Anti-inflammatory diterpenoids from the Brazilian alga Dictyota menstrualis

Algal Research 44 (2019) 101695

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Anti-inflammatory diterpenoids from the Brazilian alga Dictyota menstrualis a

T

a

Fábio do Nascimento Ávila , Luciana Gregório da Silva Souza , Pedro Bastos de Macedo Carneirob, Flávia Almeida Santosc, Greyce Luri Sasaharac, José Delano Barreto Marinho Filhod, Ana Jérsia Araújod, Ayslan Batista Barrosb, Norberto de Kássio Vieira Monteiroe, Edilberto Rocha Silveiraa, Otília Deusdênia Loiola Pessoaa,* a

Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60021-970, Fortaleza, CE, Brazil Campus Ministro Reis Velloso, Universidade Federal do Piauí, 64202-020, Parnaíba, PI, Brazil c Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, 60430-270, Fortaleza, CE, Brazil d Núcleo de Pesquisa em Biodiversidade e Biotecnologia, Universidade Federal do Piauí, 60202-020, Parnaíba, PI, Brazil e Departamento de Química Analítica e Fisico-Química, Universidade Federal do Ceará, 60461-970, Fortaleza, CE, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Dictyotaceae Marine natural product Brown alga Biological activity

The chemical investigation of the brown alga Dictyota menstrualis provided three previously unreported prenylated guaiane diterpenes derivatives named as dictyols K, M and N (1 - 3), along with five structurally known diterpenoids (4 - 8). The structures of the new compounds, including their relative stereochemistry, were established by nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS) data analysis. In addition, their structures were improved by quantum mechanical calculations. Anti-inflammatory, antimicrobial and cytotoxic assays were performed with the isolated compounds, however, only the anti-inflammatory activity was particularly prominent. The cell viability effects of 1 - 8 on murine macrophage cell line (RAW 264.7) showed IC50 values ranging from 1.12 to 2.53 μM, and the inhibitory activity against the nitric oxide production over the lipopolysaccharide-stimulated RAW 264.7 cell was observed for all compounds with IC50 values ranging to 0.12 - 0.23 μM.

1. Introduction Most drugs are derived from terrestrial sources, nevertheless, the interest in marine organisms as a promise for new bioactive compounds, has properly increased in the last two decades [1]. The marine algae have been intensively investigated for different purposes particularly for pharmaceutical and cosmetically oriented applications [2,3]. Based on a continuous coastline of about 8.500 Km long and with several ecosystems, the Brazilian seas represent a great potential for the discovery of new bioactive natural compounds. Within this context, the northeastern Brazil coastline, with several marine reserves, remains practically unexplored [4]. Firstly, our effort was to develop a study in the marine invertebrates and their associated microorganisms [5,6], but recently, we have also turned our attention to the study of the algae species themselves [7]. Brown algae (Phaeophyceae) are an important class of marine organisms with a higher chemical diversity and therapeutic potentials [8]. The Dictyotaceae family is the most representative group of the aforementioned class, since it is the third larger in number of species

⁎

[9]. This particular group is a rich and diversified source of secondary metabolites, specially of terpenoids [10]. Previous reports on the chemical studies about members of the Dictyotaceae group, revealed the isolation of more than 300 diterpenes, which were described for 35 species around the world [11,12], belonging to different classes like dolastanes, secodolastanes, dolabellanes, prenylated guaianes, xenianes and dichotomanes, many of which displaying cytotoxic, antiviral, antiprotozoal, antibacterial, antifeedant, antioxidant or anticoagulant activities [10,12–16]. The Dictyota genus can be found worldwide, including the American continent, particularly in the Brazilian coast and Caribbean Islands [17,19]. Dictyota menstrualis is a producer of diterpenes, mainly xeniane derivatives, prenylated guaianes and dichotomanes [18–21] some of which exhibit antiviral, antiplatelet and anticoagulant activities [17,20–22], as well as anti-inflammatory heterofucans [23]. The present study reports the chemical investigation of the n-hexane and the EtOAc extracts from D. menstrualis of northeastern Brazil origin, including the results of the anti-inflammatory, antimicrobial and cytotoxic assays.

Corresponding author. E-mail address: [email protected] (O.D. Loiola Pessoa).

https://doi.org/10.1016/j.algal.2019.101695 Received 1 March 2019; Received in revised form 7 October 2019; Accepted 10 October 2019 Available online 14 November 2019 2211-9264/ © 2019 Elsevier B.V. All rights reserved.

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2. Materials and methods

Table 1 1 H and 13C NMR Data for Compounds 1 – 3.

2.1. General procedures

No.

Optical rotations were measured on a Jasco P-2000 polarimeter, operating with a tungsten lamp at a wavelength of 589 nm at 20 °C. High-resolution mass spectra were recorded on a Waters Acquity UPLC system coupled with a quadrupole/time-of-flight (TOF) system (UPLC/ Qtof MSE spectrometer). The NMR spectra were performed on a Bruker Avance DRX-500 or DPX-300. In addition, a Shimadzu-UFLC semipreparative HPLC system, equipped with ternary pumps and diode array SPD-M20A UV/VIS detector, was used for the high-performance liquid chromatography (HPLC). The HPLC separations were performed using a Phenomenex C18 column (250 × 10 mm, 5 μm) and a mobile phase with the H2O, MeOH or MeCN in gradients, a flow rate of 4.72 mL/min, oven temperature at 40 °C, and monitored at 210–400 nm. The gravity columns chromatography (CC) were performed over silica gel 60 (70–230 or 230–400 mesh) or Sephadex LH-20 (Pharmacia). The TLC was performed on precoated silica gel aluminum sheets (SILICYCLE) with fluorescent indicator (254 nm), and the compounds were monitored by spraying with ceric sulfate solution (0.1 % w/v). 2.2. Algal material Specimens of the brown algae D. menstrualis (Hoyt) Schnetter, Hörning & Weber-Peukert (500 g) were collected manually at Pedra Rachada beach, municipality of Paracuru in the state of Ceará (3°23′55.6″S 39°00′47.5″W) during low tide. A voucher specimen (HMAR 2998), identified by Dr. Pedro Bastos de Macedo Carneiro, was deposited at the Herbário Professora Francisca Pinheiro (SISGEN ACA89E3).

1 (CDCl3)

2 (MeOD)

3 (MeOD)

δC

δH (J in Hz)

δC

δH (J in Hz)

δ Cb

δH (J in Hz)

1

46.1

2.62, m

48.1

2.75, m

46.5

2

34.2

37.0

2.53, m

33.5

3 4 5 6

123.8 142.2 59.2 74.6

124.9 143.8 59.0 76.9

m dd (6.4;

45.4

5.34, – 2.53, 4.09, 2.9) 2.10,

124.7 143.3 61.9 71.2

7

2.25, m, 2,21, m 5.32, br s – 2.52, m 3.89, br d (8.28) 1.83, m

m

62.8

8 9

23.3 40.4

206.2 129.1

10 11 12

152.4 37.7 76.6

5.20, m 2.89, dd (15.3, 1.8) 2.38, m – 2.10, m 4.92, m

3.08, dd (17.7, 8.7) 2.56, d (14.8, 8.5), 2.32, m 5.42, br s – 2.31, m 4.06, dd (9.4, 4.4) 2.68 dd (10.6, 4.4) – 6.21, m

163.5 29.4 33.3

– 2.33, m 1.70a; 1.20, m

13

27.8

25.5

14

120.0

125.5

2.09, m; 1.98, m 5.17, t (7.1)

15 16 17 18

134.5 18.1 16.1 107.4

19

12.7

20 8’-OAc

25.9 – – 172.1 21.4

12’-OAc

44.9

br s

1.55, m 2.63, m 2.15, m – 1.92, m 4.93, dt (14.7, 6.2) 2.28, m

73.7 43.02

5.11, br t (7.15) – 1.62, s 1.82, s 4.74, br s

120.5

2.24, t (6.5); 2.32, m 5.11, m

135.1 18.1 15.3 112.4

– 1.61, s 1.80, br s 4.83a

142.6 17.5 15.5 63.8

13.6

1.00, d (6.9)

18.1

– 1.61, 1.81, 4.30, 4.25, 0.84,

26.1 172.5 21.6 172.3 21.2

1.69, s – 2.03, br s – 2.01, br s

25.5 – – – –

1.68, s – – – –

1.02, d (6.8) 1.69, s – – – 2.05, s

2.3. Extraction and isolation

148.8 40.3 79.1 31.3

s s d (16.8) d (16.8) d (6.5)

a

Overlaping. The 13C chemical shifts of the hydrogenated carbons were determined from the internal projection of the HSQC experiment, while the non-hydrogenated carbons were obtained from the HMBC spectrum. b

The fresh algal material (500 g) after dried at room temperature was grinded and extracted with n-hexane, followed by EtOAc, to give the respective crude extracts after the solvent’s evaporation under reduced pressure. The n-hexane extract (1.6 g) was subjected to CC on silica gel by elution with an increasing mixture of n-hexane/EtOAc (100:0; 80:20; 60:40; 40:60; 40:60; 0:100) as solvents to yield six subfractions (A–F). The fraction B (1.3 g) was rechromatographed on silica gel CC, eluted with binary mixtures of n-hexane/EtOAc (100:0; 90:10; 80:20; 70:30; 60:40; 50:50; 75:25; 0:100) to yield 10 subfractions (BA-BJ), after TLC analysis. The subtractions BC (184.2 mg) and BH (300.0 mg) were both subjected to HPLC analyzes using a Gemini-phenomenex semi-preparative C-18 column (250 x 10 mm) and H20 (0.1 % TFA)/methanol (85–100 % in 5 min + 15 min MeOH pure) as mobile phase for a period of 25 min. From subfraction BC, were obtained compounds 1 (50.3 mg) and 3 (1.4 mg), while compound 2 (18.8 mg) was isolated from subfraction BH. The EtOAc extract (10.0 g) after repeated CC over silica gel, eluting with n-hexane/EtOAc, or over Sephadex LH-20, using MeOH as solvent, yielded compounds 4 (46.5 mg), 5 (43.7 mg), 6 (13.4 mg), 7 (4.8 mg), and 8 (10.0 mg), which were purified by HPLC using a semi-preparative C-18 column and a solvent system constituted by H2O (TFA 0.1 %)/MeOH in different proportions. Dictyol K (1): colorless resin; [α ]22 D -4.23 (c 0.07, MeOH); UV(MeOH) λmax(pda) 202 nm; 1H and 13C NMR (CDCl3) see Table 1; HRESIMS m/z 369.2402 [M + Na]+ (calcd for C22H34O3Na, 369.2406). Dictyol M (2): colorless resin; [α ]22 D -20.70 (c 0.07, MeOH); UV (MeOH) λmax(pda) 202 nm; 1H and 13C NMR (MeOD) see Table 1; HRESIMS m/z 427.2458 [M + Na]+ (calcd for C24H36O5Na, 427.2460). Dictyol N (3): colorless resin; [α ]21 D +28.05 (c 0.02, MeOH); UV (MeOH) λmax(pda) 241 nm; 1H and 13C NMR (MeOD) see Table 1; HRESIMS m/z 319.2271 [M+H]+ (calcd for C20H31O3, 319.2273).

3. Computational details The geometry optimization of compounds 1 - 3 were performed by using a hybrid generalized gradient approximation functional B3LYP [24–26] with 6–31 G(d,p) basis set by using the Gaussian16 package [27]. The interactions between compounds 1 - 3 and the solvents (chloroform for 1 and, methanol for 2 and 3) were evaluated at the same level of theory, including the Polarizable Continuum Model (PCM), using the integral equation formalism variant (IEF-PCM) [28–30]. The electron density was further used for Bader´s theory of atoms in molecules (AIM) [31] and for Electron Localization Function (ELF) [32] implemented in the software Multiwfn [33] version 3.7. Frequency calculations were performed to analyze vibrational modes of the optimized geometries in order to determine whether the resulting geometries are true minima or transition states, and for the Gibbs free energy calculations. The Gibbs free energy (G) for the compounds 1 - 3 was obtained from the sum G = ε0 + Gcorr, where ε0 and Gcorr correspond to total electronic energy and thermal correction, respectively. The NMR isotopic shielding constants were calculated from optimized geometries of 1 - 3 with mPW1PW91/6–31 G(d,p) level of theory using the gauge-including atomic orbitals (GIAO) approach [34–37]. Incorporation of the solvent as dielectric into GIAO NMR calculations was used to estimate the effect of the medium (CHCl3 or MeOH) on the chemical shifts of compounds 1 - 3. In order to compare the theoretical data with the experimental ones, the calculated isotopic shielding constants (σcalc) were contrasted with the reference compound 2

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tetramethylsilane (TMS): δCcalc = σTMS - σcalc, where both σTMS and σcalc were calculated at the same computational level, mPW1PW91/ 6–31 G(d,p).

3.4. Cytotoxicity assay Evaluation-MTT The cytotoxicity was evaluated against four different human cancer cell lines, provided by the National Cancer Institute U.S.A. (Bethesda, MD): leukemia (HL-60), glioblastoma (SF-268), pancreas carcinoma (PANC-1) and colon adenocarcinoma (HCT-116). These said cells were maintained in the RPMI 1640 medium supplemented with 10 % fetal bovine serum (v/v), 2 mM glutamine, 100 U/mL penicillin, and 100 μg/ mL streptomycin at 37 °C under a 5 % CO2 atmosphere. The compounds 1 - 8 were tested at concentrations ranging from 0.78 to 100 μM during 72 h and the effect of the cells proliferation, was evaluated in vitro using the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay, as described by Mosmann (1983) [38]. Doxorubicin, a standard compound with anticancer activity, was used as the positive control and the IC50 (the concentration that inhibits growth in 50 %), was calculated in accordance with the respective 95 % CI (confidence interval) by a non-linear regression using the software GraphPad Prism 5.01 version [40].

3.1. Cell culture and MTT assay for cell viability The RAW 264.7 cells (ATCC#TIB-71) were cultured in a Dulbecco’s Modified Eagle Medium (DMEM), containing 10 % fetal bovine serum, 100 units/mL of penicillin and 100 μg/mL of streptomycin at 37 °C in a 5 % CO2 and 95 % relative humidified atmosphere. Cell viability was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay (Mosmann, 1983) [38]. The RAW 264.7 cells were seeded at a density of 1 × 105 cells/well in a 96-well plate and incubated for 24 h. The cells were pre-treated with the test compounds (0.3–200 μM) for 24 h and then the MTT (1 mg/mL) was added in each cell well. The medium was removed after 4 h and the DMSO (100 μL) was added and the absorbance was detected at 570 nm using a microplate reader (Asys UVM 340, Biochrom, USA). The IC50 values were calculated from the concentration–response curves and three independent experiments were performed in triplicate.

4. Results and discussion The chromatographic procedures, over silica gel and by HPLC of the n-hexane and EtOAc extracts from D. menstrualis led to the isolation of three new prenylated guaiane diterpenes (Fig. 1), along with five others previously reported diterpenoids. The structures of the new compounds were established by interpretation of their spectroscopic data and by comparison with published data of analogous compounds previously reported [10–12]. Compound 1 had its molecular formula assigned as C22H34O3, requiring six degrees of unsaturation, based on the [M + Na]+ ion peak at m/z 369.2402 (calcd m/z 369.2406) in the HRESI mass spectrum. The 1H NMR spectrum showed signals for vinyl protons at δH 5.32 (br s, H-3) and 5.11 (t, J = 7.15 Hz, H-14), exocyclic methylidene protons at δH 4.74 (br s, 2H-18), and two oxymethine protons, one at δH 3.89 (d, J = 8.2 Hz, H-6) and the other one acetylated at 4.93 (dt, J = 14.7 and 6.2 Hz, H-12). Additionally, the 1H NMR spectrum displayed signals for five methyls, and two pairs of diasterotopic methylene protons at δH 2.63/2.15 (2H-9a/b) and 2.25/2.21 (2H-2a/b), Table 1. The 13C NMR spectrum of 1 indicated 22 carbon atoms which were classified by the APT and HSQC spectra into five methyls, four methylenes, one methylidene, eight methines and four non-hydrogenated sp2 carbons (Table 1). A detailed analysis of the 1H and 13C NMR data indicated a prenylated guaiane diterpene, which are common compounds in the Dictyota species [42]. The 1H and 13C NMR data of 1 were similar to those of the pachydictyol A [43], however, bearing an acetyl group which was positioned at C-12 by the HMBC diagnostic correlations of the oxymethine H-12 (δH 4.93, m) with the carbons at δC 172.1 (C-12`OAc), 120.0 (C-14), 27.8 (C-13) and 12.7 (C-19), instead C-8, C-9 and C-14 as reported for xeniane derivatives previously isolated from the Dictyota genus [7]. Additional important HMBC correlations, confirming the suggested structure are summarized in Fig. 2. The relative stereochemistry for C-1, C-5, C-6 and C-7 was assigned as 1R*, 5S*, 6R* and 7S* as already well-established for the perhydroazulene core of the prenylated guaiane diterpenes previously isolated from Dictyota species [44–46]. This was corroborated by the NOESY spectrum (Fig. 3) which displayed dipolar coupling for the H-6 with H-1 and H-11. The multiplicity (dt), with coupling constant values (J) of 14.7 and 6.2 Hz, for the oxymethine proton H-12 strongly suggests an anti-conformation for both 19-Me and 12-OAc. The literature shows that, in all cases where the stereochemistry of C-11 was established, the 19-Me is always in the β-position, thus the acetoxy group in C-12 must be α-positioned. Thus, the structure of 1 was designated as dictyol K in allusion to the sequence of pre-established names for previous guaiane diterpenes obtained from the Dictyota genus [10]. Compound 2 had its molecular formula C24H36O5 (seven degrees of unsaturation) established based on the adduct [M + Na]+ ion peak at

3.2. Assay for inhibition of the cellular NO production The nitrite concentration in the medium was measured as an indicator of the NO production according to the Griess reaction [39]. The RAW 264.7 cells were seeded at the density of 5 × 105 cell/wells into a 96-well plate and the cells were pre-treated with the test compounds (0.07 – 0.3 μM) for 1 h and then simultaneously stimulated with the LPS (1 μg/mL) at 37 °C for 24 h. Regarding the culture supernatant, it was mixed with an equal volume of Griess reagent (1 % sulfanilamide, 0.1 % N-[1-naphtyl]-ethylene diamine dihydrochloride, 5 % phosphoric acid) and the absorbance was measured at 540 nm. Dexamethasone (1.2–10 μM) was used as the positive control and the amount of nitrite in the samples was calculated from a standard curve of sodium nitrite. Three independent experiments were performed in triplicate. The results were presented as the IC50 values ± standard deviation (SD) and the differences in mean values between the groups were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey’s test with GraphPad Prism software (GraphPad Prism version 5.01 for Windows, San Diego, CA, USA) [40]. 3.3. Antibacterial assay The antibacterial assay method used was based in the CLSI (2015) [41] against Gram-positive bacteria (Staphylococcus aureus ATCC 29213) and Gram-negative bacteria (Escherichia coli ATCC 25922) from the collection of cultures stored in the microbiology laboratory at the Universidade Federal do Piauí, Brazil. The strains were previously seeded in Petri dishes containing the Mueller Hinton agar (Difco ™), and then incubated for 24 h at 35 ± 2 °C under aerobic conditions. Thereafter, isolated colonies were collected and suspended in a sterile saline solution (0.85 % (w/v) NaCl) until an absorbance of 0.08 to 0.13 was obtained at the wavelength of 625 nm, which corresponded to the 0.5 McFarland scale (approximately 1.5 × 108 CFU/mL). This suspension was then used to obtain the bacterial inoculum in the Mueller Hinton-MHCbroth (Difco™) with a final bacterial concentration of 5 × 105 CFU/mL (CLSI, 2015) [41]. The 96-well plate microdilution was used to determine the minimum inhibitory concentration (MICs). Therefore, the bacteria were exposed to a serial double dilution with an initial concentration of 200 μg/mL, diluted in MHB and DMSO (5 %). Oxacillin was used as a positive control for S. aureus, while Meropenem was used for E. coli. MHB, with sterile DMSO (5 %), was used as the negative control. The microplates were incubated under the same conditions as stated above. Furthermore, the MIC was defined as the lowest concentration agent that restricted a visible bacterial growth. 3

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Fig. 1. Structures of compounds, 1 – 8, isolated from Dictyota menstrualis.

(C-9) indicated an α,β-unsaturated ketone. The hydroxyl at the saturated β-carbon of the ketone moiety was assigned by the HMBC correlation of the oxymethine proton at δH 4.06 (H-6) with the carbonyl carbon at δC 206.2 (C-8). Similarly, an allylic hydroxymethylene at C10 was inferred by the HMBC correlations of the diastereotopic protons at δH 4.30 (H-18a d, J = 16.8 Hz)/4.25 (H-18b, d, J = 16.8 Hz) with the sp2 carbons at δC 163.5 (C-10) and 129.1 (C-9). The complete interpretation of the 1H and 13C NMR data were congruent with a guaiane diterpene structure named as dictyol N whose relative stereochemistry was assigned based on the NOESY spectrum, as showed in Fig. 3. In order to improve the structures of compounds 1 – 3 their optimized structures were performed by Density Functional Theory (DFT) using B3LYP/6–31 G(d,p) level of theory (Fig. 4). The 1 - 3 structures showed Gibbs free energies (G) of -6.8025 × 105 kcal mol−1 (G1), -8.2323 × 105 kcal mol−1 (G2) and -6.3093 × 105 kcal mol−1 (G3), respectively. Thus, the structure of 2 showed highest stability in comparison to the others. This stability can be attributed to the amount of intramolecular interactions as shown in the molecular graph (Fig. 5). According to the Bader´s theory of Atoms In Molecules (AIM), all properties of matter can be associated with the topology of the electronic density, ρ(r), to predict chemical bonds [47]. The Fig. 5 shows the molecular graphs of 1 - 3 with bond paths of intramolecular interactions and critical points (CPs, numbered in red) obtained by AIM theory. Based on these findings all compounds showed good stabilities, but 2 has a highest number of intramolecular bond paths and CPs when compared to 1. On the other hand, 3 has the same number of intramolecular bond paths as 2, but having a different electronic system (α,β-conjugated carbonyl) can’t be compared to each other.

m/z 427.2458 (calcd m/z 427.2460) in the HRESI mass spectrum. Its 1H and the 13C NMR data were similar to those of 1, indicating an analogue structure. The main difference between these compounds was the presence of an extra acetyl group as evidenced by the signals at δH 2.03 (s), and δC 172.5/21.6 in the 1H and 13C NMR spectra, respectively. In fact, comparison of the 1H NMR spectrum of 2 with that of 1 revealed the presence of an extra oxymethine proton at δH 5.20 (m, H-8), probably for an acetylated, replacing a proton the methylene signal at δH 1.55 (m, 2H-8) of 1. HMBC correlation of the oxymethine proton H-6 (δH 3.83, dd, J = 7.2; 2.8 Hz) with C-8 (δC 73.7) supported the O-acetyl at the C-8 position. In the NOESY spectrum, the dipolar coupling for both β-positioned H-5 and Me-19 with the oxymethine H-8 implied in an αorientation of the 8-O-acetyl group, while the NOE for H-1 with both H6 and H-7 indicated a β-position for the side chain (Fig. 3). Based of the aforementioned discussed data, the structure of 2 was established, and named as dictyol M. The molecular formula of compound 3 was assigned as C20H30O3 (six degrees of unsaturation), based on the protonated [M+H]+ ion peak at m/z 319.2271 (calcd 319.2273) in the HRESI mass spectrum. The 1H NMR spectrum of 3 displayed signals for olefin protons at δH 6.21 (H-9), 5.42 (H-3) and 5.17 (H-14), a diastereotopic oxymethylene at δH 4.30/4.25 (2H-18) and an oxymethine at 4.06 (H-6). In addition, aliphatic methines and methylenes, and four methyl groups were characterized (Table 1). The HSQC spectrum displayed signals to 16 carbon atoms classified into four methyls, four methylenes, including the oxymethylene, eight methines, one of which oxygenated and three sp2, in addition to four non-hydrogenated carbons, all of them sp2 hybridized. The chemical shifts at δC 206.2 (C-8), 163.5 (C-10) and 129.1

Fig. 2. Key COSY and HMBC correlations for 1 – 3. 4

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Fig. 3. Key NOESY correlations for compounds 1 – 3.

experimental and calculated data are in agreement. In addition to the new compounds, 1 – 3, the known acetoxycrenulide (4) [50], isoacetoxycrenulatin (5) [10], 4-hidroxycrenulide (6) [10], dictyotin A (7) [51] and dictyol C (8) [52] were also isolated. It is interesting to note that D. menstrualis collected on the northeast coast of Brazil produces diterpenes from chemical groups I (prenylated guaiane and prenylated epi-elemane) and III (xeniane and crenulidane) such as those collected in southeastern Brazil and other sites of the American Atlantic Ocean [53]. The anti-inflammatory, antimicrobial and cytotoxic activities of isolated compounds were performed. The anti-inflammatory activity was determined by the quantification of the nitrite production in the RAW 264.7 cells induced with the lipopolysaccharide (LPS). Nitric oxide (NO) is involved in important physiological processes such as neurotransmission, mitochondrial respiration, gastric motility and

The values of electronic density [ρ(r)] and Laplacian of electronic density (∇2ρ), indicate that all intramolecular interactions CPs exhibit the characteristics of closed-shell interactions [48]. Furthermore, the Electron Localization Function (ELF) values of those CPs are nearly half the ELF values for weak hydrogen bonds [49]. Table 2 shows the experimental 13C chemical shifts (δC exp) and the GIAO (Gauge-Including Atomic Orbitals) isotropic magnetic shielding for 13C (δC calc) of 1 - 3. The differences between δC exp and δC calc (ΔδC) for 1 - 3 are shown in Fig. 6. All correlations are linear and showed very good correlation coefficients (R2) of 0.9983, 0.9956 and 0.9964 for 1 3, respectively, confirming that the optimized geometries for 1 - 3 resemble with the structures shown in Fig. 1. The very good correlations (ΔδC) between the experimental 13C chemical shifts (δC exp) and GIAO/ mPW1PW91/6–31 G(d,p) calculated isotropic shielding tensors (δC calc) have confirmed the optimized geometries for 1 - 3 showing that the

Fig. 4. Structures of compounds 1 - 3 optimized by B3LYP/6–31G(d,p) level of theory. 5

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Fig. 5. Molecular graphs of compounds 1 – 3, optimized at the B3LYP/6–31G(d,p) level of theory with electronic density [ρ(r)], Laplacian of electronic density (∇2ρ), and electron localization function (ELF) from critical points (CPs) of intramolecular interactions (numbered in red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). Table 2 13 C NMR Experimental Data (δC No.

δC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

exp)

and Calculated GIAO Nuclear Magnetic Shielding (δC

1 (CHCl3) exp

46.1 34.2 123.8 142.2 59.2 74.6 45.4 23.3 40.4 152.4 37.7 76.6 27.8 120.0 134.5 18.1 16.1 107.4 12.7 25.9

calc)

for 1 - 3.

2 (MeOH) δC

calc

47.7 35.4 123.0 140.8 60.2 79.9 45.6 24.3 42.1 149.3 41.2 81.0 28.5 120.6 132.2 18.5 18.3 106.5 11.0 26.8

ΔδC

δC

1.6 1.2 −0.8 −1.4 1.0 5.3 0.2 1.0 1.7 −3.1 3.5 4.4 0.7 0.6 −2.3 0.4 2.2 −0.9 −1.7 0.9

48.1 37.0 124.9 143.8 59.0 76.9 44.9 73.7 43.0 148.8 40.3 79.1 31.3 120.5 135.1 18.1 15.3 112.4 13.6 26.1

exp

3 (MeOH) δC

calc

47.2 35.2 123.7 140.8 60.2 79.3 46.8 72.7 49.4 145.3 43.9 77.4 34.4 120.8 134.5 22.2 18.3 109.2 14.8 18.5

ΔδC −0.9 −1.8 −1.2 −3.0 1.2 2.4 1.9 −1.0 6.4 −3.5 3.6 −1.7 3.1 0.3 −0.6 4.1 3.0 −3.2 1.2 −7.6

δCexp

δC 46.5 33.5 124.7 143.3 61.9 75.2 62.8 206.2 129.1 163.5 29.4 33.3 25.5 125.5 142.6 17.5 15.5 63.8 18.1 25.5

calc

48.7 34.5 124.3 140.8 62.2 80.9 59.0 202.4 134.4 159.4 35.0 40.0 26.7 124.6 138.0 18.2 18.4 69.0 18.1 26.7

ΔδC 2.2 1.0 −0.4 −2.5 0.3 5.7 −3.8 −3.8 5.3 −4.1 5.6 6.7 1.2 −0.9 −4.6 0.7 2.9 5.2 0.0 1.2

for their inhibitory effect on the NO production in LPS-activated RAW 264.7 cells at nontoxic concentrations. Compounds 1, 2, 4, 5, 6, 7 and 8 effectively inhibited the NO production at concentrations with IC50 values of 0.16, 0.23, 0.19, 0.21, 0.16, 0.12 and 0.16 μM, respectively, which were lower than the positive dexamethasone control (1.53 μM)

inflammation [54], however the overexpression of NO contributes to inflammatory processes by promoting vascular permeability and leukocyte infiltration [55]. The effect of the compounds on the viability of the RAW 264.7 cells, showed cytotoxicity with IC50 values between 1.12–2.53 μM (Table 3). Compounds 1 - 2, 4 - 8 were further evaluated 6

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Fig. 6. Plots of the experimental chemical shifts (δC

exp)

vs the magnetic isotropic shielding (δC

IC50a ± SD (μM)

1 2 4 5 6 7 8

2.12 2.53 1.75 2.12 1.82 1.82 1.12

± ± ± ± ± ± ±

0.32 0.40 0.24 0.32 0.26 0.26 0.04

Table 4 Inhibitory Effects of the Isolated Compounds on the NO Production over LPS-Stimulated RAW 264.7 Macrophages.

1 2 4 5 6 7 8 Dexamethasone

0.16 0.23 0.19 0.21 0.16 0.12 0.16 1.53

± ± ± ± ± ± ± ±

C from GIAO/B3LYP/6–31G(d,p) level of theory.

The chemical investigation of the brown algae D. menstrualis from northeast of Brazil cost lead to the isolation of three new prenilated guaiane diterpene derivatives (1 - 3), along with five known diterpenoids (4 - 8). The diterpenes worldwide isolated from D. menstrualis display several bioactivities, but no anti-inflammatory activity has been so far reported. The isolated diterpenes showed potent anti-inflammatory activity, with the IC50 ranging from 0.12 to 0.23 μM, much smaller values than those displayed by the standard dexamethasone, a potent anti-inflammatory, immunosuppressive and anti-allergic drug currently used to treat several autoimmune disorders. Based on our results, D. menstrualis could be a valuable candidate to be investigated pursuing the isolation of new promisor prototype of anti-inflammatory drugs. Interestingly, previous studies with D. menstrualis have shown it as a potential producer of diterpenes independent of the harvesting site what makes it a powerful natural source of secondary metabolites that could be economically explored, everywhere, to the production of useful anti-inflammatory drugs.

IC50 values are defined as the concentration that results in a 50 % decrease in cell viability. The values represent the means of the results from three independent experiments with similar patterns.

IC50a ± SD (μM)

13

5. Conclusions

a

Compound

for

(Table 4). Compounds 1 - 2, 4 - 8 were assayed in vitro against Staphylococcus aureus and Escherichia coli bacteria strains. All of them were inactive, except 6 that was weakly active [MIC of 100 μg/mL (314 μM)] just against S. aureus ATCC 29213. The cytotoxicity of 1 - 8 was also evaluated against the human colon adenocarcinoma cell line HCT-116, but none of them exhibited any anticancer activity.

Table 3 Cytotoxicity of Compounds 1 - 8 on the RAW 264.7 Cells. Compound

calc)

0.05 0.03 0.08 0.46 7.67 3.19 0.07 0.03

Author’s contribution

a

IC50 values are defined as the concentration that results in a 50 % decrease production of nitric oxide. The values represent the means of the results from three independent experiments with similar patterns. Dexamethasone was used as a positive control substance for NO production.

O.D.L.P., E.R.S. and F.N.A. contributed to data interpretation, discussion of the results and write the main manuscript. F.N.A and L.G.S.S. performed the isolation and characterization of the compounds. N.K.V.M. performed the quantum mechanical calculations. F.A.S and G.L.S performed the anti-inflammatory assays. J.D.B.M.F., A.J.A. and A.B.B performed the antimicrobial assay. P.B.M.C. performed the 7

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collect and taxonomic identification of the algal material.

Biochem. Syst. Ecol. 33 (2005) 1–16, https://doi.org/10.1016/j.bse.2004.06.002. [13] É.M. Bianco, T.M. Francisco, C.B. Pinheiro, R. Bagueira De Vasconcellos Azeredo, V.L. Teixeira, R.C. Pereira, 4α-acetoxyamijidictyol - a new antifeeding dolastane diterpene from the brazilian brown alga canistrocarpus cervicornis, Chem. Biodivers. 12 (2015) 1665–1677, https://doi.org/10.1002/cbdv.201400410. [14] É.M. Bianco, T.M. Francisco, R. Bagueira, D.V. Azeredo, R.C. Pereira, R. De Blumenau, V. Konder, 10 b-Acetoxy-8a, 9a-epoxy-14b-hydroxy-7-oxodolastane – a new diterpene isolated from the Brazilian Brown Macroalga Canistrocarpus cervicornis, Helv. Chim. Acta 98 (2015) 785–794. [15] L. De Andrade Moura, A.C.M. De Almeida, T.F.S. Domingos, F. Ortiz-Ramirez, D.N. Cavalcanti, V.L. Teixeira, A.L. Fuly, Antiplatelet and anticoagulant effects of diterpenes isolated from the marine alga, Dictyota menstrualis, Mar. Drugs 12 (2014) 2471–2484, https://doi.org/10.3390/md12052471. [16] A.A. El Gamal, Biological importance of marine algae, Saudi Pharm. J. 18 (2010) 1–25, https://doi.org/10.1016/j.jsps.2009.12.001. [17] D.N. Cavalcanti, M.A.R. de Oliveira, J.C. de-Paula, L.S. Barbosa, T. Fogel, M.A. Pinto, I.C.N. de Palmer Paixão, V.L. Teixeira, Variability of a diterpene with potential anti-HIV activity isolated from the Brazilian brown alga Dictyota menstrualis, J. Appl. Phycol. 23 (2011) 873–876, https://doi.org/10.1007/s10811-0109601-z. [18] D.N. Cavalcanti, C.M. Rezende, A.C. Pinto, V.L. Teixeira, Diterpenoid constituents from the brown alga Dictyota menstrualis (Dictyotaceae, Phaeophyta), Nat. Prod. Commun. 1 (2006) 609–611. [19] F. Ortiz-Ramirez, D.N. Cavalcanti, R.C. Villaca, J.C. de Paula, Y. YoneshigueValentin, V.L. Teixeira, Chemical variation in the diterpenes from the brazilian brown alga Dictyota menstrualis (Dictyotaceae, Phaeophyceae), Nat. Prod. Commun. 3 (2007) 1879–1884. [20] H.S. Pereira, L.R. Leão-Ferreira, N. Moussatché, V.L. Teixeira, D.N. Cavalcanti, L.J. Costa, R. Diaz, I.C.P.P. Frugulhetti, Antiviral activity of diterpenes isolated from the Brazilian marine alga Dictyota menstrualis against human immunodeficiency virus type 1 (HIV-1), Antiviral Res. 64 (2004) 69–76, https://doi.org/10.1016/j. antiviral.2004.06.006. [21] H.S. Pereira, L.R. Leão-Ferreira, N. Moussatché, V.L. Teixeira, D.N. Cavalcanti, L.J. Costa, R. Diaz, I.C.P.P. Frugulhetti, Antiviral activity of diterpenes isolated from the Brazilian marine alga Dictyota menstrualis against human immunodeficiency virus type 1 (HIV-1), Antiviral Res. 64 (2004) 69–76, https://doi.org/10.1016/j. antiviral.2004.06.006. [22] L. De Andrade Moura, A.C.M. De Almeida, T.F.S. Domingos, F. Ortiz-Ramirez, D.N. Cavalcanti, V.L. Teixeira, A.L. Fuly, Antiplatelet and anticoagulant effects of diterpenes isolated from the marine alga, Dictyota menstrualis, Mar. Drugs 12 (2014) 2471–2484, https://doi.org/10.3390/md12052471. [23] I.R.L. Albuquerque, S.L. Cordeiro, D.L. Gomes, J.L. Dreyfuss, L.G.A. Filgueira, E.L. Leit, H.B. Nader, H.A.O. Rocha, Evaluation of anti-nociceptive and anti-inflammatory activities of a heterofucan from dictyota menstrualis, Mar. Drugs 11 (2013) 2722–2740, https://doi.org/10.3390/md11082722. [24] R. Fletcher, Practical Methods of Optimization, (1986), https://doi.org/10.1002/ adma.200400767. [25] C. Lee, W. Yang, R.G. Parr, Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density, Phys. Rev. B 37 (1988) 785–789, https://doi.org/10.1103/PhysRevB.37.785. [26] A.D. Becke, A new mixing of Hartree-Fock and local density‐functional theories, J. Chem. Phys. 98 (1993) 1372–1377, https://doi.org/10.1063/1.464304. [27] M.J. Frisch, G.W. Trucks, H.E. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, G.A. Petersson, F. O, J.B. Foresman, J.D. Fox, Gaussian 16, Gaussian, Inc., Wallingford CT, 2016. [28] B. Mennucci, E. Cancès, J. Tomasi, Evaluation of solvent effects in isotropic and anisotropic dielectrics and in ionic solutions with a unified integral equation method: theoretical bases, computational implementation, and numerical applications, J. Phys. Chem. B 101 (1997) 10506–10517, https://doi.org/10.1021/ jp971959k. [29] E. Cancès, B. Mennucci, J. Tomasi, A new integral equation formalism for the polarizable continuum model: theoretical background and applications to isotropic and anisotropic dielectrics, J. Chem. Phys. 107 (1997) 3032–3041, https://doi.org/ 10.1063/1.474659. [30] R. Cammi, J. Tomasi, Remarks on the use of the apparent surface charges (ASC) methods in solvation problems: iterative versus matrix-inversion procedures and the renormalization of the apparent charges, J. Comput. Chem. 16 (1995) 1449–1458, https://doi.org/10.1002/jcc.540161202. [31] P.S.V. kumar, V. Raghavendra, V. Subramanian, Bader’s theory of atoms in molecules (AIM) and its applications to chemical bonding, J. Chem. Sci. Bangalore (Bangalore) 128 (2016) 1527–1536, https://doi.org/10.1007/s12039-016-1172-3. [32] A.D. Becke, K.E. Edgecombe, A simple measure of electron localization in atomic and molecular systems, J. Chem. Phys. 92 (1990) 5397–5403, https://doi.org/10. 1063/1.458517. [33] T. Lu, F. Chen, Multiwfn: A multifunctional wavefunction analyzer, J. Comput. Chem. 33 (2012) 580–592, https://doi.org/10.1002/jcc.22885. [34] R. McWeeny, Perturbation theory for the Fock-Dirac density matrix, Phys. Rev. 126 (1962) 1028–1034, https://doi.org/10.1103/PhysRev.126.1028. [35] R. Ditchfield, Self-consistent perturbation theory of diamagnetism, Mol. Phys. 27 (1974) 789–807, https://doi.org/10.1080/00268977400100711. [36] K. Wolinski, J.F. Hinton, P. Pulay, Efficient implementation of the gauge-independent atomic orbital method for NMR chemical shift calculations, J. Am. Chem. Soc. 112 (1990) 8251–8260, https://doi.org/10.1021/ja00179a005. [37] J.R. Cheeseman, G.W. Trucks, T.A. Keith, M.J. Frisch, A comparison of models for calculating nuclear magnetic resonance shielding tensors, J. Chem. Phys. 104 (1996) 5497–5509, https://doi.org/10.1063/1.471789.

Statement of informed consent, human/animal rights No conflicts, informed consent, human or animal rights applicable. Declaration of authors All authors agree to authorship and submission of the manuscript for peer review. Declaration of Competing Interest The authors declare that they have no conflict of interests in publishing this article. Acknowledgements This work was financially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (No. 420454/20160), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico – CAPES/FUNCAP (No. 88887.113263/2015-01) and Instituto Nacional de Ciência e Tecnologia - INCT BioNat (No. 465637/ 2014-0). The authors thank Prof. Diego Veras Wilke (LaBBMar-UFC) for the cytotoxic assay, the High-Performance Computing Center (NPAD) at UFRN and the National High-Performance Processing Center of the Federal University of Ceará (UFC). References [1] V. Ruiz-Torres, J.A. Encinar, M. Herranz-López, A. Pérez-Sánchez, V. Galiano, E. Barrajón-Catalán, V. Micol, An updated review on marine anticancer compounds: the use of virtual screening for the discovery of small-molecule cancer drugs, Molecules 22 (2017) 1000–1037, https://doi.org/10.3390/molecules22071037. [2] R. Montaser, H. Luesch, Marine natural products: a new wave of drugs? Future Med. Chem. 3 (2011) 1475–1489, https://doi.org/10.4155/fmc.11.118. [3] J.-Y. Berthon, R. Nachat-Kappes, M. Bey, J.-P. Cadoret, N. Renimel, E. Filaire, Marine algae as attractive source to skin care, Free Radic. Res. Commun. 51 (2017) 555–567, https://doi.org/10.3389/fpls.2019.00756. [4] R.G.S. Berlinck, E. Hajdu, R.M. Da Rocha, J.H.H.L. De Oliveira, I.L.C. Hernández, M.H.R. Seleghim, A.C. Granato, É.V.R. De Almeida, C.V. Nuñez, G. Muricy, S. Peixinho, C. Pessoa, M.O. Moraes, B.C. Cavalcanti, G.G.F. Nascimento, O. Thiemann, M. Silva, A.O. Souza, C.L. Silva, P.R.R. Minarini, Challenges and rewards of research in marine natural products chemistry in Brazil, J. Nat. Prod. 67 (2004) 510–522, https://doi.org/10.1021/np0304316. [5] E.A. Santos, A.L. Quintela, E.G. Ferreira, T.S. Sousa, F.D.C.L. Pinto, E. Hajdu, M.S. Carvalho, S. Salani, D.D. Rocha, D.V. Wilke, M.D.C.M. Torres, P.C. Jimenez, E.R. Silveira, J.J. La Clair, O.D.L. Pessoa, L.V. Costa-Lotufo, Cytotoxic plakortides from the brazilian marine sponge Plakortis angulospiculatus, J. Nat. Prod. 78 (2015) 996–1004, https://doi.org/10.1021/np5008944. [6] L.V. Costa-Lotufo, F. Carnevale-Neto, A.E. Trindade-Silva, R.R. Silva, G.G.Z. Silva, D.V. Wilke, F.C.L. Pinto, B.D.B. Sahm, P.C. Jimenez, J.N. Mendonça, T.M.C. Lotufo, O.D.L. Pessoa, N.P. Lopes, Chemical profiling of two congeneric sea mat corals along the Brazilian coast: adaptive and functional patterns, Chem. Commun. (Camb.) 54 (2018) 1952–1955, https://doi.org/10.1039/c7cc08411k. [7] F.N. Ávila, F.C.L. Pinto, P.B.M. Carneiro, K.Q. Ferreira, D.V. Wilke, N.A.P. Nogueira, E.R. Silveira, O.D.L. Pessoa, New antiproliferative polyunsaturated epoxy-heneicosane derivatives isolated from the brown alga lobophora variegata, J. Braz. Chem. Soc. 30 (2019) 406–412, https://doi.org/10.21577/0103-5053.20180190. [8] J.W. Blunt, B.R. Copp, R.A. Keyzers, M.H.G. Munro, M.R. Prinsep, Marine natural products, Nat. Prod. Rep. 34 (2017) 235–294, https://doi.org/10.1039/ c8np00092a. [9] J.C. de Paula, M.A. Vallim, V.L. Teixeira, What are and where are the bioactive terpenoids metabolites from Dictyotaceae (Phaeophyceae), Brazilian J. Pharmacogn. 21 (2011) 216–228, https://doi.org/10.1590/S0102695X2011005000079. [10] S. Cheng, M. Zhao, Z. Sun, W. Yuan, S. Zhang, Z. Xiang, Y. Cai, J. Dong, K. Huang, P. Yan, Diterpenes from a Chinese collection of the brown alga dictyota plectens, J. Nat. Prod. 77 (2014) 2685–2693, https://doi.org/10.1021/np5006955. [11] A. Othmani, N. Bouzidi, Y. Viano, Z. Alliche, H. Seridi, Y. Blache, M. El Hattab, J.F. Briand, G. Culioli, Anti-microfouling properties of compounds isolated from several Mediterranean Dictyota spp, J. Appl. Phycol. 26 (2014) 1573–1584, https:// doi.org/10.1007/s10811-013-0185-2. [12] M.A. Vallim, J.C. De Paula, R.C. Pereira, V.L. Teixeira, The diterpenes from Dictyotacean marine brown algae in the Tropical Atlantic American region,

8

Algal Research 44 (2019) 101695

F. do Nascimento Ávila, et al.

[38] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63, https://doi.org/10.1016/0022-1759(83)90303-4. [39] X.T. Zhai, Z.Y. Zhang, C.H. Jiang, J.Q. Chen, J.Q. Ye, X. Bin Jia, Y. Yang, Q. Ni, S.X. Wang, J. Song, F.X. Zhu, Nauclea officinalis inhibits inflammation in LPSmediated RAW 264.7 macrophages by suppressing the NF-κB signaling pathway, J. Ethnopharmacol. 183 (2016) 159–165, https://doi.org/10.1016/j.jep.2016.01.018. [40] GraphPad Prism, Version 6.1; Intuitive Software for Science, (2015) San Diego, CA. [41] Clinical and Laboratory Standards Institute (CLSI), CLSI Document M100-S25; Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement, CLSI, Wayne, PA, USA, 2015. [42] D.L.R. Simas, C.R. Kaiser, L.M. Gestinari, H.M. Duarte, J. Campos de Paula, A.R. Soares, Corrigendum to “Diterpenes from the brown seaweed Dictyota caribaea (Dictyotaceae, Phaeophyceae): the ecological and taxonomic significance” [BSE (2014) 33-37], Biochem. Syst. Ecol. 57 (2014) 461–462, https://doi.org/10.1016/j. bse.2014.09.001. [43] G.S.E. Abou-El-Wafa, M. Shaaban, K.A. Shaaban, M.E.E. El-Naggar, A. Maier, H.H. Fiebig, H. Laatsch, Pachydictyols B and C: New diterpenes from Dictyota dichotoma Hudson, Mar. Drugs 11 (2013) 3109–3123, https://doi.org/10.3390/ md11093109. [44] D.R. Hirschfeld, W. Fenical, G.Y. Lin, R.M. Wing, P. Radlick, J.J. Sims, Marine natural products. VIII. Pachydictyol A, an exceptional diterpene alcohol from the brown alga, Pachydictyon coriaceum, J. Am. Chem. Soc. 95 (1973) 4049–4050, https://doi.org/10.1021/ja00793a037. [45] I.H. Hardt, W. Fenical, G. Cronin, M.E. Hay, Acutilols, potent herbivore feeding deterrents from the tropical brown alga, Dictyota acutiloba, Phytochemistry 43 (1996) 71–73, https://doi.org/10.1016/0031-9422(96)00221-X. [46] J.Y. Kim, M.A. Alamsjah, A. Hamada, Y. Fujita, F. Ishibashi, Algicidal diterpenes

[47] [48]

[49]

[50]

[51] [52]

[53]

[54]

[55]

9

from the brown alga Dictyota dichotoma, Biosci. Biotechnol. Biochem. 70 (2006) 2571–2574, https://doi.org/10.1271/bbb.60281. C.F. Matta, R.J. Boyd, The Quantum Theory of Atoms in Molecules: From Solid State to DNA and Drug Desing, Wiley, 2007, https://doi.org/10.1002/9783527610709. N.K.V. Monteiro, C.L. Firme, Hydrogen-hydrogen bonds in highly branched alkanes and in alkane complexes: a DFT, ab initio, QTAIM, and ELF study, J. Phys. Chem. A 118 (2014) 1730–1740, https://doi.org/10.1021/jp500131z. M.E. Alikhani, F. Fuster, B. Silvi, What can tell the topological analysis of ELF on hydrogen bonding? Struct. Chem. 16 (2005) 203–210, https://doi.org/10.1007/ s11224-005-4451-z. T.Z. Wang, E. Pinard, L.A. Paquette, Asymmetric synthesis of the diterpenoid marine toxin (+)-acetoxycrenulide, J. Am. Chem. Soc. 118 (1996) 1309–1318, https://doi.org/10.1021/ja9533609. M.O. Ishitsuka, T. Kusimi, A. Ichikawa, H. Kakisawa, Bicyclic diterpenes from two species of brown algae of the Dictyotaceae, Phytochemistry 29 (1990) 2605–2610. S.E.N. Ayyad, M.S. Makki, N.S. Al-Kayal, S.A. Basaif, K.O. El-Foty, A.M. Asiri, W.M. Alarif, F.A. Badria, Cytotoxic and protective DNA damage of three new diterpenoids from the brown alga Dictoyota dichotoma, Eur. J. Med. Chem. 46 (2011) 175–182, https://doi.org/10.1016/j.ejmech.2010.10.033. J. Chen, H. Li, Z. Zhao, X. Xia, B. Li, J. Zhang, X. Yan, Diterpenes from the marine algae of the genus Dictyota, Mar. Drugs 16 (2018), https://doi.org/10.3390/ md16050159. M.L. Lo Faro, B. Fox, J.L. Whatmore, P.G. Winyard, M. Whiteman, Hydrogen sulfide and nitric oxide interactions in inflammation, Nitric Oxide 41 (2014) 38–47, https://doi.org/10.1016/j.niox.2014.05.014. G. Nagy, J.M. Clark, E. Buzas, C. Gorman, M. Pasztoi, A. Koncz, A. Falus, A.P. Cope, Nitric oxide production of T lymphocytes is increased in rheumatoid arthritis, Immunol. Lett. 118 (2008) 55–58, https://doi.org/10.1016/j.imlet.2008.02.009.