Two New Meroterpenoid Derivatives From Sargassum macrocarpum With Their Anti-Oxidant Activities

Abstract

Objective: The purpose of this study was to search for novel secondary metabolites present in the marine brown alga, Sargassum macrocarpum C. Agardh. Methods: The methanolic extract of S. macrocarpum was partitioned and fractionated using a silica gel column, medium pressure liquid chromatography, and preparative-high performance liquid chromatography to obtain four compounds (1-4). These structures were elucidated using nuclear magnetic resonance spectroscopic and high resolution-electrospray ionization-mass spectroscopy methods. The isolated compounds were evaluated for their anti-oxidant activities using 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. Result: Two undescribed meroterpenoid derivatives (1 and 2), together with two known racemic meroterpenoids (3 and 4) were isolated from the methanolic extract of S. macrocarpum. All compounds were confirmed to be meroterpenoid derivatives. All these compounds exhibited ABTS and DPPH radical scavenging activities. Conclusions: The four meroterpenoids (1-4) were isolated from S. macrocarpum. Compound 3 showed the most anti-oxidant effect with IC₅₀ values of 9.9 (ABTS radical) and 22.4 μM (DPPH radical), respectively.

Keywords

Anti-oxidant NMR meroterpenoids structure determination

Introduction

The unpaired electron of a free radical renders it unstable and highly reactive.¹ Consequently, it tends to receive and donate electrons to attain structural stability. This results in the beginning of a chain reaction cascade since the attacked molecule loses an electron and becomes an unpaired electron.^2,3 The overproduction and accumulation of free radicals damages cells, biomolecules, and lipids.^4,5 The excess production of free radicals generates oxidative stress which plays a major role in the development of various diseases including leukemia, cancer, cardiovascular diseases, inflammatory diseases, and diabetes.^6–8 Sargassum macrocarpum is a perennial brown alga of the family, Sargassaceae which is distributed along the South Korean and Japanese coasts. It usually grows at depths of 10 m, is up to 3 m in length and contributes to the sea forest.⁹ In previous studies, S. macrocarpum have been reported to contain various secondary metabolites such as meroterpenoids, terpenoid lactones, furan derivative, and plastoquinones.^10–13 In this paper, the isolation, structural elucidation, and anti-oxidant activities of these compounds (1-4) (Figure 1) in the 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assays are described.

Figure 1.

Chemical structures of 1 to 4.

Results and Discussion

Compound (1), a colorless gum, had the molecular formula C₂₇H₃₈O₅. This was assigned from the negative ion peak at m/z 441.2629 [M-H]⁻ (calculated for C₂₇H₃₇O₅, 441.2641) in the HR-ESI mass spectrum. The ¹H NMR spectrum of 1 (Table 1) exhibited signals of 2 aromatic methines at δ_H 6.46 (s, H-7) and 6.30 (s, H-5), 4 olefinic methines at δ_H 6.22 (d, J = 9.7 Hz, H-4), 5.95 (t, J = 7.0 Hz, H-7′), 5.55 (d, J = 9.7 Hz, H-3), and 5.10 (t, J = 6.6 Hz, H-3′), 7 methylenes at δ_H 2.55 (m, H-6′), 2.21 (m, H-9′), 2.09 (m, H-2′), 2.04 (m, H-5′), 1.65 (m, H-1′), 1.45 (m, H-10′), and 1.43 (m, H-11′), and 5 methyls at δ_H 2.11 (s, H-18′), 1.54 (s, H-16′), 1.34 (s, H-17′), and 1.18 (s, H-13′ and H-14′). The ¹³C NMR spectrum of 1 (Table 1) indicated characteristic signals for 9 non-protonated carbons including a carbonyl at δ_C 172.1 (C-15′), 4 aromatic carbons at δ_C 148.8 (C-6), 144.9 (C-8a), 126.5 (C-8), and 121.5 (C-4a), 2 sp² quaternary carbons at δ_C 134.4 (C-4′) and 130.9 (C-8′), 2 quaternary carbons at δ_C 77.9 (C-2) and 71.4 (C-12′), 2 aromatic methines at δ_C 117.3 (C-7) and δ_C 110.5 (C-5), 4 sp² methines at δ_C 145.4 (C-7′), 130.9 (C-3), 125.2 (C-3′), and 123.1 (C-4), 7 methylenes at δ_C 43.3 (C-11′), 40.8 (C-1′), 39.2 (C-5′), 35.0 (C-9′), 28.2 (C-6′), 24.2 (C-10′), and 22.7 (C-2′), and 5 methyls at δ_C 29.4 (C-13′ and 14′), 26.1 (C-17′), 16.0 (C-16′), and 15.7 (C-18′). In the HMBC spectrum, H-18′ correlated with C-7/C-8/C-8a, H-7 with C-5/C-6/C-8a/, H-5 with C-4/C-7/C-8a, H-4 with C-2/C-5/C-4a/C-8a, and H-3 with C-2/C-4a, indicating the presence of a chromenol skeleton. The linear skeleton was elucidated by key HMBC correlations from H-1′ to C-2′, from H-3′ to C-2′, C-5′, and C-16′, from H-5′ to C-6′ and C-7′, from H-6′ to C-4′, C-5′, C-7′, and C-8′, from H-7′ to C-9′, and C-15′, from H-9′ to C-7′, C-8′, C-10′, and C-11′, and from H-13′ to C-11′, C-12′, and C-14′ and ¹H-¹H COSY correlations of H-1′/H-2′/H-3′, H-5′/H-6′/H-7′, and H-9′/H-10′. The key linkage between chromenol and the linear skeleton was determined by HMBC correlations from H-17 to C-2, C-3, and C-1′. These spectroscopic data suggested that 1 was a similar moiety to the previously reported chromenol derivative, sargachromenol.¹⁴ The main difference between 1 and sargachromenol (3) was the change in the single bond (C-11′) with the connectivity of the hydroxyl group (C-12′) in 1 (Figure 2). The double bond at C-3′ was determined to have an E-configuration by comparison with the ¹³C chemical shift in the literature (E form: below δ_C 20.0, Z form : above δ_C 20.0).^15–17 Furthermore, the double bond at C-7′ was elucidated to have a Z-configuration due to good agreement with the ¹H and ¹³C chemical shifts of sargachromenol.^14,18 Compound 1 was optically inactive. Furthermore, the chiral high performance liquid chromatography (HPLC) profile of 1 displayed 2 peaks (1a and 1b) as a racemate. To elucidate the absolute configuration at C-2 in 1a and 1b, the CD spectra showed a negative Cotton effect at 260 to 270 nm in 1a (Supplemental Figure S7).¹⁹ Therefore, we determined the R-configuration at C-2 in 1a. Therefore, the stereochemistry at C-2 in 1b was assigned as the S-configuration due to the opposite Cotton effect compared with 1a. The structures of compounds 1a and 1b were elucidated as new meroterpenoid derivatives and named (-)-macrocarquinoid H (1a) and (+)-macrocarquinoid H (1b), respectively.

Figure 2.

HMBC and ¹H-¹H COSY correlations of 1 and 2.

Table 1.

¹H and ¹³C NMR Spectroscopic Data for Compounds 1 and 2.

No.	1 ^a		2 ^a
No.	δ_C	δ_H (J in Hz)	δ_C	δ_H (J in Hz)
1
2	77.9		77.9
3	130.9	5.55, d (9.7)	130.9	5.54, d (9.7)
4	123.1	6.22, d (9.7)	123.1	6.21, d (9.7)
4a	121.5		121.5
5	110.5	6.30, s	110.5	6.30, s
6	148.8		148.8
7	117.3	6.46, s	117.3	6.45, s
8	126.5		126.5
8a	144.9		145.0
1′	40.8	1.65, m	40.8	1.64, m
2′	22.7	2.09, m	22.7	2.09, m
3′	125.2	5.10, brt (6.6)	125.2	5.10, brt (7.0)
4′	134.4		134.4
5′	39.2	2.04, t (7.2)	39.1	2.03, m
6′	28.2	2.55, m	28.3	2.58, m
7′	145.4	5.95, t (7.0)	146.3	6.01, t (7.2)
8′	130.9		130.5
9′	35.0	2.21, t (6.7)	30.6	2.26, m
10′	24.2	1.45, m	34.7	1.64, m
11′	43.3	1.43, m	75.4	4.03, t (6.3)
12′	71.4		147.1
13′	29.4	1.18, s	17.8	1.69, s
14′	29.4	1.18, s	111.5	4.92, s
				4.82, s
15′	172.1		172.6
16′	16.0	1.54, s	15.9	1.53, s
17′	26.1	1.34, s	26.0	1.33, s
18′	15.7	2.11, s	15.7	2.10, s

^aNMR spectra were recorded at 500 MHz for ¹H and 125 MHz for ¹³C in CDCl₃.

Compound (2) was obtained as a colorless gum. Its molecular formula was determined to be C₂₇H₃₆O₅ based on the negative ion peak in the HR-ESI mass spectrum at m/z 439.2486 [M-H]⁻ (calculated for C₂₇H₃₅O₅, 439.2484). The nuclear Magnetic resonance(NMR) spectra of 2 (Table 1) showed meroterpenoid signals, except for the presence of a hydroxyl group (δ_H 4.03, δ_C 75.4) and exo-methylene groups (δ_H 4.92/4.82, δ_C 147.1, and 111.5) in 2. The HMBC correlations from H-9′ to C-10′, C-11′, and C-15′, from H-11′ to C-9′, C-10′, C-12′, C-13′, and C-14′, and from H-14′ to C-11′, C-12′, and C-13′, and from C-13′ to C-11′, C-12′, and C-14′ and ¹H-¹H COSY correlations with H-9′/H-10′/H-11′ confirmed the existence of 2 main different groups (the hydroxyl group and the exo-methylene group) in 2 (Figure 2). Compound 2 was optically inactive and analyzed by chiral-HPLC and the 2 peaks were confirmed. Two peaks were obtained from 2 using semi-preparative HPLC with a chiral octadecylsilyl (ODS) column. From previously reported CD spectra, C-2 in 2a (Supplemental Figure S15) was determined to have the R-configuration.¹⁶ In addition, the S-configuration was assigned for the stereochemistry at C-2 in 2b. However, the modified Mosher's method did not react. Unfortunately, the stereochemistry at C-11′ in 2 still remains unknown. The new meroterpenoid derivatives of 2a and 2b were defined and named (-)-macrocarquinoid I (2a) and (+)-macrocarquinoid I (2b), respectively.

Compounds 3 and 4 were identified as (±)-sargachromenol and (±)-7-methyl sargachromenol, respectively.^14,20

The 4 isolated meroterpenoid derivatives (1a, 1b, 2a, 2b) were tested for their anti-oxidant activities using ABTS and DPPH radical scavenging assays. All compounds (1-4) displayed anti-oxidant activities with IC₅₀ values ranging from 9.9 to 90.0 μM (ABTS radicals) and from 22.4 to 226.2 μM (DPPH radicals), respectively (Table 2). Compounds 1a/1b and 2a/2b of the meroterpenoid derivatives showed moderate anti-oxidant activity against ABTS and DPPH radicals. Compound 3 exhibited the most potent anti-oxidative activities with IC₅₀ values of 9.9 and 22.4 μM (ABTS and DPPH radicals), compared with the positive controls (BHA and Trolox). In particular, the double bonds at C-11′ and C-12′ in compounds 3 and 4 exhibited the more significant anti-oxidative (ABTS and DPPH radicals) activities compared to the single bonds at C-11′ and C-12′ in compounds 1 and 2.

Table 2.

Anti-Oxidant Activities of Compounds 1 to 4.

Compounds	IC₅₀ (μM)^a
Compounds	ABTS	DPPH
1a	85.3 ± 0.9	222.5 ± 5.0
1b	90.0 ± 0.4	226.2 ± 8.8
2a	47.4 ± 0.8	124.7 ± 7.6
2b	39.9 ± 2.6	108.1 ± 1.5
3	9.9 ± 0.3	22.4 ± 2.1
4	39.7 ± 2.6	95.0 ± 2.3
BHA	28.5 ± 1.2	98.6 ± 1.2
Trolox	26.5 ± 0.3	66.9 ± 1.2

Abbreviations: ABTS, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid); DPPH, 1,1-diphenyl-2-picrylhydrazyl.

^aResults presented as the mean (n = 3)±SD.

Conclusion

This study reported the isolation, structure elucidation, and anti-oxidant activities of two novel meroterpenoid derivatives (1 and 2), along with two known compounds (3 and 4) from the brown alga S. macrocarpum. Compound 3 displayed the strongest anti-oxidative activities against the ABTS and DPPH radical scavenging assays compared to that of the positive controls, BHA (28.5 and 98.6 μM, respectively) and Trolox (26.5 and 66.9 μM, respectively), with IC₅₀ values of 9.9 and 22.4 μM, respectively. Furthermore, the double bonds at C-11′ and C-12′ potently increased the ABTS and DPPH radical scavenging activities.

(±)-Macrocarquinoid H (1): colorless gum; {1a : [α]_D²⁰ −17 (c 0.1, MeOH); 1b [a]_D²⁰ + 18 (c 0.1, MeOH)}; UV (MeOH) λ_max (log ε) 201 (4.3), 219 (4.2), 264 (3.5), 332 (3.3) nm; {1a : CD (MeOH, c = 1.0) (Δ ε) 250 (−3.5), 266 (−3.7), 273 (−3.8), 333 (−3.2) nm; 1b : CD (MeOH, c = 1.0) (Δ ε) 250 (3.5), 266 (3.7), 273 (3.8), 333 (3.2) nm} HRESIMS m/z 441.2629 [M-H]⁻ (calculated for C₂₇H₃₇O₅, 441.2641); for the ¹H and ¹³C NMR (500 and 125 MHz) spectroscopic data, see Table 1.

(±)-Macrocarquinoid I (2): colorless gum; {2a : [α]_D²⁰ −42 (c 0.1, MeOH); 2b [a]_D²⁰ + 15 (c 0.1, MeOH)}; UV (MeOH) λ_max (log ε) 201 (4.4), 264 (3.5), 307 (3.2), 333 (3.2) nm; {2a : CD (MeOH, c = 1.0) (Δ ε) 245(−3.1), 266 (−3.3), 274 (−3.3), 335 (−2.7) nm; 2b : CD (MeOH, c = 1.0) (Δ ε) 245(3.1), 266 (3.2), 274 (3.2), 335 (2.6); HRESIMS m/z 439.2486 [M-H]⁻ (calculated for C₂₇H₃₅O₅, 439.2484); for the ¹H and ¹³C NMR (500 and 125 MHz) spectroscopic data, see Table 1.

Experimental Section

General Experimental Procedures

The UV data were obtained using a UV-1650 UV spectrophotometer (Shimadzu, Kyoto, Japan). The NMR spectra were recorded on a Varian VNMRS 500 MHz FT-NMR spectrometer (Varian, Palo Alto, CA, USA). A J-1500 Circular Dichroism Spectrometer (Jasco, Tokyo, Japan) was used to measure the CD spectra. The specific rotation was measured on a JASCO P-2000 Digital Polarimeter (Jasco, Tokyo, Japan). The HPLC Arc system (Waters, Miliford, US) on an ODS column (Cosmosil, C₁₈-MS-II, Kyoto, Japan) and a chiral column (Daicel, Chiralcel OD-R, Osaka, Japan) were used for the isolation and purification. High resolution-electrospray ionization-mass spectroscopy were recorded on a SCIEX X500R Q-TOF LC-MS/MS spectrometer (SCIEX, Massachusetts, US). The preparative HPLC was performed using a Nextra Prep (Shimadzu, Kyoto, Japan). The Biotage Selekt MPLC system (Biotage, Sweden, Uppsala) was used for the fractionation and purification.

Plant Material

The brown alga, S. macrocarpum C. Agardh. was collected from Jeju Island (Jeju-si, Udo-myeon) in June, 2019. A voucher specimen (NP-0764) was deposited at the National Marine Biodiversity Institute of Korea (MABIK).

Extraction and Isolation

Freeze dried and powdered S. macrocarpum (1.0 kg) was extracted with methanol at 20 °C, 3 times (1 day). The extract was evaporated and partitioned using organic solvents (n-hexane, chloroform, ethyl acetate, and butanol). The chloroform-soluble layer (40 g) was subject to silica gel open column chromatography using gradient elution solvents (n-hexane:ethyl acetate:chloroform:methanol = 20:1:0:0 → 0:0:0:1) to provide 9 fractions (S1-S9). S3 (12 g) was fractionated on a MPLC using a solvent mixture of ACN:H₂O (3:7 to 10:0) to afford 4 sub-fractions (S3A-S3D). S3B (2.2 g) was continuously separated by preparative HPLC using ACN/H₂O (63:37) to obtain 3 (20.4 mg). S3C (273.0 mg) was further purified by preparative HPLC using ACN/H₂O (60:40) to yield 4 (10.2 mg). S5 (1.2 g) was fractionated by MPLC, followed by preparative HPLC using ACN/H₂O (55:45) to obtain 1 (3.5 mg) and 2 (2.5 mg), respectively. Compounds 1 and 2 were further purified by semi-preparative HPLC on an ODS chiral column using MeOH/H₂O (75:25) and MeOH/H₂O (70:30) (flow rate: 1.3 mL/min) to provide 1a (1.4 mg), 1b (1.1 mg), and 2a (0.7 mg) and 2b (0.9 mg), respectively.

Measurement of Anti-Oxidant Activities

ABTS and DPPH radical scavenging assays were conducted according to a previously reported protocol.^21,22 Briefly, ABTS was dissolved in H₂O (7 mM) and incubated with potassium persulfate (2.45 mM) in the dark. The absorbances of the samples and mixed solutions were measured at 734 nm. DPPH was dissolved in ethanol. The sample and the DPPH solution were mixed and reacted for 10 min at room temperature. The absorbance of the reaction mixture was recorded at 517 nm.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231191857 - Supplemental material for Two New Meroterpenoid Derivatives From Sargassum macrocarpum With Their Anti-Oxidant Activities

Supplemental material, sj-docx-1-npx-10.1177_1934578X231191857 for Two New Meroterpenoid Derivatives From Sargassum macrocarpum With Their Anti-Oxidant Activities by Ji-Yul Kim, Dae-Cheol Choi, Jeong Min Lee, Hyun-Soo Kim, Dae-Won Ki, Seok-Chun Ko, Mi-Jin Yim, Gun-Woo Oh, Kyung Woo Kim, Chul Hwan Kim, Kyung Lee, Kyunghwa Baek and Dae-Sung Lee in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

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

Ethical Approval

Ethical Approval in not applicable for this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the National Marine Biodiversity Institute of Korea (MABIK) Research Program (grant number 2023M00500).

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent in not applicable.

Supplemental Material

Supplemental material for this article is available online.

ORCID iDs

Ji-Yul Kim

Dae-Sung Lee

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