Chemical Constituents of the Marine Traditional Chinese Medicine of Pegasus laternarius Cuvier (Hai-E Yu)

Abstract

Pegasus laternarius Cuvier (Hai-E Yu) is a marine traditional Chinese medicine that has been used to treat cancers and reduce inflammation. Previous chemical investigations have only revealed the occurrence of high levels of protein, fatty acids, and a large number of steroids, thus more active compounds in P. laternarius still need to be further discovered. The present study aims to search for new bioactive constituents of P. laternarius with cytotoxic effects and nitric oxide (NO) inhibitory activities. In this work, 16 pure compounds from the ethyl acetate fraction of Pegasus laternarius Cuvier were successively obtained by various chromatographic techniques, and the structure of the isolates was elucidated by spectroscopic analyses. The isolated and identified compounds included one 2H-1,2,3-triazole-4-carboxylate derivative (1), two oxadiazines (2, 3), two amino acids (4, 5), three nucleosides (6–8), three steroids (9–11), and five others (12–16). All the obtained compounds were evaluated for their antitumor activity on A549 and HCT-116 cell lines along with their inhibitory effects on lipopolysaccharide-induced NO production in RAW264.7 cells. The triazole compound 1 was found to exhibit moderate cytotoxicity against two human cell lines (A549 and HCT-116) with IC₅₀ values of 44.5 and 32.9 μmol/L, respectively. The steroid compound 10 inhibited NO production with IC₅₀ values lower than 50 μmol/L. Consequently, this study provides insight into the cytotoxic and NO inhibitory activities of the marine traditional Chinese medicines in Hai-E-Yu.

Keywords

marine traditional Chinese medicine - Pegasus laternarius Cuvier - cytotoxicity - anti-inflammatory

Introduction

Marine organisms tend to produce unique secondary metabolites with specific activities due to the special marine environment characteristics of high salt, high pressure, low nutrition, lack of oxygen, and lack of sunlight, which could be considered the newest source of bioactive natural products in relation to terrestrial plants and nonmarine microorganisms.[1] Among them, a variety of species (seaweeds, shellfishes, and minerals) have been used for thousands of years as marine traditional Chinese medicines (MTCMs) to treat diseases, and modern pharmacological studies have shown that they have antitumor, anti-inflammatory, and antiviral properties.[2] Currently, it has become an important medicinal resource for the development of new drugs for the prevention and treatment of difficult medical issues. For instance, Concha Ostreae polysaccharides can boost the immune system, Sargassum has the effects on antitumor and antiviral therapy, and Hippocampus exhibits a great effect on antiaging. The in vitro cellular studies have also suggested that the water-soluble Margaritifera concha protein has a strong effect on stimulating the differentiation of bone marrow stromal cells into osteoblasts and increased osteoblast proliferation.[3] Notably, a type of sulfated polysaccharide derived from seaweed has been used clinically for cardiovascular diseases, and sodium oligomannate (GV-971), an oligosaccharide compound derived from brown algae, has been used for the treatment of Alzheimer's disease by targeting the brain–gut axis.[4] Therefore, it is of great value to discover novel compounds with diverse biological activities from MTCMs.

Pegasus laternarius Cuvier (Hai-E Yu) is an MTCM commonly used for the treatment of tumors, cough, and antidiarrheal, and is documented in Chinese Pharmacopoeia. Previous studies have shown that the extracts of P. laternarius exhibited antitumor activity, anti-lipid peroxidation effect, memory-improving effect, etc. However, the constituents of P. laternarius have less been investigated. The previous chemical investigations have only confirmed the occurrence of a high level of protein, fatty acids, and a large number of steroids. We anticipated that more active compounds in P. laternarius will be further explored. In our continuing efforts to search for cytostatic and anti-inflammatory compounds from MTCMs, the chemical investigation on P. laternarius was undertaken and resulted in the isolation and identification of 16 compounds ([Fig. 1]), among them, the occurrences of compounds 1, 6, 12, and 13 were first reported from P. laternarius. In bioassay, the 2H-1,2,3-triazole-4-carboxylate compound 1 showed moderate cytotoxicity with IC₅₀ values of 44.5 and 32.9 μmol/L on the A549 and HCT-116 cell lines, while the steroid compound 10 inhibited NO production with IC₅₀ values lower than 50 μmol/L. Herein, the isolation, structural determination, and assessment of the cytostatic and anti-inflammatory activities of the isolated compounds are further described.

Fig. 1 Chemical structures of the isolated and identified compounds 1–16.

Results and Discussion

Extraction and Isolation

The air-dried whole parts of Pegasus laternarius Cuvier (483 g) were powdered and extracted with dichloromethane/methanol (CH₂Cl₂/MeOH, 1:1) five times at room temperature. The crude extracts were concentrated by evaporation under reduced pressure to yield 73 g of dry extract. The obtained dry extracts were suspended in H₂O and partitioned successively with petroleum ether (PE), ethyl acetate (EtOAc), and n-butyl alcohol (n-BuOH, 1L × 5, five times). The EtOAc extract (10.8 g) was subjected to a silica gel column (Si CC, 200 − 300 mesh; PE/EtOAc, 20:1, 10:1, 5:1, 2:1, 1:1, v/v; CH₂Cl₂/MeOH, 15:1, 10:1, 5:1, 2:1, v/v) as an eluent to obtain six fractions, labeled as Fr. 1 to Fr. 6. The fraction Fr.3 (1.05 g) was submitted to silica gel column with PE/CH₂Cl₂ (95:5–50:50, v/v), leading to two new fractions, in which fraction Fr. 3–1 (104 mg) was purified by silica gel column with gradient of PE/EtOAc (90:10–50:50, v/v) to afford compound 9 (37.7 mg). The fraction Fr. 5 (0.64 g) was further submitted to silica gel column with PE/EtOAc (95:5–50:50, v/v) leading to two new fractions, Fr. 5–1 and Fr. 5–2, which were then subjected to preparative high-performance liquid chromatography (HPLC) with a solvent system of MeOH/H₂O (60:40, v/v) to provide compound 12 (t _R = 16 minutes, 3.4 mg) and 13 (t _R = 35 minutes, 2.8 mg), respectively. The fraction Fr. 6 (1.60 g) was subjected to a C18 reversed-phase column eluted with MeOH/H₂O (30:70–100:0, v/v) to yield five new fractions. The fraction Fr. 6–4 (159 mg) was chromatographed on Sephadex LH-20 (CH₂Cl₂/MeOH, 1:1, v/v) to afford three new fractions. The fraction Fr. 6–4-3 (65 mg) was purified by silica gel column and eluted with CH₂Cl₂/MeOH (90:10–50:50, v/v) to afford the steroid compound 10 (4.5 mg). The PE extract (38.7 g) was subjected to a silica gel column using PE/CH₂Cl₂ (98:2–0:100, v/v) as an eluent to obtain seven new fractions, S.1–S.7. The fraction S.4 (1.16 g) was submitted to silica gel column with PE/CH₂Cl₂ (90:10–0:100, v/v) leading to five new fractions. The fraction S.4–1 (59 mg) was purified by a silica gel column with a gradient of PE/CH₂Cl₂ (90:10–70:30, v/v) to afford the phthalate ester compound 14 (3.6 mg) as well as the terephthalate compound 15 (20.2 mg). The 2H-1,2,3-triazole-4-carboxylate 1 (12.8 mg) was obtained from fraction S.5. The fraction S.7 (100 mg) was submitted to the silica gel column with PE/EtOAc (98:2–50:50, v/v) leading to three new fractions. The fraction S.7–1 (30 mg) was subjected to preparative HPLC (MeOH/H₂O, 80:20, v/v) leading to the octadecatrienoic acid 16 (t _R = 20 minutes, 3.1 mg). The S.7–3 was chromatographed on Sephadex LH-20 (CH₂Cl₂/MeOH, 1:1, v/v) to afford three new fractions, in which the fraction S.7–3-2 (50 mg) was purified by silica gel column with a gradient of PE/CH₂Cl₂ (95:5–60:40, v/v) to afford nucleoside 6 (4.6 mg) and cholesterone 11 (2.2 mg). The n-BuOH alcohol (7.09 g) was subjected to a silica gel column using CH₂Cl₂/MeOH (98:2–0:100, v/v) as an eluent to obtain five fractions, Z.1–Z.5. The fraction Z.3 (1.0 g) was subjected to a Si CC (CH₂Cl₂/MeOH, 95:5–5:5, v/v) followed by semipreparative HPLC (MeOH/H₂O, 10:90, v/v) to obtain the pyrimidine-2,4-dione compounds 2 (t _R = 15 minutes, 5 mg) and 3 (t _R = 8 minutes, 13 mg). The fraction Z.3 (60 mg) was chromatographed on Sephadex LH-20 (CH₂Cl₂/MeOH, 1:1, v/v) to afford three new fractions. The fraction Z.3–2 (30 mg) was subjected to preparative HPLC (MeOH/H₂O, 10:90, v/v) leading to D-phenylalanine 4 (t _R = 12 minutes, 2.1 mg) and L-tryptophan 5 (t _R = 8 minutes, 1.8 mg). Finally, the fraction Z.5 (42 mg) was purified by preparative HPLC (MeOH/H₂O, 10:90, v/v) to yield the deoxynucleosides 7 (t _R = 9 minutes, 1.2 mg) and 8 (t _R = 16 minutes, 3.1 mg).

( R )-2-Propylhexyl 2H-1,2,3-triazole-4-carboxylate (compound 1): yellow oil, C₁₂H₂₁N₃O₂, ¹H NMR (400 MHz, CDCl₃) δ_H 8.09 (s, 1H, NH), 8.09 (s, 1H, H-5), 4.31–4.22 (m, 2H, H-1'), 1.73 (t, J = 6.2 Hz, 1H, H-2'), 0.95 (t, J = 7.5 Hz, 3H, H-3′'), 0.90 (t, J = 7.0 Hz, 3H, H-6'). ¹³C NMR (100 MHz, CDCl₃) δ_C 134.40 (C-4), 129.64 (C-5), 166.12 (C-6), 67.92 (C-1'), 39.06 (C-2'), 29.84 (C-3′), 29.13 (C-4'), 23.11 (C-5′), 14.17 (C-6'), 30.72 (C-1'), 24.13 (C-2''), 11.23 (C-3′'). The data were consistent with a reported study.[5]

Thymine (compound 2): white powder, C₅H₆N₂O₂, ¹H NMR (600 MHz, DMSO-d ₆) δ_H 10.98 (s, 1H, NH), 10.58 (s, 1H, NH), 7.24 (s, 1H, H-6), 1.72 (s, 3H, H-7). ¹³C NMR (150 MHz, DMSO-d ₆) δ_C 164.95 (C-4), 151.52 (C-2), 137.74 (C-6), 107.69 (C-5), 11.82 (C-7). The data were consistent with a reported study.[6]

Uracil (compound 3): white powder, C₄H₄N₂O₂, ¹H NMR (600 MHz, DMSO-d ₆) δ_H 11.00 (s, 1H, NH), 10.81 (s, 1H, NH), 7.38 (d, J = 7.6 Hz, 1H, H-6), 5.44 (d, J = 7.6 Hz, 1H, H-5). ¹³C NMR (150 MHz, DMSO-d ₆) δ_C 164.37 (C-4), 151.55 (C-2), 142.22 (C-6), 100.25 (C-5). The data were consistent with a reported study.[6]

D -Phenylalanine (compound 4): white powder, C₉H₁₁NO₂, ¹H NMR (400 MHz, D₂O) δ_H 7.22 (m, 1H, H-2), 7.21 (m, 1H, H-1, 3, 4 and 6), 4.05 (m, 1H, H-8), 3.18 (m, 2H, H-7a), 3.02 (m, 2H, H-7b). ¹³C NMR (100 MHz, D₂O) δ_C 174.81 (C-9), 135.47 (C-5), 129.42 (C-1 and C-3), 129.11 (C-4 and C-6), 127.64 (C-2), 56.22 (C-8), 36.82 (C-7). The data were consistent with a reported study.[7]

L -Tryptophan (compound 5): white powder, C₁₁H₁₂N₂O₂, ¹H NMR (400 MHz, CD₃OD) δ_H 7.70 (d, J = 7.9 Hz, 1H, H-1), 7.36 (d, J = 8.1 Hz, 1H, H-4), 7.19 (s, 1H, H-8), 7.12 (t, J = 7.5 Hz, 1H, H-3), 7.05 (t, J = 7.5 Hz, 1H, H-2), 3.86 (dd, J = 9.3, 4.0 Hz, 1H, H-10), 3.52 (dd, J = 15.2, 4.1 Hz, 1H, H-9a), 3.15 (dd, J = 15.2, 9.4 Hz, 1H, H-9b). ¹³C NMR (100 MHz, CD₃OD) δ_C 138.43 (C-5), 128.51 (C-6), 125.13 (C-8), 120.13 (C-3), 119.35 (C-2), 112.44 (C-4), 28.54 (C-9). The data were consistent with a reported study.[8]

2-Hydroxypurine nucleoside (compound 6): white powder, C₁₀H₁₂N₄O₅, ¹H NMR (400 MHz, D₂O) δ_H 8.26 (s, 1H, H-8), 8.16 (s, 1H, H-6), 6.05 (d, J = 5.8 Hz, 1H, H-1'), 4.43 (dd, J = 6.4, 2.6 Hz, 1H, H-3′), 4.30–4.25 (m, 1H, H-4'), 3.91 (dd, J = 12.8, 1.5 Hz, 1H, H-5′a), 3.83 (dd, J = 12.8, 3.9 Hz, 1H, H-5′b). ¹³C NMR (100 MHz, D₂O) δ_C 160.86 (C-2), 148.62 (C-4), 148.00 (C-6), 139.85 (C-8), 124.75 (C-5), 88.42 (C-1'), 85.67 (C-4'), 73.92 (C-3′), 70.53 (C-2'), 61.43 (C-5′). The data were consistent with a reported study.[9]

2'-Deoxyuridine (compound 7): white solid, C₉H₁₂N₂O₅, ¹H NMR (600 MHz, DMSO-d ₆) δ_H 7.85 (d, J = 8.1 Hz, 1H, H-6), 6.15 (dd, J = 7.5, 6.2 Hz, 1H, H-1'), 5.63 (d, J = 8.1 Hz, 1H, H-5), 4.23 (dt, J = 6.2, 3.2 Hz, 1H, H-3′), 3.79–3.76 (m, 1H, H-4'), 3.57 (dd, J = 11.9, 3.8 Hz, 1H, H-5′a), 3.53 (dd, J = 11.9, 3.8 Hz, 1H, H-5′b), 2.12–2.03 (m, 2H, H-2'). ¹³C NMR (150 MHz, DMSO-d ₆) δ_C 163.17 (C-4), 150.48 (C-2), 140.55 (C-6), 100.78 (C-5), 87.44 (C-4'), 84.15 (C-1'), 70.44 (C-3′), 61.31 (C-5′), 48.63 (C-2'). The data were consistent with a reported study.[10]

Thymidine (compound 8): white solid, C₁₀H₁₄N₂O₅, ¹H NMR (600 MHz, DMSO-d ₆) δ_H 7.69 (d, J = 1.3 Hz, 1H, H-6), 6.16 (dd, J = 7.6, 6.2 Hz, 1H, H-1'), 4.23 (dt, J = 6.2, 3.1 Hz, 1H, H-3′), 3.75 (dd, J = 3.6 Hz, 1H, H-4'), 3.61–3.52 (m, 2H, H-5′), 2.11–2.03 (m, 2H, H-2'), 1.76 (d, J = 1.3 Hz, 3H, 5-CH₃). ¹³C NMR (150 MHz, DMSO-d ₆) δ_C 163.83 (C-4), 150.54 (C-2), 136.20 (C-6), 109.45 (C-5), 87.32 (C-4'), 83.82 (C-1'), 70.51 (C-3′), 61.41 (C-5′), 39.49 (C-2') 12.33 (5-CH₃). The data were consistent with a reported study.[10]

(3β)-Cholest-5-en-3-ol (compound 9): white amorphous powder, C₂₇H₄₆O, ¹H NMR (400 MHz, CDCl₃) δ_H 5.34 (d, J = 5.4 Hz, 1H, H-6), 3.57 (m, 1H, H-3), 1.00 (s, 3H, H-19), 0.91(d, J = 6.5 Hz, 3H, H-21), 0.87 (d, J = 1.8 Hz, 3H, H-26), 0.85 (d, J = 1.8 Hz, 3H, H-27), 0.67 (s, 3H, H-18). ¹³C NMR (100 MHz, CDCl₃) δ_C 140.84 (C-5), 121.78 (C-6), 71.87 (C-3), 56.85 (C-14), 56.25 (C-17), 50.22 (C-9), 39.87 (C-16), 39.60 (C-24), 37.34 (C-1), 36.58 (C-10), 36.28 (C-22), 35.87 (C-8 and C-20), 31.99 (C-7), 31.73 (C-2), 28.31 (C-12), 28.09 (C-25), 24.37 (C-15), 23.92 (C-24), 22.90 (C-27), 22.64 (C-26), 21.17 (C-11), 19.48 (C-19), 18.80 (C-21), 11.94 (C-18). The data were consistent with a reported study.[11]

Methyl-3α,7α-dihydroxy-5β-cholan-24-oate (compound 10): colorless gelatinous solid, C₂₅H₄₂O₄, ¹H NMR (400 MHz, CDCl₃) δ_H 3.84 (m, 1H, H-7), 3.68 (s, 3H, H-1'), 3.46 (m, 1H, H-3), 0.93 (d, J = 6.5 Hz, 3H, H-21), 0.89 (s, 3H, H-19), 0.65 (s, 3H, H-18). ¹³C NMR (100 MHz, CDCl₃) δ_C 177.84 (C-24), 72.20 (C-3), 68.73 (C-7), 35.20 (C-17), 50.60 (C-14), 42.87 (C-13), 41.61 (C-5), 40.01 (C-12), 39.77 (C-4), 39.57 (C-8), 35.50 (C-1 and C-20), 35.19 (C-6 and C-10), 32.98 (C-9), 30.94 (C-2), 30.79 (C-22), 30.74 (C-23), 28.29 (C-16), 23.85 (C-15), 22.91 (C-19), 20.72 (C-11), 18.38 (C-21), 11.92 (C-18), 55.9 (C-1'). The data were consistent with a reported study.[12]

Cholest-4-en-3-one (compound 11): white solid, C₂₇H₄₄O, ¹H NMR (400 MHz, CDCl₃) δ_H 5.72 (s, 1H, H-4), 1.25 (s, 2H, H-24), 1.17 (s, 3H, H-19), 0.90 (d, J = 6.5 Hz, 3H, H-21), 0.87 (d, J = 1.8 Hz, 3H, H-27), 0.85 (d, J = 1.8 Hz, 3H, H-26), 0.70 (s, 3H, H-18). ¹³C NMR (100 MHz, CDCl₃) δ_C 199.79 (C-3), 171.85 (C-5), 123.82 (C-4), 56.19 (C-17), 55.96 (C-14), 53.90 (C-9), 42.47 (C-13), 39.71 (C-12), 39.57 (C-24), 38.69 (C-10), 36.19 (C-22), 35.83 (C-20), 35.76 (C-1), 35.70 (C-8), 34.06 (C-2), 33.03 (C-6), 32.13 (C-7), 28.25 (C-25), 28.08 (C-16), 24.25 (C-15), 23.89 (C-23), 22.88 (C-27), 22.62 (C-26), 21.10 (C-11), 18.71 (C-21), 17.46 (C-19), 12.02 (C-18). The data were consistent with a reported study.[13]

Matriisobenzofuran (compound 12): yellow powder, C₁₃H₁₄O₃, ¹H NMR (400 MHz, CDCl₃) δ_H 8.18 (d, J = 1.6 Hz, 1H, H-7), 7.94 (dd, J = 8.6, 1.8 Hz, 1H, H-5), 7.49 (d, J = 8.6 Hz, 1H, H-4), 6.66 (s, 1H, H-8), 2.66 (s, 3H, H-10), 1.70 (s, 6H, 3-CH₃). ¹³C NMR (100 MHz, CDCl₃) δ_C 197.91 (C = O, C-9), 164.94 (C-1), 157.54 (C-6), 132.93 (C-3a), 128.66 (C-7a), 125.08 (C-5), 122.54 (C-7), 111.40 (C-4), 101.17 (C-8), 69.55 (C-3), 28.94 (3-CH₃), 27.02 (C-10). The data were consistent with a reported study.[14]

6-Hydroxy-6-methylocta-3,7-dien-2-one (compound 13): colorless oil, C₉H₁₄O₂, ¹H NMR (400 MHz, CDCl₃) δ_H 6.81 (dt, J = 15.3, 7.5 Hz, 1H, H-5), 6.11 (d, J = 16.0 Hz, 1H, H-6), 5.95 (dd, J = 17.3, 10.7 Hz, 1H, H-2), 5.26 (d, J = 17.3 Hz, 1H, H-1a), 5.12 (d, J = 10.7 Hz, 1H, H-1b), 2.47 (d, J = 7.2 Hz, 2H, H-4), 2.26 (s, 3H, H-8), 1.34 (s, 3H, H-9). ¹³C NMR (100 MHz, CDCl₃) δ_C 198.54 (C = O, C-7), 144.05 (C-2), 143.36 (C-5), 134.19 (C-6), 112.77 (C-1), 72.77 (C-3), 45.16 (C-4), 27.91 (C-8), 26.91 (C-9). The data were consistent with a reported study.[15]

Bis(2-ethylhexyl) phthalate (compound 14): colorless gelatinous solid, C₂₄H₃₈O₄, ¹H NMR (400 MHz, CDCl₃) δ_H 7.71 (dd, J = 5.7, 3.3 Hz, 2H, H-3′' and H-6''), 7.53 (m, 2H, H-4'' and H-5′'), 4.31–4.16 (m, 2H, H-1 and H-1'), 1.74–1.64 (m, 2H, H-2 and H-2'), 0.92 (m, 6H, H-6 and H-6'), 0.90 (m, 6H, H-8 and H-8'). ¹³C NMR (100 MHz, CDCl₃) δ_C 167.92 (2C = O), 132.62 (C-1'' and C-2''), 131.03 (C-4'' and C-5′'), 128.96 (C-3′' and C-6''), 68.32 (C-1 and C-1'), 38.89 (C-2 and C-2'), 30.52 (C-3 and C-3′), 29.08 (C-4 and C-4'), 23.91 (C-7 and C-7'), 23.14 (C-5 and C-5′), 14.20 (C-8 and C-8'), 11.11 (C-6 and C-6'). The data were consistent with a reported study.[16]

Bis(2-ethylhexyl) terephthalate (compound 15): yellow oil, C₂₄H₃₈O₄, ¹H NMR (400 MHz, CDCl₃) δ_H 8.10 (s, 4H, H-2, 3, 5 and 6), 4.32–4.21 (m, 4H, H-1' and H-1''), 1.80–1.67 (m, 2H, H-2' and H-2''), 1.47–1.27 (m, 16H, H-3′, 5′, 6', 7' and H-3′', 5′', 6'', 7''), 0.97–0.92 (m, 6H, H-8' and H-8''), 0.92–0.88 (m, 6H, H-4' and H-4''). ¹³C NMR (100 MHz, CDCl₃) δ_C 166.08 (C-7 and C-8), 134.38 (C-1 and C-4), 129.62 (C-2, 3, 5 and 6), 67.89 (C-1'), 39.04 (C-2'), 30.70 (C-3′), 29.11 (C-4'), 24.11 (C-7'), 23.09 (C-5′), 14.15 (C-6'), 11.21 (C-8'). The data were consistent with a reported study.[17]

( S,9E,13Z,15E )-12-Hydroxyoctadeca-9,13,15-octadecatrienoic acid (compound 16): yellow oil, C₁₈H₃₀O₃, ¹H NMR (400 MHz, CD₃CD) δ_H 6.00 (dd, J = 15.2, 11.0 Hz, 1H, H-14), 5.46 (t, J = 11.0 Hz, 1H, H-15), 5.14 (dd, J = 15.2, 6.6 Hz, 1H, H-10), 0.46 (t, J = 7.5 Hz, 3H, H-18). ¹³C NMR (100 MHz, CD₃CD) δ_C 175.50 (C-1), 136.68 (C-13), 134.58 (C-16), 133.05 (C-9), 129.34 (C-15), 126.65 (C-14), 125.55 (C-10), 73.27 (C-12), 36.29 (C-11), 34.94 (C-2), 30.70 (C-7), 30.25 (C-6), 30.17 (C-4), 30.11 (C-5), 28.61 (C-3), 25.99 (C-8), 21.70 (C-17), 14.56 (C-18). The data were consistent with a reported study.[18]

All the isolates were evaluated for their antitumor activity on A549 and HCT-116 cell lines and their inhibitory effects on lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells. Inhibiting NO production in LPS-stimulated RAW 264.7 cells represents a possible way to screen agents with anti-inflammatory activity.[19] The bioassay screening results indicated that the 2H-1,2,3-triazole-4-carboxylate derivative 1 displayed cytotoxicity with IC₅₀ values at 44.5 and 32.9 μmol/L on the A549 and HCT-116 cell lines ([Table 1]), respectively. In addition, the steroid compound 10 inhibited NO production with an IC₅₀ value at 44.5 μmol/L ([Table 2]), suggesting the anti-inflammatory activity of the compound.

Table 1 Cytotoxicity of compound 1 from P. laternarius

Compds.	IC50 (μmol/L)[a]
	A549	HCT-116
1	44.5 ± 1.1	32.9 ± 0.7
Sorafenib[b]	12.6 ± 0.2	10.3 ± 0.5

^a Data were expressed as means ± standard deviation (n = 3).

^b Sorafenib was used as a positive control.

Table 2 Nitric oxide inhibition of compound 10 in RAW264.7 cells

Compds.	IC50 (μmol/L)[a]
10	44.5 ± 1.1
Dexamethasone[b]	20.3 ± 0.8

^a Data were expressed as means ± standard deviation (n = 3).

^b Dexamethasone was used as a positive control.

Conclusion

Chemical investigations on Pegasus laternarius (Hai-E Yu) were undertaken, and 16 compounds were isolated and identified. Among them, compounds 1, 6, 12, and 13 were first reported from P. laternarius. The bioassay results showed that the triazole compound 1 exhibited moderate cytotoxicity with IC₅₀ values of 44.5 and 32.9 μmol/L on the A549 and HCT-116 cell lines, respectively, while the steroid compound 10 showed NO production inhibition activity with IC₅₀ value at concentration lower than 50 μmol/L. This study provides valuable information for understanding the MTCM of Hai-E Yu and searching for anticancer and anti-inflammatory candidates from MTCMs.

Experimental Section

General Experimental Procedures

¹H and ¹³C NMR spectra were acquired on a Bruker AVANCE III 400 and 600 spectrometer. HRESIMS spectra were recorded on an Agilent G6250 Q-TOF (Agilent, Santa Clara, California, United States). All solvents used for column chromatography and HPLC were of analytical grade (purchased from Shanghai Chemical Reagents Co., Ltd., Shanghai, China) and chromatographic grade (purchased from Dikma Technologies Inc., Beijing, China), respectively. Sephadex LH-20 (Pharmacia, Peapack, New Jersey, United States) was also used for column chromatography. Commercial silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China, 100–200 and 300–400 mesh) was used for column chromatography, and precoated silica gel GF254 plates (Sinopharm Chemical Reagent Co., Shanghai, China) were used for analytical thin-layer chromatography. Reversed-phase HPLC was performed on an Agilent 1260 series liquid chromatograph equipped with a DAD G1315D detector at 210 nm (Agilent, Santa Clara, California, United States). An Agilent semipreparative XDB-C18 column (5 µm, 250 mm × 9.4 mm) was employed for the purification.

Animal Materials

The animal Pegasus laternarius Cuvier was collected from Yangjiang City, Guangdong Province, China in July 2022, and was authenticated by Dr. Lin Gong (Institute of Oceanology, Chinese Academy of Sciences). For reference and future studies, a voucher specimen of the animal was cataloged (No. 202207–16) in Laboratory 1808 of Shandong Laboratory of Yantai Drug Discovery.

Cytotoxicity Assay

This part was conducted by referring to our previous paper.[20] The materials used in the study were CCK8 kit (Shanghai Lfe-iLab Biotechnology Co., Ltd., Shanghai, China), human lung carcinoma cell line A549 and colon cancer cell line HCT-116 (Shanghai Beyotime Biotechnology, Shanghai, China), and sorafenib (Promega, https://www.promega.com.cn/). OD at 450 nm was measured to assess cell viability with the inhibitory ratios calculated as [A_(control) − A_(sample)]/A_(control) × 100%. GraphPad Prism 7 (GraphPad Software, San Diego, CA, United States) was used to calculate IC₅₀ values. All data were expressed as the mean ± standard deviation of three independent experiments.

Determination of NO Production and the Cell Viability Assay

The experiment was conducted according to a reported study.[19] Dexamethasone was used as a control drug (Promega, https://www.promega.com.cn/). The NO production level was identified by measuring the nitrite concentration in the cell culture supernatants. In brief, the RAW264.7 cells (10⁵ cells/well) were stabilized with or without 1 μg/mL of LPS for 24 hours in the presence or absence of the test compounds. Then the cell culture supernatant (100 μL) was reacted with 100 μL of Griess reagent. The viability of the remaining cells after the Griess assay was detected by colorimetric assay using CCK8. The compounds were tested for NO assay at a concentration of 50 μmol/L. If the NO inhibition of compounds in RAW264.7 cells was more than 50% at 50 μmol/L, the IC₅₀ values were tested, and the concentrations were set as 100, 50, 20, 10, 5, 1, 0.1 μmol/L, respectively.

Supporting Information

Spectroscopic characterization processes (¹H NMR and ¹³C NMR) for compounds 1–16 are included in the Supporting Information ([Figs. S1–S32] [available in the online version]).

Conflict of Interest

None declared.

Ethical Approval

None declared.

^# These authors contributed equally to this work.

• References

• 1 Montaser R, Luesch H. Marine natural products: a new wave of drugs?. Future Med Chem 2011; 3 (12) 1475-1489
• 2 Wang Y, Xing M, Cao Q, Ji A, Liang H, Song S. Biological activities of fucoidan and the factors mediating its therapeutic effects: a review of recent studies. Mar Drugs 2019; 17 (03) 183
• 3 Cao W, Liu J, Dai Y, Zhou Y, Li R, Yu P. Bibliometric analysis of marine traditional Chinese medicine in Pharmacopoeia of the People's Republic of China: development, differences, and trends directions. Evid Based Complement Alternat Med 2022; 2022: 3971967
• 4 Wang T, Kuang W, Chen W. et al. A phase II randomized trial of sodium oligomannate in Alzheimer's dementia. Alzheimers Res Ther 2020; 12 (01) 110
• 5 Nguyen NT, Dang PH, Vu NXT, Le TH, Nguyen MTT. Quinoliniumolate and 2H-1,2,3-triazole derivatives from the stems of Paramignya trimera and their α-glucosidase inhibitory activities: in vitro and in silico studies. J Nat Prod 2017; 80 (07) 2151-2155
• 6 Nollet AJH, Koomen GJ, Grose WFA, Pandit UK. Application of NMR spectroscopy in distinguishing between N1- and N3-substituted 2,4-dioxo-1,2,3,4,-tetrahydropyrimidines. Tetrahedron Lett 1969; 10 (53) 4607-4608
• 7 Tian J, Yin Y, Sun H, Luo X. Magnesium chloride: an efficient 13C NMR relaxation agent for amino acids and some carboxylic acids. J Magn Reson 2002; 159 (02) 137-144
• 8 Malta BLF, Senra DJ, Tinoco WL, Medeiros EM, Antunes ACO. Chiral recognition of 2-hydroxypropyl-alpha-cyclodextrin towards DL-tryptophan. Lett Org Chem 2009; 6 (03) 258-263
• 9 Secrist JA, Shortnacy-Fowler AT, Bennett LL, Montgomery JA. Synthesis and biologic evaluation of 8-substituted derivatives of nebularine (9-β-D-ribofuranosylpurine). Nucleosides Nucleotides 1994; 13 (05) 1017-1029
• 10 Kline PC, Serianni AS. Chiral hydroxymethyl groups: ¹H NMR assignments of the prochiral C-5′ protons of 2′-deoxyribonucleosides. Magn Reson Chem 1990; 28 (04) 24-330
• 11 Soubias O, Jolibois F, Réat V, Milon A. Understanding sterol-membrane interactions, part II: complete 1H and 13C assignments by solid-state NMR spectroscopy and determination of the hydrogen-bonding partners of cholesterol in a lipid bilayer. Chemistry 2004; 10 (23) 6005-6014
• 12 D'Amore C, Di Leva FS, Sepe V. et al. Design, synthesis, and biological evaluation of potent dual agonists of nuclear and membrane bile acid receptors. J Med Chem 2014; 57 (03) 937-954
• 13 Kontiza I, Abatis D, Malakate K, Vagias C, Roussis V. 3-Keto steroids from the marine organisms Dendrophyllia cornigera and Cymodocea nodosa. Steroids 2006; 71 (02) 177-181
• 14 Iverson CD, Zahid S, Li Y, Shoqafi A, Ata A, Samarasekera R. Glutathione S-transferase inhibitory, free radical scavenging, and anti-leishmanial activities of chemical constituents of Artocarpus nobilis and Matricaria chamomilla. Phytochem Lett 2010; 3 (04) 207-211
• 15 Abegaz BM, Herz W. A nor-monoterpene from Artemisia schimperi . Phytochemistry 1991; 30 (03) 1011-1012
• 16 Katade SR, Pawar PV, Tungikar VB. et al. Larvicidal activity of bis(2-ethylhexyl) benzene-1,2-dicarboxylate from Sterculia guttata seeds against two mosquito species. Chem Biodivers 2006; 3 (01) 49-53
• 17 Dissanayake AA, Wagner CM, Nair MG. Chemical characterization of lipophilic constituents in the skin of migratory adult sea lamprey from the Great Lakes region. PLoS One 2016; 11 (12) e0168609
• 18 Kikuchi M, Yaoita Y, Kikuchi M. Monohydroxy-substituted polyunsaturated fatty acids from Swertia japonica . Helv Chim Acta 2008; 91 (10) 1857-1862
• 19 Zhang XJ, Zhong WM, Liu RX. et al. Structurally diverse labdane diterpenoids from Leonurus japonicus and their anti-inflammatory properties in LPS-induced RAW264.7 cells. J Nat Prod 2020; 83 (09) 2545-2558
• 20 Gao Z, Wang MX, Gao CL, Chen S, Li XW. New glycerolipids from the traditional Chinese Medicine of Syngnathus acus (Hai-Long). Chem Biodivers 2023; 20 (06) e202300616

Publikationsverlauf

Eingereicht: 15. November 2023

Angenommen: 24. April 2024

Artikel online veröffentlicht:
30. Mai 2024