Da-Yuan-Yin is a Chinese traditional prescription.This study explores the therapeutic effects of the Da-Yuan-Yin decoction polyphenol fraction (DYY-4) on acute lung injury (ALI) in mice induced by lipopolysaccharide (LPS).n = 10) were orally administrated with DYY-4  or DXM (5 mg/kg), half an hour after LPS (2 mg/kg) instilled intratracheally. The protein content and the levels of inflammatory factors, the levels of complements, the mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the level of myeloperoxidase (MPO) and superoxide dismutase (SOD), the expression of the IkB kinase (IKK) and nuclear factor-kappa B (NF-κB), the lung wet-to-dry weight (W/D) ratio and lung tissue were evaluated, 24 h after LPS challenge. Network pharmacology predicted potential targets.The mice  decreased the lung W/D ratio, total protein concentration, the levels of C3, C3c and C5a, the levels of TNF-α, IL-6, and IL-1β, while increased the levels of IL-4 and IL-10. DYY-4 (60 mg/kg) decreased the levels of C5aR1, C5b-9 and COX-2 mRNA (p < 0.05), the levels of MPO and iNOS mRNA, the activation of the IKK/NF-κB pathway (p < 0.01), and increased the levels of IL-13 and SOD (p < 0.01). DYY-4 (60 mg/kg) relieved the lung tissue pathological changes and reduced the C3c deposition.DYY-4 (30, 60 mg/kg, Network pharmacology combined with animal experiments revealed the targets of DYY-4 alleviating ALI. Acute lung injury (ALI) originates from many direct and indirect injury factors. Its severe form is acute respiratory distress syndrome (ARDS). The main pathological features of ALI are diffuse lung cell injury, inflammatory cell infiltration in lung tissue and pulmonary edoema caused by pulmonary vascular injury  developed by Youke Wu, who was a famous doctor in the early Qing Dynasties, in Ancient China, is used to treat various types of inflammation . The corresponding fractions of each herbal medicine  were obtained according to the preparation of DYY-4.Chinese Medicine components of Da-Yuan-Yin, which were all planted in China, were purchased from Suzhou Tianling Traditional Chinese Medicine Beverage Co., Ltd, Jiangsu Province of China, in October 2013. Referring to Chinese Pharmacopoeia, Chinese Medicine components of Da-Yuan-Yin were authenticated by Ph.D. Chunyan Min, Suzhou Institute for Food and Drug Control, and Prof. Lili Hao, Soochow University, China. The voucher specimen (DYY-201309) is deposited in the Herbarium of Material Media, College of Pharmaceutical Science, Soochow University, China. Da-Yuan-Yin components (2000 g) were refluxed twice with water (20 L) for 2 h each time to obtain the Da-Yuan-Yin decoction water extract. After filtration, ethanol was added to obtain the supernatant of Da-Yuan-Yin decoction (DYY-CSL). DYY-CSL was added into the column loading-treated octadecyl silyl. DYY-4 was obtained by eluting with MeOH/H3/MeOH  to yield five fractions (Fr.1-5). Fr. 1 was purified with a ODS column and eluted with MeOH/H2O , further eluted by Preparative high-performance liquid chromatography (P-HPLC) with MeOH/H2O  to obtain compound 1 (8.0 mg) and compound 2 (14.0 mg). Fr. 2 was purified with a ODS column and eluted with MeOH/H2O  to obtain two fractions (Fr.2.1-2.2), then Fr.2.1 was eluted on Sephadex LH-20 with MeOH to obtain compound 3 (12.0 mg), then Fr.2.1 was eluted on P-HPLC with MeOH/H2O  to obtain compound 4 (18.0 mg) and compound 5 (20.0 mg). Fraction 3 was purified with a ODS column and eluted with MeOH/H2O  to obtain two fractions (Fr.3.1-3.2), then Fr.3.2 was eluted by P-HPLC with MeOH/H2O  to obtain compound 6 (4.0 mg). Fraction 4 was purified by P-HPLC with MeOH/H2O  to obtain compound 7 (3.0 mg). Fraction 5 was decolourized by acetone to obtain compound 8 (7.0 mg).DYY-4 (20.0 g) was fractionated on silica gel by CHClm/z range 150–2000. The principal component analysis (PCA) of DYY-4 was processed with simca-p13.0.Total polyphenol content of DYY-4 was measured by the Folin-Ciocalteu colourimetric method, and gallic acid was used as the standard  (16A6) rabbit mAb and IKKβ (L570) antibody rabbit mAb were purchased from Cell Signalling Technology Inc. . GAPDH mouse mAb was purchased from China Wuhan Sanying Biotechnology Co., Ltd. . Peroxidase-conjugated Affinipure goat Anti-mouse IgG (HtL) and Peroxidase-conjugated Affinipure goat Antirabbit IgG (HtL) were purchased from China Biyuntian Biotechnology Co., Ltd. . Sheep erythrocytes were collected in Alsevers’ solution. Anti-sheep erythrocyte antibody was obtained from rabbit antiserum and kindly provided by Prof. Yunyi Zhang . Rabbit erythrocytes were obtained from the ear vein of New Zealand white rabbits. The isotonic veronal-buffered saline (VBS2+) buffer contained 0.5 mmol/L Mg2+ and 0.15 mmol/L Ca2+. Heparin  was purchased from Shanghai Aizite Biotech Co. Ltd. All other reagents were of the highest quality available.LPS (No. 013M4029V) was purchased from Sigma-Aldrich Co., Ltd . Dexamethasone (DXM) acetate tablets (No. H33020822) were purchased from Zhejiang Xianju Pharmaceutical Co., Ltd. . Mouse TNF-α, IL-6 and IL-1β, IL-10, IL-4, IL-13 enzyme-linked immunosorbent assay ELISA (No. B1307233) kits were purchased from Shanghai Chuanfu Biotechnology Co., Ltd. . Mouse C3, C3c, C5a ELISA kits (No. E20130801A) and bicinchoninic acid  protein assay kit were purchased from Shanghai Yuanye Biotechnology Co., Ltd. . Mouse MPO, SOD and C5b-9, C5aR1 ELISA kits were purchased from Shanghai ZeYe Biotechnology Co., Ltd. . Polyclonal rabbit anti-human C3c complement (No. AB 204121) was purchased from Shanghai Ruiqi Biological Technology Co., Ltd. . Phospho-NF-κBp65 (Ser536) (93H1) rabbit monoclonal antibody (mAb), NF-κB p65 (D14E12) XPsample − ODsample background) ÷ OD100% lysis.The 1:60 diluted guinea pig serum was chosen to give submaximal lysis in the absence of complement inhibitors. Various dilutions of tested samples (200 μL) were mixed with 200 μL of guinea pig serum (Jackson Immuno Research), and 200 μL of sensitized erythrocytes (EAs) was added, then the mixture was incubated at 37 °C for 30 min. Optical density of the supernatant of the reacted mixture was measured at 405 nm (Labsystems Dragon). Results were indicated in the percentage of haemolytic inhibition . The mice were kept in a specific laboratory at 24 ± 1 °C and 60% relative humidity and received food and water n = 10): control group, DYY-4 group (mice were treated only with DYY-4 at 60 mg/kg), LPS group (the mice were treated only with LPS at 2 mg/kg to induce ALI), LPS + DYY-4 group  and LPS + DXM group . The mice were anesthetized with 20% urethane (4 mL/kg). LPS (2 mg/kg) was instilled intratracheally (i.t.) to induce ALI  for administration to mice. The mice were divided into seven groups , and its supernatants (serum) were stored at −80 °C for subsequent analysis. The levels of complement  in serum were quantified by the ELISA kit according to the manufacturer’s instructions strictly. The superior lobe of the right lung was homogenized to measure the levels of MPO and SOD by ELISA kits.Twenty-four hours after LPS challenge, the left lung was excised, blotted dry, and weighed to obtain the ‘wet’ weight, and then placed in an oven at 80 °C for 48 h to obtain the ‘dry’ weight. The ratio of the wet lung to the dry lung was calculated to assess tissue edoema. The right lung of mice was used to collect BALF, which was lavaged three times with 0.8 mL of autoclaved normal saline. The protein content and the levels of cytokines  in the supernatants of the BALF were analyzed by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. Blood samples were coagulated at room temperature for 10 min, and then centrifuged ) was used. 40 different cycling programs were established using real-time PCR which included run at 50 °C for 25 min and 75 °C for 5 min, hence at the end each analysis was run from 50-75 °C, respectively. Target cDNA levels were determined with the help of ΔCt method. Fold of suppression Expiration = 2Ct(control)-Ct(treatment). The primer sequences were designed as follows: iNOS: forward 5′-AGCAACTACTGCTGGTGGTG-3′; reverse 5′-TCTTCAGAGTCTGCCCATTG-3′; COX-2: forward 5′-CAGTTTATGTCTGTCCAGAGTTTC-3′; reverse 5′-CCAGCACTTCACCCATCAGTT-3′; β–action: forward 5′-CCCATCTATGAGGGTTACGC-3′; reverse 5′-TTTAATGTCACGCACGATTTC-3′. RT-qPCR was performed using the NovoScript® Plus All-in-one 1-st-Strand cDNA Synthesis SuperMix (gDNA Purge) kit (Novoprotein).Trizol  was used to extract the total mRNA in the lung, while the purity of mRNA preparation was checked by measuring the absorbance ratio at 260/280 nm. The experimental processes included 500 ng (1 μL) of total mRNA, 1 μL gDNA Purge, 2 × NovoScript 1g. Protein was separated by SDS/PAGE and transferred onto PVDF membrane . Membranes were probed with the following antibodies: Phospho-NF-κBp65 (Ser536) (93H1) rabbit mAb, NF-κB p65 (D14E12) XP® rabbit mAb, avidt-IKKα/β (Ser176/180) (16A6) rabbit mAb, IKKβ (L570) antibody rabbit mAb, IKKα antibody rabbit and GAPDH mouse mAb. The protein bands were measured using an ECL detection system , and semi-quantified using Image J software. The Western blot analysis results were normalized to the band intensity of GAPDH.Protein samples of lung tissue were prepared in RIPA lysis buffer with freshly added protease and phosphatase inhibitors  and then centrifuged for 10 min at 4 °C and 12000 Histological studies of the inferior lobe of right lung tissue were carried out by paraffin sections stained with haematoxylin and eosin (H&E). The 5 μm sections were deparaffinized, rehydrated, and incubated with rabbit anti-human C3c overnight at 4 °C. The complement deposits of the slices were visualized by using the chromogenic substrate solution 3, 3′-diaminobenzidine. All slides were constantly observed and imaged at an original magnification of 400×  was applied, while https://www.swisstargetprediction.ch), Pharm Mapper (http://www.lilab-ecust.cn/pharmmapper/) databases and the Uniprot databases (https://www.uniprot.org/) to predict potential targets of the main ingredients of DYY-4. We searched for disease-related targets by entering keywords ‘Viral pneumonia’ and ‘Lung injury’ into the GeneCards databases (https://www.genecards.org), OMIM databases (https://omim.org) and CTD databases (http://ctdbase.org/). The Venn diagram was used to map drug targets and disease targets to obtain the potential targets of compounds. The String databases (https://www.string-db.org/) was used to analyze the protein interaction relationship of the therapeutic targets, and the species was limited to ‘Homo sapiens’ (Combined score ≥ 0.9). The protein interaction relationship was imported into Cytoscape software 3.72 to construct protein-protein interaction (PPI) network. The ‘Network Analyzer’ tool was used to calculate the degree value of targets and the targets larger than the corresponding median values were recognized as crucial targets . The pathway terms with p < 0.05 were mapped to the KEGG human diseases in order to obtain the pneumonia-related pathways and construct component-core targets-pathway networks. We used PDB (http://www.rcsb.org/) to obtain the structural data of the top ten protein targets of DYY-4 based on PPI network analysis. The ten proteins were modified using AutoDock Tools for hydrogenation, ligands and water removal. Finally, we used AutoDock Vina 1.5.6 to perform molecular docking of the ten proteins with the compounds. We selected the receptors and ligands with strong binding energy and generated three-dimensional graphs in PyMOL to analyze their interactions.The crucial targets were used for Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment pathway analyses by using the David databases  δ: 7.72 , 7.31 , 6.80 , 6.48 , 6.34 , 5.37 , 3.04 , 2.68 ; 13C-NMR  δ: 192.1 (C-4), 165.4 (C-9), 164.1 (C-7), 157.5 (C-4′), 129.9 (C-1′), 128.4 (C-5), 127.6 , 114.9 , 113.5 (C-10), 110.3 (C-6), 102.4 (C-8), 79.2 (C-2), 43.5 (C-3). Compound 2 was identified as wogonin, EI-MS m/z: 285.07 [M + H]+. 1H-NMR  δ: 8.02 , 7.58 , 6.76 , 6.29 , 3.93 ; 13C-NMR  δ: 182.6 (C-4), 164.0 (C-5), 157.7 (C-7), 156.9 (C-2), 149.9 (C-9), 132.5 (C-4′), 131.7 (C-1′), 128.9 , 128.0 (C-8), 126.0 , 104.5 (C-3), 103.9 (C-10), 99.0 (C-6), 60.6 (OCH3). Compound 3 was identified as glycycoumarin, EI-MS m/z: 369.13 [M + H]+. 1H-NMR  δ: 7.96 , 7.14 , 6.58 , 6.38 , 6.36 , 5.22 2C=CHCH2), 3.83 , 3.36 2C = CH2CH), 1.78 2C = CHCH2), 1.68 2C = CHCH2); 13C-NMR  δ: 162.4 (C-2), 160.3 (C-4′), 158.5 (C-2′), 155.9 (C-7), 155.7 (C-9), 153.4 (C-5), 137.9 (C-4), 131.3 (C-6′), 130.8 (C-3″), 122.5 (C-3), 120.3 (C-2″), 119.4 (C-6), 114.1 (C-1′), 106.7 (C-5′), 106.5 (C-10), 102.6 (C-3′), 97.7 (C-8), 62.2 (OCH3), 24.5 (C-5″), 22.2 (C-1″), 16.6 (C-4″). Compound 4 was identified as magnolol, EI-MS m/z: 267.13 [M + H]+. 1H-NMR  δ: 7.00 , 6.99 , 6.87 , 6.00 , 5.10 , 3.32 ; 13C-NMR  δ: 152.8 , 138.3 , 131.3 , 130.0 , 127.9 , 125.9 , 115.8 , 115.2 , 38.8 . Compound 5 was identified as honokiol, mp 88.0-90.0 °C. EI-MS m/z: 267.13 [M + H]+. 1H-NMR  δ: 7.26 , 7.24 , 7.02 , 6.92 , 6.89 , 6.00 , 5.12 , 5.05 , 5.08 , 5.03 , 3.37 , 3.31 ; 13C-NMR  δ: 153.8 (C-2), 152.4 (C-4′), 138.3 (C-8), 137.2 (C-8′), 130.4 (C-5), 130.1 (C-6), 129.9 (C-4), 129.4 (C-2′), 127.8 (C-1′), 127.8 (C-6′), 127.4 (C-3′), 125.2 (C-1), 115.9 (C-9′), 115.2 (C-9), 115.2 (C-3), 114.4 (C-5′), 38.8 (C-7), 33.9 (C-7′). Compound 6 was identified as glycyrol. EI-MS m/z: 367.11 [M + H]+. 1H-NMR  δ: 10.81 , 10.02 , 7.72 , 7.17 , 6.96 , 6.78 , 5.21 2C=CHCH2), 3.90 , 3.33 2C = CH2CH), 1.77 2C = CHCH2), 1.65 2C = CHCH2);13C-NMR  δ: 159.5 (C-7), 158.2 (C-4), 157.5 (C-2), 156.9 (C-2′), 156.1 (C-4′), 153.8 (C-5), 152.9 (C-9), 130.9 (C-6′), 122.4 (C-3″), 120.5 (C-3), 119.7 (C-2″), 114.3 (C-6), 114.0 (C-1′), 102.2 (C-5′), 99.7 (C-10), 99.2 (C-3′), 98.5 (C-8), 62.4 (OCH3), 25.5 (C-5″), 22.0 (C-1″), 17.7 (C-4″). Compound 7 was identified as glyasperin F. EI-MS m/z: 355.11 [M + H]+. 1H-NMR  δ: 6.87 , 6.65 , 6.31 , 5.89 , 5.88 , 5.65 , 4.58 , 4.44 , 4.20 , 1.38 ; 13C-NMR  δ: 197.7 (C-4), 166.9 (C-7), 164.4 (C-5), 163.7 (C-9), 153.3 (C-4′), 150.7 (C-2′), 129.2 (C-6′), 129.0 (C-8′), 116.5 (C-7′), 115.5 (C-1′), 110.9 (C-3′), 108.4 (C-5′), 102.1 (C-10), 95.7 (C-6), 94.6 (C-8), 75.0 (C-9′), 70.0 (C-2), 46.7 (C-3), 26.4 . Compound 8 was identified as daucosterol which was confirmed by TLC comparison with an authentic sample.Data of the compounds were compared with the reported literature  was performed on mass spectrum data of DYY-4 and each herbal medicine . Chemical components of DYY-4 mainly came from HP-4, HQ-4, and CG-4 . Moreove50), was 0.051 ± 0.011 mg/mL and 0.063 ± 0.010 mg/mL for heparin, respectively.The anti-complementary activity of DYY-4, obtained in 50% haemolysis inhibition concentrations (CHp < 0.01). Similarly, DYY-4 at 60 mg/kg greatly decreased the level of MPO and increased the level of SOD (p < 0.01), as shown in As shown in p < 0.01) depicted in p < 0.01), as shown in p < 0.01) in BALF, while at the same concentration, the ratio of TNF-α/IL-10 and the expression of iNOS mRNA were greatly reduced, and these results were clearly shown in p < 0.05, Compared with vehicle-treated ALI model group, DYY-4 at 30 and 60 mg/kg markedly lowered the levels of TNF-α, IL-6, and IL-1β . However, DYY-4 at 60 mg/kg effectively reversed this trend compared to the LPS group (p < 0.01). In the ALI mice, the protein levels of both IKK and p-IKK showed pronounced upregulation in comparison to the control group (p < 0.01), but DYY-4 at 60 mg/kg significantly reduced expressions of IKK and p-IKK comparison to the LPS group  protected lung tissues with the reduction of the ratio of lung W/D and the protein content in BALF. DYY-4 regulated the levels of inflammatory factors  and the ratio of TNF-α/IL-10 in BALF. Meanwhile, DYY-4 (60 mg/kg) clearly reduced the levels of C3, C3c, C5a, C5aR1, and C5b-9 in serum and decreased the deposition of complement in lung tissue. DYY-4 (60 mg/kg) decreased the level of MPO and increased the level of SOD. Additionally, DYY-4 inhibited the expressions of COX-2 and iNOS mRNA. Importantly, DYY-4 (60 mg/kg) depressed the protein levels of IKK, p-IKK, the nuclear p65 and pp65. In conclusion, DYY-4 attenuated ALI induced by LPS


Penicillium setosum represents a Penicillium species recently described, with little up-to-date information about its metabolic and biological potential. Due to this scenario, we performed chemical and biological studies of P. setosum CMLD18, a strain isolated from Swinglea glutinosa (Rutaceae). HRMS-MS guided dereplication strategies and anti-leukemia assays conducted the isolation and characterization of six compounds after several chromatographic procedures: 2-chloroemodic acid (2), 2-chloro-1,3,8-trihydroxy-6- (hydroxymethyl)-anthraquinone (7), 7-chloroemodin (8), bisdethiobis(methylthio)acetylaranotine (9), fellutanine C (10), and 4-methyl-5,6-diihydro-2H-pyran-2-one (15). From the assayed metabolites, (10) induced cellular death against Kasumi-1, a human leukemia cell line, as well as good selectivity for it, displaying promising cytotoxic activity. Here, the correct NMR signal assignments for (9) are also described. Therefore, this work highlights more detailed knowledge about the P. setosum chemical profile as well as its biological potential, offering prospects for obtaining natural products with anti-leukemia capabilities. Penicillium genus has a worldwide distribution, including 483 species reported so far. Some of them are known to have a tremendous economic impact on human life due to the production of bioactive metabolites − 332.9817 .2-Chloroemodic acid (7): 1H NMR (Acetone-d6), δ (J/Hz): 12.46 (OH); 11.77 (OH); 7.19 , 6.78 ; 6.67 ; 6.07 ; 5.10 ; 4.15 . 13C NMR (DSMO-d6): 181,98 (C-10); 161.21 (C-8); 160.03 (C-1); 152.10 (C-6); 132.90 (C-10a); 132.10 (C-4a); 120.76 (C-5); 116.85 (C-8a); 114.60 (C-2); 112.59 (C-4); 62.07 (CH2). HRMS: m/z [M-H]- 319.0024 2-Chloro-1,3,8-trihydroxy-6-(hydroxymethyl)-anthraquinone (8): 1H NMR (CDCl3), δ (J/Hz): 12.00 ; 7.56 ; 7.42 ; 7.15 ; 2.30 (s). 13C NMR (CDCl3): 182.0 (C-10); 149.8 (C-3); 121.6 (C-4); 109.7 (C-5); 22.0 (Me). HRMS: m/z [M-H]- 303.0080 7-Chloroemodin (9): 1H NMR (CDCl3), δ (J/Hz): 6.57 ; 6.29 ; 5.80 ; 5.17 ; 4.69 , 3.03 ; 2.26 , 2.07 . 13C NMR (CDCl3): 170.2 (C-13), 164.5 (C-1); 139.8 (C-7); 137.8 (C-5); 109.6 (C-4), 105.9 (C-8); 71.9 (C-9); 70.6 (C-2); 60.4 (C-10); 40.6 (C-3); 21.1 (C-14); 14.8 (C-12). HRMS: m/z 557.1028 [M+Na]+ .Bisdethiobis(methylthio)acetylaranotine (10): 1H NMR (CDCl3), δ (J/Hz): 8.19 (1-NH); 7.53 ; 7.34 ; 7.16 ; 7.16 ; 6.16 ; 5.78 (13-NH); 5.23 ; 5,18 ; 4,40 ; 3.75 ; 3.27 ; 1.59 ; 1.58 . 13C NMR (CDCl3): 168.0 (C-12); 147.5 (C-15); 142.5 (C-2); 136.1 (C-8); 130.2 (C-9); 121.8 (C-6); 119.8 (C-7); 111.9 (C-16), 111.8 (C-4); 111.7 (C-5); 106.1 (C-3); 57.2 (C-11); 39.9 (C-14); 32.5 (C-10); 28.7 (C-14b); 28.6 (C-14a). HRMS: m/z 507.2750 [M-H]− . [α]20D = −79 .Fellutanine C (15): 1H NMR (CDCl3), δ (J/Hz): 5.75 ; 4.32 ; 2.34 ; 1.95 . 13C NMR (CDCl3): 164.7 (C-1); 158.1 (C-3); 116.6 (C-2); 65.9 (C-5); 29.2 (C-4); 23.0 (Me). HRMS: m/z 113,0601 [M+H]+ .4-Methyl-5,6-diihydro-2H-pyran-2-one , rice (M2), CYB (M3), YES liq (M4), YES + HBr liq (M5), and YES + Agar (M6). Methanolic micro-extracts were obtained from each medium, followed by analysis through UHPLC-HRMS. Extracts from M1 and M2 presented activity against the acute myeloid cell line (discussed in the section below). The MS data referring to the generated extracts were submitted to the GNPS platform [In order to verify how abiotic conditions  may induplatform  with theCompounds highlighted in yellow represent the chlorinated substances annotated through the GNPS; in red are those not annotated by the platform but identified as chlorinated substances because of their isotopic pattern . Green rPenicillium genus. For m/z 303.008, the substances 5-chloroemodine and 7-chloroemodine were found (error of ± 5ppm). The other chlorinated compounds present in this extract were annotated as 2-chloro-1,3,8-trihydroxy-6-hydroxymethyl-9,10-anthracenedione (7) (m/z 319.011) and 2-chloro-1,3,8-trihydroxy-2-carboxylicacid-9,10-dioxoanthracene (2) (m/z 332.993), as shown in In the bioactive methanolic fraction obtained from M2, three of those chlorinated compounds were detected, two of which were annotated by the GNPS. Regarding unannotated compounds, a search for accurate masses was performed in the Dictionary of Natural Products and Antibase (2012) databases, as well as in the current literature referring to the 10) (m/z 507.2750) was identified during the dereplication process using the Dictionary of Natural Products database (https://dnp.chemnetbase.com/), accessed on 5 August 2022. However, bisdethiobis(methylthio)acetylaranotine (9) (m/z 557.1028) was not found in any consulted database. After the employment of the purification and characterization procedures described below, its structure was defined. Additionally, these metabolites were found in a molecular family where no compounds were annotated  was found to be considered cytotoxic against acute myeloid cell lines (11), tricin (12), 5,6,2′-trimethoxyflavone (13), and hydroquinidine (14) were also annotated  [2) [7) [8), initially annotated as 5-chloroemodin, was very important once we could unequivocally identify its structure. Additionally, from the same fraction, we also purified another three substances not annotated by the GNPS platform: 4-methyl-5,6-diihydro-2H-pyran-2-one (15) [9) [10) [9) unrelated to the Penicillium genus.The methanolic extract was subjected to chromatographic procedures, such as silica column chromatography and high-pressure liquid chromatography, which led to the isolation of three chlorinated compounds: 7-chloroemodin (8) , 2-chlor8) [2) , and 2-c [2) [7) ,15. Theione (15) , bisdeth(15) [9) , and fel[9) [10)  . Nagarajan et al. (1968) reported (9), for the first time, isolated from Arachniotus aureus (Eidam) [Alternaria raphani. Gao et al. (2017) [Aspergillus sydowii. In the last two reports, we believe that the structural identification of (9) was performed probably by comparison with data reported by Nagarajan, R. et al. (1968) [While analyzing the spectroscopic data, we noticed some divergencies from our analysis and the data referring to compound , a sign at δ 6.57 was attributed to position H-7, while δ 6.29 was assigned to H-5. However, through our analysis of the COSY experiment, the sign at δ 6.29 corresponded to the hydrogen that couples with H-8 (δ 4.69) and H-9 (δ 5.80). Therefore, we believe this sign should be correctly assigned to H-7. Corroborating this proposition, in the HMBC experiment, we observed that H-7 (δ 6.29) presented a long-range correlation with C-8 and C-9 , (8), (9), and (10) and the cell death mechanism, Kasumi-1 and Jurkat cells were selected. A reduction in cell viability was observed for all compounds in both lineages after 48 h (7), (8), (9), and (10) (50 was used for both) (10) did not show a cytotoxicity effect on normal PBMC cells. In order to examine the effect of Compounds  C–F. Compor both) F. Stauroor both) G. Additi50 values at 24 hrs were used to verify the cell death mode using Annexin V-FITC and 7-AAD staining. Kasumi-1 showed predominant apoptosis staining (Anx-FITC+/7 AAD-), whereas Jurkat showed predominant double staining (late apoptosis and necrosis-like) after 24 h (Anx-FITC+/7 AAD+) (10), flow cytometry was used to quantify the cleaved form of caspases 3 (activated form of caspases). The treatment with (10) increased cleaved-caspase-3 (10) produced a reduction in cell proliferation (10) did not produce an increase in the expression of CD11b  A–C. In oaspase-3 A–D in boferation A–E and cof CD11b F.Penicillium genus, our recent survey on the anti-leukemia potential of 76 compounds related to this group of fungi [Although recently ,21, inte3) showed a strong uncoupling effect on mitochondrial respiration by inhibiting L1210 cells with an IC50 of 35 μM [10) showed potent activity against the HL-60 lineage, also an acute myeloid leukemia cell line (IC50 = 9.34 μM) [8) showed inhibitory activity against ACL, an ATP citrate lyase (IC50 = 12.6 μM) [7) was inactive  against Jurkat, K-562, and Hela [P. setosum present incipient chemical data [Among the compounds presented in of 35 μM . Prior t12.6 μM) . This enand Hela . Althougcal data . Thus, wP. setosum CMLD 18 growth in different culture media (M1-M6). Six compounds were isolated, which enabled their structure confirmation. The three isolated chlorinated anthraquinones (2), (7), and (8), as well as the diketopiperazine (9), have not been described for the genus Penicillium before, representing a contribution to the chemical description of both genus and species. Moreover, the isolation of (9) and the NMR 1D and 2D experiments allowed the unequivocal assignment of the NMR shifts for this compound. Based on our findings, it is also possible to suggest that this fungus has a biosynthetic apparatus able to produce chlorinated metabolites, as it was indicated through the dereplication analysis. Importantly, this work also demonstrated Fellutanine C (10) cytotoxic effect, inducing cell death with apoptotic features and cell arrest in Jurkat and Kasumi-1. This is the first description regarding anti-leukemia activity for this diketopiperazine up to date. Moreover, on the basis of the obtained diketopiperazines, molecular families in P.setosum CMLD 18 extracts, unknown compounds of the same group are likely biosynthesized by the microorganism. Due to the cytotoxic activity observed for Fellutanine C, further bioassays with other diketopiperazines produced by this fungus may unveil other metabolites with anti-leukemia potential.Our dereplication approaches allowed the description of 15 annotated/dereplicated compounds originating from


Correction: BMC Nephrol 23, 312 (2022)https://doi.org/10.1186/s12882-022-02932-2Table 2, Column 5, row Q2. It should be 1.01 [0.95-1.09].Following publication of the original article , the autThe original article has been corrected.


Cynanchum taihangense was carried out; twelve new seco-pregnane glycosides, cynataihosides I–L (1–4), M-T (7–14), and two known glycosides, glaucoside A (5) and atratcynoside F (6), were isolated from the 95% ethanol extract of Cynanchum taihangense. Two new aglycones were found among compounds 10, 11, 13, and 14. The structures of the glycosides were elucidated based on 1D and 2D NMR spectroscopic data, HR-ESI-MS analysis, and chemical evidence. The cytotoxicity of compounds against three human tumor cell lines  were evaluated by MTT assay. Compound 11 displayed significant cytotoxicity against THP-1 and PC-3 cell line with IC50 values of 5.08 and 22.75 μm, respectively. Compounds 3 and 14 exhibited moderate and selective cytotoxicity on HL-60 and THP-1 with IC50 values of 17.78 and 16.02 μm, respectively.For our interest in the potential biologically active and structurally unique steroidal glycosides, continued phytochemical investigation of  The 1H NMR spectrum of 1 displayed characteristic signals of steroidal glycoside with a 13,14:14,15-disecopregnane-type skeleton aglycone: two tertiary methyl groups  and 1.53 ), an oxygen-substituted methine proton , H-16)), one olefinic proton ), and one olefinic deshielded proton ). The 1H and 13C NMR signals were assigned by the HSQC spectrum. The HMBC cross-peaks of H-19/C-10, C-1, C-9, and C-5 indicated the connection of rings A and B. Furthermore, correlations of H-21/C-20, C-21, and C-17 were observed. HMBC correlations from δH 5.41 (H-6) to δC 28.2 (C-7), 37.3 (C-4), 39.2 (C-10), and 40.0 (C-8), and from δH 6.47 (H-18) to δC 114.1 (C-13), 23.6 (C-12), 55.9 (C-17), and 118.3 (C-20) proved the presence of the Δ5–6 and Δ13–18 olefinic groups. Interactions from δH 2.47 (H-8) and 5.43 (H-16) to δC 175.1 (C-14), though 2JCH and 3JCH, respectively, verified that C-16 of the furan ring and C-8 were connected through an ester carbonyl group -α-L-diginopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl. These were supported by spectroscopic analyses and acid hydrolysis. The linkage of the four sugars were determined by HMBC correlations from δH 4.96 (H-1′′′′ of β-glucopyranose) to δC 74.1 (C-4′′′ of α-diginopyranose), from δH 5.20 (H-1′′′ of α-diginopyranose) to δC 82.0 (C-4′′ of β-cymaropyranose), from δH 5.05 (H-1′′ of β-cymaropyranose) to δC 82.7 (C-4′ of β-cymaropyranose), and from δH 5.18 (H-1′ of β-cymaropyranose) to δC 85.2 (C-3) ; H-17/H-16 and H-21 (α-orientated); H-1′/H-3, H-5′, and C-3′-OCH3 (α-orientated); H-1′′/H-5′′ (α-orientated); H-3ʹʹʹ/H-5ʹʹʹ (β-orientated); H-1ʹʹʹʹ/ H-3ʹʹʹʹ, H-5ʹʹʹʹ (β-orientated) (1 was established as glaucogenin A 3-O-β-D-glucopyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside, and named cynataihoside I.Cynataihoside I (1) was afforded as light yellow amorphous gum, + ion in the HR-ESI-MS data. Detailed analysis of 1H and 13C NMR spectra revealed that the structure of 2 was similar to that of compound 1, and the difference was only in the second sugar, which was attached to the first cymarose. A comparison of the NMR data of 2 with those of cynataihoside C + ion observed in its HR-ESI-MS spectrum. The 1H NMR of 4 showed the presence of two tertiary methyl groups  and 1.54 ), one olefinic proton ), one olefinic deshielded proton ) connected with the trisubstituted double bond, and two oxygen-substituted methine protons  and 5.43 ). The data and corresponding 13C NMR , 5.51 , 5.12 , and 4.74 ), indicating the existence of four β-linked sugars. The 1H and 13C NMR signals were assigned with the help of an extensive study of HMQC, HMBC, and NOESY experiments. The existence of β-D-oleandropyranosyl, β-D-digitoxopyranosyl, β-D-thevetopyranosyl, and β-D-glucopyranosyl units was confirmed by a further comparison of the sugar chain spectroscopic data of 4 with those of β-D-oleandropyranosyl, β-D-digitoxopyranosyl, and β-D-glucopyranosyl in cynataihoside E + ion in HR-ESI-MS  indicated that the molecular formula of 7 was C28H40O8. The 1H NMR spectrum of 7 showed two tertiary methyl singlets at δH 0.84  and 1.56 , two oxygen-substituted methine protons at δH 4.25 , 3.78 , 4.07 , and 4.01 , and one olefinic proton at δH 5.45 . In the 13C NMR spectrum, four olefinic carbons at δc 140.1 (C-5), 120.2 (C-6), 103.4 (C-8), and 152.8 (C-14) were observed. The NMR data were basically consistent with those of a known steroidal aglycone 2α-hydroxyanhydrohirundigenin in sublanceoside E4 + . The 1H NMR spectrum of 9 showed the signals corresponding to tertiary methyl groups  and 1.56 ) and an olefinic proton ). In 13C NMR spectrum, 21 carbon signals were ascribed to the aglycone with a seco-pregnane skeleton. The 1H and 13C NMR data  and δH 1.52 ), two methoxyl methyl signals  and δH 3.45 ), and two anomeric protons signals  and 5.05 ) in the 1H NMR spectrum revealed that 9 had two β-form deoxysugars units. Comparing the 13C NMR data with those of the first cymarose in sublanceoside G4 + ion peak at m/z 669.3246 [M + Na]+  in HR-ESI-MS. The 1H NMR data revealed the presence of tertiary methyl groups  and δH 1.56 ), one oxygen-substituted methine proton ), four oxygen-substituted methylene protons , 3.78 , 4.04 , and 3.98 ), and one olefinic proton ). In combination with analysis of the HMQC and HMBC spectra, the 1H NMR and corresponding 13C NMR spectral data , 2.95 (H-4a), 2.37 (H-1a), and 2.33 (H-1b) to δC 205.7, as well as from δH 0.77 (H-19) to δc 137.6 (C-5), 51.5 (C-1), and 44.5 (C-9). Furthermore, correlations between δH 2.34 (H-9) and δC 102.5 (C-8), 153.4 (C-14) established the connectivity between the rings B and C. In addition, the connection of the three furan rings and their connection to the C ring were demonstrated by HMBC correlations of δH 1.56 (H-21) to δC 118.0 (C-20) and 63.3 (C-17), of δH 4.25 (H-15a) to δC 84.3 (C-16), 63.3 (C-17), and 118.0 (C-20), of δH 3.98 (H-18b) to δC 53.6 (C-13), 63.3 (C-17), and 118.0 (C-20), and of δH 4.04 (H-18a) to δC 31.4 (C-12) and 153.4 (C-14). The α-orientation of H-3 was suggested by the large coupling constant between H-3 ) and H-4  and 2.63 ), as well as the NOE correlations for H-3/H-1b (δH 2.33) and H-4a (α-orientated), and for H-19/H-4b (β-orientated). In addition, the NOE correlations for H-17/Me-21 indicated their α-orientation  and 5.06 , which implied the presence of two deoxysugar units with β-linkages. The NMR data of sugar moiety were basically consistent with those of 9 except the proton signals due to the anomeric atom connected with the aglycone, which were identical to the data of β-D-cymarose linked with the aglycone of cynataihoside F + ion, based on which the molecular formula was calculated as C34H48O11. A detailed comparison between the 1H and 13C NMR data of 11 and those of 10 in β-D-cymaropyranosyl connected with their C-3 hydroxyl group, but different terminal sugar. From the 13C NMR spectra, the outer deoxysugar units were in good agreement with the terminal β-D-digitoxopyranosyl in sublanceoside B2 + . From its 13C NMR data  and 5.34  and their corresponding anomeric carbon signals at δc 97.4 and 100.5, respectively, revealed that 12 had two sugars with β-linkages. Its NMR data of sugar moiety were a close resemblance to those of 11 except for a small increase of chemical shifts at C-1′ and C-2′ of β-D-cymaropyranosyl. Considering that the change in chemical shifts was probably caused by different aglycones linked to sugars, it was speculated that they had the same sugar units. Cymaroses and digitoxose were afforded in the acid hydrolysis experiment. In addition, the existence of β-D-cymaropyranosyl was further confirmed by comparison with the corresponding spectroscopic data of 9. The linkage positions and sequence of the two sugars were ascertained by HMBC correlations from δH 5.34  to δc 82.7 (C-4′) and from δH 5.18  to δc 84.5 (C-3) -β-D-digitoxopyranoside, and named cynataihoside R.Cynataihoside R  . The NOE13) was isolated as white amorphous powder. HR-ESI-MS of 13 showed a [M + Na]+ ion peak at m/z 975.46006  that accounted for a molecular formula of C48H72O19. Inspection of the NMR data of 13 in 10. The large coupling constant between H-3 ) and H-4  and 2.62 ), as well as the NOE correlation for H-3/H-1b (δH 2.33) and H-4a (α-orientated), and for H-19/H-4b (β-orientated) confirmed the α-orientation of H-3. Thus, 13 was composed of cynataihogenin P and a sugar chain with linked C-3 group. The 1H NMR spectrum of 13 showed three secondary methyl and three methoxyl methyl signals of deoxysugars and four anomeric proton signals at δH 5.26 , 5.05 , 4.93 , and 4.99 , which revealed the existence of four sugars with three β- and one α- form. According to the comparison of its 13C NMR data to those in cynataihoside F + .Cynataihoside I (2): Light yellow amorphous gum (MeOH), + .Cynataihoside J (3): Light yellow amorphous gum (MeOH), + .Cynataihoside K (4): Light yellow amorphous gum (MeOH), + .Cynataihoside L (7): White amorphous gum (MeOH), + .Cynataihoside M (8): White amorphous gum (MeOH), + .Cynataihoside N (9): White amorphous powder, + .Cynataihoside O (10): White amorphous powder, + .Cynataihoside P (11): White amorphous powder, + .Cynataihoside Q (12): White amorphous powder, + .Cynataihoside R (13): White amorphous powder, + .Cynataihoside S (14): White amorphous powder, + .Cynataihoside T (7 (15 mg), 8, 9, 11, 12 (2 mg), and 10 (30 mg), in MeOH  were heated separately with 0.1 N H2SO4  at 50 °C for 30 min. Then the mixture was diluted with water  and concentrated to . After that, the solution was kept at 60 °C for a further 30 min, followed by neutralizing with aqueous saturated Ba(OH)2, and the precipitates were filtered off.Acid hydrolysis was prepared by following the methods described in our previous report ,12,13. E1 (5 mg), 2 (2 mg), 3 (5 mg), 4 (2 mg), 13 (18 mg), and 14 (18 mg) in 50% 1,4-dioxane  was heated separately with 0.5 N H2SO4  at 95 °C for 3 h. Reaction mixture was cooled, neutralized with aqueous saturated Ba(OH)2, and the precipitates were filtered off [Each solution of ered off .2Cl2 and H2O. The aqueous layer was concentrated and further analyzed by TLC. The known steroidal glycosides were hydrolyzed to give deoxysugars to make a comparison. Three solvent systems, CHCl3/CH3OH (9:1 v/v), CH2Cl2/C2H5OH (9:1 v/v), and PE/acetone (3:2 v/v), were performed to reference the Rf values of oleandrose, digitoxose, cymarose, and diginose according to the literature [1–3 and 7–14, the corresponding deoxysugars Rf values were basically identical to the corresponding ones mentioned in the reports.The filtrate was partitioned between CHterature . In the The configurations of the monosaccharides were identified by the comparison of their spectral data with those in the literature and HPLC analysis.1–4, 8, and 9–14 (24 h after dissolution) were subjected to HPLC analysis under the following conditions: column, Shodex Asahipak NH2P-50 4E column ; flowrate, 0.8 mL/min; solvent, MeCN/H2O (3:1 v/v); detection, OR (Jasco OR-4090) detector. D-glucose  in compounds 1–4, 13, and 14, D-digitoxose in compounds 1–3, 11, and 12, L-diginose in compounds 1–3, and D-cymarose in 1–2 and 8–14 were identified.The concentrated aqueous layer of 3, 4, 7, and 8 was determined to be D-form by the spectroscopic data. Those oleandroses were all directly linked to their respective aglycones so that the absolute configurations could be suggested by the NMR data.Since the impurities produced during the hydrolysis process interfered with the detection, the absolute configuration of oleandrose in compounds 4, as well as the deficiency of authentic samples of thevetose, the monosaccharides of 4 were not detected completely. The configurations of the deoxysugars were further identified by the comparison of those spectral data with those in the literature.Unfortunately, owing to the failure of hydrolysis of compound -60 (human leukemic promyelocytic cell), THP-1 (human acute monocytic leukemia cell line), and PC-3 (prostate cancer cell line). Cell growth inhibition assay was performed as reported previously [An MTT assay was used to determine the cytotoxicity effect of the compounds on three cultured human cancer cell lines, including HLeviously . 5-Fluor1–4 and 7–14), were isolated from the ethanolic extract of C. taihangense by multiple separation methods. All compounds were reported for the first time from the plant. Among them, compounds 10, 11, 13, and 14 contained two new seco-pregnane-type aglycones. In addition, the cytotoxicity of the glycosides against HL-60, THP-1, and PC-3 cell lines were evaluated. Compound 11 displayed significant cytotoxicity against THP-1 and PC-3 cell line. Compounds 3 and 14 exhibited moderate and selective cytotoxic activity on HL-60 and THP-1.Fourteen seco-pregnane steroidal glycosides, including twelve new ones (


Furthermore, automated docking simulation studies were also performed to support the results obtained.Herein, a distinctive dihydroxy ionic liquid ([Py-2OH]OAc) was straightforwardly assembled from the sonication of pyridine with 2-chloropropane-1,3-diol by employing sodium acetate as an ion exchanger. The efficiency of the , MCF-7 (breast cancer), and A549 (lung cancer) by employing the MTT assay, and the findings were evaluated with the reference Doxorubicin. The results demonstrated that the majority of the developed products had potent activities at very low doses. Compounds comprising rhodanine ( Rhodanine and related compounds are known as scaffolds or templates to discover and develop drugs . In the The carcinogenic activities of rhodanines have yet to be thoroughly addressed. Aldose reductase (AR) has lately been reported to be an essential regulator of signaling pathways comprising oxidative stress-induced inflammatory diseases, notably cardiovascular diseases, cancers, sepsis, and asthma . AR accuOn binding to synthetic or natural ligands, peroxisome proliferator-activated receptors (PPARs) have displayed transcription mediators that control gene production and inhibition . PPARs aThe melting points were determined employing the Electrothermal IA9100 melting point instrument (UK), and the findings were given uncorrected. The IR spectra (KBr) from a Perkin-Elmer Spectrum One spectrometer were recorded. The NMR spectra were obtained by employing a Bruker AV instrument with 400 MHz. A Bruker Daltonics microTOF spectrometer was used to obtain high-resolution mass spectra (HRMS). The purity of substances was determined using analytical thin-layer chromatography (TLC) on an EM silica gel F254 sheet (0.2 mm). The sonication reactions were carried out in an SY5200DH–T ultrasound cleaner.1). Then, to the residue of 1 in EtOH (15.0 mL), NaOAc  was introduced. The resulting solution was sonicated at 70 °C for 30 min, following which the solvent was removed under reduced pressure to yield the corresponding IL (2) with an almost quantitative yield.In a closed vial, a mixture of pyridine  and 2-chloropropane-1,3-diol  was sonicated at 70 °C for 3 h. The obtained crude product was purified by recrystallization from acetonitrile to produce the pure product (99%) as white crystals (−1 (C=C). 1H NMR : δ = 9.17 , 8.49 , 8.20–8.17 , 5.59 , 4.81–4.80 , 3.74–3.69 , 2.23 ppm . 13C NMR : δ = 173.0 (C=O), 148.8, 147.3, 132.4 (Ar-C), 68.2 (CH), 60.0 (CH2), 26.3 ppm (CH3). HRMS, m/z: 236.0886 (M + Na). Anal. Calcd for C10H15NO4: C, 56.33; H, 7.09; N, 6.57%. Found: C, 56.39; H, 6.97; N, 6.51%.White crystals; Yield 99%; mp 88–90 °C. IR (KBr): 3412-3108 (OH), 1683, (C=O), 1598 cm2O-DABCO (60–40%-1.0 mmol), 1.0 mmol of carbonyl compounds  was added. The resulting solution was sonicated at 30 °C for 5 min (TLC). After cooling to room temperature, the obtained crystals were filtered off, dried, and crystallized from a proper solvent.To a mixture of appropriate active methylene (1.0 mmol) in IL-HH-indene-1,3(2H)-dione (5a)2-(3-Methyl-4-oxo-2-thioxothiazolidin-5-ylidene)-1−1 (C=S). 1H NMR : δ = 7.92–7.90 , 3.35 ppm . 13C NMR : δ = 195.5 (C=S), 192.3, 167.5 (C=O), 148.2, 148.1, 139.8, 135.7, 128.2 (Ar-C), 34.1 ppm (CH3). HRMS, m/z: 311.9764 (M + Na). Anal. Calcd for C13H7NO3S2: C, 53.97; H, 2.44; N, 4.84%. Found: C, 53.92; H, 2.48; N, 4.78%.Yellow crystals (EtOH); Yield 99%; mp 187–188 °C. IR (KBr): 1739, 1678 (C=O), 1593, (C=C), 1427 cmH-indene-1,3(2H)-dione (5b)2-(3-Ethyl-4-oxo-2-thioxothiazolidin-5-ylidene)-1−1 (C=S). 1H NMR : δ = 8.07–7.93 , 4.21–4.16 , 1.22–1.18 ppm . HRMS, m/z: 325.9924 (M + Na). Anal. Calcd for C14H9NO3S2: C, 55.43; H, 2.99; N, 4.62%. Found: C, 55.46; H, 2.92; N, 4.57%.Yellow crystals (dioxane); Yield 94%; mp 165–166 °C. IR (KBr): 1741, 1677 (C=O), 1601, (C=C), 1442 cmH-inden-2-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (5c)2-. 1H NMR : δ = 13.61 , 7.67–7.58 , 4.60 ppm . HRMS, m/z: 355.9663 (M + Na). Anal. Calcd for C14H7NO5S2: C, 50.45; H, 2.12; N, 4.20%. Found: C, 50.49; H, 2.10; N, 4.17%.Yellow crystals (dioxane); Yield 97%; mp 251–252 °C. IR (KBr): 3356-3012 (OH), 1739, 1708, 1685, (C=O), 1593 (C=C), 1440 cmH-indene-1,3(2H)-dione (5d)2-(3-Allyl-4-oxo-2-thioxothiazolidin-5-ylidene)-1−1 (C=S). 1H NMR : δ = 7.72-7.61 , 5.88–5.73 , 5.19–5.10 , 3.79–3.75 ppm . HRMS, m/z: 337.9924 (M + Na). Anal. Calcd for C15H9NO3S2: C, 57.13; H, 2.88; N, 4.44%. Found: C, 57.17; H, 2.80; N, 4.38%.Yellow crystals (EtOH); Yield 99%; mp 145–146 °C. IR (KBr): 1739, 1678 (C=O), 1593, (C=C), 1427 cmH-inden-2-ylidene)imidazolidine-2,4-dione (8a)5-. 1H NMR : δ = 11.01 , 8.70 , 7.92–7.90 ppm . 13C NMR : δ = 191.0 (C=S), 167.6, 160.1 (C=O), 134.5, 131.5, 127.8, 127.7, 123.2 ppm (Ar-C). HRMS, m/z: 242.0325 (M). Anal. Calcd for C12H6N2O4: C, 59.51; H, 2.50; N, 11.57%. Found: C, 59.54; H, 2.46; N, 11.50%.Brown crystals (dioxane); Yield 96%; mp 202–204 °C. IR (KBr): 3311, 3245 (NH), 1741, 1688 cmH-inden-2-ylidene)-1-methylimidazolidine-2,4-dione (8b)5-. 1H NMR : δ = 10.18 , 7.73–7.71 , 2.90 ppm . HRMS, m/z: 279.0384 (M + Na). Anal. Calcd for C13H8N2O4: C, 60.94; H, 3.15; N, 10.93%. Found: C, 60.91; H, 3.19; N, 10.87%.Brown crystals (dioxane); Yield 96%; mp 192–193 °C. IR (KBr): 3197 (NH), 1732, 1672 cmH-indene-1,3(2H)-dione (8c)2-(5-oxo-2-Thioxoimidazolidin-4-ylidene)-1−1 (C=S). 1H NMR : δ = 11.59 , 11.40 , 7.86–7.75 ppm . HRMS, m/z: 280.9992 (M). Anal. Calcd for C12H6N2O3S: C, 55.81; H, 2.34; N, 10.85%. Found: C, 55.77; H, 2.39; N, 10.81%.Yellow crystals (dioxane); Yield 95%; mp 222–224 °C. IR (KBr): 3354–3112 (NH), 1738, 1673 (C=O), 1449 cmH-indene-1,3(2H)-dione (8d)2-(2-Imino-3-methyl-5-oxoimidazolidin-4-ylidene)-1−1 (C=O). 1H NMR : δ = 10.00 , 8.40 , 7.86–7.84 , 2.79 ppm . HRMS, m/z: 279.0541 (M + Na). Anal. Calcd for C13H9N3O3: C, 61.18; H, 3.55; N, 16.46%. Found: C, 61.10; H, 3.58; N, 16.41%.Brown crystals (EtOH); Yield 95%; mp 169–170 °C. IR (KBr): 3318, 3109 (NH), 1741, 1679 cmH-pyrazol-4-ylidene)-1H-indene-1,3(2H)-dione (10a)2-. 1H NMR : δ = 11.30 , 7.67–7.58 , 2.20 ppm . HRMS, m/z: 240.0536 (M). Anal. Calcd for C13H8N2O3: C, 65.00; H, 3.36; N, 11.66%. Found: C, 64.92; H, 3.39; N, 11.60%.Red crystals (dioxane); Yield 95%; mp 195–196 °C. IR (KBr): 3389 (NH), 1738, 1671 cmH)-ylidene)-1H-indene-1,3(2H)-dione (10b)2-(5-Oxo-3-phenylisoxazol-4(5−1 (C=O). 1H NMR : δ = 7.98 , 7.88 , 7.71 , 7.39 ppm . HRMS, m/z: 303.0534 (M). Anal. Calcd for C18H9NO4: C, 71.29; H, 2.99; N, 4.62%. Found: C, 71.22; H, 3.04; N, 4.56%.Brown crystals (DMF); Yield 98%; mp 240–242 °C. IR (KBr): 1745, 1730 cmH-indene-1,3(2H)-dione (12a)2-(4-Oxochroman-3-ylidene)-1−1 (C=O). 1H NMR : δ = 8.07–7.96 , 7.79 , 7.47 , 7.30 , 4.69 ppm . HRMS, m/z: 290.0582 (M). Anal. Calcd for C18H10O4: C, 74.48; H, 3.47%. Found: C, 74.51; H, 3.43%.Brown crystals (dioxane); Yield 94%; mp 311–313 °C. IR (KBr): 1738, 1698 cmH-indene-1,3(2H)-dione (12b)2-(6-Fluoro-4-oxochroman-3-ylidene)-1−1 (C=O). 1H NMR : δ = 7.89–7.79 , 7.34–7.30 , 7.08 , 4.77 ppm . 13C NMR : δ = 194.2, 180.0 (C=O), 149.7 , 147.2, 143.9, 139.6, 137.8, 125.3 , 122.2, 121.6, 115.4, 113.8, 108  (Ar-C), 64.7 ppm (CH2). 19F NMR : δ = −125.91 ppm. HRMS, m/z: 307.0409 (M-H). Anal. Calcd for C18H9FO4: C, 70.13; H, 2.94%. Found: C, 70.17; H, 2.89%.Brown crystals (dioxane); Yield 98%; mp 277–279 °C. IR (KBr): 1741, 1726 cm50 is the concentration of the substance that delivers a 50% growth inhibitory value.Cell lines: ATCC provided the investigated cells; Colo-205 (colon cancer), MCF-7 (breast cancer), and A549 (lung cancer) via Holding firm for bio-supplies and vaccinations (VACSERA) in Cairo, Egypt. The MTT -2,5-diphenyl tetrazolium bromide) analyses were employed to evaluate the compounds’ cytotoxic effects. In 96-well plates, cells (1 × 104/well) were plated in 100.0 mL of medium/well. After overnight incubation, the cells were handled with various drug doses in RPMI 1640 with 10% FBS for 24 h. MTT was then introduced, and the cells were maintained for four hours. MTT-formazan was prepared in 150.0 mL DMSO and stirred for 10 min. At a test wavelength of 490 nm, the absorbance was determined using an ELISA reader. Every experiment was carried out three times in total. The IC®) version 2014.09. The X-ray crystallographic structures of the target AKR1B10 and PPARγ from the protein data bank  were obtained from the protein data bank. The target compounds were constructed into a 3D model using the builder interface of the MOE® program after checking their structures and the formal charges on atoms by 2D depiction.Automated docking simulation studies were performed using Molecular Operating Environment (MOE2) was assembled through the reaction of IL (1) with sodium acetate as the anion exchanger (2) was achieved by HRMS and NMR spectroscopies (See 2) was employed as a promoter in the DABCO-catalyzed Knoevenagel reactions between various cyclic active methylene derivatives and ninhydrin  and 3-methylrhodanine  as model reactants to evaluate the efficiency of the constructed ionic liquid [Py-2OH]OAc , a moderate yield of the hitherto unreported 2-(3-methyl-4-oxo-2-thioxothiazolidin-5-ylidene)-1H-indene-1,3(2H)-dione (5a) was obtained  while the targeted product (5a) was not detected in the reaction profile (TLC) when performing the reaction in an aqueous medium (2O-DABCO (3 g:3 mL:1.0 mmol), the reaction profile was quite clear, and the Knoevenagel product (5a) was generated as the sole product (6)  albeit with a prolonged time (2) utilized in the process (2O was 60:40% (4a (1.0 mmol), ninhydrin (1.0 mmol), and DABCO (1.0 mmol) in H2O-IL solvent (40–60%) at 30 °C for 5 min was identified as the optimal condition for the model reaction  , entry 36)   underwent rapid Knoevenagel reactions with ninhydrin (3) to produce hitherto unknown compounds  in nearly quantitative yields  and electron-attracting motifs  were amenable to the reaction  and afforded Knoevenagel condensation products (8a–d) in almost quantitative yields , the reactions also proceeded effectively and provided the targeted products with excellent yields  performed effective and potent reactions with ninhydrin, yielding the condensed compounds 12a and 12b in 94 and 98% yields, respectively  were evaluated utilizing IR, NMR spectroscopy as well as HRMS  in superior yield  reuse, the synthesis of 5a was selected for this assignment. The reusability of the IL (2) was studied by repeating the model reaction under optimal conditions  at optimum conditions and could be recycled up to seven cycles without a considerable loss of potency , resulting in intermediate C. Finally, DABCO-quaternary ammonium proton facilitated the removal of a water molecule and the generation of the olefinic double bond  were evaluated for their in vitro antitumor effects against an array of human cancer cell lines, including Colo-205 (colon cancer), MCF-7 (breast cancer), and A549 (lung cancer). 50 (compound concentration that causes a 50% reduction in cell proliferation) measurements for the produced compounds employing Doxorubicin as a reference drug. The antitumor activity results suggested that the majority of the investigated derivatives demonstrated moderate to excellent potency, with IC50 values ranging from 0.04 to 11.81 μM. Derivatives 5, 8, and 12 demonstrated the most antiproliferative potential toward the three cell lines (IC50: 0.04–5.51 M). Of these, the efficiency of compound 5c was approximately 3-fold more than that of Doxorubicin. Further, derivatives 10a and 10b were especially effective at inhibiting Colo-205 cell proliferation.Utilizing the MTT test , the new5–12 could be categorized into four classes: A (5a–d), B (8a–d), C , and D . Regarding series A, derivative 5c with an acetic acid motif was observed to be the most potent molecule of all series against the three investigated cells (IC50 varied between 0.04 and 0.06 μM) and demonstrated superior in vitro inhibition effects than Doxorubicin. Mechanistically, such carboxylic acids suppressed tube-forming activity induced by vascular endothelial growth factor (VEGF) and thus reduced the tumor growth [5d exhibited similar antitumor activity to the reference drug (IC50 varied between 0.15 and 0.21 μM) against the three examined cell lines, confirming the beneficial effect of the allyl group in the treatment of cancer [5a or ethyl, 5b) to the rhodanine nucleus reduced the in vitro inhibitory effects against three cell lines marginally. When the rhodanine motif was replaced with an imidazole nucleus, the resultant series (class B) effectively contributed to the anticancer activity of the assessed strains. In this series, introducing an alkyl substituent such as methyl (8b or 8d) instead of hydrogen atom (8a or 8c) resulted in a slight diminishing in the inhibitory activities while the introduction of the thione motif (8c) resulted in a remarkable improvement in the cytotoxicity, particularly toward Colo-205 with IC50 of 0.89 μM. This might be due to the existence of a polar thiocarbonyl moiety that may enhance their interaction with the hydrophilic binding sites [50 (10a (class C) showed significantly high activity (for A549 and Colo-205), which was slightly decreased upon being replaced by an isoxazole ring (10b). This might be attributed to the introduction of a more bulky moiety such as the benzene ring (in derivative 10b), leading to an enhancement in the steric hindrance and therefore reducing the interaction to the target [12a and 12b (class D) represented a noteworthy exception, demonstrating comparable antiproliferative potency to the reference drug, indicating that the presence of a chromone ring might considerably enhance potency toward the examined strains. In this series, replacing the hydrogen atom (12a) with a substituent such as a fluoro (F) atom (12b) resulted in a significant increase in activities , respectively, by Molecular Operating Environment (MOE®) version 2014.09  or chromane (12) motifs are necessary for improving the cytotoxic activities. Of them, compound 5c was three times more potent than Doxorubicin, indicating that it could serve as a promising and new candidate for further research. The newly synthesized compounds are presently being studied in our lab as potential precursors to design interesting spiro compounds.An eco-friendly, promising, and recyclable ionic liquid was synthesized for the first time from pyridine and 2-chloro-1,3-propanediol using sodium acetate as an ion exchanger. Under ultrasonic irradiation, the designed ionic liquid was employed as a promoter for DABCO-catalyzed Knoevenagel reactions in water. Under these conditions, a new series of Knoevenagel products were obtained in excellent yields. This approach offers significant features including gram-scale practicality, superior environmental criteria, simplicity of work-up, and facile recycling of ionic liquid, making it a sustainable and green approach for synthesizing multifunctional Knoevenagel products. The antiproliferative potencies of the newly obtained compounds were evaluated against three tumor cells viz Colo-205, MCF-7, and A549. The results demonstrate that all of the evaluated compounds displayed various activities for the cancer cell lines studied. Furthermore, SAR assessment showed that compounds containing rhodanine (


Their structures and absolute configurations were determined by extensive spectroscopic analysis, QM-NMR, and TDDFT-ECD calculations. In addition, compound 3 exhibited better anti-inflammation activity compared to the indomethacin as a positive control in zebrafish at 20 μM.Five new cembranes, named sarcoeleganolides C–G ( Sarcophyton (family Alcyoniidae) are widely regarded as an important source of cembranoids +, implying seven degrees of unsaturation. The 1D NMR data , six methylenes , four methines (two olefinic and two oxygenated), and six quaternary carbons . These data indicate that compound 1 was a cembrane-type diterpenoid.Sarcoeleganolide C  and C-4 (δC 61.5) and HMBC correlations from H3-18 to C-3, C-4, and C-5. Based on the above data, together with the key HMBC correlation from H3-19 to C-7, C-8, and C-9; H3-20 to C-11, C-12, and C-13; H3-17 to C-1, C-15, and C-16; H-14a (δH 2.74) to C-1, C-2, and C-15 the connection of the carbon skeleton was permitted. Thus, compound 1 was deduced as a cembranoid possessing a trisubstituted epoxide. In the NOESY spectrum of 1 (3-19/H-6a (δH 2.20), H3-20/H-10a (δH 2.26) indicate that the Δ7 and Δ11 double bonds could be of an E-configuration. The NOESY correlation of H-2/H3-18 indicates that these protons were on the same side. In addition, considering the geometry of the 3-(E)-olefin in co-isolates, the epoxide of 1 should be in an anti-relationship between H-3 and H3-18, which was further confirmed by the 13C NMR chemical shift calculation for the DP4+ calculations , a colorless oil, had a molecular formula of C21H30O4 on the basis of its HRESIMS ion peak at m/z 347.2221 [M + H]+, requiring seven degrees of unsaturation. The 1H and 13C NMR data of 2 (7), a known cembranoid previously isolated from the soft coral Sarcophyton trochliphroum. In fact, the structure of 2 was truly similar to 7, with the exception of a methoxyl at C-2 in 2 instead of the proton in 7. This deduction was further proven by the HMBC correlation (3-21 (δH 3.14) to C-2, along with the significant downfield shift observed for C-2 (δC 108.3). Then, the relative configurations of 2 were deduced on the basis of the NOESY experiment (2-5 (δH 2.20 and δH 2.20), and H3-19/H-6a (δH 2.35) established the E geometry of the Δ3 and Δ7 double bonds. The NOESY correlations of H3-21/H-13a (δH 1.68), and H-11/13a (δH 1.68) indicate that these protons were all co-facial. Moreover, the NOESY correlation of H3-20/H-13b (δH 1.89) suggests these protons were on the opposite side. Finally, the absolute configuration of 2 was defined by TDDFT-ECD calculations (Sarcoeleganolide D (ata of 2  resemblerelation  from theperiment . The NOEulations .3), a colorless oil, possessed the molecular formula C22H30O5, as indicated by its HRESIMS ion peak at m/z 397.1991 [M + Na]+. The comparison of the 1D NMR data , revealing the major difference in configurations between 3 and 6. The E geometry of the Δ3 double bonds was established by the observed NOESY correlations of H-3/H-5a (δH 2.38) and H-2/H3-18. Based on the above data, the NOESY correlations of H3-18/H-5b (δH 2.04) indicate the inverse orientation of H-2 and H-3, which was further confirmed by the coupling constants . The diagnostic NOESY correlations of H-13a (δH 1.09)/H-11, and H-13a/H-2 assigned H-11 and H-2 were all co-facial. The NOESY correlations of H-6/H-3, H-6/H-9a (δH 2.61), and H3-20/H-9a suggest that H3-20, H-3, and H-6 were on the same side of the ring system. Hence, the relative configurations of 3 were deduced, and finally, the absolute configurations of 3 were defined by TDDFT-ECD calculations (Sarcoeleganolide E (NMR data  of 3 andata of 3  indicatespectrum . By the ulations .4) was obtained as a colorless oil. The HRESIMS ion peak at m/z 369.2034 [M + Na]+ suggests the molecular formula was C21H30O4, suggestive of seven degrees of unsaturation. The 1H NMR spectrum (3 hybridized), five methylenes , four methines (three olefinic and one oxygenated), and seven quaternary carbons . By analysis of these data above, compound 4 was speculated to be a cembrane nucleus.Sarcoeleganolide F (spectrum  and HSQC4 was established by the 1H-1H COSY and HMBC experiments (1H-1H COSY correlations. The HMBC correlations from H3-21 (δH 3.21) to C-6 (δC 73.4) indicate the presence of a methoxy group located at C-6. A trisubstituted double bond located at C-2 and C-3 was proven by HMBC correlations from H-3 (δH 5.23) to C-2 (δH 148.2). Combined with the significant HMBC correlations from H3-17 to C-1, C-15, and C-16; H3-18 to C-3, C-4, and C-5; H3-19 to C-7, C-8, and C-9; H3-20 to C-11, C-12, and C-13; and H2-14 to C-1, C-2, and C-15, the planar framework of 4 was established. The relative configurations of 4 were deduced by the NOESY spectrum (3-19/H-6 (δH 4.30) and H-11/H2-13 (δH 2.33) established the E geometries of the Δ7 and Δ11 double bonds, and the NOESY cross-peaks of H-3/H2-14 (δH 2.54) established the Z geometry of the Δ2 double bonds. By the 13C NMR chemical shift calculations for the DP4+ calculations  and H-6/H-5a. Finally, the absolute configurations of 4 were defined by TDDFT-ECD calculations  was isolated as a colorless oil with a molecular formula of C20H28O3, established by the HRESIMS ion peak at m/z 339.1928 [M + Na]+. A survey of the literature revealed that the 1D NMR data of compound 5 , H-2/H3-18, and H-7/H2-9 (δH 2.03) indicate that the geometries of Δ3 and Δ7double bonds were of an E-configuration. By the NOESY correlations of H-2/H-14a (δH 2.26) and H-11/H-14a (δH 2.26), the relative configurations were defined as 2S* and 11S*. Finally, the absolute configurations of 5 were defined by TDDFT-ECD calculations  were evaluated for anti-inflammatory activity in CuSO4-induced transgenic fluorescent zebrafish. CuSO4 can produce an intense acute inflammatory response in the neuromast and mechanosensorial cells in the lateral line of zebrafish, stimulating the infiltration of macrophages m/z 317.2114 [M+H]+ . For 1H NMR and 13C NMR data, see Sarcoeleganolide C (2): colorless oil; [α]m/z 347.2221 [M + H]+ . For 1H NMR and 13C NMR data, see Sarcoeleganolide D (3): colorless oil; [α]m/z 397.1991 [M + Na]+ . For 1H NMR and 13C NMR data, see Sarcoeleganolide E (4): colorless oil; [α]m/z 364.2481 [M + NH4]+  and 369.2034 [M + Na]+ . For 1H NMR and 13C NMR data, see Sarcoeleganolide F (5): colorless oil; [α]m/z 339.1928 [M + H]+ . For 1H NMR and 13C NMR data, see Sarcoeleganolide G (1–5) were tested for anti-inflammatory activities with zebrafish models. Three days post-fertilization (dpf) healthy macrophage fluorescent transgenic zebrafish were used as animal models to evaluate the anti-inflammatory effects of 1–5.Healthy macrophage fluorescent transgenic zebrafish (Tg: zlyz-EGFP) was provided by the Biology Institute of the Shandong Academy of Science . Zebrafish maintenance and the anti-inflammation assay were carried out as previously described . Each zeSarcophyton elegans collected from the South China Sea, five new cembranes, named sarcoeleganolides C–G (1–5), and three known analogs, trocheliolide B (6), (−)-sartrochine (7), and 7α-hydroxy-Δ8(19)-deepoxysarcophine (8), were isolated. In addition, their structures and absolute configurations (1–5) were determined by extensive spectroscopic analysis, QM-NMR, and TDDFT-ECD calculations. Among them, compound 3 showed better anti-inflammatory activity, compared to the indomethacin as the positive control at 20 μM in the zebrafish model. This research enriches the chemical libraries of soft coral Sarcophyton elegans and provides a basis for developing new drugs.In our search for soft coral


Pueraria lobata (Willd.) Ohwi by column chromatography and preparative thin-layer chromatography; the structures were identified by spectroscopic analysis and compared with data reported in the literature. Seven compounds were isolated and identified from Pueraria lobata for the first time: Linoleic acid, Sandwicensin, Isovanillin, Ethyl ferulate, Haginin A, Isopterofuran, 3′.7-Dihydroxyisoflavan. The other 10 compounds were structurally identified as follows: Lupenone, Lupeol, β-sitosterol, Genistein, Medicarpin, Coniferyl Aldehyde, Syringaldehyde. All compounds were evaluated for their ability to inhibit SW480 and SW620 cells using the CCK-8 method; compound 5 (Sandwicensin) had the best activity, and compounds 6, 9, 11 and 12 exhibited moderate inhibitory activity. In addition, the targets and signaling pathways of Sandwicensin treatment for CRC were mined using network pharmacology, and MAPK3, MTOR, CCND1 and CDK4 were found to be closely associated with Sandwicensin treatment for CRC; the GO and KEGG analysis showed that Sandwicensin may directly regulate the cycle, proliferation and apoptosis of CRC cells through cancer-related pathways.Fourteen compounds were isolated from  Colorectal cancer (CRC) is the second most common cause of cancer death and the third most common cancer worldwide . CRC incPueraria lobata is the dried root of Pueraria lobata (Willd.) Ohwi, one of the most important herbal medicines native to East Asia, South America and Europe −.1H-NMR , δ: 5.35 , 2.77 , 2.34 , 2.04 , and 0.89 . 13C-NMR , δ: 180.1 (C-1), 34.32 (C-2), 24.86 (C-3), 29.24 (C-4), 29.31 (C-5), 31.68 (C-6), 29.74 (C-7), 27.39 (C-8), 128.04 (C-9), 130.35 (C-10), 25.78 (C-11), 130.15 (C-12), 128.2 (C-13), 27.34 (C-14), 29.5 (C-15), 32.08 (C-16), 22.72 (C-17), 14.22 (C-18). Compound 3 was identified as Linoleic acid after comparison with the literature −.1H-NMR , δ: 1.65 , 1.75 , 3.28 , 3.52 , 3.62 , 3.80 , 4.23 , 5.23 , 5.45 , 6.40 , 6.41 , 6.56 , 7.01 , 7.41 . 13C-NMR , δ: 132.56 (C-1), 113.44 (C-1a or C-10), 109.71 (C-2), 158.7 (C-3 or C-10a), 103.15 (C-4), 156.77 (C-4a or C-9), 66.65 (C-6), 40.12 (C-6a), 119.41 (C-6b), 121.69 (C-7), 103.68 (C-8), 156.98 (C-9 or C-4a), 113.29 (C-10 or C-1a), 158.65 (C-10a or C-3), 77.98 (C-11a), 23.02 (C-12), 122.39 (C-13), 131.65 (C-14), 25.95 (C-15), 17.88 (C-16), 56.07 (OCH3). Compound 5 was identified as Sandwicensin after comparison with the literature [Compound terature . 6: MS m/z: 269.07 [M+H]−.1H-NMR , δ: 7.02 , 6.92 , 5.59 , 6.51 , 5.71 . 13C-NMR , δ: 185.91 (C-4), 163.52 (C-7), 161.26 (C-4′), 156.52 (C-9), 156.39 (C-5), 151.88 (C-2), 130.94 , 122.2 (C-1′), 121.14 (C-3), 116.42 , 105.22 (C-10), 100.72 (C-8), 91.85 (C-6). Compound 6 was identified as Senistein by comparison with the literature [Compound 7: MS m/z: 153.06 [M+H]−.1H-NMR , δ: 9.80 , 7.41 , 7.40 , 7.02 , 3.94 . 13C-NMR , 191.19 (-CHO), 129.83 (C-1), 108.95 (C-2), 152.02 (C-3), 147.41 (C-4), 114.61 (C-5), 127.7 (C-6), 56.18 (-OCH3). Compound 7 was identified as Isovanillin after comparison with the literature [Compound terature .8: MS m/z: 221.03 [M+H]−.1H-NMR , δ: 7.61 , 7.07 , 7.03 , 6.92 , 6.29 , 4.26 , 3.93 , 1.33 . 13C-NMR , δ: 167.44 (C-1), 115.76 (C-2), 144.81 (C-3), 127.15 (C-4), 109.38 (C-5), 146.87 (C-6), 148.03 (C-7), 114.82 (C-8), 123.12 (C-9), 60.52 (C-10), 14.51 (C-11), 56.06 (C-12). Compound 8 was identified as Ethyl Ferulate by comparison with the literature [Compound 9: MS m/z: 269.18 [M+H]−.1H-NMR , δ: 3.56–3.47 , 3.63 , 3.79 , 4.24 , 5.51 , 6.36 , 6.49–6.37 , 6.64 , 7.07 , 7.42 . 13C-NMR , δ: 131.99 (C-1), 112.43 (C-1a), 109.32 (C-2), 157.13 (C-3), 101.78 (C-4), 156.69 (C-4a), 66.71 (C-6), 39.58 (C-6a), 125.1 (C-7), 119.5 (C-7a), 107.83 (C-8), 161.12 (C-9), 98.52 (C-10), 160.77 (C-10a), 78.79 (C-11a), 55.52 (C-12). Compound 9 was identified as Medicarpin by comparison with the literature [Compound 10: MS m/z: 177.01 [M+H]−.1H-NMR , δ: 9.65 , 7.40 , 7.12 , 7.07 , 6.96 , 6.59 , 3.95 . 13C-NMR , δ: 193.75 (C-9), 153.22 (C-7), 149.12 (C-4), 147.11 (C-3), 126.79 (C-1), 126.57 (C-8), 124.20 (C-6), 115.09 (C-5), 109.60 (C-2), 56.15 (OCH3-C). Compound 10 was identified as Coniferyl aldehyde by comparison with the literature [Compound terature .11: MS m/z: 299.10 [M+H]−.1H-NMR , δ: 6.94 , 6.72 , 6.54 , 6.42 , 5.00 , 3.94 , 3.81 . Compound 11 was identified as Haginin A after comparison with the literature [Compound terature .12: MS m/z: 285.08 [M+H]−.1H-NMR , δ: 7.60 , 7.40 , 7.11 , 6.99 , 6.82 , 6.76 , 3.99 (2′-OCH3), 3.93 (3′-OCH3). Compound 12 was identified as Isopterofuran after comparison with the literature [Compound terature .13: MS m/z: 241.09 [M+H]−.1H-NMR , δ: 2.92 , 3.15 , 3.96 , 4.29 , 6.40 , 6.61–7.01 , 7.04–7.23 . Compound 13 was identified as 3′7-Dihydroxyisoflavan after comparison with the literature [Compound terature .14: MS m/z: 181.31 [M+H]−.1H-NMR , δ: 9.82 , 7.15 , 3.82 . 13C-NMR , δ: 190.92 (CHO), 147.46 , 140.88 (C-4), 128.54 (C-1), 107.57 , 56.65 (OCH3). Compound 14 was identified as Syringaldehyde by comparison with the literature [Compound terature .Pueraria lobata for the treatment of CRC, we isolated and purified CH2Cl2 extracts to obtain compounds 1–14, as shown in 1-14 on the growth of SW480 and SW620 cells were determined by the CCK-8 method. As shown in 5, 6, 9, 11, 12 showed dose-dependent inhibition of SW480 and SW620 cell growth, while the other compounds had no significant inhibitory effect. The effect of compound 5 was the most obvious among these compounds, indicating that this compound is the main active ingredient of Pueraria lobata in the treatment of CRC.To further determine the material basis of http://www.cytoscape.org, accessed on 20 September 2022) to draw a component–target network map. As shown in In order to clarify the mechanism of Sandwicensin in the treatment of CRC, we performed target prediction as well as pathway analysis using network pharmacology; no target was found after searching “Sandwicensin” using the TCMSP database, and 112 predicted targets of “Sandwicensin” were retrieved from the Swiss Target Prediction database. A total of 11,967 disease targets were collected in five databases: DisGeNET, GeneCards, OMIM, TTD and Drugbank. Disease and component targets were mapped and a Venn diagram was drawn, as shown in http://www.cytoscape.org, accessed on 20 September 2022) for network analysis and then sorted according to the degree values, as shown in The PPI network was constructed by uploading the targets of Sandwicensin for CRC in the String database and excluding the free nodes. As shown in p < 0.05), as shown in p < 0.05), as shown in n = 11) and the cell cycle . Notably, Sandwicensin treatment of CRC was strongly associated with cell cycle and apoptosis. This indicates that Sandwicensin may directly regulate the cycle, proliferation and apoptosis of tumor cells through cancer-related pathways.To further elucidate the functional process of Sandwicensin for the treatment of CRC, the three parts, namely biological process (BP), cellular component (CC) and molecular function (MF), of the intersecting targets were analyzed using GO enrichment analysis. The results showed that a total of 53 enrichment results were obtained. We selected the top 20 biological functions (Cyclin D1 (CCND1), or G1/S-specific cyclin-D1, is a protein encoded by the human CCND1 gene ,31. The 1H and 600 MHz for 13C in CDCl3. Column chromatographic silica gel (300 mesh) and TLC plates (GF254) were purchased from Qingdao Marine Chemical Inc. . The other solvents were purchased from Shanghai Titan Scientific Co., Ltd. .Recording of 1D-NMR spectra was performed on a Bruker AV-600 spectrometer  with a single NMR probe at 300 MHz for Pueraria lobata (Willd.) Ohwi were purchased at Beiyao Pharmaceutical Group Co., Ltd., Jilin Province, China, in May 2021 and authenticated by Li-Li Weng, a professor at Changchun University of Traditional Chinese Medicine.DMEM high glucose medium (C11995500BT) and fetal bovine serum FBS (30044333) were from Gibco. PBS (SH30256), trypsin (SH30042), and penicillin–streptomycin dual antibody (SV30010) were all from Hyclone. Cell Counting Kit-8 kit (IV08-500) was from Invigentech. Propidium iodide (CF0031) was from Dingguo.Pueraria lobata (10 kg) was extracted with 70% EtOH  and concentrated under reduced pressure to obtain 4.9 kg of crude ethanol extract. The crude ethanol extract of Pueraria lobata was dissolved according to a certain proportion , and then liquid–liquid extraction was performed with dichloromethane, ethyl acetate and n-butanol in turn , followed by concentration under reduced pressure to obtain the extract of each layer. Dichloromethane extracts were separated by silica gel column chromatography (CC) and gradient elution with petroleum ether–ethyl acetate  to obtain nine fractions (D-1→D-9).1 (51.1 mg).Fr.D-2 (0.75 g) was further separated by a normal phase silica gel column (2.0 × 60 cm) with gradient elution with petroleum ether–dichloromethane and petroleum ether–ethyl acetate  to afford a total of 16 fractions (D-2-1→D-2-16). Fr.D-2-9 (0.135 g) was further separated on a reversed-phase silica gel column (2.5 × 35 cm) with gradient elution with methanol–purified water and methanol–acetone  to afford compound 2 (86.7 mg). Fr.D-4-3 (2.803 g) was further separated by reversed-phase silica gel column (2.5 × 45 cm) and gradient elution with methanol–purified water  to afford compounds 3 and 4 . Fr.D-4-4 (0.483 g) was further separated by reversed-phase silica gel column (2.5 × 45 cm) and gradient elution with methanol–purified water  to give compound 5 (8.9 mg). Fr.D-4-4-8 (0.0082 g) was further separated by a normal phase silica column (1.2 × 20 cm) and eluted with petroleum ether–ethyl acetate  to give compound 6 (9.2 mg).Fr.D-4 (4.01 g) was further separated by a normal phase silica gel column (2.5 × 30 cm) and gradient elution with petroleum ether–dichloromethane  to afford a total of four fractions (D-4-1→D-4-4). Fr.D-4-2 (0.969 g) was further separated by reversed-phase silica gel column (2.5 × 30 cm) and gradient elution with methanol–purified water  to give compound 7 and 8 . Fr.D-6-7 (0.205 g) was further separated on a reversed-phase silica gel column (2.5 × 45 cm) with gradient elution with methanol–purified water  to give compound 9 (24.5 mg).Fr.D-6 (2.04 g) was further separated by a normal phase silica gel column (2.5 × 30 cm) and gradient elution with petroleum ether–dichloromethane  to give a total of nine fractions (D-6-1→D-6-9). Fr.D-6-3 (0.177 g) was separated on a reversed-phase silica gel column (2.5 × 45 cm) with gradient elution with methanol–purified water  to afford compounds 10–12 .Fr.D-7 (3.91 g) was further separated by a normal phase silica gel column (2.5 × 30 cm) and gradient elution with petroleum ether–ethyl acetate  to afford a total of four fractions (D-7-1→D-7-4). Fr.D-7-2 (1.4 g) was further separated by reversed-phase silica gel column (3.5 × 45 cm) and gradient elution with methanol–purified water  to give compounds 13 (9.47 mg). Fr.D-8-5-14 (0.016 g) was further separated by preparing thin layer plates (20 × 20 cm) with dichloromethane:methanol = 20:1 as developing solvent to give compound 14 (12 mg).Fr.D-8 (7.13 g) was further separated by a normal phase silica gel column (5 × 45 cm) and gradient elution with petroleum ether–ethyl acetate  to give a total of five fractions (D-8-1→D-8-5). Fr.D-8-2 (0.197 g) was further separated by reverse silica gel column (2.5 × 45 cm) and gradient elution with methanol–purified water  to give compound Test article solution was spotted on preparative TLC , with dichloromethane:methanol = 20:1 as developing solvent; the TLC plate was taken out after developing, the solvent was evaporated to dryness, the plated was observed under an ultraviolet lamp and 10% sulfuric acid was used to develop color.NMR spectroscopy was performed by Jilin University.2 and 95% air). The medium was changed every other day, and the cells were subcultured when they reached 80% confluence.SW480 and SW620 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C in a humidified incubator  value was measured at 450 nm to calculate the cell survival rate.The cells were inoculated in 96-well plates at a density of 5 × 10http://lsp.nwu.edu.cn/tcmsp.php, accessed on 20 September 2022) to collect the targets of this component; the structure of Sandwicensin was drawn in the Swiss Target Prediction database , and the predicted targets corresponding to this component were collected [“Sandwicensin” was entered in the TCMSP database , GeneCards database (https://www.genecards.org/), Disgenet database , TTD database and Drugbank database and then logged in UniProt  database, and the gene names and Uniprot IDs of the targets were corrected [“Colorectal cancer” and “colorectal carcinoma” for all targets of the disease were collected in the OMIM database  was used to construct the “component–target” relationship network. This network can be used to analyze the mechanism of action of Sandwicensin in the treatment of CRC [The targets after component and disease mapping are the key targets of Sandwicensin for the treatment of CRC. After the component and disease target mapping, Cytoscape 3.2.1 software  [Using the “Multiple Proteins” function of the STRING database (er 2022) , we selehttps://david.ncifcrf.gov/, accessed on 20 September 2022) for GO function enrichment analysis and KEGG pathway enrichment analysis. The information was collated separately, and bubble maps were plotted in the Microbioxin database.The key targets of Sandwicensin for CRC were entered into the Math Escape online analysis tool . Statistical significance was determined based on p values . All results are expressed as mean ± SD, n = 3.The experimental data were entered into Graphpad Prism 7 and analyzed by Pueraria lobata, including 7 compounds that were isolated from this plant for the first time. In addition, compounds 5, 6, 9, 11 and 12 showed good anti-tumor activity, with compound 5 having the best biological activity. This indicates that compound 5 is the main bioactive component of the anti-tumor activity of Pueraria lobata. Moreover, we used network pharmacology to mine and analyze the targets and signaling pathways of Sandwicensin for the treatment of CRC, and the results showed that Sandwicensin exerts its anti-tumor effects mainly through regulating several cell cycle-related proteins  and several proliferation-related proteins .In the present study, 14 compounds were isolated and purified from the dichloromethane extract of


Guitarra abbotti, whose 1-O-alkyl-sn-glycerol ethers (AGEs) have not been investigated so far, led to the isolation of a complex lipid fraction containing, along with previously known compounds, six new lipids of the AGE type. The composition of the AGE fraction as well as the structures of 6 new and 22 previously known compounds were established using 1H and 13C NMR, GC/MS, and chemical conversion methods. The new AGEs were identified as: 1-O-(Z-docos-15-enyl)-sn-glycerol (1), 1-O-(Z-docos-17-enyl)-sn-glycerol (2), 1-O-(Z-tricos-15-enyl)-sn-glycerol (3), 1-O-(Z-tricos-16-enyl)-sn-glycerol (4), 1-O-(Z-tricos-17-enyl)-sn-glycerol (5), and 1-O-(Z-tetracos-15-enyl)-sn-glycerol (6). The isolated AGEs show weak cytotoxic activity in THP-1, HL-60, HeLa, DLD-1, SNU C4, SK-MEL-28, and MDA-MB-231 human cancer cells. A further cytotoxicity analysis in JB6 P+ Cl41 cells bearing mutated MAP kinase genes revealed that ERK2 and JNK1 play a cytoprotective role in the cellular response to the AGE-induced cytotoxic effects.The cytotoxicity-bioassay-guided fractionation of the ethanol extract from the marine sponge  O-alkyl-sn-glycerol ethers (AGEs); for a review, see [sn-1 position. Previously, it was established that all the natural AGEs are enantiomerically pure, with an S configuration of the asymmetric carbon in the glycerol moiety [It is well known that many marine invertebrates, including sponges ,11,12,13iew, see ,32. AGE l moiety ,34. The l moiety ,36. In ml moiety ,30,31,32l moiety  as well l moiety ,20. Bothl moiety . Among ol moiety ,43,44,45l moiety , antibacl moiety ,30,47,48l moiety , and antl moiety  activitil moiety , the sidl moiety ,53,54, ol moiety ,56,57,58l moiety ,59. Addil moiety ,61, whicl moiety , the imml moiety ,64,65,66l moiety ,68, are l moiety , and impl moiety . The seaGuitarra abbotti, which exhibited a cytotoxic activity against human leukemia THP-1 cells in a screening assay. The AGEs of this sponge have never been studied before. A bioassay-guided fractionation of the crude extract led to the isolation of an AGE mixture containing 6 new  exhibit very similar fragmentations to compounds isolated earlier from other biological sources [To establish the composition and the structures of the isolated AGEs, including the percentages of the compounds, the AGEs were converted to their corresponding TMS ethers and further analyzed with GC/MS. The GC data and characteristic MS fragmentation of the TMS derivatives of AGEs are shown in  sources ,30,31,3222-C24, were identified as new compounds: 1-O-(Z-docos-15’-enyl)-sn-glycerol (1); 1-O-(Z-docos-17’-enyl)-sn-glycerol (2); 1-O-(Z-tricos-15’-enyl)-sn-glycerol (3); 1-O-(Z-tricos-16’-enyl)-sn-glycerol (4); 1-O-(Z-tricos-17’-enyl)-sn-glycerol (5); and 1-O-(Z-tetracos-15’-enyl)-sn-glycerol (6)  , Table 21–6, the 22 previously known AGEs (10 saturated and 12 unsaturated) were identified in the isolated mixture: 1-O-(tetradecanyl)-sn-glycerol (7); 1-O-(pentadecanyl)-sn-glycerol (TR 16.58 of TMS derivarive (TMS-d), 8); 1-O-(pentadecanyl)-sn-glycerol ; 1-O-(hexadecanyl)-sn-glycerol ; 1-O-(hexadecanyl)-sn-glycerol ; 1-O-(heptadecanyl)-sn-glycerol ; 1-O-(heptadecanyl)-sn-glycerol ; 1-O-(heptadecanyl)-sn-glycerol ; 1-O-(octadecanyl)-sn-glycerol ; 1-O-(octadecanyl)-sn-glycerol ; 1-O-(Z-hexadec-7’-enyl)-sn-glycerol ; 1-O-(Z-hexadec-9’-enyl)-sn-glycerol ; 1-O-(Z-hexadec-11’-enyl)-sn-glycerol ; 1-O-(Z-hexadec-13’-enyl)-sn-glycerol ; 1-O-(Z-octadec-9-enyl)-sn-glycerol ; 1-O-(Z-octadec-11-enyl)-sn-glycerol ; 1-O-(Z-octadec-12-enyl)-sn-glycerol ; 1-O-(Z-octadec-13-enyl)-sn-glycerol ; 1-O-(Z-cos-11’-enyl)-sn-glycerol ; 1-O-(Z-cos-13’-enyl)-sn-glycerol ; 1-O-(Z-cos-15’-enyl)-sn-glycerol ; and 1-O-(Z-tetracos-17’-enyl)-sn-glycerol  [Apart from the 6 new compounds, glycerol ; 1-O- ,32.50 values are shown in The cytotoxicity of the isolated AGE mixture was evaluated using the MTS viability assay in seven human cancer cell lines . The calWe used dominant negative mutant (DNM) JB6 Cl41 cells to elucidate the roles of the three major MAP kinases (MAPKs) in the cytotoxic effects of the isolated AGE mixture. The cytotoxic activity of the isolated AGE fraction against the normal mouse epidermal cell line JB6 Cl41 and its stable transfectants JB6 Cl41 DN-JNK1, JB6 Cl41 DN-p38, and JB6 Cl41 DN-ERK2 cells is shown in G. abbotti without any prior derivatization or hydrolysis. A comparison with the literature data [1H NMR spectrum of the AGE mixture  made it possible to correlate the signals at δ 3.55–3.86 ppm with the protons of the glyceride group, those at δ 3.46 ppm with the protons at C1’ of the alkyl chains, those at δ 1.57 ppm with the protons at C2’ of the alkyl chains, those at δ 1.22–1.38 ppm with the protons of other CH2 groups in the alkyl chains, those at δ 5.34 ppm with the protons at double bonds, those at δ 2.01 ppm with the allylic protons, and those at 0.85–0.88 ppm with protons of the terminal methyl groups of the alkyl chain. Thus, the analysis of the NMR spectra showed that the mixture consists of saturated and unsaturated AGEs, the side chains of which may contain n- and iso- terminal methyl groups -sn-glycerol (1), 1-O-(Z-docos-17’-enyl)-sn-glycerol (2), 1-O-(Z-tricos-15’-enyl)-sn-glycerol (3), 1-O-(Z-tricos-16’-enyl)-sn-glycerol (4), 1-O-(Z-tricos-17’-enyl)-sn-glycerol (5), and 1-O-(Z-tetracos-15’-enyl)-sn-glycerol (6)  .Different studies suggest that in a living organism AGEs serve as precursors and can be enzymatically converted into various types of biologically active ether lipid compounds . At the MAP kinases play roles in various biological processes ,86,87. DThe inhibitory effect of AGEs on protein kinase C  suggests50 = 35.9 µg/mL  at 300 and 75 MHz, respectively, in CDCl3 with tetramethylsilane as an internal standard. The The GC/MS data for the TMS derivatives were obtained using a Hewlett Packard GC HP6890 instrument  with an HP5973 mass-selective detector. The injector and transfer line temperatures were 270 °C. A Hewlett Packard HP-5MS capillary column , 30 m × 0.25 mm, phase layer 0.25 µm, was used at 100 °C with a 2 °C/min ramp to 270 °C, which was held for 30 min. The column contained 5% phenylmethylsiloxane and it was used as the mobile phases at a flow rate of 1 mL/min. The sample was dissolved in chloroform at a concentration of 10 mg/mL. The injection volume was 0.2 μL, and the split ratio was 15:1. The mass spectra were recorded at 70 eV.The GC/MS data for the DMDS-derived AGEs were obtained using a Shimadzu GCMS-QP5050 instrument . An MDN-5S capillary column , 30 m × 0.25 mm, phase layer 0.25 µm, was used at 200 °C with a 2 °C/min ramp to 300 °C, which was held for 45 min. The split ratio was 15:1, and the flow rate was 1 mL/min. The injector temperature was 270 °C. The mass spectra were recorded at 70 eV.Low-pressure column liquid chromatography was performed using KSK silica gel . Sorbfil silica gel plates  were used for TLC. HPLC was performed using an Agilent 1100 instrument  equipped with a differential refractometer on a Diasorb-60-Silicagel (250 × 4.6 mm) column . Cells were counted using an Olympus inverted research microscope . The absorption of MTS/farmazan was measured spectrophotometrically using the μQuant microplate reader .Minimum essential medium (MEM), DMEM, and RPMI medium were purchased from BioloT ; fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific ; penicillin/streptomycin was purchased from Bio-Whittaker ; and L-glutamine was purchased from Mediatech Inc. . The Cell Titer 96 Aqueous One Solution Reagent (MTS) kit for the cell viability assay was purchased from Promega .Guitarra abbotti  was collected by dredging at the depth of 109 m at 48°00′08′′ N, 153°20′07′′ E, Kuril Islands, the Sea of Okhotsk, Pacific Ocean. A voucher specimen is kept in the collection of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry. The taxonomic identification was performed by V.B. Krasokhin.The marine sponge 2O  and extracted with 3 × 200 mL of n-hexane. The aqueous-ethanolic and combined n-hexane fractions after evaporation were subjected to an evaluation of cytotoxic activity against human leukemia THP-1 cells by the MTS method and showed IC50 = 0.6 and 0.5 mg/mL, respectively. The n-hexane fraction was selected for the further isolation of anticancer compounds. This fraction (3.525 g) was evaporated and subjected to column chromatography on a silica gel column (diameter/length = 6:14 cm) using an n-hexane/AcOEt gradient as an eluent with n-hexane to AcOEt ratios of 19:1, 9:1, 4:1, 3:2, 1:1, 2:3, 1:4, 1:9, and 1:19 (v/v), then with 100% EtOAc and then with 100% EtOH. A 200 mL volume of eluent was used for the elution of each fraction. A subsequent evaluation of the cytotoxic activity revealed that the fraction eluted with n-hexane/AcOEt  possessed the highest cytotoxicity towards THP-1 cells, IC50 = 125 μg/mL. This fraction (299 mg) was further subjected to the same silica gel column chromatography using 330 mL of n-hexane/AcOEt  and 400 mL of n-hexane/AcOEt  as eluents. The collected fractions (10 mL each) were examined using TLC ) as a chromatographic system, with the further detection of spots on the TLC chromatograms using H2SO4/EtOH . Following the TLC analysis, three fractions were obtained, i.e., (i) fraction #1 ; (ii) fraction #2 (one violet spot on the TLC with Rf = 0.2); and (iii) fraction #3 . The examination of the cytotoxic activity against human THP-1 cells revealed fraction #2 to be the most active, with IC50 = 62.5 μg/mL. Further, fraction #2  was additionally purified by HPLC using a Diasorb-60-Silica gel column and n-hexane/AcOEt  as the eluent. As a result, the more active mixture (IC50 = 35.9 μg/mL in THP-1 cells) of the purified 1-O-alkylglycerol ethers (AGEs) was obtained .Animal materials  were extracted with EtOH (2 L) immediately after collection. After evaporation in vacuo, the ethanol extract was re-dissolved in 200 mL of EtOH/HThe chemical nature of the isolated purified fraction was established using their NMR spectra. Generally, the next spectroscopic information was obtained. 1H NMR  δ 0.85 d, J = 6.6 Hz, iso-CH3; 0.88 t, J = 6.7 Hz, n-CH3; 1.22-1.38, m, (CH2)n, aliphatic chain; 1.57, quint, J = 6.9 Hz, 2H-2’; 2.01, q, J = 6.3 Hz, 2H allylic protons; 3.46, td, J = 6.6 Hz, J = 1.6 Hz, 2H-1’; 3.55, dd, J = 9.2 Hz, J = 3.5 Hz, 2H-1; 3.64 dd, J = 11.4 Hz, J = 5.2 Hz, 1H-3a; 3.72 dd, J = 11.4 Hz, J = 3.8 Hz, 1H-3b; 3.86, m, 1H-2; 5.34, t, J = 4.6 Hz, 2H, olefinic protons.13C NMR  δ 14.10 (CH3); 22.64 (CH2); 26.05 ; 27.19 (CH2); 28.97 (CH2); 29.07 (CH2); 29.30 (CH2); 29.34 (CH2); 29.44 (CH2); 29.54 (CH2); 29.66 (CH2); 31.76 (CH2); 31.90 (CH2); 64.24 (CH2-3); 70.40 (CH-2); 71.84 (CH2-1); 72.48 (CH2-1’); 129.88 (CH=CH).1–6) were new natural products (see below).These data indicate the AGE nature of the compounds in the isolated mixture. These compounds contain a glycerol moiety linked by an ether bond with a fatty alcohol residue. In the analyzed compounds, the fatty alcohol residues contain either normal or iso-ends, and some of them have an additional disubstituted double bond. The majority of the identified metabolite compounds have previously been isolated from different biological sources. However, six of them  at 60 °C for 1 h to convert the AGEs to their trimethylsilyl derivatives (TMS-d). The obtained TMS-d were analyzed by the GC/MS method . The mas7–28) are given below. The mass spectra data of the TMS-d for the previously known compounds (O-(tetradecanyl)-sn-glycerol (7), TR 16.07, m/z 417, 285, 205, 147, 117, 103, 73; 57; TMS-d of 1-O-(pentadecanyl)-sn-glycerol (8), TR 16.58, m/z 431, 356, 299, 205, 147, 117, 103, 73; 57; TMS-d of 1-O-(pentadecanyl)-sn-glycerol (9), TR 16.87, m/z 431, 356, 299, 205, 147, 117, 103, 73; 57; TMS-d of 1-O-(hexadecanyl)-sn-glycerol (10), TR 17.35, m/z 445, 370, 313, 205, 147, 117, 103, 73, 57; TMS-d of 1-O-(Z-hexadec-7’ or 9’ or 11’ or 13’-enyl)-sn-glycerol (17 or 18 or 19 or 20), TR 17.51, m/z 458, 443, 355, 311, 265, 205, 147, 117, 103, 73, 55; TMS-d of 1-O-(Z-hexadec-7’ or 9’ or 11’ or 13’-enyl)-sn-glycerol (17 or 18 or 19 or 20), TR 17.59, m/z 458, 355, 311, 265, 205, 147, 117, 103, 73, 55; TMS-d of 1-O-(hexadecanyl)-sn-glycerol (11), TR 17.64, m/z 445, 370, 313, 205, 147, 117, 103, 73, 57; TMS-d of 1-O-(heptadecanyl)-sn-glycerol (12), TR 18.10, m/z 459, 384, 327, 205, 147, 117, 103, 73, 57; TMS-d of 1-O-(heptadecanyl)-sn-glycerol (13), TR 18.18, m/z 459, 384, 327, 205, 147, 117, 103, 73, 57; TMS-d of 1-O-(heptadecanyl)-sn-glycerol (14), TR 18.38, m/z 459, 384, 327, 205, 147, 117, 103, 73, 57; TMS-d of 1-O-(octadecanyl)-sn-glycerol (15), TR 18.80, m/z 473, 398, 341, 205, 147, 117, 103, 73, 57; TMS-d of 1-O-(Z-octadec-9’ or 11’ or 12’ or 13’-enyl)-sn-glycerol (21 or 22 or 23 or 24), TR 18.93, m/z 486, 471, 293, 205, 147, 117, 103, 73, 55; TMS-d of 1-O-(Z-octadec-9’ or 11’ or 12’ or 13’-enyl)-sn-glycerol (21 or 22 or 23 or 24), TR 18.99, m/z 486, 471, 293, 205, 147, 117, 103, 73, 55.; TMS-d of 1-O-(octadecanyl)-sn-glycerol (16), TR 19.10, m/z 473, 398, 341, 205, 147, 117, 103, 73, 57; TMS-d of 1-O-(Z-cos-11’ or 13’ or 15’-enyl)-sn-glycerol (25 or 26 or 27), TR 20.38, m/z 514, 321, 205, 147, 117, 103, 73, 55; TMS-d of 1-O-(Z-tetracos-17’-enyl)-sn-glycerol (28), TR 23.43, m/z 480, 467, 390, 377, 205, 147, 117, 103, 73, 55.TMS-d of 1-1–6) (For the six new identified compounds (1–6) , the masO-(Z-docos-15’-enyl)-sn-glycerol (1), retention time (TR) 21.74, m/z 542, 527, 395, 349, 205, 147, 117, 103, 73, 55; TMS-d of 1-O-(Z-docos-17’-enyl)-sn-glycerol (2), TR 21.81, m/z 542, 527, 395, 349, 205, 147, 117, 103, 73, 55; TMS-d of 1-O-(Z-tricos-15’-enyl)-sn-glycerol (3) or 1-O-(Z-tricos-16’-enyl)-sn-glycerol (4), or 1-O-(Z-tricos-17’-enyl)-sn-glycerol (5), TR 22.56, m/z 363, 205, 147, 117, 103, 73, 55; TMS-d of 1-O-(Z-tetracos-15’-enyl)-sn-glycerol (6), TR 23.35, m/z 480, 467, 390, 377, 205, 147, 117, 103, 73, 55.TMS-d of 1-2O/Py  at RT overnight to convert the AGEs to their diacetate derivatives. Then, 2 mL of EtOH was added, and the sample was dried in vacuo.First, 1.3 mg of the dried AGE mixture was treated with 0.4 mL of Ac2O (60 mg/mL) and incubated at RT overnight. Then, 5 mL of n-hexane was added, and the mixture was washed with 5 mL of an aqueous 5% solution of Na2S2O3 × 5H2O until the color of iodine disappeared. The n-hexane fraction was separated, and the reaction products were extracted one more time from the polar fraction using n-hexane (1 mL). The combined n-hexane fractions were dried over sodium sulfate, evaporated in vacuo, and re-dissolved in hexane for further analysis.First, 1.2 mg of dried AGE acetate mixture (see above) was mixed with 0.2 mL of dimethyldisulfide (DMDS) and 0.05 mL of iodine solution in Et1–6:Mass spectra of the DMDS-d of the new AGEs, O-(Z-docos-15’-enyl)-sn-glycerol (1), TR 70.67, m/z 576, 529, 431, 371, 311, 285, 263, 255, 206, 159, 145, 97, 83, 69, 55; DMDS-d of 1-O-(Z-docos-17’-enyl)-sn-glycerol (2), TR 72.07, m/z 576, 529, 459, 399, 339, 313, 291, 283, 234, 159, 117, 97, 83, 69, 55; DMDS-d of 1-O-(Z-tricos-15’-enyl)-sn-glycerol (3), TR 77.50, m/z 590, 436, 371, 341, 311, 173, 159, 145, 117, 97, 83, 69, 55; DMDS-d of 1-O-(Z-tricos-16’-enyl)-sn-glycerol (4), TR 77.90, m/z 590, 436, 385, 341, 297, 159, 145, 117, 97, 83, 69, 55; DMDS-d of 1-O-(Z-tricos-17’-enyl)-sn-glycerol (5), TR 78.67, m/z 590, 436, 399, 341, 283, 159, 145, 131, 117, 97, 83, 69, 55; DMDS-d of 1-O-(Z-tetracos-15’-enyl)-sn-glycerol (6), TR 86.27, m/z 604, 450, 371, 311, 255, 207, 173, 159, 109, 97, 83, 69, 55.DMDS-d of 1-Mass spectra of the DMDS-d of the previously known AGEs:O-(Z-hexadec-7’-enyl)-sn-glycerol (17), TR 44.69, m/z 492, 319, 259, 173, 159, 143, 95, 89, 81, 69, 61, 55; DMDS-d of 1-O-(Z-hexadec-9’-enyl)-sn-glycerol (18), TR 45.00, m/z 492, 347, 287, 171, 159, 145, 95, 89, 81, 69, 61, 55; DMDS-d of 1-O-(Z-hexadec-11’-enyl)-sn-glycerol (19), TR 45.69, m/z 492, 375, 315, 199, 159, 117, 95, 89, 81, 69, 61, 55; DMDS-d of 1-O-(Z-hexadec-13’-enyl)-sn-glycerol (20), TR 47.69, m/z 492, 403, 343, 227, 159, 95, 89, 81, 69, 61, 55; DMDS-d of 1-O-(Z-octadec-9’-enyl)-sn-glycerol (21), TR 51.10, m/z 520, 347, 287, 227, 173, 159, 95, 81, 69, 55; DMDS-d of 1-O-(Z-octadec-11’-enyl)-sn-glycerol (22), TR 51.40, m/z 520, 375, 315, 255, 145, 159, 95, 81, 69, 55; DMDS-d of 1-O-(Z-octadec-12’-enyl)-sn-glycerol (23), TR 51.62, m/z 520, 389, 329, 269, 131, 159, 95, 81, 69, 55; DMDS-d of 1-O-(Z-octadec-13’-enyl)-sn-glycerol (24), TR 52.13, m/z 520, 403, 343, 283, 117, 159, 95, 81, 69, 55; DMDS-d of 1-O-(Z-cos-11’-enyl)-sn-glycerol (25), TR 58.48, m/z 548, 375, 315, 255, 199, 159, 117, 109, 95, 83, 69, 55; DMDS-d of 1-O-(Z-cos-13’-enyl)-sn-glycerol (26), TR 59.39, m/z 548, 403, 343, 227, 159, 117, 109, 95, 83, 69, 55; DMDS-d of 1-O-(Z-cos-15’-enyl)-sn-glycerol (27), TR 60.29, m/z 548, 431, 371, 255, 199, 159, 117, 109, 95, 83, 69, 55; DMDS-d of 1-O-(Z-tetracos-17’-enyl)-sn-glycerol (28), TR 87.30, m/z 604, 459, 399, 339, 283, 159, 145, 97, 83, 69, 55.DMDS-d of 1-+ Cl41 mouse epidermal cell line and its stable transfectants JB6 Cl 41 DN-JNK1, JB6 Cl 41 DN-p38, and JB6 Cl 41 DN-ERK2, which have the knockout JNK1 p38 and ERK2 genes, respectively, were cultured as monolayers at 37 °C and 5% CO2 in MEM containing 5% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin. The human cancer cell lines HL-60 (promyelocytic leukemia), THP-1 (monocytic leukemia), HeLa (cervix carcinoma), SNU C4 (colon cancer), DLD-1 (colon cancer), MDA-MB-231 (breast adenocarcinoma), and SK-MEL-28 (melanoma) were obtained from the American Type Culture Collection . The HL-60, THP-1, HeLa, DLD-1, SNU C4, and SK-MEL-28 cancer cell lines were cultured at 37 °C and 5% CO2 in RPMI medium containing 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin. The MDA-MB-231 cancer cell line was cultured at 37 °C and 5% CO2 in DMEM containing 10% FBS, 2mM L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin. HL-60 and THP-1 cells were cultured as suspensions, and the other cell lines were cultured as monolayers. Information regarding the genetic background of these cell lines is available online at the ATCC website.The JB6 P+ Cl41, JB6 Cl 41 DN-JNK1, JB6 Cl 41 DN-p38, and JB6 Cl 41 DN-ERK2 cell lines was evaluated using the MTS assay [The effect of the obtained AGE mixture on the viability of the THP-1, HL-60, HeLa, DLD-1, SNU C4, SK-MEL-28, MDA-MB-231, JB6 PTS assay . Briefly50 values.The statistical analyses were performed using Statistica 6.0  or GraphPad Prism v.9.1.1 software . The results of two independent experiments, each performed in triplicate, were used for the analyses. Significant differences from control were calculated using Student’s t-test. The method of regressions was used to calculate the ICO-alkylglycerol ethers were identified in the AGE fraction isolated from the marine sponge Guitarra abbotti. Their structures were established using 1H and 13C NMR spectroscopy and a GC/MS analysis of their TMS and DMDS derivatives as well as a comparison with the literature data. The isolated AGE fraction consisted of both saturated and unsaturated AGEs, which presented at a nearly equimolar ratio. The isolated AGEs exhibited a rather weak cytotoxic activity towards seven human cancer cell lines. Moreover, the active MAP kinases ERK2 and JNK1 were shown to play a cytoprotective role in the cellular response to the AGE-induced cytotoxic effects.In conclusion, 6 new and 22 previously known 1-