Paralemnolins X and Y, New Antimicrobial Sesquiterpenoids from the Soft Coral Paralemnalia thyrsoide

The organic extracts of the Red Sea soft coral Paralemnalia thyrsoides has led to the identification of two neolemnane-type sesquiterpenoids: paralemnolins X and Y (1, 2). In addition to these newly characterized compounds, ten known metabolites (3–12) were isolated. Previously reported compounds were elucidated by literature comparison of spectroscopic data (1D and 2D NMR as well as MS data). In vitro antimicrobial activity was investigated for compounds (1–12) against Staphylococcus aureus, Escherichia coli, Candida albicans and Aspergillus niger. Compound 5 showed antimicrobial activity against all assayed microorganisms.


Introduction
While natural product research includes plant, marine animal and microbe sources, more recently, marine flora and fauna have afforded an exciting number of potential therapeutic agents that are currently in preclinical and clinical evaluation due to promising biological activities with in vivo and in vitro assays [1][2][3][4][5][6][7]. Such drug leads have also significantly contributed to our understanding of cellular biochemical processes. Typical marine skeletons combined with robust biological activities have attracted the attention of pharmacists, chemists and biologists [8][9][10][11].
With the high endemic biota observed in the Red Sea, this marine region serves as an epicenter for marine biodiversity. Indeed, the Red Sea is home to over 40% of the 180 soft coral species that have been discovered worldwide [12]. Soft corals of the genus Lemnalia and Paralemnalia, in particular, have been discovered to be a significant source of bioactive chemicals such as sesquiterpenoids of the nardosinane, neolemnane and africanane types [13][14][15][16]. Norsesquiterpenes have been found to be abundant in Paralemnalia thyrsoides (Alcyonaceae) soft coral [14,[16][17][18][19][20][21]. Previous chemical studies of P. thyrsoides resulted in the isolation of sesquiterpenoids known as paralemnolins A-W [18,19,[21][22][23].
Here we report on two neolemnane-type sesquiterpenoids isolated from the Formosan soft coral P. thyrsoides.

Coral Material, Extraction and Separation
A collected Red Sea soft coral Paralemnalia thyrsoide (Hurghada in March 2017) was identified by Montaser A. Alhammady (co-author) with a voucher specimen (08RS1075) deposited in the National Institute of Oceanography and Fisheries (NIOF), Egypt.
Nutrient agar medium was used for bacterial cultivation followed by suspension in nutrient broth medium at 37 • C for 24 h. The fungus culture was grown on potato dextrose agar medium at 28 • C for 4 days, and then suspended in potato dextrose broth. The turbidity of the suspension was adjusted to that of the standard 0.5 McFarland solution.

Minimum Inhibitory Concentration Determination
The MIC values were determined by the broth microdilution assay (NCCLS, 2008) [28] with slight modifications. Each sample was initially dissolved in DMSO, and subsequently diluted with broth media to reach the desired final concentration. Five-fold dilutions were prepared in a 96-well plate. The microbial suspensions were added into each well, then incubated at 37 • C for 24 h for bacteria and at 28 • C for 72 h for fungi. The MIC value was determined as the lowest concentration of the sample that inhibited microbial growth. The assay was carried out in nutrient broth medium for bacteria and potato dextrose broth medium for fungi. The assay was performed according to Hammer et al. (1999) [29], with slight modifications. Briefly, 1 mg of the pure compound was dissolved in 50 µL DMSO, and 10 µL was added as the initial concentration in the first column of the sterile polystyrene 96-well plates. Then, 190 µL of the tested microbial suspension adjusted to 5 × 10 5 CFU/mL was added. Serial dilutions were performed by the addition of 100 µL of the first column to the second one, and so on. The final volume was adjusted to 200 µL on each well by the addition of the microbial suspension to obtain final concentrations of the tested compounds ranging from 100 to 3.125 µg. Microbial growth controls were made by replacing the tested compound with the same volume of DMSO (in order to eliminate the possible antibacterial effect of the solvent). Sterility controls were prepared by using broth media alone. The plates were covered with a sterile plate sealer, carefully mixed and incubated at 37 • C for 24 h for bacteria and 28 • C for fungi. Microbial growth was indicated by the turbidity. The absence of microbial growth was interpreted as antimicrobial activity. The MIC value was taken as the lowest concentration of the test agent that caused complete inhibition (100%) of microbial growth [30].