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


 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.


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. 4Therefore, 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 IC 50 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 IC 50 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.

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 IC 50 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 IC 50 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 antiinflammatory candidates from MTCMs.

Experimental Section
General Experimental Procedures 1 H and 13 C 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 Â 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.Chemical Constituents of the MTCM of Pegasus laternarius Cuvier (Hai-E Yu) Sun et al.

Cytotoxicity Assay
This part was conducted by referring to our previous paper. 20The 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) À A (sample) ]/A (control) Â 100%.GraphPad Prism 7 (GraphPad Software, San Diego, CA, United States) was used to calculate IC 50 values.All data were expressed as the mean AE standard deviation of three independent experiments.

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

Table 1
Cytotoxicity of compound 1 from P. laternarius a Data were expressed as means AE standard deviation (n ¼ 3).b Sorafenib was used as a positive control.

Table 2
Nitric oxide inhibition of compound 10 in RAW264.7 cells a Data were expressed as means AE standard deviation (n ¼ 3).b Dexamethasone was used as a positive control.Pharmaceutical Fronts © 2024.The Author(s).