Two rare antioxidative prenylated terpenoids from loop-root Asiatic mangrove Rhizophora mucronata (Family Rhizophoraceae) and their activity against pro-inflammatory cyclooxygenases and lipoxidase

Abstract Two new biogenic prenylated terpenoids were isolated from the methanol extract of Rhizophora mucronata. The extended C20 sesquiterpenoid with prenylated guaiane framework was characterised as (4E, 8Z)-3, 3a, 6, 7-tetrahydro-3, 9-dimethyl-5-(6-methylheptan-2-yl) cycloocta[b]furan-2-(9aH)-one (1). (35E)-1,2,3,5,6,6-icosahydro-4,4,8b,10,14,17,20,20-octamethylpicen-3-yl-34,35-dimethyloct-31-enoate (2) represents the first example of naturally occurring C40 prenylated oleanane-type triterpenoid, whereas one 4,5-dimethyloct-5-enoate side chain remains attached at C-3 position of the oleanane framework formed by the E-ring closure of C30 saccharide moiety. The structures of the compounds were elucidated using NMR and mass spectrometric analysis. Compound 1 was found to have significantly greater antioxidant activities (IC50 ~ 0.75 mg/mL) compared to 2 (IC50 > 0.80 mg/mL). No significant differences in anti-cyclooxygenase-2 of these compounds were discernable (IC50 0.8 – 0.9 mg/mL), whilst compound 1 showed greater anti-5-lipoxidase activities (IC50 ~ 0.8 mg/mL) those that of 2 (IC50 0.96 mg/mL). Bioactivities of the prenylated terpenoids were inversely proportional to lipophilic and bulk descriptors.


Introduction
Mangrove plants are rich source of secondary metabolites such as steroids, alkaloids, phenolics, triterpenes, saponins, tannins and flavonoids, which were reported to have toxicological, pharmacological and ecological importance. These species have greater capacities of salt tolerance as they adapted to live in anoxic conditions in the coastal ecosystems (Nebula et al. 2013). Rhizophora mucronata Lam. (family Rhizophoraceae) is one of the predominantly available mangrove plants grown in the coastal regions of India. It has been used as traditional medicine in the treatment of diabetes, diarrhoea, dysentery, blood in urine, fever, angina, diabetes and the Indo-Chinese use, and the roots for angina and haemorrhage were well documented. In India, bark used for diabetes and old leaves used as decoction at childbirth (Kusuma et al. 2011), and was reported for bioactive triterpenoids, such as 4-methoxy cinnamoyl-15-hydroxyl-β-amyrin, adian-5-en 3-ol, lupeol (Rohini & das 2010), pentacyclic terpenoids from R. stylosa ) and secolabdane diterpenoids from R. mucronata (anjaneyulu & Rao 2001). Earlier in vitro studies revealed that extract of R. mucronata demonstrates the significant anti-inflammatory and antiarthritic activities due to the presence of active principles, such as triterpenoids, alkaloids, polyphenolic content and flavonoids (Kumari et al. 2015). Polysaccharide extracts from the leaves were reported for anti-HIV activity (Premanathan et al. 1999). However, there are no reports of the occurrence of naturally occurring antioxidative and anti-inflammatory prenylated type sesqui-and triterpenoids from this mangrove species. The present study was aimed to isolate and characterise two rare biogenic prenylated terpenoids from the methanol extract of R. mucronata and their evaluation for antioxidative and anti-inflammatory properties by various in vitro models. These terpenoids included one new prenylated guaiane sesquiterpenoid with an uncommon five-membered lactone ring, (4E,8Z)-3,3a,6,7-tetrahydro-3,9-dimethyl-5-(6-methylheptan-2-yl) cycloocta[b]furan-2(9aH)-one (1) and prenylated oleanane-type triterpenoid, designated as (35E)-1,2, 3,5,6,6-icosahydro-4,4,8b,10,14,17,20,20-octamethylpicen-3-yl-34,35-dimethyloct-31-enoate (2). structure-activity relationship analysis was used to correlate different physicochemical parameters that significantly contribute towards the target bioactivities of the prenylated terpenoids. This study established the potential of prenylated terpenoids as potential lead molecules for use in pharmaceutical and functional food industries.
Compound 2, a new derivative of the naturally occurring C 40 prenylated oleanane-type triterpenoid, was isolated as yellow amorphous powder upon repeated column chromatography using silica gel as adsorbent. Its mass spectrum exhibited a molecular ion peak at m/e 579 (HREsIMs m/e 579.5264 [M + H] + ; d 0.0 amu), which in combination with its 1 H and 13 C NMR data indicated the elemental composition as C 40 H 66 o 2 . It satisfied 8° of unsaturation in which 3° of unsaturation were due to the double bonds, whilst 5° of unsaturation were from the ring systems. The 1 H NMR spectrum showed signals for eight methyl groups, which were positioned at the quaternary carbons as singlets at δ 0.73, 0.83, 0.85, 0.89, 0.93, 0.95, 0.97, 1.02 and 1.08 (Table s1). These assignments were found to be close with olean-18-ene as reported in the literature (Taye et al. 2015). an additional methyl signal was detected as a singlet at δ 1.12, corresponding to the carbon at δ 26.04 (C-39), which was assigned to be located in the side chain. The signal at δ 4.50 has been assigned to be due to H-3 (1H, t), and the chemical shift experienced a downfield shift from the typical value of δ 3.60, possibly due to the presence of the electronegative group (assigned to o-C=o) at its vicinity. This attachment was confirmed by the HMBC correlations between δ 4.50 (assigned to H-3) and δ 173.68 (assigned to carbonyl carbon). In the 1 H-1 H CosY spectrum, couplings were apparent between the protons at δ 1.73, 1.04 (assigned to be as H-1)/δ 2.03, 1.61 (H-2)/δ 4.50 (H-3), which along with the HMBC correlations between the proton at δ 4.50 (H-3) and the carbon at δ 27.53 (assigned to C-2)/δ 39.82 (C-4) supported the presence of substituted cyclohexane ring a with a possible oxygenation (assigned to o-C=o) at the C-3 position (Figure 1(B)) The 13 C NMR chemical shifts exhibited the signal for the characteristic double bond at δ 148.67 (assigned to C-18), δ 129.77 (C-19) along with the olefinic proton signal at δ 4.87 (H-19). These chemical shifts were found to be similar to those of the reported olean-18-ene (osorio et al. 2012) exhibiting olefinic signature peaks (Yang et al. 2006 -32), which support the presence of 4, 5-dimethyloct-5-enoate network (Figure s5). auxiliary olefinic bond at δ 145.2 (assigned to C-35) was confirmed by the strong HMBC correlations between δ 1.14 (H-39)/C-35, δ 1.94 (H-34) and δ 5.18 (H-36) with δ 145.2 (C-35). spatial arrangement of the angular methyl groups between the ring junctions was identified from the NoE spectra. The stereochemistry at δ 80.59 (assigned to C-3) was established by coupling constants of the proton at δ 4.50 (dd, J = 5.52,7.36 H-3) has mutual NoE correlations with δ 0.88 (assigned as H-40) and δ 0.84 (H-5) which was at the α-face of the molecule (axial configuration) (osorio et al., 2012) and has no NoE interactions with the protons at δ 1.08 (H-25) and δ 0.97 (H-26) and δ 1.02(H-28), thereby indicating that these groups must be equatorial and on the β-side of the molecule. The relative stereochemistries of the chiral centres at δ 55.59 (assigned to C-5), 51.14 (C-9) were α-side and 39.1 (C-13), and 47.56 (C-34) were in β-side orientation deduced from the NoEsY spectrum of the compound. Its mass spectrum exhibited a molecular ion peak at m/e 578, which appeared to undergo elimination of 4, 5-dimethyloct-5-enoate (m/e 169) to yield octamethylpicene at m/e 409 (Figure s6). The latter appeared to undergo C-ring cleavage to yield the base peak at m/e 189. The appearance of base peak at m/e 189 was in accordance with the literature values of oleananetype triterpenoid (Madureira et al. 2004).

Antioxidative and anti-inflammatory activities
Compound 1 with the extended C 20 guaiane sesquiterpenoid recorded greater antioxidant activity as determined by in vitro dPPH and aBTs radical scavenging properties (IC 50 0.73-0.76 mg/mL) than 2 (IC 50 0.85-0.89 mg/mL) with C 40 prenylated oleanane triterpenoid framework. Likewise, compound 1 exhibited greater anti-inflammatory property as described by the anti-CoX-2 and anti-5-LoX activities (IC 50 0.87 and 0.85 mg/mL, respectively) than those exhibited by 2 (IC 50 0.92 and 0.96 mg/mL, respectively) ( Table 1). Both compounds 1 and 2 exhibited lesser activity against CoX-1 isoform (IC 50 1.77 and 1.86 mg/mL, respectively) and their selectivity indices remained significantly greater (anti-CoX-2 IC 50 to anti-CoX-1 IC 50 , 0.4-0.5). No significant differences in anti-cyclooxygenase-2 of these compounds were discernable (IC 50 0.8-0.9 mg/mL), whilst compound 1 showed greater anti-5-lipoxidase activities (IC 50 ~ 0.8 mg/mL) than those of 2 (IC 50 0.96 mg/mL). The physicochemical parameters such as polarisability, steric and hydrophobic descriptors (lipophilicity, partition coefficients) were reported to have predominant roles to influence the biological activities (Cinq-Mars et al. 2008). The ability of any molecule to penetrate biological membranes is a primary factor in controlling the interaction of compounds with biological systems and is dependent on lipophilicity factors as determined by the partition coefficient between 1-octanol and water (log P ow ). although no significant differences in the polarisability depicting the electronic descriptor (32-38 × 10 −24 cm 3 ) in 1 and 2 were apparent, the anti-inflammatory activity (in IC 50 ) of the former was lesser (0.85-0.87 mg/mL) than the latter (0.92-0.96 mg/mL) due to the greater hydrophobic values of 2 (log P ow 9.29) those that recorded in 1 (log P ow 5.22). The larger size (MR > 150 cm 3 /mol) and greater lipophilic descriptor values of 2 might prevent the access to the catalytic cleft of 5-LoX and CoX-2 resulting in lesser anti-inflammatory activity than 1. This leads demonstrated in the present study will be significant in explaining the pharmacophore-fit in the macromolecular receptor site and exploring the primary site and mode of action of this class of the substituted terpenoid compounds. sesquiterpenoids with free radical scavenging properties were isolated from the marine macroalga Ulva fasciata (Chakraborty & Paulraj 2010). Previous study revealed the antioxidant and antimicrobial properties of different solvent extracts R. mucronata (Imdadul et al. 2011). The anti-inflammatory sesquiterpenes from Annona reticulata L. bark (Chavan et al. 2012), antioxidant sesquiterpenes from Cedrus deodara (Roxb.) Loud. (Chaudhary et al. 2015), new oleanane-type triterpene saponins from Glycyrrhiza glabra (Wei et al. 2014) and anti-inflammatory novel lindenane sesquiterpenes were isolated from Chloranthus fortunei (Zhang et al. 2012). an olean triterpene 3β-estearioxy-olean-12-ene was isolated from the medicinal plant Acacia brachypoda exhibited anti-inflammatory properties (da Rocha et al. 2015).

Chemicals and instrumentation
Fourier transform infra-red spectrometer 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 uV-vis spectrometer (Varian Cary, usa). The GC-Ms analysis was performed in electronic impact ionisation mode in a PerkinElmer Clarus 680 GC-Ms fitted with an Elite 5 Ms nonpolar, 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. The temperature was programmed initially at 50 °C for 2 min., then increased with a rate of 10 °C min -1 to 180 °C and kept for 2 min, and raised at 4 °C/min 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.

Plant material and preparation of crude extracts
The mangrove used in this study was R. mucronata (Family Rhizophoraceae) freshly collected from the Vallarpadam area of Kochi (Kerala state of India) located between south west coast of India (9°59′24.0″ North and 76°15′18.0″ East). a voucher specimen (No. CMFRI/MoEs/dFs/ aC 1144) was deposited in Marine Biodiversity Museum at CMFRI. The samples (1000 g) were washed in running water and transported to the laboratory before being shade dried (35 ± 3 °C) for 36 h. The shade-dried leaves were powdered (400 g) and extracted with aqueous methanol (50-60 °C, 3 h) followed by partitioning with n-hexane (500 mL × 3). The n-hexane fraction was filtered (Whatman No. 1 filter paper) through anhydrous Na 2 so 4 , and the pooled filtrate was concentrated (50 °C) in rotary vacuum evaporator (Heidolph Instruments, Germany) to furnish the n-hexane extract (24 g).

Structure-activity relationship analysis
The structure-antioxidant activity relationship analysis was carried out using different physicochemical parameters. The structural descriptors were calculated or taken from aCd Chemsketch (version 8.0) and Chemdraw ultra 8.0 databases: steric (molar volume, MV; parachor, Pr; molar refractivity, MR), hydrophobic (logarithmic scale of the octanol-water partition coefficient, log P ow ) and electronic descriptor variables (topological polar surface area, tPsa; polarisability, Pl).

Statistical analysis
statistical evaluation was carried out with the statistical Program for social sciences 13.0 (sPss Inc, Chicago, usa, ver. 13.0). analysis was 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.

Conclusion
Bioactivity-guided chromatographic fractionation of the n-hexane fraction obtained from the aqueous methanol extract of the leaves of mangrove R. mucronata afforded two terpenoids: one with an extended guaiane sesquiterpenoid and the other containing C 40 prenylated oleanane-type triterpenoid framework with potential antioxidative and anti-inflammatory activities. The bioactivities of the C 20 prenylated sesquiterpenoid (1) were greater than the oleanane triterpenoid (2). The antioxidant activities of these compounds were inversely proportional to the bulk and lipophilicity parameters.