Sterols from the Green Alga Ulva australis

Three new sterols, (24R)-5,28-stigmastadiene-3β,24-diol-7-one (1), (24S)-5,28-stigmastadiene-3β,24-diol-7-one (2), and 24R and 24S-vinylcholesta-3β,5α,6β,24-tetraol (3), together with three known sterols (4–6) were isolated from the green alga Ulva australis. The structures of the new compounds (1–3) were elucidated through 1D and 2D nuclear magnetic resonance spectroscopy as well as mass spectrometry. Compounds 4–6 were identified as isofucoterol (4), 24R,28S and 24S,28R-epoxy-24-ethylcholesterol (5), and (24S)-stigmastadiene-3β,24-diol (6) on the basis of spectroscopic data analyses and comparison with those reported in the literature. Compounds 4–6 were isolated from U. australis for the first time. These compounds, together with the previously isolated secondary metabolites of this alga, were investigated for their inhibitory effects on human recombinant aldose reductase in vitro. Of the compounds, 24R,28S and 24S,28R-epoxy-24-ethylcholesterol (5), 1-O-palmitoyl-3-O-(6′-sulfo-α-d-quinovopyranosyl) glycerol, (2S)-1-O-palmitoyl-3-O-[α-d-galactopyranosyl(1→2)β-d-galactopyranosyl] glycerol, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, and 8-hydroxy-(6E)-octenoic acid weakly inhibited the enzyme, while the three new sterols, 1–3, were almost inactive.


Introduction
Marine organisms have recently received much attention in the search for structurally interesting compounds with a wide range of pharmacological activities to develop new medicines or health foods [1][2][3][4][5].Approximately 8000 species of marine algae have been identified and grouped into different classes, including brown algae (Phaeophyta), red algae (Rhodophyta), and green algae (Chlorophyta).Ulva australis Areschoug is a green alga belonging to the family Ulvaceae and is widely distributed along the coasts of the Yellow Sea and the Bo Sea of China.As an edible seaweed, it contains high nutritional value minerals, vitamins, and noncaloric dietary fibers [6].Ulva australis is consumed by local inhabitants of Asia [7] and has been authorized for human utilization by French authorities [8].In addition to edibility, the decoction of this alga has been accepted as a natural traditional medicine for hyperlipidemia, sunstroke, and urinary diseases [9].The chemical and pharmacological studies of U. australis available in the literature are mostly concerned with the algal polysaccharides.These sulfated polysaccharides have been described to possess diverse biological activities, such as antihyperlipidemic [10], antioxidant [11], antiviral [12], immunomodulatory [13], and anti-radiation activities [14].However, small-molecule chemical components of U. australis and structure identification have only received limited attention [15][16][17].It has been recently reported that 3-hydroxy-4,7-megastigmadien-9-one, isolated from this alga, attenuates lipopolysaccharide-induced downregulating mitogen-activated protein kinase and NF-κB pathways [7,18].In our search for aldose reductase inhibitors from marine algae, we found that bromophenols isolated from the red alga Symphyocladia latiuscula exhibited significant human recombinant aldose reductase inhibitory activity [19].The extract from U. australis exhibited weak human recombinant aldose reductase inhibitory activity, which is in agreement with the previous report [20].Our continued interest in discovering new secondary metabolites from marine algae led us to isolate three new sterols, (24R)-5,28-stigmastadiene-3β,24-diol-7-one (1), (24S)-5,28-stigmastadiene-3β,24-diol-7-one (2), and 24R and 24S-vinylcholesta-3β,5α,6β,24-tetraol (3), together with three known sterols, isofucoterol (4), 24R,28S and 24S,28R-epoxy-24-ethylcholesterol (5), and (24S)-5,28-stigmastadiene-3β,24-diol (6), from the green alga U. australis (Figure 1).The structures of these compounds were identified by NMR spectroscopy, mass spectrometry, and compared with those reported in the literature.The configurations of compounds 1 and 2 were determined on the basis of the chemical shift differences between H-27 and H-26 and between H-27 and H-21.Herein, we also report on the evaluation of the human recombinant aldose reductase inhibitory activities of the compounds isolated by us from this alga.

Results and Discussion
Compound 1 was isolated as a white amorphous solid.The molecular formula was determined to be C29H46O3 by HR-EI-MS at m/z 442.3445 (calcd.for C29H46O3 442.3447).The sterol nature of this compound was deduced from a combination of 13 C NMR (Table 1) and distortionless enhancement by polarization transfer spectra.The 13   2) suggested that compound 1 possessed a 24-hydroxy-24-vinyl side chain [21,22].The above data thus
Compound 2 was isolated as a white amorphous solid.The 13 C NMR spectrum of compound 2 displayed 29 signals (Table 1), including five quaternary carbons (one ketone carbonyl carbon, one olefinic carbon, and one oxygen-bearing carbon), nine methine carbons (two olefinic carbons and one oxygen-bearing carbon), 10 methylene carbons (one olefinic carbon), and five methyl groups.The molecular formula was established as C 29 H 46 O 3 using HR-EI-MS (m/z 442.3439 [M] + , calcd.for C 29 H 46 O 3 442.3447).Comparison of the 1 H and 13 C NMR data of compound 2 with those of compound 1 showed that both compounds shared the same sterol skeleton nucleus and side chain, the only difference being that the signal of H-27 in compound 2 appeared at δ H 0.91 (d, J = 7.2 Hz), whereas the signal of H-27 in compound 1 displayed at δ H 0.89 (d, J = 8.0 Hz).Stereochemistry at C-24 of compound 2 was established to be S, since the signal attributed to H-27 at δ H 0.909 (3H, d, J = 7.2 Hz) was very close to that of H-21 at δ H 0.931 (3H, d, J = 6.8 Hz) in the 1 H NMR spectrum of compound 2. The chemical shift difference between H-27 and H-21 of compound 2 was 0.022 ppm, whereas the chemical shift difference between H-27 and H-26 of compound 2 was 0.038 ppm, which was in accordance with those of 24S-saringosterol.The chemical shift difference between H-27 and H-21 of 24S-saringosterol was 0.019 or 0.020 ppm, whereas the chemical shift difference between H-27 and H-26 24S-saringosterol was 0.027 or 0.027 ppm in the previous reports [21,22].Thus, compound 2 structure was established, and named (24S)-5,28-stigmastadiene-3β,24-diol-7-one.demonstrated that compound 1 was a Δ 5 -3β-hydroxyl 7-oxysterol derivative, similar to the ring system of decortinone (Table 1), a known sterol previously obtained from the green alga Codium decorticatum [23], and the side chain of 5,28-stigmastadiene-3β,24-diol (Table 1), a known sterol previously isolated from the brown alga Sargassum fusiforme [24].The stereochemistry of compound 1 at C-24 was established to be R, since the signals attributed to H-27 at δ 0.890 (3H, d, J = 8.0 Hz) were close to those of H-26 at δ 0.871 (3H, d, J = 7.2 Hz) in the 1 H NMR spectrum of compound 1.The chemical shift difference between H-27 and H-26 of compound 1 was 0.019 ppm, whereas the chemical shift difference between H-27 and H-21 of compound 1 was 0.041 ppm, which was in accordance with those of 24R-saringosterol.The chemical shift difference between H-27 and H-26 of 24R-saringosterol was 0.020 or 0.020 ppm, whereas the chemical shift difference between H-27 and H-21 24R-saringosterol was 0.032 or 0.033 ppm in the previous reports [21,22].Thus, compound 1 was elucidated with the structure as shown in Figure 1, and named (24R)-5,28-stigmastadiene-3β,24-diol-7-one.

Algal Material
The wild green alga Ulva australis was collected at the coast of Dalian, China, in October 2002 and identified by Professor Zi'ang Yao (School of Life Science and Technology, Dalian University, Dalian, China).A voucher specimen (20021001) was deposited at the Department of Natural Medicine and Pharmacognosy, School of Pharmacy, Qingdao University, Qingdao, China.

Human Recombinant Aldose Reductase Inhibitory Activity Assay
The human recombinant aldose reductase inhibition assays were performed according to the method modified by our group [19].The assay was performed at 25 • C in a 200 mM sodium phosphate buffer solution (pH 6.2) (700 µL), containing 1.5 mM NADPH solution (100 µL), 100 mM DL-glyceraldehyde solution (100 µL), and 3 × 10 −2 unit/mL human recombinant aldose reductase solution (100 µL), in the total volume of 1.0 mL.The effects of each sample on the enzyme activity were determined by adding 3 µL of test sample solution (final concentration 3 µg/mL dissolved in 100% DMSO) to the reaction mixture.An appropriate blank control mixture and positive control mixture were prepared.The reaction was initiated by the addition of DL-glyceraldehyde solution and the rate of NADPH oxidation was followed by recording the decrease in the absorbance at 340 nm on a UV spectrophotometer (SHIMADZU UV 1600, Kyoto, Japan).Inhibition percentage (%) was calculated as [1 − (∆A s − ∆A b )/(∆A c − ∆A b )] × 100, where A s is the decreased absorbance of the sample, A c and A b are the decreased absorbances without a sample as a positive control and without a sample and enzyme as a blank control, respectively.An overview about the effects of these substances on human recombinant aldose reductase inhibitory activity is given in Table 2.

Conclusions
Phytochemistry investigation of the green alga U. australis led to the isolation of three new sterols and three known sterols.
These compounds, together with the previously isolated secondary metabolites of this alga, were investigated for their inhibitory effects on aldose reductase in vitro.Of the compounds, 24R,28S and 24S,28R-epoxy-24-ethylcholesterol ( 5

Table 2 .
Human recombinant aldose reductase inhibitory activities of the isolated compounds.

Table 2 .
Human recombinant aldose reductase inhibitory activities of the isolated compounds.