Isogloiosiphone B, a novel acetal, and hydrophobic compounds as β-glucuronidase inhibitors derived from the red alga Neodilsea yendoana

Abstract

A novel acetal named isogloiosiphone B was isolated from the red alga Neodilsea yendoana, along with three known hydrophobic compounds as β-glucuronidase inhibitors. The acetal was determined as a naturally occurring compound from the extraction experiments with several kinds of solvent. The acetal showed the highest inhibition against β-glucuronidase among the compounds examined.

Keywords:

• Acetal
• β-glucuronidase
• inhibitor
• Neodilsea yendoana
• alga

Beta-glucuronidase (EC 3.2.1.31) is not only found in anaerobic intestinal bacteria [1], but also in mammalian organs and body fluids [2–5]. The bacterial enzyme catalyzes hydrolysis of β-glucuronosyl-O-bond [6] to generate β-glucuronic acid and aglycons. The aglycons can be re-absorbed into body to delay eliminating toxic compounds from body as glucuronides [7]. Excess expression of β-glucuronidase in human is related with diverse pathological symptoms [8–13]. Hence, β-glucuronidase inhibitors are highly expected to sustain healthy condition. Neodilsea yendoana Tokida (Dumontiaceae, Gigartinales) is a red macroalga abundantly distributed along the coast of North Japan. Several studies have revealed that N. yendoana comprises amino acids [14], rhodoic acid [15], polyunsaturated fatty acid [16], and yendolipin [17]. The present paper aims to isolate novel potential β-glucuronidase inhibitors from N. yendoana.

Methanol extract of air-dried N. yendoana (1.73 kg), collected along the coast of Hakodate, Japan in 2016, was separated to ethyl acetate-soluble fraction (1.30 g) by organic solvent partitioning with guidance of results by slightly modified Escherichia coli β-glucuronidase inhibition assay using p-nitrophenyl β-D-glucuronide (Supplemental method 1) [18]. The fraction was chromatographed twice on silica gel and Sephadex LH-20 to obtain three inhibitory fractions. Final purification was performed using HPLC (ULTRON VX-ODS, 5 μm; 250 × 4.6 mm) eluted with MeCN-H₂O = 15:85 and MeCN-H₂O = 9:1 to obtain novel compound 1 (1.43 mg), and phytal (2, 2.02 mg), which was first isolated from this alga, cholesterol (3, 3.08 mg), and 22-dehydrocholesterol (4, 3.31 mg), respectively (Supplemental method 2). Compounds 2–4 were identified by comparison of literature data [19,20].

Novel compound 1 was obtained as colorless oil. Physicochemical data for 1 were as follows: UV λ_max (log ε) (MeOH) 206 (2.60); IR ν_max (CHCl₃): 1769, 1081 cm⁻¹; [α]_D²⁷ = 0 (c1.32, CHCl₃); FI-MS m/z 146.07 (M⁺); FI-HR-MS m/z 146.05861 (M⁺, calcd 146.05791 for C₆H₁₀O₄); ¹H NMR (500 MHz, acetone-d₆) and ¹³C NMR (125 MHz, acetone-d₆): see Table 1. IR spectrum of 1 showed C=O and C-O stretching peaks. ¹H NMR spectrum of 1 indicated the presence of one hydroxy, three methylene, and one methoxy proton signals, where the hydroxy proton was deduced from no cross-peak in HSQC spectrum. ¹³C NMR spectrum of 1 revealed one carbonyl, one quaternary, three methylene, and one methoxy carbon signals which were assisted with HSQC experiment. DQF COSY experiment of 1 revealed two couples of correlations (H-4/H-5 and H-6/hydroxy proton). The latter correlation was assigned as a hydroxymethyl group. All the proton and carbon signals of compound 1 were assigned according to HMBC cross-peaks of C-2 with Hs-5 and 6, and methoxy protons; ketonic carbon (C-3) with Hs-4, 5 and 6; C-4 with Hs-5; C-5 with H-4; and C-6 with the hydroxy proton. Consequently, compound 1 was elucidated as an isomer of known compound gloiosiphone B (5) [21], a structural analog of laurencione [22], isolated from the red alga Gloiosiphonia verticillaris (Gloiosiophoniaceae, Gigartinales). The ¹³C NMR signal at C-2 of 1 was shifted to downfield compared to the signal of gloiosiphone B while the signal at C-6 of 1 was shifted to up-field. This difference was contributed to substituting position of methoxy group. Thus, compound 1 was identified as 2-hydroxymethyl-2-methoxyoxolan-3-one, a novel acetal named isogloiosiphone B (Figure 1). Compound 1 possesses an asymmetric center in its structure. However, 1 might be a racemic mixture because specific rotation of 1 was optically inactive. With the consideration that compound 1 might be an artifact from its simple acetal structure, the alga was extracted with methanol, ethanol, and acetone. Compound 1 was isolated from all the extracts using different solvents (Figures S7–S9). Thus compound 1 was determined as a naturally occurring compound, not an artifact. Plausible precursor of isogloiosiphone B and gloiosiphone B would be 1,5-dihydroxypentane-2,3-dione. After cyclization of the precursor to corresponding hemiacetal, both compounds may be derived by methylation at different positions.

Table 1. ¹H (500 MHz) and ¹³C NMR (125 MHz) data of compound 1 and literature data [21] of gloiosiphone B (5).

Position	Compound 1^a		Gloiosiphone B (5)^b
	δC	δH (mult, J in Hz)	δC	δH (mult, J in Hz)
2	101.9		96.0
3	211.7		208.8
4	36.3	2.55 (2H, m)	34.2	2.88 (2H, m)
5	64.2	4.33 (1H, m)	62.5	4.32 (1H, m)
		4.27(1H, m)		4.33 (1H, m)
6	63.1	3.62 (2H, d, 5.7)	72.8	3.62 (1H, d)
				3.90 (1H, d)
−OCH3	50.6	3.12 (3H, s)	59.9	3.45 (3H, s)
−OH		3.87 (1H, t, 5.7)		3.40 (1H, s)

^a measured in acetone-d₆.

^b measured in chloroform-d.

Figure 1. Compounds 1–4 isolated from N. yendoana as β-glucuronidase inhibitors and gloiosiphone B (5).

The isolated compounds 1–4 were evaluated for inhibitory effects against E. coli β-glucuronidase (Table 2, Supplemental method 3). Isogloiosiphone B (1) showed the highest inhibitory activity in a competitive manner (Figure S19) among the isolated compounds and the positive control D-glucaro-1,4-lactone [23]. Compound 1 might easily access to active site of the enzyme. Additionally, this is the first report that phytal (2), cholesterol (3) and 22-dehydrocholesterol (4) exhibited β-glucuronidase inhibitory activity even though their inhibitory activities were weak.

Table 2. β-Glucuronidase inhibitory activities of compounds 1–4.

Compound	IC50 (μM)	Ki (μM)	Inhibition mode
1	57.0 ± 0.56	8.3 ± 1.3	Competitive
2	103.7 ± 1.02	210.7 ± 13.7	Competitive
3	> 250	421.2 ± 7.1	Competitive
4	89.5 ± 0.44	32.7 ± 5.5	Non-competitive
D-Glucaro-1,4-lactone	77.9 ± 1.40	2.5 ± 2.1	Competitive

In summary, we isolated compounds 1–4 as β-glucuronidase inhibitors from the red alga N. yendoana. Compound 1 was determined as a novel naturally acetal and showed strong β-glucuronidase inhibitory activity. In addition, this is the first report on isolation of phytal from the alga and elucidation of β-glucuronidase inhibitory activity by the hydrophobic compounds.

Author contribution

D. Z. performed all experiments and prepared draft. H. K. made experimental plans and organized all of the studies. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by authors.

Supplemental data

The supplemental material for this paper is available at https://doi.org/10.1080/09168451.2017.1403885.

Acknowledgment

We thank to Dr. Eri Fukushi and Mr. Yusuke Takata, GC-MS & NMR Laboratory, Faculty of Agriculture, Hokkaido University, for measurements of MS and NMR spectra.