First report of spiro-compounds from marine macroalga Gracilaria salicornia: prospective natural anti-inflammatory agents attenuate 5-lipoxygenase and cyclooxygenase-2

Abstract The inflammation pathology is an orchestrated biological process and the dual inhibition of pro-inflammatory enzymes 5-lipoxygenase and cyclooxygenase-2 has been found to be an effective approach against inflammation. This study involves the characterisation of two previously undescribed spiro[5.5]undecanes, 3-(hydroxymethyl)-7-(methoxymethyl)-3,11-dimethyl-9-oxospiro[5.5]undec-4-en-10-methylbutanoate (1) and 4-ethoxy-11,11-dimethyl-7-methylene-8-(propionyloxy)spiro[5.5]undec-2-en-104,106-dihydroxytetrahydro-2H-pyran-10-carboxylate (2) with potential anti-inflammatory properties, from seaweed Gracilaria salicornia by extensive-spectroscopic-experiments. These metabolites recorded prospective bioactivities against 5-lipoxygenase (IC50 < 2.80 mM), whereas their selectivity indices were significantly greater (∼1) than ibuprofen (0.89) (p < 0.05), which attributed selective anti-inflammatory potencies of the studied spiro[5.5]undecane derivatives against inducible cyclooxygenase-2 than constitutive cyclooxygenase-1. Radical scavenging potential of spiro[5.5]undec-2-en-104,106-dihydroxytetrahydro-2H-pyran-10-carboxylate analogue (2) against oxidants, 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis-3 ethylbenzothiozoline-6-sulfonic acid were found to be greater (IC50 < 1.25 mM) than commercial standard, α-tocopherol (IC50 1.42–1.79 mM). The greater hydrogen-bonding interactions and binding affinity of 2 (−10.13 kcal/mol) with 5-LOX appropriately corroborated its prospective anti-inflammatory activity.


Introduction
Spiro frameworks represented as a significant class of naturally occurring compounds characterised with highly potent bioactive properties and has been known to be present in various phytochemicals with their wide range of pharmacological applications (Leonid et al. 2012). These spirocyclic compounds are of recent interest due to their remarkable conformational features and their structural implications on biological systems, and were largely found in various biologically active natural products and pharmaceuticals (Bartoli et al. 2011). The stereochemistries of spiranes with six-membered rings have been extensively studied (Cismas et al. 2005). The non-halogenated rearranged spiro-compound, 1, 4,9-trimethyl-5-methylidenespiro[5.5]undec-1,7-diene-4,9-diol and halogenated spiro-fused compounds, such as 2-bromo-1,1,9-trimethyl-5methylidenespiro[5.5]undec-7-ene-3,9-diol and 2-bromo-9-bromomethylidene-1, 1-dimethyl-5-methylene spiro [5.5]undec-7-ene were previously reported from a red seaweed, Laurencia scoparia with bio-potentials (Davyt et al. 2001). Also, the halogenated and non-halogenated spiro-compounds, which were classified as compositacins, were reported from red seaweed, Laurencia composita (Yu et al. 2017). In order to access the versatile motifs of spiro-compounds with unique structural features, it has become one of the prominent research areas to the natural product chemists.
Phytochemical investigation on red seaweeds reported different biological activities (Makkar and Chakraborty 2017;Makkar and Chakraborty 2018a). A large amount of scientific research has been reported on specialised metabolite chemistry of red seaweeds of the genus Gracilaria, whereas related reports on Gracilaria salicornia (C. Agardh) E. Y. Dawson were relatively rare. It has been documented that the organic extracts of Gracilaria sp. (family Gracilariaceae) were comprised of secondary metabolites with anti-inflammatory and antioxidant potentials (Antony and Chakraborty 2018;Makkar and Chakraborty 2018b;Makkar and Chakraborty 2018c). The red seaweed G. salicornia were ubiquitous in the Indian coastline, although bioactive compounds from this species were not extensively investigated. In the present study, the organic ethyl acetate:methanol (EtOAc:MeOH) extract of G. salicornia collected from southern coastline of Indian peninsular was fractionated by repeated chromatographic techniques, which resulted in the identification of two unprecedented spiro-fused analogues through the extensive spectroscopic experiments. The pharmacological activities were evaluated by various anti-inflammatory and antioxidant assays, whereas the physicochemical parameters of the titled compounds were used to corroborate their structural characteristics with bioactive potentials. The in silico mode of pro-inflammatory 5-lipoxygenase (5-LOX) inhibition by spiro-compounds were assessed by molecular modelling analysis to correlate with their in vitro anti-inflammatory properties.

Antioxidative and anti-inflammatory activities
Antioxidant potentials of the spiro-compounds (1) and (2) isolated from G. salicornia were investigated by in vitro radical quenching activities against DPPH and ABTS þ ( Table S2). The compound 2 exhibited higher DPPH and ABTS þ radical quenching activities (IC 50 1.13 and 1.24 mM, respectively) than those displayed by 1 and standard antioxidant agent a-tocopherol (IC 50 !1.40 mM). The compound 2 registered significantly higher activities against pro-inflammatory 5-LOX (IC 50 1.91 mM) than that displayed by 1 (IC 50 2.78 mM, p < 0.05). The selectivity index of the titled compounds were also found to be higher (IC 50 anti-COX-1/IC 50 anti-COX-2 $ 1) than that exhibited by the non-steroidal anti-inflammatory agent ibuprofen (0.89) (p < 0.05). This appropriately suggested the greater attenuating activities of the studied spiro-compounds against inducible pro-inflammatory cyclooxygenase-2 (COX-2) than cyclooxygenase-1 (COX-1). The molecular descriptor variables of the studied spiro-compounds appeared to play predominant roles in attributing the target bioactivities. Antioxidative and antiinflammatory activities were found to be directly proportional towards the electronic (topological polar surface area, tPSA and polarisability, Pl) and hydrophilic-lipophilic balance (log P ow , logarithm of octanol/water partition-coefficient), and not the molecular bulk. The tPSA of 2 was found to be greater (111.52) than that of 1 (72.83) and a-tocopherol (29.46). The total numbers of electron-rich centres were greater in 2 and that appeared to result in its higher polarisability (48.07) and antioxidant potentials (Table S2). Therefore, the electronic factors appeared to play significant role to govern the bioactive potentials ). The log P ow of 2 was found to be within the desirable range ($2.2), which might cause a suitable lipophilic-hydrophobic relation (Lipinski 2004) and receptor binding (Huuskonen et al. 2000) leading to higher target bioactivities than other studied compounds.

In silico molecular modelling studies
The attenuating potential of the studied spiro derivatives against the pro-inflammatory enzymes has been considered as one of the effective approaches to monitor the disturbances associated with inflammatory pathologies. The titled compounds were subjected to molecular docking analysis using pro-inflammatory enzyme 5-LOX ( Figure  S25), wherein the effective inhibition of compounds (1-2) was initially assessed by the RMSD reports in accordance with their decreasing order of binding energies (Table  S3). Among the spiro-compounds, compound 2 recorded least binding energy of À10.13 kcal/mol subsequent to that exhibited by 1 (À10.00 kcal/mol). Likewise, the constant of enzyme inhibition (Ki) was lesser for 2 (37.80 nM) towards 5-LOX enzyme than that exhibited by 1 (46.89 nM). Also, intermolecular energy was lesser for 2 (À11.51 kcal/mol).

Sample collection and preparation of organic extract
The fresh seaweed samples G. salicornia were acquired from Mandapam region (Gulf of Mannar, situated between 8 48 0 N, 78 9 0 E and 9 14 0 N, 79 14 0 E) of southern coastline of peninsular India. Samples of seaweeds (voucher specimen number AC. 3.1.1.4) were subjected to repeated washing with running water before being dried for 2-4 days at room temperature. The shade-dried seaweeds were kept in air-tight containers for further experiments. Shade-dried seaweed samples were powdered (1 Â 10 3 g), and were subjected to solvent extraction with equal volume (1:1 v/v, 500 mL) of EtOAc and MeOH at 60-65 C for 3-6 h. The organic crude extract was filtered through Whatman No. 1 by anhydrous sodium sulfate (Na 2 SO 4 , 100 g), and the clarified filtrate was concentrated by rotary vacuum evaporator (Heidolph, Instruments GmbH & Co., Schwabach, Germany) at about 50 C for obtaining the crude viscous mass (33 g) of G. salicornia. The latter was fractionated by chromatographic techniques.

Statistical analysis
The triplicate results were tabulated with mean ± standard deviation. Significant differences (p 0.05) obtained among their means were documented by using one-way analysis of variance (ANOVA; Statistical Program for Social Sciences, USA, ver. 13.0).

Conclusion
A large amount of scientific research has been reported on the specialised metabolite chemistry of the red algae of the genus Gracilaria, whereas related reports on Gracilaria salicornia are relatively rare. In the present study, the organic EtOAc:MeOH extract of G. salicornia was fractionated by repeated chromatographic techniques to obtain two unprecedented spiro fused analogues. Among the isolated compounds, the one with spiro[5.5]undec-2-en framework (2) exhibited higher pro-inflammatory enzyme inhibitory activities (IC 50 1.91 mM) over that bearing oxospiro[5.5]undec-4ene moiety (1) (IC 50 ! 2.78 mM). The spiro analogues isolated from G. salicornia registered greater radical scavenging potentials against ABTS þ (IC 50 < 1.75 mM) than that exhibited by a-tocopherol (IC 50 > 1.75 mM). Structure-activity relationship analyses showed that higher polarisability, tPSA and lesser steric bulk of spiro[5.5]undec-2-en (2) predominantly contributed towards the comparatively radical quenching and proinflammatory enzyme inhibitory activities than those displayed by the oxospiro[5.5]undec-4-en analogue (1). Molecular docking analysis inferred that the spiro derivative (2) exhibited binding energy of -10.13 kcal/mol with efficient interactions of 5-LOX enzyme, which corroborated its greater inhibitory potential. In particular, spiro[5.5]undec-2-ene analogue was found to be an effective inhibitor of 5-LOX along with potential antioxidative properties, and therefore, might be utilised as valuable pharmacophore lead against oxidant species and inflammation pathology.