Natural Compounds from the Marine Brown Alga Caulocystis cephalornithos with Potent In Vitro-Activity against the Parasitic Nematode Haemonchus contortus

Eight secondary metabolites (1 to 8) were isolated from a marine sponge, a marine alga and three terrestrial plants collected in Australia and subsequently chemically characterised. Here, these natural product-derived compounds were screened for in vitro-anthelmintic activity against the larvae and adult stages of Haemonchus contortus (barber’s pole worm)—a highly pathogenic parasitic nematode of ruminants. Using an optimised, whole-organism screening system, compounds were tested on exsheathed third-stage larvae (xL3s) and fourth-stage larvae (L4s). Anthelmintic activity was initially evaluated on these stages based on the inhibition of motility, development and/or changes in morphology (phenotype). We identified two compounds, 6-undecylsalicylic acid (3) and 6-tridecylsalicylic acid (4) isolated from the marine brown alga, Caulocystis cephalornithos, with inhibitory effects on xL3 and L4 motility and larval development, and the induction of a “skinny-straight” phenotype. Subsequent testing showed that these two compounds had an acute nematocidal effect (within 1–12 h) on adult males and females of H. contortus. Ultrastructural analysis of adult worms treated with compound 4 revealed significant damage to subcuticular musculature and associated tissues and cellular organelles including mitochondria. In conclusion, the present study has discovered two algal compounds possessing acute anthelmintic effects and with potential for hit-to-lead progression. Future work should focus on undertaking a structure-activity relationship study and on elucidating the mode(s) of action of optimised compounds.


The collection, extraction and purification of compounds 1 and 2:
Collection: A sample of the southern Australian marine sponge Dactylospongia sp. (voucher code 2004_48) was collected by Mr Richard Goudie at several sites in Gunnamatta Beach, Victoria on the 26/11/2004. The sponge was collected via diving using self-contained underwater breathing apparatus (SCUBA) at a depth of 8 to 15 metres and identified by Ms Lisa Goudie as Demospongiae, Dictyoceratida, Thorectidae, Dactolospongia sp.

Extraction:
The marine sponge specimen 2004_48 (80 g, wet weight) was extracted using 3:1 methanol (MeOH)/dichloromethane (DCM) to yield 1.5 g of a brown gum. The gum was sequentially solvent partitioned into DCM followed by MeOH respectively.
Semi-preparative reversed phased HPLC: Reversed phased semi-preparative HPLC was carried out on the DCM extract (960 mg) on a Dionex P680 (solvent delivery module) equipped with a Dionex UVD340U PDA detector and a Foxy Jr. automated fraction collector. An isocratic method (100% CH3CN) was employed using a Phenomenex Luna (2) 100 Å C18 250 x 10 mm (5 µ m) column. The automatic fraction collector was programmed to collect based on elution time and a peak threshold.
This resulted in the isolation of the compounds 1 (35.8 mg) and 2 (49.8 mg).

The collection, extraction and purification of compounds 3 and 4:
Collection: The sample of the brown alga Extraction: The C. cephalornithos specimen (240 g, wet weight) was extracted using 3:1 MeOH/DCM, followed by trituration into DCM and MeOH (soluble extracts).
Column Chromatography: The DCM extract (0.77 g), was subjected to silica flash chromatography using a 20% stepwise elution profile from 100% petroleum spirits followed by DCM, ethyl acetate (EtOAc) and finally to MeOH. Following collection of the 100% MeOH fraction a final elution was performed using 100% MeOH containing 0.1% trifluoracetic acid (TFA) to yield a total of 11 fractions.

The collection, extraction and purification of compound 5:
For details about the collection, extraction and compound purification, refer to ref. [36].

The collection, extraction and purification of compounds 6 and 7:
For details about the collection, extraction and compound purification, refer to ref. [37]. Chromatography: The compounds in the DCM fraction (868 mg) were separated by a flash silica column to give a total of fifteen fractions. Fraction ten, which was eluted from the silica column using 40% DCM and 60% EtOAc, was subjected to semi-preparative HPLC and performed on a Varian Pro Star 210 solvent delivery system, using a Phenomenex C18 100 Å 250 x 10 mm (5 μ) column with a Varian Pro Star 335 PDA detector at a flow rate of 3.5 mL/min using 50% CH3CN/H2O resulting in the isolation of compound 8 (1.3 mg).

Collection: The medicinal plant
Structure determination and purity verification of isolated natural products: All natural products isolated were elucidated via NMR spectroscopy (1D and 2D) and mass spectrometry and confirmed by comparison to data reported in the literature. 1 H (500 MHz), 13 C (125 MHz), and 1D NOE spectra were acquired in CDCl3 and DMSO-d6 on a 500 MHz Agilent DD2 NMR spectrometer with referencing to solvent signals (δH 7.26; δC 77.0 and δH 2.50; δC 39.5 ppm, respectively). Two-dimensional NMR