Anti-inflammatory decurrencyclics A-B, two undescribed nor-dammarane triterpenes from triangular sea bell Turbinaria decurrens

Abstract Intertidal triangular sea bell Turbinaria decurrens (Bory de Saint-Vincent) (family Sargassaceae) belongs to one of the largely abundant genus of marine brown alga. Bioactivity-directed chromatographic purification of the organic extract of T. decurrens afforded two new nor-dammarane triterpenoids named as decurrencyclic A-B. Decurrencyclic B showed superior attenuation properties against cyclooxygenase-2 (IC50 13.98 μM) and 5-lipoxygenase (IC50 3.02 μM) in contrast with decurrencyclic A. Decurrencyclic B showed higher inhibition potential against COX-2 than that revealed by the anti-inflammatory agent, ibuprofen (IC50 70.44 μM). The higher selectivity index of decurrencyclics (1.39–1.57) acknowledged their selective attenuation property against inducible cyclooxygenase-2. In-silico molecular modeling analysis of decurrencyclic B with the inflammatory enzymes showed least binding energy of −14.55 kcal mol−1. These reports have proven that decurrencyclic B could be a potential therapeutic lead for use against inflammatory pathogenesis. Graphical Abstract


Introduction
Terpenoids comprise the largest class of phytochemicals that are extensively available from marine algae. Among their derivatives, triterpenoids are natural products encompassing about thirty carbon atoms, resulting from an acyclic precursor (squalene), and constitute as one of the most copious secondary metabolites found in the marine organisms, for example sponges, fungi, sea cucumbers, and marine algae. It has been reported that more than 20,000 triterpenoids are isolated from marine algae belonging to the different chemical classes, such as dammarane, lanostane, squalene, lupine, hopane, ursane, oleanane, etc. Cyclosadol and 3-epicyclomusalenol are two cycloartane-type triterpenoids isolated from brown algae Kjellmaniella crassifolia (Wu et al. 2012). Cytotoxic squalenoid triterpenoids, such as laurenmariannol, (21a)-21-hydroxythyrsiferol, dehydrothyrsiferol, isodehydrothyrsiferol and 10-epidehydrothyrisiferol (Norte et al. 1996) were characterised from the red alga of Laurencia species. Antioxidative abeo-oleanenes and anti-inflammatory polyether triterpenoids were isolated from seaweed Gracilaria salicornia (Chakraborty and Antony 2019; Antony and Chakraborty 2020). Three anti-diabetic cycloartane type triterpene sulfates were isolated from Vietnamese marine alga Tricleocarpa fragilis (Tran et al. 2021).
Inflammation is a defensive mechanism of body to antigenic stimulation or physical injuries, wherein cyclooxygenases (COX-1/2) and 5-lipoxygenase (5-LOX) are enzymes responsible for converting arachidonic acid to prostaglandin H 2 (PGH 2 ) and leucotrienes (LTs), respectively, and are involved in inflammatory process (Martel-Pelletier et al. 2003). An increasing number of reports have showed that extracts and compounds isolated from marine algae have potential anti-inflammatory activity (Antony and Chakraborty 2020;Chakraborty and Dhara 2020). Several marine algal metabolites like glycosterol, phlorotannins, terpenoids, and polysaccharides were reported to have anti-inflammatory properties (Fernando et al. 2016). These algal compounds hold potential immune modulatory capacities, and could prompt the immune system activities to regulate undesirable responses like inflammation.
Turbinaria is a brown algae, which is ubiquitous in the coastal zones of the Indo-Pacific, and is one of the most common genus of the phylum Phaeophyceae. This genus has been widely used for a number of biological studies, and among those Turbinaria decurrens (Bory de Saint-Vincent) (family Sargassaceae) has proven to be an active bioresource of compounds demonstrating cytotoxic, anti-viral, antioxidant, anti-hyperglycemic, and anti-obesity activities (Rushdi et al. 2020). T. decurrens obtained from the Pameungpeug Beach of Java was reported to possess ectoine, fucoxanthin, bengazole C, tanacetol A, pseudoanguillosporin, and embelin (Stranska-Zachariasova et al. 2017). Ectoine was reported as an inhibitor associated with neurodegenerative diseases, and could inhibit prompted signaling accountable for pro-inflammatory pathologies in the epithelial cells of lung (Kanapathipillai et al. 2005). Presence of nor-dammarane type terpenoids could find mention in the marine-originated fungus Aspergillus fumigatus concomitant with a Sinularia sp, a species of marine soft coral (Afiyatullov et al. 2012). The plants of the genus Viburnum have also reported nor-dammarane type triterpenoids with cytotoxic and radical scavenging activities (Tu et al. 2008;Wang and Wang 2013). In this study, we have defined the isolation and structural characterization of two new nor-dammarane type tritrpenoids, named as decurrencyclic A-B (Figure 1) from the intertidal triangular sea bell T. decurrens (marine brown alga), available in the coastal zones of the Peninsular India. The structures of the isolated compounds were characterised using one and two-dimensional nuclear magnetic resonance (NMR), Fourier-transform infra-red (FT-IR), and mass spectrometric studies. The compounds were screened for their anti-inflammatory and radical scavenging properties using different in vitro assays. Molecular modeling analysis was executed to reveal the binding affinities and interactions of decurrencyclic A-B in the active sites of the inflammatory enzyme.

General
Marine macroalgae have been used traditionally as a food supplement in several countries, and lately the search for pharmacological compounds from bioactive natural sources has paved the way for growth of macro-algal industries. Among many other bioactive compounds isolated, terpenes have presented many biological activities (Chakraborty and Dhara 2020). Structural investigation of triterpenoid compounds from T. decurrens was not yet reported, but there were several reports of triterpenes isolated from brown algae. The present work envisaged on the structural elucidation of triterpenoids obtained from T. decurrens and assessing their biological activities.

Spectroscopic depiction
The organic extract of T. decurrens was fractionated by recurrent column chromatography to isolate two undescribed triterpene nor-dammarane derivatives, called as decurrencyclic A-B (Figure 1). Structural elucidation of these compounds was performed by spectroscopic analyses (mass, FT-IR, and 1D/2D NMR) (Figures S1-S30).

Decurrencyclic A
The triterpene decurrencyclic A (compound 1) was characterized as 6b, 16b-diacetoxy-3,7,25-trihydroxy-nor-dammara-22-en-21,24-lactone, and its molecular formula was deduced as C 34 H 52 O 9 by mass spectroscopic analysis fHRESIMS m/z 605.3691 [M þ H] þ for [C 34 H 53 O 9 ] þ g ( Figure S1) and FT-IR/NMR experiments (Table S2; Figures S2-S10), accounting for nine degrees of unsaturation. The IR spectrum exhibited absorption bands at 3330 (-OH), 1745 (-CO-CH 3 ), and 1648 (C ¼ C) cm À1 ( Figure S10). The 13 C NMR spectrum ( Figure S3) along with 135 DEPT (distortionless enhancement by polarization transfer) spectrum ( Figure S4) determined the C-atoms in 1 as composed of nine methyls, five CH 2 , twelve CH (five oxygenated and two olefinic), eight non-protonated C-atoms, and three ester C ¼ O groups (d 171.9, 175.4 and 181.5). Characteristic 1 H NMR signals and its multiplicities for methyl groups were recorded at d 0.68 (s), 0.76 (s), 0.88 (s), 0.95 (s), 0.96 (s), 1.30 (s), 1.36 (s), 2.06 (s), and 2.32 (s) ( Figure S2). Of these, chemical shift values at d 2.06 and 2.32 inferred that those might be connected to the carbonyl carbon of ester group. From this data, it could be concluded that the third ester carbon must be a lactone. The 1 H-13 C correlation from heteronuclear multiple bond correlations (HMBC) spectrum deduced the correlation between methyl groups at d 2.06 and 2.32 to the corresponding ester carbonyls (d 175.4 and 181.5) supported the attribution ( Figure S7). Calculated proton integral value in the olefinic region (d 5.06-5.36) attributed the presence of one double bond, whereas the C ¼ C was located between C-23 and C-24 on account of the HMBC correlations of H-23 and H-24 to C-24 and C-23, respectively. Of the total nine degrees of unsaturation calculated from the molecular formula, two were found as methyl esters, one as a lactone and one double bond, whilst the rest were concluded as five rings. These ring structures were deduced with the help of 1 H-1 H COSY-45 ( Figure S5), HMBC ( Figure S7), and TOCSY ( Figure S8) correlations. TOCSY showed a total of five spin systems, which corresponded to the five rings metioned earlier. H-1, H-2 and H-3 consists in one system, H-5, H-6 and H-7 were found to be in another spin system, pointing that these two systems can be regarded as two rings. Similarly, H-9, H-11, and H-12 were found to be correlated while H-13, H-15, H-16, and H-17 belonged to same spin system. The fifth spin system consisted of H-22, H-23, and H-24 which were included as another ring. The linkage patterns of each ring were deduced from HMBC correlations obtained. The HMBC correlation from H-1 to C-10 and H-5 to C-10 revealed the first two ring skeletons as cyclohexane. The weak HMBC correlation from H-13 to C-8 indicated the presence of a quaternary carbon in between. Hence, it was suggested that the third ring forms a cyclohexane extending from C-9 to C-14. Similarly, HMBC peaks between H-13 to C-17 revealed a five membered ring. The characteristic 13 C and 1 H NMR signals of the skeleton were found to be similar to that of an earlier reported nor-dammarane 6b,16b-diacetoxy-25-hydroxy-3,7-dioxo-29-nordammara-1,17(20)-dien-21,24-lactone (Afiyatullov et al. 2012) except for the side chains. Thus, it was inferred that the skeleton of the carbon was composed of 27 carbons with a nor-dammarane structure, and was numbered accordingly (Table S2). HSQC (heteronuclear single quantum coherence) ( Figure S6) was used for assigning the protons attached with each carbons, and were given as follows; d 1.05,1.70 (H-1)/37.6 (C-1), d 1.61, 1.72 (H-2)/33.9 (C-2),  (Tu et al. 2008). This constitutes the fifth ring in the molecule. The acetyl moiety at C-6 and C-16 were confirmed by the HMBC correlations of H-6 (d 3.67) to C-31 (d 175.4) and H-34 (d 2.32) to C-33 (d 181.5). NOE couplings were apparent between H-3/H-5, H-5/H-6, H-6/H-7, H-7/H-16 and H-16/H-17, which represented that these protons to be on the same side of the molecule, and was denoted as b-oriented ( Figure S9). The mass spectrum ( Figure S11) showed a molecular ion peak at m/z 604.9, and other fragment peaks at m/z 518, 506, and 70 were due to the losses of two acetic acid fragments, water molecules, and finally resulting in isoprene units ( Figure S12). The chemical ionization mass spectrum (CI-MS) spectrum showed a peak at m/z 605.3 corresponding to the [M þ 1] peak ( Figure S13). Purity of the compound was analyzed using HPLC chromatogram ( Figure S14) and the UV spectrum recorded a peak at 264 nm ( Figure S15). Based on these data the structure of the compound was deduced as a nor-dammarane type of triterpenoid.

Decurrencyclic B
Decurrencyclic B (compound 2) was obtained as a viscous oily substance. Its positive HR-ESI-MS spectrum ( Figure S16) ), and 1639 (-C ¼ C-) cm À1 ( Figure  S25). The 13 C NMR spectrum ( Figure S18, Table S2) along with 135 DEPT ( Figure S19) inferred the C-atoms in compound 2 as composed of nine methyls, five CH 2 , eleven CH (four oxygenated and two olefinic), eight non-protonated C-atoms, and three ester C ¼ O groups (d 171.8, 175.4 and 173.9). Characteristic 1 H NMR signals and their multiplicities for methyl groups were recorded at d 0.57 (s), 0.67 (s), 0.75 (s), 0.88 (s), 0.97 (s), 1.30 (s), 1.36 (s), 1.45 (s), and 2.04 (s) ( Figure S17). These NMR data were fairly comparable to those of decurrencyclic A (1) except for the side chains. A comparison of the chemical shift values of compounds (1) and (2) is tabulated (Table S3). From the chemical shift values of 1 H NMR, the signal at d 2.04 might be connected to the carbonyl carbon of ester group. From this data, it could be concluded that the third ester carbon must be a lactone, whereas the other ester might be connected to a CH moiety. The 1 H-13 C correlation from HMBC spectrum of the methyl group at d 2.04 to the corresponding ester carbonyl (d 175.4) supported the attribution ( Figure S22). Calculated proton integral value in the region (d 5-8) attributed the presence of one double bond, wherein the C ¼ C was located between C-22 and C-23 as a result of the HMBC correlation of H-23/C-22. Characteristic 13 C and 1 H NMR signals were found to be similar to that of an earlier reported nor-dammarane 3b,12b-dihydroxy-25,26,27-trinordammara-22-en-24,20-olide (Wang and Wang 2013) except for the side chain substitutions. The distinct difference was that the ketone carbon signal at C-3 was replaced by an oxygenated methine (d 75.0), which was supported by the spin-spin correlations in 1 H-1 H COSY (between H3 (d 3.14)-H2 (d 1.61, 1.73), H2 (d 1. The HMBC correlation of H-21/C-22, H-23/C-22, and H-23/C24 supported this assumption. This was also supported by the TOCSY experiment ( Figure S23) as it disclosed a total of six spin systems, of which four of them corresponding to each skeletal ring. The fifth spin system was identified as furanone moiety as it displayed correlation between H-22 and H-23 as same as in the COSY spectrum. The acetyl moiety at C-6 and C-16 were confirmed by the HMBC correlations of H-6 (d 3.66) to C-31 (d 175.4) and H-16 (d 2.29) to C-33 (d 173.9). The HMBC correlations of H-35 (d 1.30) and H-36 (d 1.36) to C-34 attributed the presence of an isopropyl group next to acetyl group corresponding to the sixth system observed in TOCSY. HSQC experiment ( Figure S21) was used for assigning protons attached to the side carbons, and were attributed as:  Figure S24). NOE coupling was apparent between H-3/H-5, H-5/H-6, H-6/H-7, H-7/H-16, and H-16/H-17, which represented that these protons to be on the same side of the molecule, and was denoted as b-oriented. Thus, the structure of compound was established as, 6b-acetoxy-16b-isopropoxy-3,7-dihydroxy-25,26,27-trinordammara-22-en-24,20-olide. The mass spectrum ( Figure S26) showed a molecular ion peak at m/z 574.3, and other fragment peaks at m/z 414, 448, 394, 350, and 70 were due to the losses of acid fragments, lactone cleavage, water molecules, carbon dioxide, and finally resulting in isoprene units ( Figure S26). The CI-MS spectrum exhibited a signal at m/z 575.3 owing to the [M þ 1] peak ( Figure S28). Purity of the compound was analyzed using HPLC chromatogram ( Figure S29) and UV spectrum recorded a peak at 280 nm ( Figure S30).

Biosynthesis of isolated compounds
Triterpenes are isoprenoids, which are biosynthesized through mevalonic acid pathway. It initiates with the combination of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the presence of farnesyl pyrophosphate synthase to form farnesyl pyrophosphate (FPP) ( Figure S31), which gets converted to squalene in presence of squalene synthase. The action of squalene epoxidase on squalene yields 2,3-oxidosqualene. In triterpene synthesis, this oxidosqualene substrate is then folded to a different conformation, the chair-chair-chair (C-C-C) conformation, and prior to the cyclization to form dammarane cation (Thimmappa et al. 2014). This dammarane cation, in presence of hydroxylase, oxidase and acetyl CoA yields the triterpene derivatives decurrencyclic A-B ( Figure S32).

Bioactive potential of decurrencyclics A-B
The radical scavenging potential displayed by decurrencyclic B (IC 50 $ 80 lM) was comparable to that of commercially available standard (IC 50 $ 90 lM). There were preceding reports for antioxidant activities of dammarane triterpernes (Cao et al. 2015). The hydroxyls, ester and lactone functionalities in the compounds could assist in the delocalization of electron, while radical scavenging can be attributed to their promising antioxidant properties. Decurrencyclic B possesses an a,b-unsaturated lactone moiety, which could easily transfer H-atoms. Thus, it explains the higher antioxidant activity of decurrencyclic B compared to decurrencyclic A. Decurrencyclic B exhibited inhibition property against inflammatory COX-2 and 5-LOX (IC 50 13.98 and 3.02 lM, respectively). The anti-inflammatory activities of decurrencyclic B were higher compared to those displayed by decurrencyclic A (IC 50 > 3.9 lM) (Table S4), whereas the anti-inflammatory selectivity index was superior for the former (1.57) than that of the latter (1.39). Noticeably, a lesser selectivity index of synthetic anti-inflammatory agent ibuprofen (0.14) corresponded to selective inhibition towards COX-1, which is a constitutive enzyme resulting in several side effects related to gastrointestinal disarrays (Laneuville et al. 1995). Hence, decurrencyclic B with a greater specificity towards COX-2 and higher selectivity index could be utilized for the development of natural antiinflammatory leads. The 3,4-seco-dammarane triterpenoid saponins isolated from the leaves of Cyclocarya paliurus was previously reported for its COX-2 inhibition property (Liu et al. 2020).

Structure-activity correlation analysis and in silico molecular modeling of decurrencyclics A-B
The electronic properties of decurrencyclic A were not significantly different with those of decurrencyclic B (Table S3), despite the fact the bioactivities of the latter were greater. This could be explained by the reasonably higher steric bulkiness of decurrencyclic A (P 1320.9 cm 3 , MV 491.1 cm 3 ) than those noted for decurrencyclic B (P 1265.8 cm 3 , MV 477.3 cm 3 ). Hydrophobicity-lipophobicity balance (log P ow ) of decurrencyclics was within the allowable threshold limit (log P ow 3.4-4.4) consequently accrediting its drug-likeness characteristics and well-adjusted lipophilic-hydrophilic nature (Kujawski et al. 2012). Further, the in silico molecular modelling studies of the studied triterpene compounds were performed utilizing 5-LOX enzyme. Even though both decurrencyclic A and B showed only one hydrogen bond each with ARG 438.B (4.680 A ) and ARG 438.A (3.370 A ) residues in the active site of the enzyme (Table S5, Figure S33), the lesser binding energy and docking score of decurrencyclic B (À14.55 and À17.82 kcal mol À1 , respectively) than those in decurrencyclic A (À14.50 and À17.79 kcal mol À1 , respectively) with the active site of 5-LOX could support the potential anti-inflammatory properties of the former. Both these compounds displayed a lesser binding energy compared to the standard zileuton (À7.62 kcal mol À1 ), which could be substantiated with its attenuation potential against pro-inflammatory 5-LOX.

Development of algal organic extract
The study material of T. decurrens were collected (voucher specimen number of CMFRI/SUB24/06) from the Mandapam region (8 48  material S1). The samples (14 kg) were thoroughly cleaned and shade-dried on blotting paper before being powdered ($1 kg), and depigmented with n-hexane (250 mL Â 3). The left over algal residue was extracted with ethyl acetate-methanol (EtOAc: MeOH, 1:1 v/v; 300 mL Â 2) at about 60 C for 6 hours. The organic extract was dehydrated (over Na 2 SO 4 , 60 g) and concentrated by using a rotary vacuum evaporator (below 50 C) (Heidolph, Germany) to result into a viscous residue, termed as crude organic extract of T. decurrens (20.6 g, yield based on dry weight 2.06%).

Bioactivities of decurrencyclics A-B and molecular docking analysis
Anti-inflammatory activities of studied nor-dammarane analogues and column subfractions fractionated from the solvent extract of T. decurrens were examined by their inhibition potential against COX-1/2 (Larsen et al. 1996) and 5-LOX (Baylac and Racine 2003). Selectivity index (SI) with regard to anti-inflammatory properties was designed as the ratio of the attenuation property of decurrencyclics against COX-1 to that against COX-2 (COX-1 inhibitory IC 50 /COX-2 inhibitory IC 50 ). Antioxidant properties were assessed by the assays against stable DPPH and ABTS þ radicals (Wojdylo et al. 2007), wherein the results of inhibition properties against the enzymes and oxidants (free radicals) were tabulated, and expressed in IC 50 (concentration of samples at that they inhibit 50% of radical/enzyme activities; mg mL À1 /lM) values. Structure-activity relationship studies were directed with molecular descriptors, for example hydrophobic (octanol-water partition coefficient/log P ow ), polar/electronic (topological polar surface area/tPSA, polarizability/PI), and bulk/steric (molar volume/MV, molar refractivity/ MR, parachor/P) descriptors using ChemDraw Ultra (Cambridge Soft Corporation, USA; version 12.0) and ACD ChemSketch (version 2019.1; Advance Chemistry Development, Inc., Toronto, Canada). Molecular docking with 5-LOX was carried out by AutoDock 4 (AutoDock Tools version-1.5.6) with the crystal structure) of the target protein (PDB ID 3v99; resolution 2.25 Å) (Gilbert et al. 2012).

Statistical analysis
Statistical Program for Social Sciences 13.0 (SPSS Inc, Chicago, USA, version 13.0) was used to perform the statistical calculations. Analyses were performed in triplicate and the significant means of all parameters were examined by analysis of variance (ANOVA). The level of significance for all analyses was P < 0.05.

Conclusion
Two undescribed nor-dammarane triterpene derivatives, called as decurrencyclic A and B, with potential anti-inflammatory properties, were purified by chromatographic fractionation of the organic extract of T. decurrens. Decurrencyclic B displayed significant inhibition against pro-inflammatory 5-LOX comparable to the standard zileuton. Similarly, the compound could effectively inhibit COX-2 enzyme over COX-1 thus rendering an acceptable selectivity index. These results showed that decurrencyclic B could be developed as a promising therapeutic lead against inflammatory diseases.