An unprecedented antioxidative isopimarane norditerpenoid from bivalve clam, Paphia malabarica with anti-cyclooxygenase and lipoxygenase potential

Abstract Context: The yellow-foot bivalve clam, Paphia malabarica Chemnitz (Veneridae) is distributed in the southwest coastal regions of India. The ethyl acetate-methanol extract of this species exhibited significant antioxidant and anti-inflammatory activities. Objectives: To purify and characterize the bioactive compound from P. malabarica along with in vitro assays. Materials and methods: The edible portion of P. malabarica was freeze dried (1.20 kg, yield 20.0%) and extracted with ethyl acetate and methanol (1:1 v/v, 500 mL ×3) by sonication (8 h). The antioxidant activity against DPPH/ABTS+ and anti-inflammatory potential against cyclooxygenase-1,2 (COX-1, 2)/5-lipoxygenase (5-LOX) enzymes were carried out with varying concentrations (0.25–2.00 mg/mL) to determine the IC50 values. The crude extract was chromatographically fractionated and the fraction showing greater potential was further fractionated to yield the pure compound, which was characterized by extensive NMR, IR and mass spectroscopic analyses. Results and discussion: The fractionation of crude extract of P. malabarica was followed by structural characterization of the new rearranged isopimarane derivative, 18 (4 → 14), 19 (4 → 8)-bis-abeo C19 norditerpenoid. The isopimarane derivative displayed comparable antioxidant activity with α-tocopherol (IC50 DPPH scavenging activity ∼0.6 mg/mL), whereas anti-inflammatory (anti-5-LOX) effect of the title compound was significantly greater (IC50 0.75 mg/mL) than ibuprofen (IC50 0.93 mg/mL). In addition, the greater selectivity index (anti-COX-1IC50/anti-COX-2IC50 0.85) explained the lesser side effects of the isopimarane norditerpenoid than the nonsteroidal anti-inflammatory drug-based therapies. Conclusions: The isopimarane derivative isolated from P. malabrica can be a natural substitute to commercial drugs in future.


Introduction
The oxidative stress in the cell organelles stimulate several unfavourable effects in our body leading to various ailments, particularly ageing, hypertension, inflammatory reactions, diabetes, cancer, etc., that were found to depend on the accumulation of reactive oxygen species (ROS) (Lushchak 2011). Previous studies reported that the antioxidative agents were able to suppress the pro-inflammatory cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymes that were responsible for inducing inflammatory responses through the release of inflammatory prostaglandins (PGE 2 and PGF 2 a) and leukotrienes (LTB 4 ) (Mitchell et al. 1994;D'Orazio et al. 2012). Thus, there is increased interest in pharmacological agents that can control or quench the free radicals from accumulating in the biological systems. The existing synthetic pharmacophores and nonsteroidal anti-inflammatory drugs (NSAIDs), which were reported to inhibit the free radical intermediates and pro-inflammatory mediators were recommended for limited usage due to their adverse effects (Schnitzer et al. 1999). The selective inhibition of inflammatory enzymes, such as COX-2/5-LOX, and oxidative stress inducing factors using naturally available pharmacological compounds can significantly lessen the adverse effects of the synthetic NSAIDs, corticosteroids and painkillers.
The natural products from marine organisms, such as molluscs or bivalves, which are adapted to the adverse living conditions in the oceanic ecosystem, have been considered as potential antioxidants. These organisms were reported to biosynthesize bioactive secondary metabolites as an adaptive mechanism, and these were recognized as valuable pharmacophores for use against various oxidative stress and inflammatory disorders (Gonzalez et al. 2015;Chakraborty et al. 2016). The bioactive properties of bivalves, mainly anti-inflammatory, antioxidant, antitumour properties etc. were reported in the previous literatures (Benkendorff 2010;Nagash et al. 2010;Chakraborty et al. 2016). The secondary metabolites from marine organisms belonged to different classes, such as heterocyclics, terpenes or steroids, and their activities were found to be closely related with their chemical structures. Isopimarane and pimarane metabolites were classified as significantly important class of diterpenoids with interesting pharmacological properties, such as antidiabetic, antioxidant, anti-HIV and antimicrobial activities, and were reported in marine organisms (Porto et al. 2009;Sun et al. 2012;Xia et al. 2015). Although rare in occurrence, three cytotoxic isopimarane diterpenoids from Excoecaria acerifolia Didr. (Euphorbiaceae) (Huang et al. 2013) and brominated pimaranes from marine algae, Laurencia obtusa (Hudson) Lamouroux (Rhodomelaceae) (Takeda et al. 1990) were reported in previous literature. An ent-pimarane diterpenoid tedanol was isolated from the marine sponge Tedania ignis (Duchassaing & Michelotti, (Tedaniidae), and was reported to possess potential anti-inflammatory activity against pro-inflammatory COX-2 enzyme (Costantino et al. 2009). The bioactive diterpenoids with pimarane skeletons were also described from the marine molluscs (sea hares), Aplysia dactylomela Rang, (Aplysiidae) (Schmitz et al. 1982) and Aplysia pulmonica Gould, (Aplysiidae) (Bian et al. 2014). The commercially available anti-inflammatory lipid extract of New Zealand green-lipped mussel Perna canaliculus Gmelin, (Mytilidae) known as Lyprinol V R and anti-inflammatory supplement Cadalmin TM Green Mussel extract (Cadalmin TM GMe) from Asian green mussel Perna viridis Linn. (Mytilidae) are prominent examples of pharmacologically effective agents from bivalve molluscs (Whitehouse et al. 1997;Chakraborty et al. 2014).
The bivalve clam P. malabarica considered in the present study are predominantly available seafood resource distributed in the coastal waters of southwestern India. Previous studies at our laboratory reported this species as a valuable source of balanced nutritional elements, such as C 20-22 n-3 polyunsaturated fatty acids, essential amino acids and minerals . Antioxidant, anti-inflammatory, antidiabetic and antihypertension potentials of the crude solvent extracts of P. malabarica were documented in our earlier study . As a sequel of our previous studies, it is of interest to isolate and characterize the bioactive pharmacophores that are responsible to impart potential antioxidative and anti-inflammatory properties. Based on this background, the present paper revealed the isolation and characterization of a new rearranged isopimarane norditerpenoid derivative from yellow-foot bivalve clam, P. malabarica, based on comprehensive spectroscopic analyses including mass and two-dimensional nuclear magnetic resonance spectroscopic experiments (2D NMR). In connection with that, the antioxidant [1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2 0azino-bis-3-ethylbenzothiozoline-6-sulfonic acid diammonium salt (ABTS þ )] and anti-inflammatory (COX-1, COX-2 and 5-LOX) properties of this C-19 norditerpene were carried out to establish its pharmacological potential.

General experiments
The reagents and solvents were procured from E-Merck (Darmstadt, Germany), and were of spectroscopic/chromatographic/analytical grades. Fourier-transform infrared spectrum (FTIR) (on KBr) was recorded in a Perkin-Elmer Series 2000 FTIR spectrophotometer with scanning between 4000 and 400 cm À1 . 1D (500 MHz for 1 H, 125 MHz for 13 C, Distortionless enhancement by polarization transfer, DEPT) and 2D ( 1 H-1 H COSY, correlation spectroscopy; HSQC, heteronuclear singlequantum correlation spectroscopy; HMBC, heteronuclear multiple-bond correlation spectroscopy; and NOESY, nuclear overhauser effect spectroscopy) NMR spectra were recorded on a Bruker AVANCE III 500 MHz (AV 500) spectrometer (Bruker, Karlsruhe, Germany) in CDCl 3 as aprotic solvent at ambient temperature with TMS as internal standard (d 0 ppm). Gas chromatography-mass spectrometric (GC-MS) analyses were performed in electronic impact (EI) ionization mode in a Perkin-Elmer Clarus 680 GC-MS fitted with a Elite 5 MS nonpolar, bonded phase capillary column (50 m Â 0.22 mm i.d. Â 0.25 lm film thicknesses). UV spectrum and in vitro spectroscopic assays were obtained on UV-Vis spectrophotometer (50 Varian Cary, Walnut Creek, CA). Analytical HPLC experiments were performed with a SPD M20A DAD (diode array detector, Kyoto, Japan) connected to a LC-20AD pump and C 18 reverse phase column (Luna 250 x 4.6 mm, 5 lm, Phenomenex, Torrance, CA). The samples were freeze-dried by the lyophilization technique using Martin Christ alpha 1-4 LD Plus freezedrier (Martin Christ, Osterode, Germany) and the concentration of solvent extracts was carried out by using a rotary vacuum evaporator (Heidolf, Schwabach, Germany). The chromatographic purification was carried out by flash chromatography (Biotage AB SP1-B1A, Sweden) on a silica gel (230-400 mesh, 12 g). Thin-layer chromatography (TLC) and preparatory TLC (PTLC) were carried out on pre-coated silica gel plates (Merck, Kieselgel-60F 254 ).

Sample collection and preparation of crude extracts
The clam samples, P. malabarica (10 kg) were freshly collected from Ashtamudi Lake (8 59 0 N and 76 36 0 E) situated along the southwest coast of India and a voucher with specimen number ICAR/CRP-HF/AC 368 was deposited in the repository of the Indian Council of Agricultural Research Consortium Research Platform on Health Food. The edible portion of the test material (6 kg) was separated from the shells before being homogenized and freeze dried by lyophilization. The dried powder (1.20 kg, yield 20.0%) was extracted with equal proportion of ethyl acetate (EtOAc) and methanol (MeOH) (1:1, v/v, 500 mL Â3) solvents at 40 C and the extracts were filtered over anhydrous sodium sulphate (Na 2 SO 4 , 100 g), before being evaporated in vacuo by using a rotary evaporator (50 C) to get a dark brown viscous residue (55.0 g, yield on dry basis 4.58%) of P. malabarica .

Determination of antioxidant and anti-inflammatory activity
The antioxidant activities were evaluated using 1,1-diphenyl-2picrylhydrazyl (DPPH) (Chew et al. 2008) and 2,2 0 -azino-bis-3ethylbenzothiozoline-6-sulfonic acid diammonium salt (ABTS þ ) radical scavenging assays (Vijayabaskar & Shiyamala 2012). In vitro anti-inflammatory properties were determined by the percentage inhibition of pro-inflammatory cyclooxygenases (COX-2, COX-1) (Larsen et al. 1996) and 5-lipoxygenase (5-LOX) (Baylac & Racine 2003) enzymes. The free radical scavenging activities (DPPH and ABTS þ ) and enzyme inhibitory activities (COX-2, COX-1 and 5-LOX) of the tilted compound/standards with varying concentrations (0.25-2.00 mg/mL) were expressed as inhibition using the equation, inhibition (%) ¼ {(absorbance of control À absorbance of sample/standards)/absorbance of control Â100}. The plots of inhibitory activities on radicals or enzymes were recorded and IC 50 (concentration of samples at which it inhibits/scavenge 50% of enzyme/radical activities) values were calculated from the graph. The IC 50 values (mg/mL) were determined from the linear regression curve of percentage inhibitions against the different concentrations of the compound or standards. The plot of scavenging or enzyme inhibitory activities were recorded and the IC 50 (concentration of samples at which it inhibits/scavenge 50% of enzyme/radical activities) values were calculated from the graph. The structure-activity relationship analyses were carried out by calculating the hydrophobic descriptor (logarithm of octanol-water coefficient, log P ow ), molar refractivity (MR) and polarizability (total polar surface area, tPSA) factors of the purified compounds using ChemDraw Ultra 8.0 database.

Statistical analysis
One-way analysis of variance (ANOVA) was carried out with the Statistical Program for Social Sciences 13.0 (SPSS Inc., Chicago, IL, ver. 13.0) to assess significant differences between the means. The values were given as mean of triplicates ± standard deviation. The means of all triplicate parameters were examined for significance by ANOVA and the significant differences were represented as p < 0.05.
The titled C 19 isopimarane norditerpenoid exhibited no significant difference in scavenging DPPH and ABTS þ free radicals (IC 50 0.65 and 0.78 mg/mL, respectively) compared to those displayed by the commercially available antioxidant, a-tocopherol (0.63 and 0.73 mg/mL, respectively) (p > 0.05). The anti-inflammatory effect (against 5-LOX enzyme) was significantly greater for the title compound (IC 50 0.75 mg/mL) compared to synthetic NSAID ibuprofen (IC 50 0.93 mg/mL; p < 0.05) ( Table 2). It is of note that the NSAIDs are used for moderating the pathogenesis due to inflammatory pain and arthritis (Quan et al. 2008), although these drugs were reported to cause deleterious side effects, such as gastric ulcers, CVD and toxicosis on the various organs (Schnitzer et al. 1999). Notably, the adverse implications of NSAIDs were reported to be due to greater anti-COX-1 properties. COX-1 is a constitutive enzyme of gastrointestinal mucosa, a greater inhibition of this enzyme was found to be associated with gastro-intestinal ulcers in susceptible individuals. On the other hand, COX-2 and 5-LOX were reported to be the inducive pro-inflammatory enzymes, and their expression was found to be upregulated in response to inflammatory stimuli. Apparently, simultaneous inhibition of COX-2 and 5-LOX is vital to arrest the inflammatory response in affected individuals. A greater selectivity index (anti-COX-1 IC50 /anti-COX-2 IC50 ) of the pharmacophores also signifies their greater selectivity and safety profile. Table 2. Antioxidant and anti-inflammatory activities of the title compound from P. malabarica and the commercially available antioxidants and anti-inflammatory agents (a-tocopherol and ibuprofen).
The antioxidative and anti-inflammatory activities of the isolated norditerpenoid from the marine bivalve clam, P. malabarica were correlated with the lipophilic/hydrophobic (log P ow ), steric effect (MR) and polarizability (tPSA) factors that explained its structure-activity relations and drug-target interactions (Ajay et al. 1998). The hydrophobicity value (log P ow ) for the titled C 19 isopimarane norditerpenoid and a-tocopherol were calculated as 6.30 and 9.98, respectively, which was the ratio of 1-octanol to water partition coefficient. The antioxidant activity of title compound and a-tocopherol were comparable, even though the acceptable lipophilic levels of the former demonstrated its convenience and utility as a safer pharmacophore. Also, the steric descriptor for a-tocopherol (139.21) and isopimarane norditerpenoid (125.96) explained the relatively lesser bulk hindrance of the latter. The olefinic centres in the isopimarane along with the oxygenated and carboxylated side chain attached to the skeleton at C-3 enhanced its electronic properties and these electronegative functional moieties appeared to influence its antioxidative and anti-inflammatory activities. The commercially available NSAIDs, such as ibuprofen, were reported to inhibit both pro-inflammatory COX-1 and COX-2. The lesser selectivity ratio (anti-COX-1 IC50 /anti-COX-2 IC50 ) of the NSAIDs also explained the larger selective inhibition of constitutive COX-1 that can cause severe side effects such as gastric-related health problems (Laneuville et al. 1994). Therefore, the search for compounds with specificity towards anti-COX-2 was preferred due to the lesser gastrointestinal difficulties as well as safer therapeutic profiles (Sprangler 1996). The naturally available norditerpenoid derivative reported in the present study was found to possess specific inhibition towards COX-2 activity than COX-1, and therefore, can be suggested as better anti-inflammatory lead molecule.
Previous reports of isopimarane and their derivatives from the natural resources envisaged their bioactive potential and pharmacological effects. The potential antioxidative property of entpimara-8(14), 15-diene from fungus was demonstrated by the DPPH radical scavenging activity (Bromann et al. 2014). The antibacterial potentials of rare pimarane derivatives with cyclopropane rings at C-3 and C-4 from the isolates of A. pulmonica were reported (Bian et al. 2014). The titled C 19 isopimarane norditerpenoid enclosed with a straight chain of 3-methoxy propyl pentanoate at the C-3 position of isopimarane skeleton, thus accounted for its potential activity. The ent-pimarane derivative isolated from the sponge (T. ignis) exhibited strong in vivo inhibitory properties towards COX-2 and inflammatory cytokineinducible nitric oxide synthase (iNOS) expression (Costantino et al. 2009). The pimarane diterpenoid, libertellenones, as a potent anticancer agent (Oh et al. 2005) and a C-19 diterpenoid pimarane from Ephemerantha fimbriata (Blume) P.F. Hunt & Summerh. (Orchidaceae) ) were studied. The isolated isopimarane norditerpenoid can be a potential lead pharmacophore for therapeutic investigations. Also, the synthetic derivatives of particular compound with bioactive and safer functional groups with suitable physical properties and fewer side effects can be developed in future medications.