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Abstract

Objectives

Isolation, structure determination, and antimicrobial activities evaluation of compounds from the methanol extract of the marine microalga Thraustochytrium pachydermum TSL10.

Methods

Using chromatographic methods to isolate compounds from the methanol extract of the T pachydermum. The chemical structures were elucidated by analyses of HR-ESI-MS and NMR spectral data. The antimicrobial activities of compounds were evaluated using the dilution turbidimetric broth method as the standard protocol published by the Clinical and Laboratory Standard Institute.

Results

One new sterol, (24R)-ergosta-7,22-diene-3β,5α,9α-triol (1), and 16 known compounds, ergosta-7,22-diene-3β,5α-diol (2), cerevisterol (3), (22E,24S)-5α,6α-epoxy-ergosta-8(9),22-diene-3β,7α-diol (4), ergosterol (5), 5α,8α-epidioxyergosta-6,22-diene-3β-ol (6), linoleic acid (7), 4-((9Z,12Z)-octadeca-9,12-dienoyloxy)butanoic acid (8), 1-linoleoyl-sn-glycero-3-phosphocholine (9), cibotiglycerol (10), gingerglycolipid B (11), oleic acid (12), 4-((9Z)-octadec-9-enoyloxy)butanoic acid (13), (2S)-1-O-(9Z)-octadec-9-enoyl)-3-O-(6-sulfo-D-quinovopyranosyl)glycerol (14), 6′-O-linoleylsucrose (15), gingerglycolipid C (16), and (S)-3-stearoyl-sn-glycerol (17) were identified from the marine microalga Thraustochytrium pachydermum TSL10. Sterols 1–6 were found to exhibit significant antimicrobial activities on Gram-( + ) bacteria (Enterococcus faecalis ATCC29212, Staphylococcus aureus ATCC25923, and Bacillus cereus ATCC14579) and the yeast (Candida albicans ATCC10231) with MIC values ranging from 32 to 256 µg/mL.

Conclusions

The results suggested sterols 1–6 could be potential antimicrobial agents against Gram-( + ) bacteria.

Graphical Abstract

This is a visual representation of the abstract.

Keywords

Thraustochytriaceae (24R)-ergosta-7 marine microalgae antimicrobial

Introduction

Microalgae have been known as one of the largest and most diverse photosynthetic organisms. They are potential sources of bioactive compounds such as fatty acids, carotenoids, chlorophylls, phycobiliproteins, and vitamins. The compounds from microalgae have exhibited antibacterial, antifungal, antiviral, antitumor, and anti-inflammatory effects.¹

Thraustochytrium species (Thraustochytriaceae family) are suitable for fermentation technology and produce fatty acids and other value-added biocomponents.^2,3 The marine microalga Thraustochytrium pachydermum was isolated from Truong Sa Island, Truong Sa Archipelago, Vietnam in 2021 and is available in GenBank (accession number OL721615).⁴ The chemical constituents and biological activities of T pachydermum have not been studied so far. Herein, we report one new sterol and 17 known metabolites from the marine microalgae T pachydermum TSL10 and their antibacterial activities.

Results and Discussion

Compound 1 was obtained as a white powder. Its molecular formula, C₂₈H₄₆O₃, was defined by a pseudo-molecular ion peak at m/z 429.3376 [M + H]⁺ (calcd for [C₂₈H₄₇O₃]⁺, 429.3374) on the HR-ESI-MS. The ¹H-NMR spectrum of 1 revealed proton signals of 6 methyl groups [2 singlets at δ_H 0.62 (s, H-18) and 1.01 (s, H-19)] and 4 doublets at δ_H 1.03 (H-21), 0.82 (H-26), 0.84 (H-27), and 0.92 (H-28), (each, 3H, d, J = 6.6 Hz)], 3 olefinic protons at δ_H 5.65 (1H, br d, J = 1.8 Hz, H-7), 5.17 (1H, dd, J = 15.6, 8.4 Hz, H-22), and 5.24 (1H, dd, J = 15.6, 7.8 Hz, H-23), and one carbinol proton at δ_H 4.06 (1H, m, H-3). The ¹³C-NMR and HSQC spectra of 1 showed signals of 30 carbons, including 6 methyl carbons at δ_C 12.3 (C-18), 20.5 (C-19), 21.1 (C-21), 19.7 (C-26), 20.0 (C-27), and 17.6 (C-28), 7 methylenes at δ_C 25.5 (C-1), 30.1 (C-2), 37.1 (C-4), 28.8 (C-6), 29.1 (C-11), 34.9 (C-12), 22.4 (C-15), and 27.9 (C-16), 9 methines at δ_C 67.2 (C-3), 119.9 (C-7), 51.8 (C-14), 56.0 (C-17), 40.3 (C-20), 135.1 (C-22), 132.5 (C-23), 42.8 (C-24), and 33.1 (C-25), and 5 non-protonated carbons at δ_C 74.7 (C-5), 79.7 (C-9), 41.8 (C-10), and 45.3 (C-13), and 1 carbon (C-8, did not show in the ¹³C-NMR spectrum). Analysis of the ¹H- and ¹³C-NMR spectra of 1 (Table 1) indicated its structure was similar to that of ergosta-7,22-diene-3β,5α-diol (2),⁵ except for an addition of one hydroxyl group at C-9. The HMBC correlations between H-19 (δ_H 1.01) and C-1 (δ_C 25.5)/C-5 (δ_C 74.7)/C-9 (δ_C 79.7)/C-10 (δ_C 41.8), H-4 (δ_H 1.76 and 2.09) and C-2 (δ_C 30.1)/C-3 (δ_C 67.2)/C-5 (δ_C 74.7)/C-6 (δ_C 28.8)/C-10 (δ_C 41.8), H-7 (δ_H 5.73) and C-5 (δ_C 74.7)/C-9 (δ_C 79.7)/C-14 (δ_C 51.8) confirmed positions of the hydroxyl groups at C-3, C-5, and C-9, and the double bond at C-7/C-8 (Figure 1). The HMBC correlations between H-21 (δ_H 1.03) and C-17 (δ_C 56.0)/C-20 (δ_C 40.3)/C-22 (δ_C 135.1) and between H-28 (δ_H 0.92) and C-23 (δ_C 132.5)/C-24 (δ_C 42.8)/C-25 (δ_C 33.1) and COSY cross-peaks of H-21 (δ_H 1.03)/H-20 (δ_H 2.03)/H-22 (δ_H 5.17)/H-23 (δ_H 5.24)/H-24 (δ_H 1.86) indicated the double bond at C-22/C-23. The large coupling constant of H-22 and H-23 (J = 15.6 Hz) indicated the E configuration of the double bond at C-22/C-23. The NOESY correlations between H-19 (δ_H 1.01) and H _β -1 (δ_H 1.51)/H _β -2 (δ_H 1.48)/H _β -4 (δ_H 1.76), between H _β -4 (δ_H 1.76) and H _β -6 (δ_H 1.91) suggested the orientation of the hydroxyl group at C-5 to be α. The NOESY correlation between H-18 (δ_H 0.62) and H-19 (δ_H 1.01) proved the orientation of the hydroxyl group at C-9 to be α. The configuration of C-24 in 1 was deduced to be R by comparing the chemical shifts of C-24 (δ_C 42.8) and C-28 (δ_C 17.6) of 1 with those of 5α,8α-epidioxy-24S-methylcholesta-6,22E-diene-3β-ol (δ_C 43.0 and 18.0)⁶ and 5α,8α-epidioxy-24R-methylcholesta-6,22E-dien-3β-ol (δ_C 42.8 and 17.6).⁷ Thus, new compound 1 was elucidated to be (24R)-ergosta-7,22-diene-3β,5α,9α-triol.

Figure 1.

The key HMBC, COSY, and NOESY correlations of compound 1.

Table 1.

¹H- and ¹³C-NMR Spectral Data for 1 in CDCl₃.

C	δ _C	δ_H (mult., J in Hz)
1	25.5	1.51 (m, β)/2.33 (m, α)
2	30.1	1.48 (m, β)/1.85 (m, α)
3	67.2	4.06 (m)
4	37.1	1.76* (β) 2.09 (br d, 13.8, α)
5	74.7	-
6	28.8	1.73* (α)/1.91* (β)
7	119.9	5.65 (br d, 1.8)
8	ND	-
9	79.7	-
10	41.8	-
11	29.1	1.31 (m)
12	34.9	1.73 (m, α) 1.88 (m, β)
13	45.3	-
14	51.8	2.75 (m)
15	22.4	1.48 (m, β) 1.59 (m, α)
16	27.9	1.33 (m, β) 1.80 (m, α)
17	56.0	1.44 (m)
18	12.3	0.62 (s)
19	20.5	1.01 (s)
20	40.3	2.03 (m)
21	21.1	1.03 (d, 6.6)
22	135.1	5.17 (dd, 15.6, 8.4)
23	132.5	5.24 (dd, 15.6, 7.8)
24	42.8	1.86 (m)
25	33.1	1.48 (m)
26	19.7	0.82 (d, 6.6)
27	20.0	0.84 (d, 6.6)
28	17.6	0.92 (d, 6.6)

Abbreviation: ND, not determined.

*Overlapped signals.

The known compounds were determined to be ergosta-7,22-diene-3β,5α-diol (2),⁵ cerevisterol (3),⁸ (22E,24S)-5α,6α-epoxy-ergosta-8(9),22-diene-3β,7α-diol (4), ergosterol (5),⁹ 5α,8α-epidioxyergosta-6,22-diene-3β-ol (6),⁷ linoleic acid (7),¹⁰ 4-((9Z,12Z)-octadeca-9,12-dienoyloxy)butanoic acid (8),¹¹ 1-linoleoyl-sn-glycero-3-phosphocholine (9),¹² cibotiglycerol (10),¹³ gingerglycolipid B (11),¹⁴ oleic acid (12),¹⁵ 4-((9Z)-octadec-9-enoyloxy)butanoic acid (13),¹⁶ (2S)-1-O-(9Z)-octadec-9-enoyl)-3-O-(6-sulfo-D-quinovopyranosyl)glycerol (14),¹⁷ 6′-O-linoleylsucrose (15),¹⁸ gingerglycolipid C (16),¹⁴ and (S)-3-stearoyl-sn-glycerol (17)¹⁹ by comparing their NMR data with those reported in the literature (Figure 2).

Figure 2.

Chemical structures of compounds 1-17.

All isolated compounds were evaluated for their antimicrobial activities against microorganisms, Gram-( + ) bacteria (Enterococcus faecalis ATCC29212, Staphylococcus aureus ATCC25923, and Bacillus cereus ATCC14579), Gram-(-) bacteria (Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, and Salmonella enterica ATCC13076), and yeast (Candida albicans ATCC10231) using dilution turbidimetric broth method as the standard protocols published by the Clinical and Laboratory Standard Institute (Table 2). Streptomycin and cycloheximide were used as positive controls. Streptomycin inhibited Gram-( + ) bacteria (E faecalis, S aureus, and B cereus), Gram-(-) bacteria (E coli, P aeruginosa, and S enterica) with MIC values ranging from 32 to 256 µg/mL, respectively. Cycloheximide inhibited the yeast C albicans with MIC value of 32 µg/mL. All isolated sterols 1–6 showed significant antimicrobial activities on Gram-( + ) bacteria (E faecalis, S aureus, and B cereus) and yeast C albicans with MIC values ranging from 32 to 256 µg/mL (Table 2). The remaining compounds (fatty acid derivatives) exhibited weak or no antimicrobial activities. However, most compounds did not inhibit the growth of Gram-(-) bacteria (E coli, P aeruginosa, and S enterica) (MIC values > 256 µg/mL). The results suggested sterols 1-6 could be potential antimicrobial agents against Gram-( + ) bacteria.

Table 2.

Antimicrobial Activities of Compounds 1-17.

Compounds	MIC (μg/mL)
	Gram ( + )			Gram (-)			Yeast
	Enterococcus faecalis	Staphylococcus aureus	Bacillus cereus	Escherichia coli	Pseudomonas aeruginosa	Salmonella enterica	Candida albicans
1	256	64	256	-	-	-	256
2	256	128	256	-	-	-	256
3	128	32	128	-	-	-	256
4	256	64	128	-	-	-	64
5	128	128	256	-	-	-	256
6	128	64	64	-	-	-	64
7	-	-	-	-	-	-	-
8	-	-	-	-	-	-	-
9	128	-	256	-	-	-	-
10	256	-	-	-	-	-	-
11	-	-	-	-	-	-	-
12	-	-	256	-	-	-	-
13	-	-	-	-	-	-	-
14	-	-	-	-	-	-	-
15	-	-	-	-	-	-	-
16	-	-	-	-	-	-	-
17	128	-	256	-	-	128	256
Streptomycin	256	256	128	32	256	128
Cyclohexamide							32

(-) compound did not exhibit antimicrobial activities (MIC >256 µg/mL).

Material and Methods

General See Supplemental Material.

Microalgae Samples

See Supplemental Material.

Biomass Cultivation

An aeration rate when growing biomass was always maintained at 0.5 L air/L/min after the air was filtered through a 0.2 mm filter (Advantec, JP020AN). Biomass cultivation has changed on a large scale of 30 L fermentor: while the dissolved oxygen was maintained above 10% with the manual stirring speed from 300 to a maximum of 400 rpm, at 32 °C÷37 °C. Glucose concentrations of 9%, and 1% industrial yeast extract were used to grow the strain T pachydermum TSL10. After 120 h of cultivation, biomass was filtered and dried.

Extraction and Purification of Compounds

Thraustochytrium pachydermum (2.2 kg) was ultrasonically extracted with methanol (MeOH) at 40 °C 3 times (each 8.8 L, 3 h). The MeOH layer was evaporated in vacuo to give a dark solid residue (420 g). This crude extract was suspended with water (2.0 L) and partitioned with CH₂Cl₂ to obtain CH₂Cl₂ soluble fraction (TPD, 102 g) and water layer. The TPD fraction was roughly separated on a silica gel column chromatography (CC), eluting with CH₂Cl₂/MeOH (100:0, 40:1, 20:1, 10:1, 5:1, vol/vol) to give 5 fractions, TP1A (21.8 g), TP1B (15.7 g), TP1C (8.4 g), TP1D (25.9 g), and TP1E (20.3 g). The TP1B fraction (15.7 g) was chromatographed on a silica gel CC using CH₂Cl₂/MeOH/water (4/1/0.05, vol/vol/vol) as eluent to give 5 fractions, TP2A (1.3 g), TP2B (6.7 g), TP2C (0.6 g), TP2D (2.8 g), and TP2E (2.5 g).

TP2A (1.3 g) fraction was chromatographed on a silica gel CC and an eluent of CH₂Cl₂/acetone (7/1, vol/vol). to yield compounds 1 (3.8 mg), 3 (12.7 mg), and 4 (4.1 mg). The TP2C fraction (0.6 g) was also loaded on a silica gel column and eluted with CH₂Cl₂/acetone (10/1, vol/vol) to obtain compounds 2 (3.3 mg) and 17 (4.0 mg). The TP2E fraction (2.5 g) was first fractionated on an RP-18 CC eluting with acetone/water (2/1, vol/vol) to give 2 fractions, TP2E1 (300 mg) and TP2E2 (500 mg). TP2E1 (300 mg) fraction was chromatographed on an HPLC (solvent eluent of 40% acetonitrile in water) to yield compound 10 (3.6 mg, t_R 34.7 min). TP2E2 fraction (0.5 g) was chromatographed on an HPLC (solvent eluent of 90% MeOH in water) to yield compound 15 (4.8 mg, t_R 43.0 min).

The TP1C fraction (8.4g) was repeatedly chromatographed on a silica gel CC, eluting with n-hexane/CH₂Cl₂/acetone (3/1/0.1, vol/vol/vol) to obtain 3 fractions TP3A (2.6 g), TP3B (1.5 g), and TP3C (1.8 g). Compound 5 (8.2 mg) was purified from the TP3A fraction using a silica gel CC and solvent eluent of n-hexane/acetone (5/1, vol/vol). The TP3B fraction was chromatographed on an RP-18 column eluting with acetone/water (5/1, vol/vol) to yield compound 6 (50.0 mg). Compounds 8 (4.6 mg) and 13 (36.0 mg) were isolated from the TP3C fraction on a silica gel CC and solvent eluent of n-hexane/ethyl acetate (4/1, vol/vol). The TP1E fraction (20.3 g) was first fractionated on a silica gel CC eluting with CH₂Cl₂/MeOH/water (3/1/0.1, vol/vol/vol) to give 5 fractions, TP4A (0.5 g), TP4B (1.8 g), TP4C (2.0 g), TP4D (2.1 g), and TP4E (1.9 g). TP4A fraction (0.5 g) was chromatographed on an HPLC with a solvent eluent of 90% MeOH in water to yield compound 9 (4.6 mg, t_R 50.3 min). Compound 14 (6.1 mg, t_R 36.1 min) was isolated from the TP4B fraction on an HPLC (solvent eluent of 40% acetonitrile in water). The TP4D (2.1 g) fraction was chromatographed on an HPLC (solvent eluent of 90% acetonitrile in water) to yield compounds 11 (7.0 mg, t_R 31.1 min) and 16 (33.9 mg, t_R 43.5 min). Finally, compounds 7 (9.9 mg) and 12 (14.5 mg) were isolated from the TP4E fraction (1.9 g) on an RP-18 column eluting with MeOH/water (9/1, vol/vol).

Ergosta-7,22-diene-3β,5α,9α-triol (1)

White amorphous powder; $[α]_{D}^{25}$ : + 35.7 (c 0.1, CHCl₃); HR-ESI-MS: m/z 431.3525 [M + H]⁺ (calcd. for [C₂₈H₄₇O₃]⁺, 431.3520), IR (KBr) ν_max 3427, 2957, 2919, 1677, 1454, 1160, 1007 cm⁻¹; ¹H- and ¹³C-NMR (CDCl₃): see Table 1.

Antimicrobial Assays

See Supplemental Material.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231157145 - Supplemental material for Chemical Constituents From the Marine Microalgae Thraustochytrium pachydermum

Supplemental material, sj-docx-1-npx-10.1177_1934578X231157145 for Chemical Constituents From the Marine Microalgae Thraustochytrium pachydermum by Nguyen Thi Thu Thuy, Hoang Thi Minh Hien, Nguyen Cam Ha and Le Thi Thom, Dang Diem Hong, Nguyen Van Thinh, Nguyen Trong Dan, Nguyen Dang Hoi, Vu Thi Loan, Hoang Duc Quang, Dan Thi Thuy Hang, Phan Van Kiem, Nguyen Tien Dat, Nguyen Xuan Nhiem in Natural Product Communications

Footnotes

Acknowledgments

This work was supported by the Project KCB-TS-07.

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 KCB-TS-07.

ORCID iDs

Nguyen Xuan Nhiem

Phan Van Kiem

Supplemental Material

Supplemental material for this article is available online.

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