New sterols with anti-inflammatory potentials against cyclooxygenase-2 and 5-lipoxygenase from Paphia malabarica

Abstract Marine bivalves occupy a leading share in the total edible molluscs at the coastline regions of south-eastern Asia, and are found to possess significant nutritional and biological potential. Various in vitro evaluation (antioxidant and anti-inflammatory) guided purification of ethyl acetate–methanol (EtOAc–MeOH) extract of bivalve clam, Paphia malabarica characterised two new sterol derivatives as 23-gem-dimethylcholesta-5-en-3β-ol (1) and (22E)-241,242-methyldihomocholest-5,22-dien-3β-ol (2) collected from the south-west coast of Arabian Sea. Their structures were unambiguously assigned on the basis of 1D, 2D NMR spectroscopy and mass spectrometry. The antioxidant and anti-inflammatory activities of 2 as determined by DPPH/ABTS+ radical scavenging and anti-cyclooxygenase-2/5-lipoxygenase assays were significantly greater (IC50 < 1 mg/mL) than 1 (IC50 > 1 mg/mL). Structure–activity relationship analysis revealed that the bioactivities of these compounds were directly proportional to the electronic and lipophilic parameters. This is the first report of the occurrence and characterisation of 23-gem-dimethyl-3β-hydroxy-Δ5-cholestane nucleus and C-30 dihomosterol from marine organisms.


Introduction
The bivalves are low-cost sources of protein, amino acids, essential minerals, vitamins and low saturated fat . The importance of clam aquaculture has competitively increased against finfish fisheries with concerns as an alternative to relatively expensive food supplements, nutraceuticals and synthetic drugs available in the pharmaceutical stores . Considering the underutilisation of these species, exploring bioactive compounds and development of any biologically useful products has duel benefits as health products and their commercial farming in coastal habitats. The bioactive secondary metabolites produced by marine bivalve molluscs were identified as popular pharmacophores against inflammations ) and various oxidative stress-induced diseases (Ouyang 2006;Chakraborty et al. 2014). Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) have been implicated to be induced by various inflammatory stimuli, such as cytokines, prostaglandins (such as PGE 2 , PGF 2 α), leukotrienes (such as LTB 4 ) under inflammatory responses (Mitchell et al. 1993) and the antioxidants were reported to suppress COX-2, 5-LOX enzymes, thus inhibit the release of inflammatory prostaglandins or cytokines (D' Orazio et al. 2012). The (24R)-24-propylcholesterol was isolated from Hiptage benghalensis, a plant species reported for suppression of PGE 2 inflammatory effects as well as anti-iNOS/anti-COX-2 potentials (Hsu et al. 2015).
Sterols are known as the bioactive lipid metabolites, and were found as major constituents in marine invertebrates, such as sponges, corals, bryozoans and molluscs (Goad & Scheuer 1978). More than 200 sterols of 3β-hydroxy-Δ 5 (or saturated) cholestane nucleus and a C 8 -C 10 side chain occurring in marine organisms were reported (Sarma et al. 2005). In particular, sponges and molluscs were extensively investigated for different types of steroids than invertebrates of other phyla (Joosse 1978;Sica 1980). Among different groups of steroids, poly-oxygenated tetracyclic nucleus with varying degrees of unsaturation and atypical side chain substitution (D'Auria et al. 1993), steroidal alkaloids, 3β-cholestane esters, steroid-amino acid conjugates, steroids with spiro A/B ring system (Su et al. 2007) and bicyclo [4.4.1] or bicyclo[4.3.1] A/B steroids were prevalent among the compounds isolated from marine organisms (Amagata et al. 2003;Wang et al. 2015).
As part of our ongoing program towards the isolation of biologically active compounds from marine molluscs, the crude EtOAc-MeOH extract (1:1 v/v) of the bivalve yellow-foot mollusc Paphia malabarica (family, Veneridea), collected from Arabian Sea, was screened for antioxidative and anti-inflammatory properties by various in vitro assays. The EtOAc-MeOH extract showed promising antioxidative and anti-inflammatory activities in the initial screening as determined by DPPH/ABTS + radical scavenging and anti-cyclooxygenase-2/5-lipoxygenase assays. We herein describe the 1 H NMR guided isolation and structure elucidation of two new sterol derivatives, 23-gem-dimethylcholesta-5-en-3β-ol (1) and (22E)-24 1 ,24 2methyldihomocholest-5,22-dien-3β-ol (2) from the EtOAc-MeOH extract (1:1 v/v) of P. malabarica. Structure-activity relationship analysis was used to correlate different physicochemical parameters that significantly contribute towards the antioxidative and anti-inflammatory properties of the title compounds.

Bioactivities a 1 2
Antioxidant activity dPPh scavenging activity 1.01 p ± 0.02 0.81 q ± 0.09 aBts + scavenging activity 1.12 p ± 0.06 0.98 q ± 0.02 Anti-inflammatory activity coX-2 inhibition activity 1.15 p ± 0.03 0.92 q ± 0.08 5-loX inhibition activity 1.02 p ± 0.07 0.96 q ± 0.01 composition of C 29 H 50 O as 23-gem-dimethylcholesta-5-en-3β-ol (1) with 5° of unsaturation containing one double bond and four-ring systems. Previous works on 1 H and 13 C NMR spectroscopy established the characteristic chemical shifts of various types of protons and carbons of steroids; and based on these data steroid shift assignments were made for the present study. The absence of the characteristic aromatic proton signals in the 1 H NMR spectrum confirmed that the four cyclic rings were not of aromatic origin. The 1 H NMR spectrum contains several overlapping second-order multiplets with the type ABCDEF. The olefinic signals appeared at δ 140.76 and 121.72 in the 13 C NMR spectrum indicating a double bond. The relative downfield shift at δ C 140.76 referred to quaternary carbon adjacent to a double bond in the cyclic ring. The highly deshielded 1 H signal at Δ 5 (H-6, δ 5.35, dd), which was found to be a double doublet due to the adjacent δ H 1.98 and 1.52 protons attached to carbon (C-7, δ 31.91) with coupling constant of 5.24 and 3.36 Hz. The presence of an olefinic group in the carbocyclic ring was identified by comparison with cholestane analogues as detailed in a previous literature (Reich et al. 1969). An earlier study of steroid derivatives explained the olefinic proton resonances in the region δ 5.0-5.6 (br, J = 5 Hz) due to the >C=CH-CH 2 skeleton (Goad & Akihisa 1997). The proton at H-3 (δ 3.50) attached to tertiary carbon (δ 71.82) bearing the hydroxyl group (-OH) appeared as a quintet at the downfield position of the 1 H NMR spectrum. The downfield shift of carbinol carbon at C-3 was the result of a greater electron-withdrawing power of the hydroxyl group. It is of note that for the axial cyclohexanols, there is a δ 1 downfield shift of C-3, probably because of smaller 1, 3-interaction of the -OH group with the H-3 relative to the hydroxyl group. Further specific deuteration of the hydroxyl proton was used for identifying the C-3 carbon. The signal for deuterated -OH proton essentially disappeared. The spectroscopic analysis of 1 H NMR, 13 C NMR along with 1 H-1 H COSy, HSQC and HMBC relations allowed the elucidation of a cholestane network with δ 5.35 (H-6) as double bond (δ C 121.72) and δ 3.50 (H-3) hydroxyl (δ C 71.8), which was consistent with the literature study (Tian et al.  (attributed to C-2), δ 71.82 (C-3), δ 140.76 (C-5), δ 121.72 (C-6), δ 36.52 (C-10) and those from H-6 (δ 5.35) to δ 42.31 (C-4), δ 31.91 (C-7), δ 36.52 (C-10) supported the bicyclic framework. The three high-field methine (-CH-) protons, δ 0.95, 1.10 and 1.02 were assigned to carbons at C-9, C-14 and C-17 positions, respectively. The two quaternary carbons (C-10 and C-13) were attributed to characteristic chemical shift and signal pattern of steroids (Reich et al. 1969 ; H-14 (δ 1.10) to C-16 (δ 28.33); H-15 (δ 1.56) to C-16 (δ 28.33) unambiguously described the attachment of cylcopentane (D) moiety in compound 1 as also supported by previous literature (Díaz-Marrero et al. 2013). The C-4 proton has been flanked on both side by downfield shifting of functional groups (OH at C-3 and C=C at C-5, 6) and the two H-4 protons are pulled downfield to δ 2.25-2.29 away from the pack of overlapped resonances in the 1 H NMR spectrum. The 13 C NMR signal at the far downfield region (δ 140.76) was less intense (shorter) than other peak at δ 121.72, because of slow relaxation, it must be a quaternary carbon. It is of note that the proximity of protons is the primary means of relaxation of 13 C nuclei, and therefore, the carbons lacking proton relax much more slowly, and give less intense peaks, if the relaxation delay is short (RD = 1.7 s). The more substituted carbon (at C-5) shifted more downfield relative to C-6 due to the steric crowding effects. The resonances of the cholestane side chain (C-20 to C-27) can readily be identified by comparison with 2, 6-dimethyloctane as a model compound as detailed in a previous literature (Reich et al. 1969) and confirmed by 1 H-1 H COSy. The 1 H-1 H COSy connections between H-17 (δ 1.02) and H-20 (δ 1.35) along with their HSQC values confirmed the attachment of side chain, C-20 at C-17 (Díaz-Marrero et al. 2013). The HMBC relations from H-17 (δ 1.02) to C-21 (δ 18.72); H-21 (δ 0.92) to C-13 (δ 42.52) and C-20 (δ 36.27) further supported the presence of side chain attachment to the sterol moiety. The two quaternary carbons, C-10 and C-13 were assigned using a long-range 1 H-13 C correlation (HMBC) spectrum. A two bond correlation to the methyl protons in each case yields the assignment of C-10 at δ 36.52 and C-13 at δ 42.52. The 1 H and 13 C connectivity deduced from HSQC and HMBC experiments confirmed the side chain framework. The structure contained four singlets which made the compound different from other reported steroids from molluscs (Santalova et al. 2007 41, 11.87, 28.01, 29.72, 18.72, 19.31 and 22.82, respectively, based on the HSQC experiments. The numbers of carbon atoms were confirmed as 29 through 13 C NMR and DEPT analysis in which 7 -CH 3 , 10 -CH 2 and 8 -CH groups with a total proton integral of 51.93. The methyl groups, including the two gem-dimethyl groups give rise to tall, sharp peak at the upfield region. The 1 H, 13 C NMR and HMBC correlations were specified (Table S1). The configurations at the individual carbons were determined using their J values and detailed NOESy experiments ( Figure S17A). The relative configuration of the carbons (C-3 and C-8) in 1 were deduced by 1 H coupling constants, J 3a, 2a = J 3a, 2b = 4.2 Hz and J 8b, 9a = 12.3 Hz. In NOESy, the proton H-3 (δ 3.50) exhibited correlation with H-6 (δ 5.35)/Hα-4 (δ 2.29), and therefore, have been considered as α protons, which in turn indicated the β-disposition of hydroxyl group at C-3 (Tian et al. 2011;Sun et al. 2013). The methyl groups (H-18 and H-19) of the cholestane derivative were correlated with Hβ-4 (δ 2.25)/Hβ-12 (δ 2.01)/H-8 (δ 0.91)/H-20 (δ 1.35), which apparently suggested their β-orientation, and these attributions were supported by the literature reports (Calderón et al. 2004;Tian et al. 2011). Based on these interpretations, the compound was characterised as 23-gem-dimethylcholesta-5-en-3β-ol.
The molecular ion peak at m/e 414 (C 29 H 50 O + , [M] + ) appeared to undergo elimination of one molecule of water to yield 23-gem-dimethylcholesta-2,5-diene (1a) (m/e 396). One of the most common types of fragmentation in C-17 substituted steroids is that the loss of side chain leads to an abundant ion at m/e 255 in the corresponding spectra. The side chain elimination (2, 4, 4-trimethylheptane) from the fragment ion at m/e 396 yielded the fragment with m/e 255 (1s), which on subsequent rearrangement yielded the fragments at m/e 163 (1t), 95 (1v), 81 (1w) and 69 (1x). The molecular ion peak at 69 (1x) was found to be the base peak and corresponding to penta-1, 4-diene. The fragment ion at m/e 345 was formed from m/e 399 (23-gem-dimethylcholesta-2-ene), through a Retro-Diels-Alder mechanism. Further decomposition of the ion at m/e 399 was perceived to be accompanied by the loss of the C-18 methyl group and results in an ion at m/e 203. The molecular ion peak at m/e 264 (1y) resulted from the fragmentation of ions through elimination of water molecule and side chain. The olefinic (C=C) and alkyl (C-H) groups IR stretching vibrations were represented by the 1664 and 2945 cm −1 absorption bands, respectively. The distinctive absorption at 3427 cm −1 indicated O-H stretching vibration. The FTIR absorption bands at 1374, 1332 (C-H rocking), 1243, 1188 (C-C stretch), 881, 835, 733 cm −1 (=C-H bend) substantiated the structure of substituted cholestane.
The molecular ion peak at m/e 426 (C 30 H 50 O ·+ , [M] + ) appeared to undergo elimination of one molecule of water and an isopropyl group to yield 24 1 ,24 2 -methyldihomocholest-5,22-trien-3β-ol (2h) (m/e 409), which underwent side chain elimination followed by rearrangement at ring D to afford a fragment with m/e 229 (2i). The fragment ion at m/e 357 were formed from m/e 411 (24 1 ,24 2 -methyldihomocholest-5,22-dien-3β-ol), through a Retro-Diels-Alder mechanism. Fragmentation of the ion at m/e 366 (2b) was perceived to be accompanied by the loss of a C-6 fragment (assigned to hex-1-ene) resulted in an ion at m/e 285 (2c), which on subsequent rearrangement yielded the fragments at m/e 177 (2d), 135 (2e), and 69 (2f). It is of note that the fragment ion at m/e 69 (C 5 H 8 ·+ ) appeared as base peak of 2. The IR spectrum revealed broad absorption band at v max 3427 cm −1 attributed to hydroxyl and v max 1664 cm −1 to olefinic (C=C) functionalities.
The antioxidant and anti-inflammatory activities of 1 as inferred by DPPH/ABTS + radical scavenging and anti-cyclooxygenase-2/5-lipoxygenase assays were significantly lesser (IC 50 > 1 mg/mL) than 2 (IC 50 < 1 mg/mL) (Table 1). Commercially available antioxidative agent α-tocopherol has been used as standard in the present study. It is of note that no significant differences in the DPPH and ABTS + radical scavenging activities of compound 2 (IC 50 < 1 mg/mL) and α-tocopherol (IC 50 < 1 mg/mL) were recorded (P < 0.05). Selective inhibition of lipoxygenase (5-LOX) is a favored system to deter inflammatory stimuli . The commercially available synthetic anti-inflammatory drug ibuprofen has been used to compare the results. Notably, 5-LOX inhibitory activity of ibuprofen (IC 50 0.93 mg/ mL) showed no significant difference (P < 0.05) compared to the title compounds (IC 50 0.96-1.02 mg/mL), particularly (22E)-24 1 ,24 2 -methyldihomocholest-5,22-dien-3β-ol (2) (IC 50 0.96 mg/mL) isolated from P. malabarica. Structure-activity relationship analysis revealed that the bioactivities of the title compounds purified in this study were directly proportional to the electronic and lipophilic parameters. The electronic factor such as polarizibility (Pl) was found to significantly contribute towards the antioxidant and anti-inflammatory activities of the compounds 1 and 2. It is of note that the compound 2 was found to possess an additional olefinic group at C22=C23 position, and therefore might contribute towards greater bioactive characteristics. The antioxidant activity of 1 was found to be significantly lesser (IC 50 1.01 mg/mL) than 2 (IC 50 0.81 mg/mL) apparently due to the absence of an additional double bond in the former, resulting in lesser electron delocalisation. This resulted in higher electronic properties of 2 (Pl 53.21 × 10 −24 cm 3 ) than 1 (Pl 51.23 × 10 −24 cm 3 ). This hypothesis has been supported by studies conducted previously to find that the radical scavenging activities of a compound proportionately increases with the presence of double bonds due to effective electron transfer through the process of electron delocalisation (Cai et al. 2006). Similar reasons might be attributed to the greater anti-inflammatory activity of 2 than that of compound 1. The antioxidant activities of the substituted cholestane derivatives were also found to be directly proportional to their hydrophobic character as determined by octanol-water coefficient (log P ow ). The greater the value of log P ow , the greater the molecular hydrophobicity of the molecules. The hydrophobic character of 1 (log P ow 8.19) is lesser than those of 2 (log P ow 8.23). The lowering of activity of 1 might be explained due to the decrease in its log P ow , the membrane permeability properties and the reactivity towards DPPH free radical. It has been hypothesised that the free radical DPPH can interact with the compounds with higher hydrophobic coefficients (greater log P ow value) and therefore, showed positive correlation with the scavenging efficiency towards lipophilic DPPH. Since antioxidants have to diffuse into liposomes to react with lipophilic free radicals (such as peroxides and DPPH as the in vitro model), it is reasonable to expect that a greater hydrophobicity of antioxidants is relevant to their radical scavenging efficiency in liposomes and cells. Based on these results, it can be inferred that hydrophobicity is particularly important factor in determining the antioxidant and anti-inflammatory activities. In particular, the presence of gem-dimethyl groups at C-22 position of the cholestane framework attributed to greater steric restrictions of 1, resulting in lesser target bioactivities than 2.
More significantly, the marine-derived steroids with their diverse structures were found to exhibit interesting therapeutic properties (Goad & Akihisa 1997;Whitson et al. 2009). Anti-inflammatory properties of steroidal compounds isolated from marine invertebrates against pro-inflammatory COX-2 and cytokines were reported in earlier literature for example, the inhibitory effect of diunsaturated C-27 polyhydroxy sterols isolated from marine gastropod, Trimusculus peruvianus (Chao et al. 2008;Su et al. 2008;Díaz-Marrero et al. 2013;Thao et al. 2013). Consequently, the detection and identification of COX-2/5-LOX-specific inhibitors could have a potentially profound impact on the treatment of a number of inflammatory diseases and disorders.

General procedures
All reagents and solvents were of spectroscopic or chromatographic or analytical grade from Merck (Darmstadt, Germany). Fourier-transform infrared (FTIR) spectra (KBr) scanned between 4000 and 400 cm −1 (Perkin-Elmer Series 2000 FTIR spectrophotometer). 1D and 2D NMR spectra were analysed on a Bruker Avance DPX 500 (500 MHz) spectrometer in CDCl 3 aprotic solvent with TMS (standard). The GC-MS (Perkin-Elmer Clarus 680 GC-MS fitted with Elite 5 MS non-polar) analyses were performed in electronic impact (EI) ionisation mode in bonded phase capillary column (50 m × 0.22 mm i.d. × 0.25 μm film thicknesses) (Chakraborty et al. 2014). uV spectra were acquired on a Varian Cary 50 uV-vis spectrometer (Varian Cary, uSA) . The melting point of the compounds were determined using Melting Point Apparatus (VMP-DS, Veego, Mumbai, India) and the angle of rotation of the compounds were recorded on a polarimeter (AP-300, ATAGO, Japan).

Animal material and extraction
According to , P. malabarica (10 kg) samples were freshly collected from Ashtamudi Lake (8°59′ N and 76°36′ E) situated along the south-west coast of India. A voucher specimen has been deposited in the repository of the project titled 'Development of nutraceutical supplements from marine molluscs, macroalgae and shrimps' (grant number ICAR/ CRP-HF/2016) under the ICAR Consortium Research Platform funded by the Indian Council of Agricultural Research, New Delhi, India, with a voucher specimen No. ICAR/CRP-HF/AC 368. The edible flesh (6 kg) separated from the cleaned shell-on samples were ground and freeze dried by lyophilisation (Martin Christ alpha 1-4 LD Plus freeze-drier, Germany). The lyophilised powder (1200 g, yield 20.0%) was extracted with EtOAc-MeOH (1:1, v/v, 500 mL × 3) at 40 °C followed by sonication (8 h) under an inert atmosphere of N 2 . The extracts were filtered over anhydrous Na 2 SO 4 (100 g), before being evaporated in vacuo using a rotary vacuum evaporator (50 °C) (Heidolph, Germany) to dryness to get a dark brown oily viscous residue, which was referred to as the crude extract of P. malabarica (55.0 g, yield on dry basis 4.58%).

Antioxidative and anti-inflammatory assay
The antioxidant and anti-inflammatory activities of purified compounds (1-2) isolated from P. malabarica were carried out. The antioxidant properties were evaluated by DPPH (Chew et al. 2008) and ABTS + radical decolorisation assay (Vijayabaskar & Shiyamala 2012;Chaudhary et al. 2015). In vitro anti-inflammatory properties (Chavan et al. 2012) were determined by inhibition of COX-2 (Larsen et al. 1996) and 5-LOX (Baylac & Racine 2003) enzymes. The plot of scavenging and pro-inflammatory enzyme inhibitory activities were recorded, and the results were expressed as IC 50 (the concentration of samples at which it inhibits/scavenge 50% of enzyme/radical activities and expressed in mg/mL) value. The structure-activity relationship analysis was carried out using different physicochemical parameters of the purified compounds. The structural descriptors were acquired from ChemDraw ultra (8.0 database), steric (molar volume, molar refractivity), hydrophobic (log P ow : logarithmic value of the octanol-water partition coefficient) and polarisability (electronic descriptor) factors.

Statistical analysis
Statistical Program for Social Sciences 13.0 (SPSS, uSA, ver. 13.0) was assessed for calculating significant differences between the means (One way analysis of variance, ANOVA) of triplicates ± standard deviation of all assays and were represented as P < 0.05.

Conclusion
To the best of our knowledge, 23-gem-dimethyl-3β-hydroxy-Δ 5 -cholestane nucleus and C-30 dihomosterol (1 and 2) represent the first examples of steroids possessing the 23-gem-dimethyl derivative of sterol and C-30 dihomosterol system from a natural source. The C 30 dihomocholest-dien-3β-ol from P. malabrica has potential bioactive potential as natural antioxidant and anti-inflammatory pharmacophore. The extensive biochemical analyses and bioassay guided purification followed by identification of bioactive secondary metabolites from this species can positively influence the clam agriculture, seafood exports and pharmaceutical fields in the future.