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Abstract

Objective: In our ongoing effort to search for the novel secondary metabolites from the marine algae, chemical investigation of a methanolic extract of Sargassum micracanthum led to the isolation of a novel butenolide (1) and a known compound (2). Methods: The methanolic extract of S. micracanthum was partitioned and subjected to medium pressure column chromatography and preparative-HPLC to yield two compounds (1 and 2). Their structures were established based on comprehensive spectroscopic data (1D NMR, 2D NMR, and HRESIMS). These compounds (1 and 2) were evaluated for the production of the NO in lipopolysaccharide (LPS)-induced RAW264.7 cells and pro-inflammatory cytokines such as IL-6, IL-1β, TNF-α, and IL-10. Results: A new compound (1) was determined to be a new butenolide derivative, and a known compound (2) were identified as 2-hydroxy-(5E,9E)-6,10,14-trimethylpentadeca-5,9-dien-12-one. Compounds 1 and 2 showed inhibitory activities in a dose-dependent manner on LPS-induced NO production in RAW264.7 cells and pro-inflammatory cytokines. Conclusion: A new butenolide, sargassumin C (1), and 2-hydroxy-(5E,9E)-6,10,14-trimethylpentadeca-5,9-dien-12-one (2) were isolated from the brown alga, S. micracanthum. Compound 2 was more effective than 1 on NO production and pro-inflammatory cytokines.

Keywords

butenolide sargassumin C structure elucidation

Introduction

Sargassum is a genus of brown algae, commonly distributed worldwide.¹ Sargassum micracanthum (Kutzing) Endlicher is a member of the family Sargassaceae, which comprises about 193 species.² The species has a coriaceous thallus up to 50 cm in length and grows on rocks in the low intertidal to upper subtidal zones of open coasts.³ Previous studies have reported that Sargassum species contain compounds with various biological activities, such as anti-oxidative, cholinesterase inhibitory, neuroprotective, anti-cancer, anti-inflammatory, and anti-viral activities.^4-7 The compounds include chromanes, chromenes, meroterpenoids, farnesylactones, and plastoquinones.^8-11 Chemical investigation of a methanol extract of S. micracanthum led to the isolation of one novel butenolide (1) and a known compound (2). All isolates were evaluated for their anti-inflammatory activities on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 cells and pro-inflammatory cytokines. This paper deals with the isolation and structural elucidation of compounds 1 and 2 (Figure 1), and their anti-inflammatory activities.

Figure 1.

Structures of compounds 1 and 2.

Results and Discussion

Compound 1, obtained as a colorless oil with a specific rotation value of −11.1 ([α]_D, c = 0.15, 25°C, MeOH), exhibited an UV maximum (log ε) at 213 (3.5) nm. The IR spectrum of 1 showed important absorption bands at 3353 cm⁻¹ (hydroxyl group) and 1738 cm⁻¹ (carbonyl group). Its molecular formula was established as C₁₄H₂₂O₄ based on its HR-ESI-MS (m/z 253.1448, calcd for C₁₄H₂₁O₄ [M-H]⁻, 253.1440). The ¹H NMR spectrum of 1 revealed signals for two olefinic methines at δ_H 5.77 and 5.22, one oxygenated methine at δ_H 3.77, four methylenes at δ_H 2.13/1.95, 2.13/1.80, 2.06, and 1.46, and three methyls at δ_H 2.04, 1.62, and 1.17. The ¹³C NMR spectrum of 1 showed the presence of 14 carbon resonances for one carbonyl at δ_C 170.3, one acetal at δ_C 108.1, two sp² quaternary carbons at δ_C 166.8 and 134.4, two sp² methines at δ_C 125.9 and 118.2, one oxygenated methine at δ_C 68.0, four methylenes at δ_C 39.0, 34.1, 33.2, and 24.6, and three methyls at δ_C 23.7, 16.0, and 12.7 (Table 1). Analysis of its HMBC spectrum confirmed a furanone skeleton with cross-peaks of H-14 to C-2, C-3, and C-4, of H-2 to C-1 and C-4. The compound's partial linear structure was elucidated based on ¹H-¹H COSY correlations of H-5/H-6 and H-8/H-9/H-10/H-11/H-12, and HMBC correlations of H-5 to C-4 and C-6, H-13 to C-6, C-7, and C-8, H-10 to C-8, C-9, and C-11, and H-12 to C-10 and C-11. These data suggest the presence of a butenolide derivative (Figure 2). Compound 1 was similar in structure to sargassumin A except for the presence of a hydroxyl group (C-11) in 1, which replaced the ketone moiety in sargassumin A.¹² The E-configuration at C-13 in 1 was elucidated by comparing the carbon chemical shift (Z-form: above δ_C 20.0). The stereochemistry at C-4 in 1 was determined to be R on the basis of a positive Cotton effect at 210 nm.¹³ Furthermore, it appears to be a reduced form of sargassumin A. However, compound 1 (0.1 mg) had not reacted with Mosher's reagent for 3 days. The structure of 1 was determined to be a novel butenolide derivative named as sargassumin C.

Figure 2.

HMBC and ¹H-¹H COSY correlations of compound 1.

Table 1.

¹H- and ¹³C-NMR Spectroscopic Data of 1 in CDCl (δ in ppm and J values in (H_z) in Parentheses)).

	1
Position	δ_H (J in Hz)	δ _C
1		170.3
2	5.77 (d, J = 1.3 Hz)	118.2
3		166.8
4		108.1
5	2.13 (m)	34.1
	1.80 (m)
6	2.13 (m)	33.2
	1.95 (m)
7		134.4
8	5.22 (t, J = 6.6 Hz)	125.9
9	2.06 (m)	24.6
10	1.46 (m)	39.0
11	3.77 (m)	68.0
12	1.17 (d, J = 7.4 Hz)	23.7
13	1.62 (s)	16.0
14	2.04 (d, J = 1.3 Hz)	12.7

Compound 2 was identified as 2-hydroxy-(5E,9E)-6,10,14-trimethylpentadeca-5,9-dien-12-one (2) by comparing its NMR and mass spectrometric data with the previous literature.¹⁴ The absolute configuration at C-2 in 2 was unknown. However, in our study, we have characterized this by means of a modified Mosher's method.^15,16 Analysis of Δδ_Η (δ_S − δ_R) values revealed a R configuration (Supporting information Figure C1).

To investigate the anti-inflammatory activities of compounds 1 and 2 from S. micracanthum, production of the NO and pro-inflammatory cytokines such as IL-6, IL-1β, TNF-α, and IL-10 were measured in LPS-stimulated RAW264.7 cells. The production of nitrite and pro-inflammatory cytokines was significantly increased by LPS treatment compared with the LPS non-treated group of RAW264.7 cells. First, we determined the cytotoxicity of compounds 1 and 2 on RAW264.7 cells. Compounds 1 and 2 (62.5, 125, and 250 μM) did not show any cytotoxicity towards LPS-stimulated RAW264.7 cells (Figure 3). We measured the level of nitrite as an indicator of NO production. The production of NO was significantly inhibited in a dose-dependent manner after treatment with compounds 1 and 2. Finally, we investigated the production of pro-inflammatory cytokines by treating LPS-stimulated RAW264.7 cells with compounds 1 and 2. LPS-induced the pro-inflammatory cytokines IL-6, IL-1β, TNF-α, and IL-10 for 24 h, but the levels of these were dose-dependently reduced by compounds 1 and 2 (Figure 4). Compound 2 was more effective in reducing the production of NO and pro-inflammatory cytokines compared with compound 1.

Figure 3.

The inhibitory effects of compounds 1 and 2 on NO production in LPS-induced RAW264.7 cells and cell viability.

Figure 4.

The effect of compounds 1 and 2 on IL-1β, IL-6, TNF-α, and IL-10 productions. Results were expressed as mean ± SD ^#the non-treated sample (p < 0 .01), *treated sample (p < 0.01).

Conclusions

A new butenolide, sargassumin C (1), and 2-hydroxy-(5E,9E)-6,10,14-trimethylpentadeca-5,9-dien-12-one (2) were isolated from the brown alga, Sargassum micracanthum. Compounds 1 and 2 showed inhibitory activities in a dose-dependent manner on LPS-induced NO production in RAW264.7 cells and pro-inflammatory cytokines of IL-6, IL-1β, TNF-α, and IL-10.

Sargassumin C (1)

Colorless oil

$[a]_{D}^{25}$ : −11.1 (c 0.15, CHCl₃)

IR (film) ν_max : 3353, 2920, 2849, 1738, 1644, 1450, 1114, 1015 cm⁻¹.

UV (MeOH) : λ_max (log ε) 213 (3.5) nm.

HRESIMS

m/z 253.1448 [M-H]⁻ (Calcd for C₁₄H₂₁O₄ [M-H]⁻, 253.1440, Δ = + 0.8 mmu)

¹H (500 MHz) and ¹³C NMR (125 MHz) spectra are shown in Table 1.

2-hydroxy-(5E,9E)-6,10,14-trimethylpentadeca-5,9-dien-12-one (2)

Colorless oil

$[a]_{D}^{25}$ : −9.6 (c 0.1, CHCl₃)

HRESIMS

m/z 303.2294 [M+H]⁺ (Calcd for C₁₈H₃₃O₂ [M+H]⁺, 303.2300, Δ = - 0.6 mmu)

¹H NMR (500 MHz, CDCl₃) : δ 5.20 (1H, t, J = 6.4 Hz, H-9), 5.13 (1H, t, J = 6.4 Hz, H-5), 3.78 (1H, m, H-2), 2.98 (2H, s, H-11), 2.26 (1H, d, J = 6.8 Hz, H-13), 2.11 (2H, m, H-4), 2.09 (1H, m, H-14), 2.06 (2H, m, H-8), 2.00 (2H, m, H-7), 1.60 (3H, s, 6-CH₃), 1.58 (3H, s, 10-CH₃), 1.48 (2H, m, H-3), 1.17 (3H, d, J = 6.1 Hz, H-1), and 0.87 (6H, d, J = 6.4 Hz, 14-CH₃ and 15)

¹³C NMR (125 MHz, CDCl₃) : δ 209.8 (C-12), 135.4 (C-6), 129.6 (C-9), 129.2 (C-10), 124.4 (C-5), 68.1 (C-2), 54.6 (C-11), 50.7 (C-13), 39.3 (C-3 and C-7), 26.8 (C-8), 24.6 (C-14), 24.5 (C-4), 23.6 (C-1), 22.7 (C-15 and 14-CH₃), 16.6 (10-CH₃), and 16.1 (6-CH₃).

Experimental

General Experimental Procedures

NMR spectra were recorded in CDCl₃ using a JNM-ECZ500R FT-NMR spectrometer (JEOL, Tokyo, Japan). Optical rotation was measured on a JASCO P-2000 Digital Polarimeter (Jasco, Tokyo, Japan). The CD spectrum was recorded on a JASCO J-715 spectropolarimeter. The UV spectrum was measured on a Thermo Scientific multiskan GO spectrophotometer (Thermo Fisher Scientific, Massachusetts, US), and the IR spectrum on a JASCO FT-IR 4100 spectrometer (Jasco, Tokyo, Japan). For medium-pressure liquid chromatography (Buchi, Flawil, Switzerland), a C₁₈ column was used (40 g, Biotage, Uppsala, Sweden), and for high performance liquid chromatography (Waters Acquity Arc, Waters, Milford, US) a C₁₈ column (4.6 μm × 250 mm and 10.0 μm × 250 mm, Cosmosil, Kyoto, Japan) was used for the isolation process. HRESIMS was performed with a SCIEX X500R Q-TOF LC-MS/MS spectrometer (SCIEX, Massachusetts, US).

Plant Material

Sargassum micracanthum was collected from Udo-myeon, Jeju Island, Korea, in June 2018 and identified by Dr Jeong Chan Kang (Research institute for Basic Sciences, Jeju National University). A voucher specimen (MABIK AL00079431) was deposited at the National Marine Biodiversity Institute of Korea (MABIK).

Extraction and Isolation

Dried whole plants (450 g) of Sargassum micracanthum were ground and extracted with MeOH (2 L × 3) at room temperature for 1 day. The MeOH extract was partitioned with n-hexane, chloroform (CHCl₃), ethyl acetate (EtOAc), and n-butanol, respectively. The CHCl₃-soluble portion was mixed with the EtOAc-soluble portion due to their similar HPLC profiles. The combined extract was subjected to medium-pressure liquid chromatography (MPLC) eluting with ACN/H₂O (10:90 to 100:0) to obtain sub-fractions (1-6). Sub-fraction 1 was further separated by MPLC, followed by semi-HPLC eluting with 30% ACN in water to give compound 1 (2.0 mg). Sub-fraction 4 was further purified by semi-HPLC by eluting with 65% ACN in water to provide compound 2 (2.6 mg).

NO and ELISA Assay

The production of NO and pro-inflammatory cytokines such as IL-6, IL-1β, TNF-α, and IL-10 was assessed using Griess reagent (Thermo Fisher, Massachusetts, US) and an ELISA kit (Abcam, Cambridge, UK), according to the manufacturer's instructions. Briefly, RAW264.7 cells were seeded onto either 24-well plates (1.5 × 10⁵ cells/well) or 6-well plates (6 × 10⁵ cells/well), and incubated for 18 h at 37 °C. After incubation, the cells were treated with various concentration of compounds 1 and 2 in the presence of LPS (1 μg/mL) for 24 h. The culture supernatant was collected and used to measure the level of NO and pro-inflammatory cytokines. The absorbance of the reaction solution was determined using a microplate reader (SpectraMax i3x, Molecular Devices, San Jose, California, US). All the absorbance measurement results were expressed as the mean ± standard error of the mean. Statistical analysis was performed using IBM SPSS software (IBM SPSS Statistics for Windows, Version 22.0, Armonk, New York, US).

Preparation of (S)- and (R)-MTPA Esters (2a and 2b) of 2

Compound 2 (1.0 mg) was treated with (R)-(-)-α-methoxy − α-(trifluoromethyl)phenylacetic acid chloride (5 μL) in pyridine (500 μL). The mixture was added 4-(dimethylamino)pyridine (1.0 mg). The reaction mixture was stirred at 25 °C for 12 h. The product was isolated by semi-HPLC by eluting with 95% ACN (flow rate: 2.5 mL/min) to yield (S)-MTPA ester (2a, 0.3 mg). The (R)-MTPA ester (2b, 0.3 mg) was acquired from compound 2 (1.0 mg) by using the same procedure.^{15, 16}

(S)-MTPA ester (2a)

Colorless oil

HRESIMS

m/z 519.2690 [M+Na]⁺ (Calcd for C₂₈H₃₉F₃O₄Na [M+Na]⁺, 519.2698, Δ = −0.8 mmu)

¹H NMR (500 MHz, CDCl₃)

δ 5.19 (1H, t, J = 6.8 Hz, H-9), 5.12 (1H, m, H-5), 5.02 (1H, m, H-2), 2.99 (2H, s, H-11), 2.26 (2 h, d, J = 6.8 Hz, H-13), 2.10 (2H, m, H-4), 2.09 (2H, m, H-14), 1.97 (2H, m, H-7), 1.91 (2H, m, H-8), 1.67 (2H, m H-3), 1.58 (3H, s, 10-CH₃), 1.43 (3H, s, 6-CH₃), 1.32 (3H, s, H-1), 0.87 (6H, d, J = 6.8 Hz, 14-CH₃ and 15).

(R)-MTPA ester (2b)

Colorless oil

HRESIMS

m/z 519.2688 [M+Na]⁺ (Calcd for C₂₈H₃₉F₃O₄Na [M+Na]⁺, 519.2698, Δ = −1.0 mmu)

¹H NMR (500 MHz, CDCl₃)

δ 5.20 (1H, t, J = 6.8 Hz, H-9), 5.11 (1H, m, H-5), 5.07 (1H, m, H-2), 2.99 (2H, s, H-11), 2.26 (2 h, d, J = 6.8 Hz, H-13), 2.11 (2H, m, H-4), 2.10 (2H, m, H-14), 2.04 (2H, m, H-8), 2.00 (2H, m, H-7), 1.73 (2H, m, H-3), 1.58 (3H, s, 10-CH₃), 1.55 (3H, s, 6-CH₃, overlap), 1.24 (3H, s, H-1), 0.87 (6H, d, J = 6.8 Hz, 14-CH₃ and 15).

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221137411 - Supplemental material for Sargassumin C, a Novel Butenolide from Sargassum micracanthum

Supplemental material, sj-docx-1-npx-10.1177_1934578X221137411 for Sargassumin C, a Novel Butenolide from Sargassum micracanthum by Ji-Yul Kim, Gun-Woo Oh, Jeong Min Lee, Hyun-Soo Kim, Dae-Won Ki, Seok-Chun Ko, Mi-Jin Yim, Kyung Woo Kim, Dae-Sung Lee and Kyunghwa Baek in Natural Product Communications

Footnotes

Acknowledgements

This research was supported by the National Marine Biodiversity Institute of Korea Research Program 2022M00500.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the the National Marine Biodiversity Institute of Korea Research Program, (grant number 2022M00500).

Supplemental Material

Supplemental material for this article is available online.

ORCID iDs

Dae-Sung Lee

Ji-Yul Kim

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Supplementary Material

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Sargassumin C,a Novel Butenolide from Sargassum micracanthum

Abstract

Keywords

Introduction

Results and Discussion

Conclusions

Sargassumin C (1)

2-hydroxy-(5E,9E)-6,10,14-trimethylpentadeca-5,9-dien-12-one (2)

Experimental

General Experimental Procedures

Plant Material

Extraction and Isolation

NO and ELISA Assay

Preparation of (S)- and (R)-MTPA Esters (2a and 2b) of 2

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221137411 - Supplemental material for Sargassumin C, a Novel Butenolide from Sargassum micracanthum

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

Supplemental Material

ORCID iDs

References

Supplementary Material