Introduction

Structurally diverse and complex naturally-derived macrocycles have demonstrated a notable record of efficiency as pharmaceutical agents, and were reported to occupy an increasingly significant role in the treatment of a range of serious diseases (Montaser and Luesch 2011). Macrocyclic lactone polyethers of marine origin were found to include a sequence of compounds, which were reported to exhibit valuable pharmaceutical properties (Huryn and Wipf 2014). Though previous studies reported the incidence of naturally occurring macrocycles possessing more than fifty atoms in the ring system, a recent work of literature suggested that fourteen, sixteen and eighteen-member scaffolds were the most common naturally occurring macrocyclic framework (Frank et al. 2007). A series of macrocyclic lactones having unique cytotoxic properties were isolated from the dinoflagellate Amphidium sp. (Oguchi et al. 2007). A doubly oxygen-bridged eighteen-member macrocycle Leucascandrolide A displaying cytotoxicity against human tumor cell lines was reported from the calcareous sponge Leucascandra caveolata (Ambrosioa et al. 1999).

As a part of our ongoing program to study the biologically active food ingredients from various gastropod species, we have attempted to study the detailed structures of the secondary metabolites prevalent in the Buccinid gastropod Babylonia spirata. An extensive distribution of B. spirata (family Babylonidae) was seen from the Western Indian Ocean to Bali, Indonesia, in the east (Altena et al. 1981; Fraussen and Stratmann 2013), and was found to form one of the targeted catches in the southwestern coast of Indian peninsular due to its increased demand from around the globe. Though scanty reports on the bioactive potentials of B. spirata were existent (Govindarajalu et al. 2016; Kuppusamy and Ulagesan 2016), detailed studies on their secondary metabolite chemistry in search of novel pharmacophore leads are unprecedented to the best of our knowledge.

Herein we described the isolation and structural characterization of a previously uncharacterized sixteen-member polyether macrocyclic lactone from the ethyl acetate-methanol fraction (EtOAc-MeOH) of the gastropod B. spirata collected from the south-western coast of Indian peninsular region. The potential anti-inflammatory and antioxidant activities of the isolated compound were tested using in vitro pro-inflammatory enzyme (5-lipoxygenase, 5-LOX) inhibitory and radical scavenging assays, respectively. The bioactivity results were compared against various commercial standards. Various physicochemical descriptors were used to correlate the target bioactivities of the studied compound with its structural parameters.

Materials and methods

Chemicals and instrumentation

The reagents and solvents were of spectroscopic or chromatographic or analytical grade from E-Merck (Darmstadt, Germany). The spectrophotometric measurements were acquired using Varian Cary 50 UV-visible spectrophotometer (Varian Cary, USA). Fourier-transform infrared (FTIR) spectrum was recorded using a Perkin–Elmer Series 400 FTIR spectrophotometer assembled with an air cooled DTGS (deuteratedtriglycinesulfate) detector scanning between 4000 and 400 cm−1. Optical rotations were measured on an ATAGO AP-300 polarimeter. The one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectral experiments were measured in an aprotic solvent (deuterated chloroform, CDCl3) on a Bruker AVANCE DPX 500 (500 MHz) spectrometer, with tetramethylsilane as an internal standard. Chemical shifts (δ) were expressed in ppm and coupling constants in Hz. Electron impact mass spectrum was recorded on a GC-MS (Perkin-Elmer Clarus 680 GC-MS fitted with Elite 5 MS non-polar) in bonded phase capillary column (50 m × 0.22 mm i.d. × 0.25 μm film thicknesses). Electrospray ionization mass spectra were acquired on a liquid chromatography-mass spectrometry system (Applied Biosystems QTrap 2000, Applied Biosystems, Darmstadt, Germany) in dual modes (positive and negative modes). Silica gel (60–120 mesh, E-Merck, Germany; 230–400 mesh, Biotage, Sweden) were used for a range of column chromatography techniques and pre-coated plates of GF254 were utilized for thin layer chromatographic separation (TLC). Analytical high pressure liquid chromatograph (HPLC) instrument (Shimadzu Corporation, Nakagyo-ku, Japan) connected to RP-C18 (bonded reverse-phase; Phenomenex, Torrance, USA; Luna 250 × 4.6 mm, 5 µm) fitted with a binary gradient pump (Shimadzu LC-20AD) column and photodiode array detector (SPD-M20A, Kyoto, Japan) was used to analyze the homogeneity of the studied compound.

Sample collection, pre-treatment, and extraction

The samples of gastropod B. spirata (Family Babylonidae) (Fig. 1a) were freshly collected from the Neendakara area of Kollam (Kerala State of India) situated between south-west coast of India (latitude 8° 56′ North and longitude 76° 32′ East) and identified with the CMFRI specimen voucher number DB.24.1.1. The live samples were immediately transported to the laboratory, where the edible parts were carefully removed from the shells and washed with distilled water to remove associated debris, and the tissues (5 kg) were thereafter ground properly. The ground tissues were lyophilized and powdered (1.5 kg) before being sonicated with ethyl acetate-methanol solvent system (EtOAc/MeOH 1:1 v/v, 500 mL X 2, RT, 8 h) and refluxed at 50–60 °C for a period of 4 h. The pooled fractions were filtered (using Whatman No.1) and dried over anhydrous Na2SO4 (40 g) before being evaporated (45 °C) to dryness using a rotary evaporator (Heidolph Instruments GmbH andCo., Schwabach, Germany) under reduced pressure to yield dark yellow viscous residues of crude EtOAc-MeOH extract of B. spirata. The extraction processes were repeated thrice, and the triplicate values were calculated to estimate the yield (18 ± 0.50 g/kg, yield on dry weight basis 1.8 ± 0.08%).

Fig. 1
figure 1

a Representative shell-on samples of Babylonidae gastropod Babylonia spirata. b Structure of 10, 14-dihydroxy-4-(1′-propyl-1′(Z)-hexen-1′-yl)-7,12-dioxapentadecanolide isolated from B. spirata

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Chromatographic purification of the polyether macrocyclic lactone from the solvent extract of B. spirata

The crude EtOAc-MeOH fraction (50 g) of B. spirata was adsorbed on silica gel (60–120 mesh, 10 g), and loaded into a glass column (5 × 150 cm) packed with silica gel (60–120 mesh sized, 100 g). An initial elution of the column was carried out with the solvent n-hexane to remove the pigmented and waxy material. The eluent polarity (n-hexane/EtOAc 9:1 to 2:8, v/v) was gradually increased to provide 13 fractions of 25 mL each, which were reduced to 6 groups (SB1-SB6) based on TLC (n-hexane/EtOAc, 9:1, v/v) analysis. The pooled fractions were then submitted for bioactivity screening against the antioxidant assays, such as DPPH and ABTS+ radical decolorization along with the anti-inflammatory assay using 5-LOX inhibitory potential. The fraction SB1 (96.3 mg) was found to be active, and was analyzed by RP-C18-HPLC {Waters X-bridge 150 × 2 mm; ACN/H2O (+0.1% formic acid) [5:95] to [95:5] linear gradient}. Out of the six sub-fractions, SB11–SB16 (15 mL each), which were collected, the sub-fraction SB15 was further subjected to a Waters X-bridge semi-preparative column under the same conditions. The sub-fraction SB153 collected out of it was fractionated over preparatory TLC on silica gel GF254 using n-hexane/EtOAc (2:8, v/v) to afford 10,14-dihydroxy-4-(1′-propyl-1′ (Z)-hexen-1′-yl)-7,12-dioxapentadecanolide as a pure compound. The homogeneity of the compound was substantiated by RP-C18-HPLC (MeOH/ACN, 3:2 v/v, Rt 3.7 min) analysis.

Spectral analysis of 10, 14-dihydroxy-4-(1′-propyl-1′(Z)-hexen-1′-yl)-7, 12-dioxapentadecanolide

Pale yellow liquid; UV (MeOH) λmax (log ε): 217 (1.17) nm; Rf: 0.40 (EtOAc/n-hexane 3:2, v/v); Rt (HPLC, MeOH:ACN 3:2 v/v): 3.7 min.; IR (KBr, cm−1) νmax (ν = stretching, δ = bending, ρ = rocking vibrations) 957.00 (alkene C-Hδ), 1054.00 (C-O-Cν), 1372.00 (C-Hρ), 1460.00 (C-Hδ), 1745.20 (C = Oν), 2937.36 (C-Hν), 3410.00 (OHν); 1H NMR (500 MHz, CDCl3): δH 4.16 (1H, d, 6.9 Hz, H-1), 2.35 (2H, t, 7.5 Hz, H-2), 1.64 (2H, m, H-3), 2.17 (1H, p, 4.85 Hz, H-4), 1.56 (2H, m, H-5), 3.66 (2H, t, 5.30 Hz, H-6), 3.71 (2H, t, 7.49 Hz, H-8), 1.5 (2H, m, H-9), 3.86 (1H, p, 5.5 Hz, H-10), 3.52 (2H, d, 5.00 Hz, H-11), 3.61 (2H, d, 8.00 Hz, H-13), 3.93 (1H, p, 5.43 Hz, H-14), 4.20 (1H, d, 4.9 Hz, H-15), 5.34 (1H, t, 5.50 Hz, H-2′), 2.02 (2H, m, H-3′), 1.31 (2H, m, H-4′), 1.26 (2H, m, H-5′), 0.88 (3H, 6.7 Hz, H-6′), 2.02 (2H, m, H-1′a), 1.30 (2H, m, H-1′b), 0.88 (3H, t, 6.7 Hz, H-1′c); 13C NMR (125 MHz, CDCl3): δC174.5 (C, C-1), 34.2 (CH2, C-2), 25.0 (CH2, C-3), 36.3 (CH, C-4), 33.5 (CH2, C-5), 71.8 (CH2, C-6), 64.4 (CH2, C-8), 29.6 (CH2, C-9), 70.5 (CH, C-10), 72.6 (CH2, C-11), 71.9 (CH2, C-13), 70.3 (CH, C-14), 65.2 (CH2, C-15), 141.5 (C, C-1′), 130.0 (CH, C-2′), 27.3 (CH2, C-3′), 29.7 (CH2, C-4′), 22.7 (CH2, C-5′), 14.1 (CH3, C-6′), 32.0 (CH2, C-1′a), 20.9 (CH2, C-1′b), 14.1 (CH3, C-1′c); 1H-1H-COSY, and HMBC data (Table 1); HR (ESI) MS m/z found 401.2899 [M + H]+, calcd for C22H41O6 401.2903.

Table 1 NMR spectroscopic data of 10, 14-dihydroxy-4-(1′-propyl-1′(Z)-hexen-1′-yl)-7, 12-dioxapentadecanolide in CDCl3a
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Anti-inflammatory and antioxidant activities

In vitro anti-inflammatory property of the studied compound was evaluated by studying the inhibition of 5-LOX (Baylac and Racine 2003) enzymes. The antioxidant properties were assessed by DPPH and ABTS+ radical decolorization assay (Chew et al. 2008; Wojdylo et al. 2007). The results were expressed as IC50 (the concentration of samples at which 50% of enzyme/radical activities were inhibited/scavenged, presented as molar units) values.

Structure–activity relationship analysis

Different physicochemical parameters of the newly isolated macrocyclic lactone were analyzed so as to relate its bioactivity with the proposed structure (Walters and Murcko 1998; Joy and Chakraborty 2017; Makkar and Chakraborty 2018). The structural descriptors, such as partition coefficient for octanol-water system (log Pow), categorized as hydrophobic; topological polar surface area and polarizability as electronic descriptor variable; and parachor, molar volume, and molar refractivity, which were categorized as steric (or bulk) descriptors, were acquired from the ChemDraw ultra 11 (8.0 database) and ACD Chemsketch (version 8.0).

Statistical analysis

Statistical Program for Social Sciences 13.0 (SPSS, USA, ver. 13.0) was accessed 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.

Results and discussion

General

Bioactivity-guided repeated chromatographic purification of EtOAc-MeOH fraction resulted in the isolation of a previously undescribed polyether macrocyclic lactone, characterized as 10, 14-dihydroxy-4-(1′-propyl-1′(Z)-hexen-1′-yl)-7,12-dioxapentadecanolide (Table 1, Fig. 1b). The structure of the purified compound was proposed on the basis of the comprehensive interpretation of the 1H NMR and 13C NMR, including two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) experiments (heteronuclear single-quantum correlation spectroscopy, HSQC; correlation spectroscopy, 1H-1H COSY (Fig. 2a); heteronuclear multiple-bond correlation spectroscopy, HMBC (Fig. 2b); nuclear overhauser effect spectroscopy, NOESY (Fig. 3); and mass spectral analysis (Fig. S1-S10).

Fig. 2
figure 2

a 1H-1H COSY spectrum of the sixteen-membered polyether macrocyclic lactone isolated from B. spirata. The key 1H-1H COSY cross-peaks were displayed by bold-faced bonds. b HMBC spectrum of the studied compound. The selected HMBC correlations were shown as double-barbed arrows

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Fig. 3
figure 3

NOESY spectrum of the studied compound. The selected NOESY correlations were shown as double-barbed arrows

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Structural characterization of the polyether macrocyclic lactone from B. spirata

The purified compound 10, 14-dihydroxy-4-(1′-propyl-1′(Z)-hexen-1′-yl)-7,12-dioxapentadecanolide (Fig. 1) was obtained as a pale yellow liquid. The molecular ion peak appeared at m/z 400 [M]+ {HR (ESI) MS m/z found 401.2899 [M + H]+, calcd. for C22H41O6 401.2903}, which in combination with its one-dimensional (1D) and two-dimensional (2D) NMR data, described the elemental composition of the compound as C22H40O6. The 13C-NMR and DEPT spectra specified the presence of 22 carbon resonances counting those of one carbonyl carbon at δC 174.5, one methine olefinic carbon at δC 130.0, one non-protonated olefinic carbon at δC 141.5, two oxygenated carbons at δC 70.5 and 70.3, one methine carbon at 36.3, fourteen methylene carbons at δC 65.2, 71.9, 72.6, 29.6, 64.4, 71.8, 33.5, 25.0, 34.2, 27.3, 29.8, 22.7, and 32.0, along with two methyl carbons at δC 14.1. The typical 13C NMR signal recorded at δC 174.5 was assigned to the ester carbonyl carbon. The significantly deshielded methylene protons at δH 4.20 (H-15) displayed strong HMBC cross-peaks with the carbonyl carbon at δC 174.5, which supported that it should be adjacent to the electronegative oxygen atom of the ester functionality. Further HMBC couplings from δH 4.20 (H-15) to the methine carbon at δC 70.3 (C-14) held that the deshielded proton was flanked by the latter on the other side. The two deshielded methine protons at δH 3.93 (corresponding to δC 70.3, HSQC) and δH 3.86 (corresponding to δC 70.5, HSQC) were geminal to hydroxyl groups (Oguchi et al. 2007). Four methylene signals appeared at δH 3.61, 3.52, 3.71 and 3.66 displayed HSQC correlation with the carbon traces at δC 71.9, 72.6, 64.4, and 71.8, respectively. These carbon signals have been allocated the positions at C-13, C-11, C-8, and C-6 that were adjacent to the electronegative ether functionality, and thus appeared at the downfield positions. The long range correlations between δH 3.61 (H-13) and δC 72.6 (C-11), and that between δH 3.66 (H-6) and δC 64.4 (C-8) supported the location of the ether bridges in the structure. The methine signal at δC 36.3 was in agreement with the deshielded proton signal at δH 2.17, which referred to a possible olefinic group in the vicinity. This attribution was appropriately validated by the HMBC cross-peak between the carbon signal and the olefinic proton at δH 5.34. The carbonyl group at the C-1 position of the studied compound caused intense deshielding of the methylene proton at δH 2.35, which appeared as a triplet, and therefore, has been consigned the C-2 position of the structure. Detailed HMBC couplings from δH 4.20 (H-15) to δC 70.3 (C-14), 174.5 (C-1); δH 3.93 (H-14) to 65.2 (C-15); δH 3.61 (H-13) to δC 72.6 (C-11); δH 3.52 (H-11) to δC 71.9 (C-13), 29.6 (C-9); δH 3.71 (H-8) to δC 70.5 (C-10); δH 3.66 (H-6) to δC 64.4 (C-8); δH 1.56 (H-5) to δC 71.8 (C-6); δH 2.35 (H-2) to δC 25.0 (C-3), 174.5 (C-1) established the closed loop of the 10, 14-dihydroxy-7, 12-dioxapentadecanolide moiety in the compound. The one bond 1H-1H COSY recorded three spin systems, δH 4.20 (H-15)/3.93 (H-14)/3.61 (H-13); δH 3.52 (H-11)/3.86 (H-10)/1.5 (H-9)/3.71(H-8); δH 3.66 (H-6)/1.56 (H-5)/2.17 (H-4)/1.63 (H-3)/2.35 (H-2) within the dioxapentadecanolide moiety. The attachment of an alkenyl side chain to the macrocyclic lactone moiety was predicted from the long range HMBC cross-peaks between δH 1.56 (H-5) to δC 141.8 (C-1′) and δH 5.34 (H-2′) to δC 36.3 (C-4). Further HMBC couplings found within the alkenyl chain were δH 5.34 to δC 27.3 (C-3′); δH 2.02 to δC 29.7 (C-4′); δH 1.31 to δC 22.7 (C-5′); δH 1.26 to δC 29.7 (C-4′), 14.1 (C-6′); δH 0.88 to δC 22.7 (C-5′); δH 1.30 to δC 14.1 (C-1′c); and δH 0.88 to δC 32.0 (C-1′a). The three spin systems δH 5.34 (H-2′)/2.02 (H-3′); δH 1.26 (H-5′)/0.88 (H-6′), and that at δH 2.02 (H-1′a)/1.30 (H-1′b)/0.88 (H-1′c) additionally attributed the side chain structure. The relative stereochemistries of the protons at C-14, C-10, and C-4 were deduced from the NOESY interactions. The proton resonances at δH 3.86 (H-10) and 3.93 (H-14) displayed NOE cross-peaks with δH 4.16 (H-15α), and these were disposed in the similar plane, and therefore, arbitrarily considered as α-oriented. The methine proton at δH 2.17 (H-4) was found to interact with the methylene proton at δH 4.20 (H-15β), which assigned their β-orientation. The intense NOE cross-peaks between the olefinic proton at δH 5.35 (H-2′) and the methylene protons at δH 2.00 (H-1′a) apparently indicated their spatial proximity.

The molecular ion peak m/z at 400, with the molecular formula of C22H40O6 were indicative of three degrees of unsaturation, out of which, one each was due to the ester carbonyl and olefinic moieties, and the remaining was accounted for the macrocyclic ring structure. The presence of a C9H18 fragment ion at m/z 125 and the series of low mass fragments at m/z 111 (1c), 85 (1d) and 71 (1e) assigned the presence of an uninterrupted alkyl side chain comprising of nine carbon atoms. The major fragment at m/z 329 (1a) attributed to the 4-(but-2-en-2-yl)-10, 14-dihydroxy-7, 12-dioxapentadecanolide ion, which might have formed via the allylic cleavages of the double bond in the side chain. The base peak was recorded at m/z 85 (1 l) corresponding to 4-hydroxy-1-oxobut-3-en-2-ylium ion.

The presence of ester carbonyl in the compound was appropriately supported by an intense band at 1745 cm−1 in the FTIR spectrum. The strong infrared absorption bands between 1250 and 1050 cm−1 (C–O–C stretch) were indicative of the presence of the ether bridges in the studied compound (Joy and Chakraborty 2016). The bands pertaining to the stretching vibration of the hydroxyl groups were present at 3410 cm−1, and the deuterium exchange {D2O (deuterium oxide)-1H NMR} experiment additionally confirmed the occurrences of hydroxyl groups in the compound.

Anti-inflammatory and antioxidant activities

The pharmacological properties encompassing anti-inflammatory and antioxidant potential of the purified compound were depicted in Table 2, and their activities were compared with the commercially available antioxidative agents and non-steroidal anti-inflammatory drugs (NSAIDs). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS.+) radical scavenging activities (IC50 0.058 × 10−2 M and 0.065 × 10−2 M, respectively) of the studied compound were significantly greater than those obtained for the commercially available antioxidants BHA (IC50 0.14 × 10−2 M and 0.189 × 10−2 M, respectively, P < 0.05), and BHT (IC50 0.123 × 10−2 M and 0.118 × 10−2 M respectively, P < 0.05), and ranked better than α-tocopherol (IC50 0.139-0.169 × 10−2 M). The studied compound displayed significantly greater anti-5-lipoxidase activities (5-LOX, IC50 0.073 × 10−2 M) than the non steroidal anti-inflammatory drugs ibuprofen (IC50 0.436 × 10−2 M), sodium salicylate (IC50 1.062 × 10−2 M), and aspirin (IC50 0.211 × 10−2 M) (P < 0.05). There has been earlier report that the column purified fractions of the methanol extract of the Babylonidae Babylonia zeylanica could significantly reduce the carrageenan-induced paw edema in albino rats when compared to the control diclofenac sodium (Santhi et al. 2012). Likewise, the gastropod molluscs Trochus tentorium and Drupa margariticola were found to be potent inhibitors of exudative and proliferate phase of inflammation (Chellaram and Edward 2009a, b).

Table 2 In vitro antioxidant and anti-inflammatory activities of the polyether macrocyclic lactone purified from B. spirata
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The lipophilic parameter of octanol-water partition coefficient (log Pow) of the studied compound recorded a value as low as 2.76, when judged against the commercial antioxidants, such as BHA (log Pow 3.22), BHT (log Pow 5.54), and α-tocopherol (log Pow 9.98), which could be corroborated with the higher antioxidant characteristics of the former. Notably, the lipophilic parameter log Pow was found to be one of the principal parameters used in the rational drug design, and has been a primary physico-chemical determinant regulating the bioavailability of the pharmacophores (Kujawski et al. 2012). It is also significant to mention that the optimum range of log Pow for an effective drug possessing adequate lipophilic-hydrophobic characteristics was found to be 2-5 (Lipinski and Hopkins 2004). It is of note that the compound has demonstrated significantly greater anti-inflammatory property (IC50 0.073 × 10−2 M) than the commercial NSAIDs in spite of its greater steric bulk compared to the latter. This recognized that the electronic parameters of the isolated polyether macrocyclic lactone have primarily contributed towards its anti-inflammatory activity. Though the NSAIDs are employed to alleviate the pathogenesis due to inflammatory pain and arthritis (Quan et al. 2008), they were noted for their probable adverse effects, such as gastro-intestinal complications, and toxicosis on various organs, such as kidney, heart, and liver (Schnitzer et al. 1999). The alkenic, hydroxyl, ester and ether moieties within the sixteen-membered polyether macrocyclic lactone might have contributed towards increasing the total polar surface area and polarizability index, which appeared to increase its potential against inflammatory responses.

Conclusions

Bioactivity-guided chromatographic fractionation of the ethyl acetate: methanol extract of the Buccinid gastropod mollusc Babylonia spirata afforded an unprecedented sixteen-membered marocyclic lactone polyether. The studied compound was characterized as 10, 14-dihydroxy-4-(1′-propyl-1′(Z)-hexen-1′-yl)-7, 12-dioxapentadecanolide with potential antioxidative and anti-inflammatory activities. The title compound might be considered as novel pharmacophore lead that has significantly greater antioxidant activities, as evidenced by the DPPH (IC50 0.058 × 10−2 M) and ABTS (IC50 0.065 × 10−2 M) radical scavenging activity than the commercial antioxidant α-tocopherol (IC50 0.139-0.169 × 10−2 M) and superior anti-inflammatory activity (IC50 0.073 × 10−2 M) than the NSAIDs aspirin (0.211 × 10−2 M), ibuprofen (0.436 × 10−2 M) and sodium salicylate (IC50 1.062 × 10−2 M). The target bioactivities of the title compound were primarily dictated by the lipophilicity and electronic parameters. This result suggested that the macrocyclic polyether lactone may be subjected to further biomedical investigation so as to be used as natural antioxidant and anti-inflammatory lead in the pharmaceutical and food applications.