
==== Front
Adv Biomed Res
Adv Biomed Res
ABR
Adv Biomed Res
Advanced Biomedical Research
2277-9175
Wolters Kluwer - Medknow India

ABR-13-22
10.4103/abr.abr_103_23
Original Article
Cytotoxic Sulfoquinovosyl Glycerols from the Seaweed Sargassum Angustifolium from Persian Gulf
Sajjadi Seyed Ebrahim 1
Ghobeishavi Sahar 1
Yegdaneh Afsaneh 1
1 Department of Pharmacognosy and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
Address for correspondence: Prof. Afsaneh Yegdaneh, Department of Pharmacognosy and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran. E-mail: yekdaneh@pharm.mui.ac.ir
2024
28 3 2024
13 2219 3 2023
11 7 2023
15 7 2023
Copyright: © 2024 Advanced Biomedical Research
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
Backgrounds:

Seaweeds are an important source of marine organisms that produce a lot of bioactive compounds.

Materials and Methods:

In this research, the seaweed Sargassum angustifolium was collected from Bushehr province of Persian Gulf of Iran. The seaweed was extracted by methanol:ethyl acetate (1:1) using maceration method. The compounds were isolated with different column chromatography and HPLC(High Performance Liquid Chromatography) by silica gel and hexane:ethyl acetate as mobile phase.

Results:

The isolated compounds were elucidated structurally by various 1 and 2 D-NMR and MS spectra. Besides the cytotoxicity test was done against HeLa using standard MTT assay and normal cells.

Conclusion:

It afforded four known sulfoquinovosyl diacylglycerides and fucosterol. Compounds 1-5 showed cytotoxic effects against HeLa and HUVEC cell lines, with IC50 values of 12.2 ± 2.3, 25.8 ± 3.7, 14.9 ± 2.6, 9.8 ± 1.2 μM, and 5.6 ± 1.2, respectively.

Cytotoxicity test
monogalactosyldiacylglycerol
Persian gulf
seaweed
sulfoquinovosyl diglyceride
==== Body
pmcINTRODUCTION

Marine environment is a unique source of biologically active secondary metabolites. Isolated compounds from marine resources are different from terrestrial metabolites because of special physical and chemical conditions in the marine habitat.[1] So marine organisms such as algae, sponges, fungi, corals, and ascidians contain potentially active metabolites with characteristic chemical structures. There are more than 2,400 compounds in the field of marine natural plants isolated only from seaweeds of different regions of oceans.[2]

A number of research showed that marine seaweeds are potent resources for drug development. They contain important kinds of secondary metabolites such as steroids, terpenoids, phlorotannins, amino acids, phenolic compounds, and halogenated structures particularly ketones, glycolipids, and cyclic polysulphides.[345] New structures and unique mechanisms of action of marine natural products have led to the identufucation and structure elucidation of substances with, antimicrobial, antiviral, antioxidant, antitumor, antidepression, anti-inflammatory, and anti-Alzheimer activities.[6789]

Glycoglycerolipids are one of the important groups of lipid compounds present in the marine seaweeds. These compounds are famous because of their especial activities, such as antitumor and anti-inflammatory activities along with improving the intestinal condition.[1011] Marine algae synthesize three important types of glycolipids: monogalactosyldiacylglycerides, digalactosyldiacylglycerides, and sulfoquinovosyldiacylglycerides (SQDG). A number of seaweeds can convert simple polyunsaturated fatty acids into complex oxylipins[12] and these derivatives are extremely useful to keep homeostasis in mammalian systems. Besides glycolipids are abnormally produced in diseases such as psoriasis, asthma, arteriosclerosis, ulcers, and cancer.[13] Therefore, it is an interesting area to elucidate the structures of glycoglycerolipid molecules from seaweeds resources.

Sargassum is a genus of more than 250 species in Sargassaceae family that is widespread in different tropical and temperate oceans geographically. Until now, different glycerolipids have been isolated from sargassum species worldwide. Recent data have exhibited that more than 150 species of marine algae are present in the coastal area of Persian Gulf and Oman Sea of Iran.[14] There are only limited studies on the phytochemistry of the marine seaweeds of Iran, especially sargassum species. So, in this study, we have selected the brown seaweed Sargassum angustifolium to isolate its glycolipids.

MATERIALS AND METHODS

General

TLC: GF254 silica gel plates (Merck, Germany, 20 × 20 cm); detection by spraying with 10% cerium sulphate and heating. Column chromatography (CC): silica gel 63-200 μm. HPLC: Agilent 1100 Series with a silica column (YMC Co., Ltd., Kyoto, Japan) and UV-Vis detector. NMR: Bruker AV-400 (1H) and AV-100 (13C), EI-MS spectra: Varian MAT 112 or MAT 312 spectrometers. The GC-MS was Agilent Technologies 6890N GC with a mass-selective detector 5973 Network MSD and a silica-capillary GC column HP-5MS (30 m × 0.25 mm; i.d. 0.25 μm film, Agilent Technologies, Inc.).

Authentication of plant material

The algae was collected from Bushehr Province in 2012 and identified by Agricultural and Natural Resources Research Center of Bushehr (voucher specimens coded as 2662).

Extraction and fractionation of lipids

The powder of dried S. angustifolium was extracted with EtOAc/methanol 1:1 (v/v) solvent at room temperature. The extracts were filtered and dried and partitioned to hexane, dichloromethane, butanol, and water through Kupchan method. The Hexane partition was fractionated by normal phase MPLC with a gradient solvent system from pure hexane to 100% EtOAc. The eluates were monitored by 1HNMR and TLC and divided into 14 fractions (Frs. 1-14). Fraction F14 was purified on a silica gel columnand chloroform/methanol solvent with increasing amounts of methanol (95:5, 90:10, 80:20, 50:50 v/v) and 100% methanol. The eluates were combined into 12 final fractions. Fractions F14i, F14j, F14k, and F14l were further isolated by HPLC separation yielded the pure compounds 1, 2, 3, 4, and 5.

Alkaline hydrolysis

A 12% solution of each compounds (2-5) was treated with NaOMe (0.5M in MeOH) and stirred for 5 h at room temperature. After this time, the mixture was further neutralized with Dowex 50 W × 4 and the resin filtered. The filtrate was dissolved in hexane, concentrated and analyzed by GC-MS. The column oven temperature was 80°C for 1 min and then increased up to 310°C with a rate of 15°C/min (flow rate 0.8 mL/min).

In vitro cytotoxicity assay

The HeLa (epitheloid cervix carcinoma) cell line and HUVEC (and human umbilical vein endothelial cells) were obtained from the Pasteur Institute of Iran. Cells were incubated in an incubator with 5% CO2 at 37°C. The cells were fed with Roswell Park Memorial Institute medium and Dulbecco’s Modified Eagle’s medium, supplemented with FBS (10%) and penicillin-streptomycin (100 IU/mL and 100 μg/mL).

Compounds 2-5 were tested about cytotoxic effects using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] against HeLa as well as HUVEC cells. Briefly, a cell suspension of 2 × 105 cells/mL in 96-well plates were incubated overnight. The dried samples were dissolved in dimethyl sulfoxide (DMSO) (less than 1% final concentration of DMSO in the palate). 20 μL of various concentrations of compounds or partitions were added and incubated at 37°C in a humidified atmosphere for 72 h. After that cells were incubated with 20 μL of MTT solution (5 mg/mL) at 37°C for 3 h. The medium was removed and 150 μL of DMSO was added to dissolve MTT-formazan crystals. Finally, the absorbance at 570 nm was measured by a plate reader. Following equation was used to calculate the cell survival:[151617]

% Cell survival = (Absorbance in treated wells - Absorbance in blank well)/(Absorbance in negative control or untreated well – Absorbance in blank well).

RESULTS

Compound 1

White powder, MW (g/mol): 412 (M + H); 1H NMR (400 MHz, CDCl3): 5.38 (1H, br. d, J = 5.3 Hz, H-6), 5.2 (1H, q, J = 6.7 Hz, H-28), 3.58 (1H, m, H-3), 1.52 (3H, br s, H-21), 1.05 (3H, s, H-19), 1.03 (3H, br s, H-21),1.02 (3H, d, J = 1.2 Hz, H-27), 0.99 (3H, d, J = 1.2 Hz, H-26), 0.75 (3H, s, H-18). 13C-NMR (100 MHz, CDCl3): 146.5 (C-24), 140.6 (C-5),121.3 (C-6), 115.8 (C-28), 71.4 (C-3), 56.8 (C-14), 55.2 (C-17), 50.6 (C-9), 42.7 (C-13), 42.1 (C-4), 39.5 (C-12), 37.5(C-1), 36.4 (C-10), 36.3 (C-20), 35.5 (C-22), 34.8 (C-25),31.6 (C-7,8), 31.7 (C-2), 28.1 (C-16), 25.5 (C-23), 24.2 (C-15), 22.1 (C-26), 22.0 (C-27), 21.3 (C-11), 19.5 (C-19), 18.7(C-21), 131.2 (C-29), 11.8 (C-18).

Compound 2

White powder, MW (g/mol): 792 (M + H); 1H NMR (400 MHz, DMSO): 4.32 (1H, dd, J = 2.4,12 Hz, H-1), 4.12 (1H, dd, J = 7.6,12 Hz, H-1), 5.12 (1H, m, H-2), 3.86 (1H, dd, J = 6,10.8 Hz, H-3), 3.41 (1H, dd, J = 6,10.8 Hz, H-3), 4.56 (1H, d, J = 3.6 Hz, H-1ʹʹʹ), 3.18 (1H, dd, J = 6,9.6 Hz, H-2ʹʹʹ), 3.36 (1H, t, J = 9.2 Hz, H-3ʹʹʹ), 2.92 (1H, t, J = 9.2 Hz, H-4ʹʹʹ), 3.75 (1H, ddd, J = 4.8,5.6,10.4 Hz, H-5ʹʹʹ), 2.58 (1H, dd, J = 6,14 Hz, H-6ʹʹʹ), 2.57 (1H, dd, J = 6.2,13.9 Hz, H-6ʹʹʹ), 2.25 (4H, m, H-2ʹ,2ʹʹ), 1.49 (4H, m, H-3ʹ,3ʹʹ), 1.2-1.3 (m, H-4ʹ-7ʹ, 12ʹ-15ʹ, 4ʹʹ-15ʹʹ), 5.31 (2H, t, J = 4.8 Hz, H-9ʹ,10ʹ), 0.84 (6H, t, J = 6.8 Hz, H-16ʹ,16ʹʹ), 1.97 (4H, m, H-8ʹ,11ʹ).

13C-NMR (100 MHz, DMSO): 172.3,173.5 (C-1ʹ,1ʹʹ), 129.5 (C-9ʹ,10ʹ), 98.2 (C-1ʹʹʹ), 74.2 (C-5ʹʹʹ), 72.8 (C-4ʹʹʹ), 71.5 (C-3ʹʹʹ), 69.6 (C-2ʹʹʹ), 68.5 (C-2), 64.5 (C-3), 62.5 (C-1), 54.6 (C-6ʹʹʹ), 33.4,33.5 (C-14ʹ,14ʹʹ), 31.2 (C-2ʹ,2ʹʹ), 28.9-29 (C-4ʹ-7ʹ,12ʹ,13ʹ,4ʹʹ-13ʹʹ), 28.4 (C-8ʹ,11ʹ), 24.4 (C-3ʹ,3ʹʹ), 22.0 (C-15ʹ,15ʹʹ), 13.9 (C-16ʹ,16ʹʹ).

Compound 3

White powder, MW (g/mol): 826 (M + H); 1H NMR (400 MHz, MeOH): 4.32 (1H, dd, J = 2.8, 12 Hz, H-1), 4.12 (1H, dd, J = 2.8, 12 Hz, H-1), 5.17 (1H, m, H-2), 3.86 (1H, dd, J = 5.6, 10.8 Hz, H-3), 3.63 (1H, dd, J = 5.2, 10.8 Hz, H-3), 4.12 (1H, d, J = 7.6 Hz, H-1ʹʹʹ), 3.62 (1H, dd, J = 7.2, 9.6 Hz, H-2ʹʹʹ), 3.36 (1H, t, J = 9.2 Hz, H-3ʹʹʹ), 2.92 (1H, t, J = 9.2 Hz, H-4ʹʹʹ), 3.75 (1H, ddd, J = 4.8, 5.6, 10.4 Hz, H-5ʹʹʹ), 2.58 (1H, dd, J = 6, 14 Hz, H-6ʹʹʹ), 2.57 (1H, dd, J = 6.2,13.9 Hz, H-6ʹʹʹ), 2.21 (4H, m, H-2ʹ,2ʹʹ), 1.50 (4H, m, H-3ʹ,3ʹʹ), 1.2-1.3 (m, H-1ʹ,1ʹʹ,4ʹ-7ʹ,12ʹ-17ʹ, 4ʹʹ-15ʹʹ), 5.23 (2H, t, J = 4.8 Hz, H-9ʹ,10ʹ), 0.84 (6H, t, J = 6.8 Hz, H-18ʹ,16ʹʹ), 1.97 (4H, m, H-8ʹ,11ʹ). 13C NMR (100 MHz, MeOH): 174.7,175 (C-1ʹ,1ʹʹ), 130.9,130.8 (C-9ʹ,10ʹ), 105.3 (C-1ʹʹʹ), 76.8 (C-5ʹʹʹ), 74.8 (C-4ʹʹʹ), 72.4 (C-3ʹʹʹ), 71.8 (C-2ʹʹʹ), 70.2 (C-2), 68.7 (C-3), 64.0 (C-1), 62.4 (C-6ʹʹʹ), 35,35.1 (C-16ʹ,14ʹʹ), 33.1 (C-2ʹ,2ʹʹ), 30.8 (C-8ʹ,11ʹ), 30.2-30.6 (C-4ʹ-7ʹ, 12ʹ-15ʹ, 4ʹʹ-13ʹʹ), 26.0 (C-3ʹ,3ʹʹ), 23.7 (C-17ʹ,15ʹʹ), 14.5 (C-18ʹ,16ʹʹ).

Compound 4

Crystallin, MW (g/mol): 302 (M + H); 1H NMR (400 MHz, CDCl3): 3.54 (1H, dd, J = 2.8, 11.2 Hz, H-1), 3.65 (1H, dd, J = 2.8, 11.2 Hz, H-1), 5.24 (1H, m, H-2), 4.13 (1H, ddd, J = 5.2, 11.6, 12.4 Hz, H-3), 2.2 (2H, t, J = 7.6 Hz, H-2ʹ), 1.47 (2H, m, H-3ʹ), 1.2-1.3 (m, H-4ʹ-13ʹ), 0.83 (6H, t, J = 6.8 Hz, H-14ʹ), 1.58 (2H, m, H-1ʹ). 13C NMR (100 MHz, CDCl3): 174.4 (C-1ʹ), 70.1 (C-2), 65.5 (C-3), 63.4 (C-1), 34.2 (C-12ʹ), 31.8 (C-2ʹ), 29.6-29.8 (C-4ʹ-11ʹ), 24.8 (C-3ʹ), 22.7 (C-13ʹ), 14.1 (C-14ʹ).

Compound 5

White powder, MW (g/mol): 836 (M + H); 1H NMR (400 MHz, DMSO): 4.26 (1H, dd, J = 2.8, 12 Hz, H-1), 4.0 (1H, dd, J = 7.6, 12 Hz, H-1), 5.04 (1H, m, H-2), 3.3 (1H, dd, J = 6, 16 Hz, H-3), 3.80 (1H, dd, J = 6,10.6, H-3), 4.48 (1H, d, J = 4 Hz, H-1ʹʹʹ), 3.10 (1H, dd, J = 6,9.6, H-2ʹʹʹ), 3.21 (1H, t, J = 9.6, H-3ʹʹʹ), 2.82 (1H, t, J = 9.2 Hz, H-4ʹʹʹ), 2.90 (1H, s, H-11ʹ), 3.68 (1H, ddd, J = 4.8, 5.6, 10.4 Hz, H-5ʹʹʹ), 2.41, 2.47, 2.19 (4H, m, H-2ʹ,2ʹʹ), 1.40 (4H, m, H-3ʹ,3ʹʹ), 1.1-1.2 (m, H-4ʹ-18ʹ,4ʹʹ-15ʹʹ), 0.76 (6H, t, J = 6.8 Hz, H-19ʹ,16ʹʹ). 13C NMR (100 MHz, DMSO): 172.3, 172.5 (C-1ʹ,1ʹʹ), 98.2 (C-1ʹʹʹ), 74.2 (C-5ʹʹʹ), 72.8 (C-4ʹʹʹ), 71.5 (C-3ʹʹʹ), 69.6 (C-2ʹʹʹ), 68.5 (C-2), 64.5 (C-3), 62.6 (C-1), 54.5 (C-6ʹʹʹ), 42.07 (C11ʹ), 33.4, 33.5 (C-14ʹʹ,16ʹ), 31.2 (C-2ʹ,2ʹʹ), 29 (C-4ʹ-7ʹ, 12ʹ-15ʹ, 4ʹʹ-13ʹʹ), 28.4 (C-8ʹ), 24.4 (C-3ʹ,3ʹʹ), 22.0 (C-17ʹ,15ʹʹ), 13.9 (C-19ʹ,16ʹʹ).

Cytotoxic activity

The cytotoxic activities of all compounds was shown in Table 1.

Table 1 Cytotoxic activity of isolated compounds

Sample	Compound 1	Compound 2	Compound 3	Compound 4	Compound 5	
IC50 (μg/ml)	12.2±2.3	25.8±3.7	14.9±2.6	9.8±1.2	5.6±1.2	

DISCUSSION

Sargassum species are rich sources of different primary and secondary metabolites.[181920] Assignment of all 13C- and 1H-NMR signals was done by careful analysis of 1H-1H COSY, DEPTHMBC, and HSQC spectra [Figure 1]. About compound 2, a glycerol one spin moiety [δH 4.32 and 4.14 (δC 62.7); δH 5.18 (δC70.2); δH 3.88 and 3.64 (δC 68.7)] was recognized.

Figure 1 Structure of compounds 1-5

The presence of acyl groups on the sn-1 and sn-2 positions of the glycerol moiety was identified by HMBC (Heteronuclear Multiple Bond Coherence) cross-peaks [δH/δC: 5.18 (Hsn-2)/173.5, 175 (COO); 4.34 and 4.13 (Hsn-1)/172.5, 172.6 (COO); 2.29 (α-CH2)/172.5, 172.6 (COO)]. Therefore, terminal methyl signals of two fatty acyl groups were shown by the second spin system signals (6H, t, J = 6.8 Hz, δC 14.6).

The relatively small coupling constant of the anomeric proton (H-1ʹʹʹ), J = 3.7 Hz, presented the α orientation of the glycosidic part, and the large vicinal coupling constants between H-2ʹʹʹ/H-3ʹʹʹ, H-3ʹʹʹ/H-4ʹʹʹ, and H-4ʹʹʹ/H-5ʹʹʹ (J = 9.6 Hz), showed the glucopyranosyl structure of the sugar unit.

Besides, the 1H- and 13C-NMR characteristic chemical shifts of carbon C-6ʹʹʹ(δc 54.6) and methylene protons H-6ʹʹʹ (δ 2.91 and 2.55) indicated the presence of a sulphonyl group attach to the sugar (C-6ʹʹʹ), instead of glucose.[212223]

All 1H- and 13C-NMR characteristic data are in agreement with the structure of 6-deoxy-6-sulpho-α-D-glucopyranosyl-1,2-O-diacyl-glycerols. Alkaline hydrolysis with NaOMe in MeOH was done for identification of acyl substituents at sn-1 and sn-2. The hydrolysis was followed by GC/MS analysis. The composition of the fatty acid methyl esters was elucidated as methyl myristate, methyl oleate, and methyl palmitate, being the last in greater proportion.

Compound 5 was the most potent isolated compound as shown in Table 1. Comparing the structures and cytotoxic activity of isolated compounds shows that presence of a double bond in the side chains of fatty acid may reduce the cytotoxic activity. Structure activity relationship studies of SQDG has shown that the cytotoxicity is probably dependent on the fatty acid chain, besides each of the SQMG/SQDG was a stronger inhibitor than the fatty acid alone. The inhibitory effect could be influenced by the chain size of fatty acids too. The sulfate moiety in the quinovose is also important for the inhibition.[24]

Financial support and sponsorship

This article was financially supported by the Vice Chancellor of Research, Isfahan University of Medical Sciences, and Iran National Science Foundation (INSF).

Conflicts of interest

There are no conflicts of interest.

Acknowledgments

The authors are thankful to the Vice Chancellor of Research, Isfahan University of Medical Sciences, and Iran National Science Foundation for financial support. The results of this paper are part of a Pharm. D. thesis.
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