiliaFlashV R P60, SiliCycle Inc., USA), Sephadex LH-20 (Pharmacia, USA) and Diol-functionalized silica gel (Sigma, USA). Analytical TLC was carried out on silica gel F 254 aluminum sheet (20 Â 20 cm, Fluka) or Silica 60 RP-18 F 254S aluminum sheet (20 Â 20 cm, Merck). The detection of the spots was made possible by visualization under UV-254 nm and by spraying with 1% vanillin in H 2 SO 4 -EtOH (10:90), followed by heating at 110 C for 2 min. Analytical grade solvents (Fischer chemicals) were used for the isolation and the purification procedures.

Coral material
The octocoral Litophyton arboreum was collected by hand using scuba at Neweba, Red Sea, Egypt, in May 2016, at a depth of 10 m, and was kept at À 20 C for five weeks until extraction. This soft coral was identified by Dr. Khalid Eldamhougy, Professor of Marine Invertebrates, Faculty of Science, Al-Azhar University, Cairo, Egypt. A voucher specimen was deposited (ZM-0045314) at the Department of Zoology, Faculty of Science, Al-Azhar University, Cairo, Egypt.

Anti-malarial assay
A suspension of red blood cells infected with D6 or W2 strains of P. falciparum (200 lL, with 2% parasitemia and 2% hematocrit in RPMI 1640 medium supplemented with 10% human serum and 60 lg/mL amikacin) is added to the wells of a 96-well plate containing 10 lL of test samples diluted in the medium at various concentrations. The plate is placed in a modular incubation chamber that maintains an environment of 90% N 2 , 5% O 2 , and 5% CO 2 and incubated at 37 C for 72 h. Parasitic LDH activity, as a measure of growth, is determined as described earlier (Kumarihamy et al. 2020). Briefly, 20 lL of the incubation mixture is mixed with 100 lL of the Malstat TM reagent and incubated at room temperature for 30 min. 20 lL of a 1:1 mixture of NBT/ PES (Sigma, St. Louis, MO) is then added, and the plate is further incubated in the dark for 1 h. The reaction is then stopped by the addition of 100 lL of a 5% acetic acid solution. The plate is read at 650 nm using the EL-340 Biokinetics Reader (Bio-Tek Instruments, Vermont). IC 50 values are computed from the dose response curves. Artemisinin and chloroquine are included in each assay as the drug controls. DMSO (0.25%) is used as vehicle control.

Conclusion
In the current investigation of Litophyton arboreum; a widely distributed coral species in the Red Sea, we isolated an undescribed sesquiterpene; litoarbolides A (1), in addition to fourteen known metabolites. They encompassed sesquiterpenoids, steroids, and ceramides. Most of them were firstly reported from the organism of the current study, adding value to the diverse structural chemotypes of sesquiterpenes and steroids from marine invertebrates. However, a few of the isolated compounds displayed weak anti-malarial activity against CQ-resistant (W2) strains of P. falciparum, including compound 3 and 4, consistent with previous studies. Nevertheless, only metabolite 7 displayed activity for both D6 and W2 strains. The current study may lead to in-depth study of these compounds from the medicinal chemistry point of view due to their characteristic structure features. Furthermore, ecological studies and the biosynthetic pathways of 1 in soft corals warrant further research.