Unprecedented antioxidative cyclic ether from the red seaweed Kappaphycus alvarezii with anti-cyclooxygenase and lipoxidase activities

Abstract An unprecedented non-isoprenoid oxocine carboxylate cyclic ether characterised as (3S, 4R, 5S, 6Z)-3-((R)-hexan-2′-yl)-3,4,5,8-tetrahydro-4-methyl-2H-oxocin-5-yl acetate was isolated from the ethyl acetate–methanol extract of the red seaweed Kappaphycus alvarezii. The structure, as well as its relative stereochemistry, was proposed on the basis of extensive spectral data. The antioxidative activity of the isolated metabolite was found to have significantly greater as determined by 1, 1-diphenyl-2-picrylhydrazyl and 2,2′-azino-bis-3-ethylbenzothiozoline-6-sulfonic acid radical scavenging activities (IC50 ~ 0.3 mg/mL) compared to α-tocopherol (IC50 > 0.6 mg/mL) and was comparable to the synthetic antioxidants butylated hydroxytoluene and butylated hydroxyanisole (IC50 ~ 0.35–0.34 mg/mL). The compound exhibited greater activity against COX-2 (cyclooxygenase-2) than COX-1 (cyclooxygenase-1) isoform, and therefore, the selectivity index remained significantly lesser (anti-COX-1IC50: anti-COX-2IC50 0.87) than synthetic anti-inflammatory drugs (0.02–0.44). No significant difference of in vivo 5-lipoxidase activity (IC50 0.95 mg/mL) than ibuprofen (IC50 0.93 mg/mL) indicated the potential anti-inflammatory properties of the title compound.


Introduction
Oxidative stress-induced molecular damages of living cells are caused due to the uncontrolled production of free radicals in the living cells. Traditionally, seaweeds are considered as valuable marine flora, which are preferred delicacies in the South-east Asian countries, particularly Japan, China, Korea and Indonesia due to their potential antioxidant and therapeutic properties (Wang et al. 2009). Species of the red seaweeds (class Rhodophyceae) were proven to be rich sources of structurally unique and biologically active secondary metabolites for applications in functional food and pharmaceuticals (Kladi et al. 2004). Antioxidative compounds obtained from these species were identified as phylopheophylin (Cahyana et al. 1992(Cahyana et al. ), phlorotannins (yan et al. 1996, terpenoids (Chakraborty & Paulraj 2010) and fucoxanthin (yan et al. 1999). The bioactive compounds extracted from the seaweeds are used as safer anti-inflammatory therapeutics as well (Shu et al. 2013). The vast majority of the red seaweed-derived bioactive compounds were diterpenes (Rochfort & Capon 1996), sesquiterpenes (Amico et al. 1991) and C 15 non-terpenoids containing ether rings of different sizes including halogenated cyclic ether enynes and related allenes (Erickson 1983;König & Wright 1997;Iliopoulou et al. 2002). The red seaweeds were reported for its antioxidant potential and in vitro antiproliferative activity in cancer cell lines (Chakraborty et al. 2015). Kappaphycus alvarezii (doty ex P.C. Silva, 1996) (class Rhodophyceae, family Solieriaceae, order Gigartinales) is economically significant and predominantly farmed red seaweed in shallow tropical marine habitats around the South-east Asian countries, particularly Philippines, Taiwan, Malaysia, Indonesia and India (Ask & Azanza 2002;Chandrasekaran et al. 2008). despite the fact that red seaweeds, particularly Laurencia spp, have been studied extensively with respect to secondary metabolite chemistry (Amico et al. 1991;Rochfort & Capon 1996;Manta 2001;Iliopoulou et al. 2002), studies on members of the genus K. alvarezii have been rare for the isolation of novel intriguing structures. As part of our ongoing programme aimed at the isolation of biologically active compounds from marine organisms of the Gulf of Mannar of south-east coast of India, we herein describe the isolation and structure elucidation of new non-isoprenoid oxocine carboxylate cyclic ether of the red seaweed K. alvarezii, collected from the south-east coast of Gulf of Mannar of India.
the samples were analysed in triplicate (n = 3) and expressed as mean ± standard deviation. Means followed by the different superscripts (a-c) within the same row indicate significant differences (p < 0.05).
* the bioactivities were expressed as Ic 50 values (mg/ml). ** selectivity index has been calculated as the ratio of anti-coX-1(Ic 50 ) to that of anti-coX-2 (Ic 50 ). contribute to the greater antioxidant activity of the title compound than α-tocopherol. The log P ow of the title compound was comparable to those of bhA (log P ow 3.22), and bhT (log P ow 5.54), and therefore, their antioxidative properties were not different (IC 50 0.25-0.26 mg/ mL). The free radical scavenging activity of bioactive leads depends on the electron-transferring groups, such as -COOMe, -Oh and -Nh, which can easily abstract free radicals and can convert from highly reactive species to non-reactive compounds (Cai et al. 2006). The radical scavenging activities were also reported to increase with the presence of double bonds due to effective electron transfer through electron delocalisation (Pietta 2000), and these reasons might be attributed to the significant antioxidative property of the title compound. Cyclooxygenase (COX-1) has been known as a constitutive enzyme, which is essentially required for various metabolic functions. It is intriguing to note that aspirin, sodium salicylate and ibuprofen are commonly available non-steroidal anti-inflammatory drugs (NSAIds), which recorded greater inhibitory properties towards COX-1 than COX-2 isoform. The title compound exhibited greater activity against COX-2 than against COX-1 isoform, and therefore, the selectivity index remained significantly lesser (anti-COX-1 IC50 : anti-COX-2 IC50 0.87) than the synthetic NSAIds (0.02-0.44). No significant difference of in vitro 5-lipoxidase (5-LOX) activity (IC 50 0.95 mg/mL) than ibuprofen (IC 50 0.93 mg/mL) indicated the potential anti-inflammatory properties of the title compound (Table 1). Sodium salicylate was found to be a weaker inhibitor of both COX isoforms (anti-COX-2 IC 50 2.65 mg/mL, anti-COX-1 IC 50 1.93 mg/mL) and demonstrated significantly lesser activity against 5-LOX (anti-COX-1 IC 50 1.75 mg/mL). Likewise, the log P ow and tPSA of (3S, 4R, 5S, 6Z)-3-((R)-hexan-2′-yl)-3,4,5,8tetrahydro-4-methyl-2H-oxocin-5-yl acetate were recorded as 3.63 and 35.53, respectively, whilst the values were found to be comparable with those noted for ibuprofen (3.75 and 37.30, respectively). It is, therefore, the selectivity indices of the title compound, and ibuprofen recorded greater values (0.87 and 0.44, respectively) than aspirin (0.02), and consequently, the title compound appeared to be safer than that of the synthetic NSAIds. The title compound purified from the red seaweed might therefore be used as a potential selective inhibitor of COX-2 with significantly lesser side effect profiles, such as renal and gastric damage than the present therapies by using NSAIds used to combat inflammatory disorders.
There were reports of closely related structures, such as laurefurenynes A-F and cyclic ether acetogenins isolated and characterised from red seaweed Laurencia sp. The 1 h NMR of C 15 -acetogenin derived from red seaweed Laurencia sp revealed the presence of a cis-ene-yne functionality (h-4 δ 6.05, h-3 δ 5.57, h-1 δ 3.10), which was proved by the respective 13 C NMR signals at C-4 (δ 139.9), C-3 (δ 111.3), C-2 (δ 80.0) and C-1 (δ 82.4) (Wael et al. 2010). The signals obtained at δ 4.93 and δ 4.43 indicated the two hydroxyl groups were at Oh-7 and Oh-12 (Wael et al. 2010), which are absent in the title compound (non-isoprenoid oxocine carboxylate cyclic ether) isolated from the ethyl acetate-methanol extract of the red seaweed K. alvarezii. The IR spectrum of laurefurenynes A-F broadly belongs to the group of C 15 cyclic ether acetogenins that were isolated from Laurencia sp, showing an intense absorption bands attributed to a terminal acetylene functional group (3315 and 2107 cm −1 ), which is not present in the title compound isolated from K. alvarezii. These groups of compounds exhibited non-selective cytotoxic activity, presumably due to the terminal acetylene. The C 15 acetogenins bearing cyclic ether skeletons have been isolated as the main secondary metabolites from red seaweed Laurencia sp (Erickson 1983) and were reported to be antimicrobial (König & Wright 1997), antifeedant (Kurata et al. 1998(Kurata et al. ), anthelmintic (davyt et al. 2001) and cytotoxic (Juagdan et al. 1997). A range of chlorinated compounds, C 15 acetogenin en-ynes, which are structurally similar to the title compound, were isolated from Laurencia glandulifera and were reported to be moderately cytotoxic towards various human tumour cell lines (Kladi et al. 2009). however, there is no literature report for the antioxidant and anti-inflammatory activities of the laurefurenynes A-F and cyclic ether acetogenins isolated and characterised from red seaweeds. Anti-inflammatory potential of the chromene sargachromanol G from the Korean seaweed Sargassum siliquastrum (Fucales) (yoon et al. 2012); halogenated compounds from the red seaweed Laurencia snackeyi (Vairappan et al. 2013) and the porphyrin derivatives; and pheophorbide and pheophytin from the seaweed Sargassum japonica were reported in previous literature (Islam et al. 2013). Antioxidative compounds from seaweeds were identified as phylopheophylin in Eisenia bicyclis (Cahyana et al. 1992), phlorotannins in Sargassum kjellamanianum (yan et al. 1996) and fucoxanthin in Hijikia fusiformis (yan et al. 1999). Laureatin, isolaureatin and deoxyprepacifenol are other related compounds obtained from the red alga Laurencia nipponica (Masuda et al. 1997). They exhibited significant insecticidal activity against the mosquito larvae Culex pipens pallens (Watanabe 1989). however, there is no literature report for the antioxidant and anti-inflammatory activities of the laurefurenynes A-F and cyclic ether acetogenins isolated and characterised from red seaweeds.

General experimental procedures
FTIR spectra of the compounds under Kbr pellets were recorded in a Thermo Nicolet Avatar 370 in the IR range between 4000 and 400 cm −1 . uV spectra were obtained on a Varian Cary 50 ultraviolet visible (uV-VIS) spectrometer (Varian Cary, uSA). The gas chromatographymass spectrometry (GC-MS) analyses were performed in electronic impact (EI) ionisation mode in a PerkinElmer Clarus 680 GC-MS fitted with an Elite 5 MS non-polar, bonded phase capillary column (50 m × 0.22 mm i.d. × 0.25 μm film thicknesses). helium (he) was used as the carrier gas, and the flow rate used was 1 mL min −1 . The temperature was programmed initially at 50 °C for 2 min., then increased at a rate of 10 °C min −1 to 180 °C and kept for 2 min and raised at 4 °C min −1 to 280 °C and held for 15 min. ESI-MS spectra were acquired on a liquid chromatography-mass spectrometry system (Applied biosystems QTrap 2000, Applied biosystems, darmstadt, Germany). 1 h and 13 C NMR spectra were recorded on a bruker AVANCE III 500 Mhz (AV 500) spectrometer (bruker, Germany) in CdCl 3 as aprotic solvent at ambient temperature with TMS as the internal standard (δ 0 ppm). All the reagents and solvents used in this study were of analytical grade and purchased from E-Merck.

Seaweed material, extraction and chromatography
The entire algal thalli (10 kg dry weight) of K. alvarezii were freshly harvested intertidally at Gulf of Mannar in Mandapam region located between 8º48′ N, 78º9′ E and 9º14′ N, 79º14′E on the south-east coast of India. The samples were washed in running water for 10 min, transported to the laboratory before being shade dried (35 ± 3 °C) for 36 h and powdered (1000 g). The thalli of seaweed were placed as a monolayer on the adsorbent papers and inverted after every 3 h under aeration.

Antioxidant and anti-inflammatory activities
Antioxidant activities were measured using the stable 1, 1-diphenyl-2-picryl-hydrazil (dPPh · ) (Lim et al. 2007;Chaudhary et al. 2015) and 2, 2′-azino-bis-3-ethylbenzothiozoline-6-sulphonic acid (AbTS ·+ ) radical cation decolorisation assays (Chakraborty et al. 2015). In brief, dPPh (100 μM) was dissolved in methanol to generate the stock solution. The purified fractions (1 mL in methanol) were mixed with dPPh solution (1 mL) before being placed in the dark for 10 min at room temperature. The reduced absorbance of the mixture was measured at 517 nm against a reagent blank by using a uV-vis spectrophotometer. The AbTS radical scavenging activity was determined by dissolving AbTS in deionised water to a concentration of 7 μM. The AbTS solution was mixed with potassium persulphate (K 2 S 2 O 8 , 2.45 μM) and kept in dark at room temperature for 12-16 h. The resultant AbTS solution was diluted with MeOh to get an absorbance of 0.70 at 734 nm. diluted AbTS ·+ solution 3 (mL) was mixed with 30 μL of the title compound, and the absorbance was noted after 6 min at 734 nm. The percentage of dPPh and AbTS ·+ scavenging activities was calculated by the following equation: dPPh · /AbTS ·+ scavenging activity (%) = ((A 0 − A 1 )/A o ) × 100, where A 0 is the absorbance of control and A 1 is the absorbance of the sample. The IC 50 value (mg/mL) calculated by plotting radical scavenging activity on dPPh · /AbTS ·+ was recorded.

Statistical analysis
Statistical evaluation was carried out with the Statistical Program for Social Sciences 13.0 (SPSS Inc, Chicago, uSA, ver. 13.0). Analyses were carried out in triplicate, and the means of all parameters were examined for significance by analysis of variance (ANOVA). The level of significance for all analyses was P ≤ 0.05.

Conclusions
bioactivity-guided chromatographic fractionation of the ethyl acetate: methanol extract obtained from the thalli of the red seaweed K. alvarezii afforded an unprecedented non-isoprenoid oxocine carboxylate cyclic ether characterised as (3S, 4R, 5S, 6Z)-3-((R)-hexan-2′-yl)-3,4,5,8-tetrahydro-4-methyl-2H-oxocin-5-yl acetate with potential antioxidative and anti-inflammatory activities. The title compound may be considered as new-generation pharmacophore lead that becomes available to selectively inhibit COX-2 and therefore has significantly greater selectivity than the NSAIds. The target bioactivities of the title compound were directed by the electronic and lipophilicity parameters. The newly reported non-isoprenoid oxocine carboxylate cyclic ether has therefore bioactive potential as natural antioxidant and anti-inflammatory lead for use in the pharmaceutical and food applications.