The chemical structures of the synthesized compounds were identified by NMR, HR-MS, and IR data. Moreover, the cytotoxicity of the obtained compounds was determined. The dStigMAs possessing a carbonate linker showed potent cytotoxicity to cells isolated from the small intestine and colon epithelium and liver, whereas the opposite results were obtained for compounds containing a succinate linker.Plant sterols, also referred as phytosterols, have been known as bioactive compounds which have cholesterol-lowering properties in human blood. It has been established that a diet rich in plant sterols or their esters alleviates cardiovascular diseases (CVD), and also may inhibit breast, colon and lung carcinogenesis. Phytosterols, in their free and esterified forms, are prone to thermo-oxidative degradation, where time and temperature affect the level of degradation. Looking for new derivatives of phytosterols with high thermo-oxidative stability for application in foods, our idea was to obtain novel structured acylglycerols in which two fatty acid parts are replaced by stigmasterol residues. In this work, asymmetric  distigmasterol-modified acylglycerols (dStigMAs) were synthesized by the covalent attachment of stigmasterol residues to  Nigella sativa oil to 13.8 mg/g in crude rice bran oil +: 535.3763, found: 535.3759.Colorless crystals, m.p. 156–158 °C. Yield, 6.41 g, 86%. TLC: 1H-NMR , 13C-NMR , COSY and HMQC spectra of dStigS-PA are presented.On Rf = 0.58 . 1H-NMR  δ: 0.69 , 0.79 , 0.80 , 0.84 , 0.87 , 0.91–0.98 , 0.99–1.06 , 1.01 , 1.02 , 1.07–1.32 , 1.36–1.64 , 1.65–1.75 , 1.81–1.89 , 1.91–2.09 , 2.27–2.34 , 2.56–2.67 C–C2H–C2H–C(O)O– a and b), 4.15 , 4.19 , 4.29 , 4.30 , 4.55–4.65 , 5.01 , 5.15 , 5.27 , 5.34–5.38 . 13C-NMR  δ: 12.02 (C-18′ a and b), 12.23 (C-29′ a and b), 14.11 (C-16″), 18.97 (C-27′ a and b), 19.28 (C-19′ a and b), 20.99 (C-11′ a and b), 21.07 (C-26′ a and b), 21.22 (C-21′ a and b), 22.68 (C-15″), 24.33 (C-15′ a and b), 24.82 (C-3″), 25.39 (C-28′ a and b), 27.71 (C-2′ a and b), 28.90 (C-16′ a and b), 28.93, 29.09, 29.12, 29.18, 29.27, 29.30, 29.51, 29.70 and 29.75 (–O(O)C–CH2–CH2–C(O)O– a and b, C-4″, C-5″, C-6″, C-7″, C-8″, C-9″, C-10″, C-11″, C-12″, C-13″), 31.82 (C-7′ a and b), 31.86 , 31.91 (C-14″), 33.98 (C-2″), 36.56 (C-10′ a and b), 36.94 (C-1′ a and b), 38.03 (C-4′ a and b), 39.60 (C-12′ a and b), 40.50 (C-20′ a and b), 42.17 (C-13′ a and b), 50.00 (C-9′ a and b), 51.22 (C-24′ a and b), 55.90 (C-17′ a and b), 56.76 (C-14′ a and b), 61.87 (C-3), 62.36 (C-1), 69.31 (C-2), 74.38 (C-3′ a and b), 122.69 (C-6′ a and b), 129.26 (C-23′ a and b), 138.29 (C-22′ a and b), 139.52 (C-5′ a and b), 171.36, 171.43, 171.52 and 171.89 (–O(O)C–CH2–CH2–C(O)O– a and b), 173.28 (C-1″). IR (ATR) υmax 2926, 2852, 1732, 1465, 1366, 1154, 972 cm−1. ESI-HRMS (m/z) calcd for C85H138O10Na [M + Na]+: 1342.0188, found: 1342.0178.Colorless crystals, m.p. 91–93 °C. Yield, 1.78 g, 89%. TLC: 1H-NMR , 13C-NMR , COSY, and HMQC spectra of dStigS-OA are presented.In Rf = 0.57 . 1H-NMR  δ: 0.69 , 0.79 , 0.80 , 0.84 , 0.88 , 0.91–0.98 , 0.99–1.06 , 1.01 , 1.02 , 1.07–1.35 , 1.36–1.64 , 1.65–1.75 , 1.81–1.89 , 1.91–2.09 , 2.27–2.34 , 2.56–2.67 C–C2H–C2H–C(O)O– a and b), 4.15 , 4.19 , 4.29 , 4.30 , 4.55–4.65 , 5.01 , 5.15 , 5.27 , 5.32–5.38 . 13C-NMR  δ: 12.02 (C-18′ a and b), 12.23 (C-29′ a and b), 14.11 (C-18″), 18.96 (C-27′ a and b), 19.28 (C-19′ a and b), 20.99 (C-11′ a and b), 21.07 (C-26′ a and b), 21.22 (C-21′ a and b), 22.67 (C-17″), 24.33 (C-15′ a and b), 24.79 (C-3″), 25.39 (C-28′ a and b), 27.16 and 27.20 (C-8″ and C-11″), 27.71 (C-2′ a and b), 28.90 (C-16′ a and b), 28.92 and 29.18 (–O(O)C–CH2–CH2–C(O)O– a and b), 29.09, 29.12, 29.27, 29.30, 29.51, 29.70 and 29.75 , 31.82 (C-7′ a and b), 31.86 , 31.88 (C-16″), 33.96 (C-2″), 36.56 (C-10′ a and b), 36.93 (C-1′ a and b), 38.03 (C-4′ a and b), 39.60 (C-12′ a and b), 40.50 (C-20′ a and b), 42.17 (C-13′ a and b), 49.99 (C-9′ a and b), 51.21 (C-24′ a and b), 55.89 (C-17′ a and b), 56.75 (C-14′ a and b), 61.88 (C-3), 62.37 (C-1), 69.29 (C-2), 74.37 (C-3′ a and b), 122.69 (C-6′ a and b), 129.25 (C-23′ a and b), 129.70 and 129.97 (C-9″ and C-10″), 138.29 (C-22′ a and b), 139.50 (C-5′ a and b), 171.36, 171.43, 171.52 and 171.89 (–O(O)C–CH2–CH2–C(O)O– a and b), 173.24 (C-1″). IR (ATR) υmax 2927, 2852, 1732, 1459, 1377, 1154, 972 cm−1. ESI-HRMS (m/z) calcd for C87H140O10Na [M + Na]+: 1368.0344, found: 1368.0348.Colorless wax. Yield, 1.60 g, 85%. TLC: 1H-NMR , 13C-NMR , COSY, and HMQC spectra of dStigC-PA are presented.In Rf = 0.62 . 1H-NMR  δ:0.70 , 0.79 , 0.80 , 0.84 , 0.88 , 0.91–0.98 , 0.99–1.06 , 1.01 , 1.02 , 1.07–1.33 , 1.36–1.76 , 1.84–2.09 , 2.32 , 2.35–2.45 , 4.20 , 4.24 , 4.36 , 4.37 , 4.42–4.53 , 5.01 , 5.10 , 5.15 , 5.37–5.41 . 13C-NMR  δ: 12.02 (C-18′ a and b), 12.25 (C-29′ a and b), 14.13 (C-16″), 18.96 (C-27′ a and b), 19.23 (C-19′ a and b), 20.99 (C-11′ a and b), 21.09 (C-26′ a and b), 21.21 (C-21′ a and b), 22.69 (C-15″), 24.32 (C-15′ a and b), 24.80 (C-3″), 25.40 (C-28′ a and b), 27.57 and 27.59 (C-2′ a and b), 28.90 (C-16′ a and b), 29.10, 29.28, 29.37, 29.48, 29.68 and 29.72 , 31.78 (C-7′ a and b), 31.86 , 31.92 (C-14″), 34.00 (C-2″), 36.50 (C-10′ a and b), 36.79 (C-1′ a and b), 37.89 (C-4′ a and b), 39.57 (C-12′ a and b), 40.50 (C-20′ a and b), 42.16 (C-13′ a and b), 49.95 (C-9′ a and b), 51.21 (C-24′ a and b), 55.87 (C-17′ a and b), 56.75 (C-14′ a and b), 61.82 (C-3), 65.12 (C-1), 72.48 (C-2), 78.41 and 78.50 (C-3′ a and b), 123.03 and 123.06 (C-6′ a and b), 129.24 (C-23′ a and b), 138.29 (C-22′ a and b), 139.14 and 139.18 (C-5′ a and b), 153.61 and 154.06 (–OC(O)O– a and b), 173.25 (C-1″). IR (ATR) υmax 2927, 2853, 1739, 1456, 1370, 1243, 1159, 972 cm−1. ESI-HRMS (m/z) calcd for C79H130O8Na [M + Na]+: 1229.9663, found: 1229.9666.Colorless crystals, m.p. 72–74 °C. Yield, 1.72 g, 94%. TLC: 1H-NMR , 13C-NMR , COSY, and HMQC spectra of dStigC-OA are presented.In Rf = 0.62 . 1H-NMR  δ: 0.69 , 0.79 , 0.80 , 0.84 , 0.88 , 0.91–0.98 , 0.99–1.06 , 1.01 , 1.02 , 1.07–1.33 , 1.36–1.76 , 1.84–2.09 , 2.32 , 2.35–2.45 , 4.20 , 4.25 , 4.33–4.39 , 4.42–4.53 , 5.01 , 5.10 , 5.15 , 5.31–5.36 , 5.37–5.41 . 13C-NMR  δ: 12.01 (C-18′ a and b), 12.25 (C-29′ a and b), 14.13 (C-18″), 18.95 (C-27′ a and b), 19.23 (C-19′ a and b), 20.98 (C-11′ a and b), 21.09 (C-26′ a and b), 21.20 (C-21′ a and b), 22.68 (C-17″), 24.32 (C-15′ a and b), 24.76 (C-3″), 25.40 (C-28′ a and b), 27.16 and 27.20 (C-8″ and C-11″), 27.56 and 27.59 (C-2′ a and b), 28.91 (C-16′ a and b), 29.06, 29.09, 29.17, 29.31, 29.52, 29.70 and 29.75 , 31.77 (C-7′ a and b), 31.85 , 31.89 (C-16″), 33.96 (C-2″), 36.49 (C-10′ a and b), 36.78 (C-1′ a and b), 37.87 and 37.91 (C-4′ a and b), 39.56 (C-12′ a and b), 40.51 (C-20′ a and b), 42.15 (C-13′ a and b), 49.94 (C-9′ a and b), 51.20 (C-24′ a and b), 55.86 (C-17′ a and b), 56.74 (C-14′ a and b), 61.81 (C-3), 65.16 (C-1), 72.47 (C-2), 78.42 and 78.50 (C-3′ a and b), 123.05 (C-6′ a and b), 129.23 (C-23′ a and b), 129.71 and 129.96 (C-9″ and C-10″), 138.29 (C-22′ a and b), 139.15 (C-5′ a and b), 153.61 and 154.05 (–OC(O)O– a and b), 173.22 (C-1″). IR (ATR) υmax 2928, 2853, 1744, 1459, 1369, 1239, 1162, 972 cm−1. ESI-HRMS (m/z) calcd for C81H132O8Na [M + Na]+: 1255.9819, found: 1255.9822.Colorless wax. Yield, 1.61 g, 78%. TLC: Distigmasterol-modified acylglycerols were examined for their cytotoxicity in normal human cells originating from the digestive system. The cells were treated for 48 h with increasing concentrations of dStigMAs, and cell proliferation, metabolic activity, and viability were assayed using the MTT test. The cytotoxic effects of the analyzed dStigMAs on CCD 841CoN colon mucosa cells, FHs 74 Int small intestine epithelial cells, and THLE-2 hepatocytes are presented in 50) values were calculated at 0.32 µg/mL (266 nM/mL) for dStigC-PA and 0.47 µg/mL (381 nM/mL) for dStigC-OA. Lower cytotoxicity of dStigMAs containing a carbonate linker was observed in colon and liver cell cultures. Following cell exposure to the compounds at the maximum dose, colon and liver cell proliferation and viability were reduced to 53.8% (dStigC-PA) and 59.4% (dStigC-OA) (Distigmasterol-modified acylglycerols (dStigMAs) possessing a carbonate linker showed potent cytotoxicity to the cells isolated from the small intestine A and coltigC-OA) B, and totigC-OA) C, respectigC-OA) A–C.Phytosterols and their fatty acid esters naturally occur in vegetable oils and fats, where they consist up to 90% of the unsaponifiable fraction. These bioactive components reduce blood cholesterol levels in humans due to blockage of cholesterol absorption. Moreover, these compounds are often added in large amounts to various food products, the last are often treated by thermal processes such as frying. Thermal processes and long-term storage of food products enriched in plant sterols and their esters caused their degradation to oxidized derivatives, dimers, oligomers, and low molecular compounds. Some of these degradation products had adverse effects on cell cultures, while low molecular weight compounds may also negatively affect sensory perception of foods. Degradation products of stigmasterol induced apoptotic death of colon cells (CCD 841 CoN), measured by fluorescence cytometric tests . The impThe obtained structured lipids being a hybrid of monoacylglycerols with a plant sterol could be new functional components characterized higher stability than free and esterified phytosterols. The thermo-oxidized stability of phytosterol esters depended on the unsaturation level of fatty acids moiety. The polyunsaturated fatty acids promoted the degradation of esters and the formation of phytosterol oxidation products . Using sBecause of that cytotoxicity of synthesized dStigMAs was determined. The analysis of their biological properties showed that hybrids with carbonate linkers characterized higher cytotoxicity than compounds with succinate linkers. It is crucial for consumers′ health and safety to establish plant sterol derivatives that will be thermo-oxidative stable and to produce benign degradation products when these bioactive compounds are used to enrich food products and to control cholesterol levels in human blood and directly affect global heart problems. Before using them as food components, a more detailed analysis should be performed.N,N′-dicyclohexylcarbodiimide (DCC), 3-palmitoyl-sn-glycerol, acetic acid, ethanol-free chloroform, ethyl acetate, methanol, n-hexane, dichloromethane, diisopropyl ether, toluene, and pyridine were purchased from Sigma-Aldrich . 1-Oleoyl-sn-glycerol was purchased from Carbosynth Ltd.,  and glyceryl heptadecanoate was acquired from Larodan .Stigmasterol, stigmasteryl chloroformate (StigCF), succinic anhydride, 4-(dimethylamino)pyridine (DMAP), sn-glycerol, 1-oleoyl-sn-glycerol and stigmasteryl chloroformate (StigCF) were used for the synthesis of dStigMAs. Stigmasteryl hemisuccinate (StigHS) was obtained as described below.Commercially available 3-palmitoyl-4, filtered, and concentrated in vacuo. The crude product was purified by recrystallization from methanol at 4 °C.To a solution of stigmasterol  in dry pyridine (100 mL), succinic anhydride , and DMAP  were added. The reaction mixture was stirred at 60 °C for 6 h, and then at r.t. overnight. When stigmasterol reacted completely (TLC), the reaction mixture was washed with 10% HCl solution (250 mL) and extracted with ethyl acetate (3 × 100 mL). The organic layer was washed with saturated NaCl solution (3 × 100 mL), dried over MgSOsn-glycerol or 1-oleoyl-sn-glycerol; 0.5 g,) and stigmasteryl hemisuccinate  in ethanol-free chloroform (30 mL), DMAP (2.6 equiv.) and DCC (2.6 equiv.) were added. After 24 h of stirring at 20 °C, the reaction was monitored using TLC. The mixture was filtered and the filtrate was diluted with 100 mL of chloroform and washed with 100 mL of 0.5 M HCl. The organic layer was washed with brine, dried over anhydrous MgSO4, and concentrated in vacuo. The crude 1,2-distigmasterylsuccinoyl-3-palmitoyl-sn-glycerol (dStigS-PA) and 2,3-distigmasterylsuccinoyl-1-oleoyl-sn-glycerol (dStigS-OA) were purified by column chromatography .To a solution of the corresponding monoacylglycerol  and DMAP (2.6 equiv.) in ethanol-free chloroform (15 mL), the solution of stigmasteryl-chloroformate  in ethanol-free chloroform (10 mL) was added dropwise. The reaction mixture was stirred at 20 °C. When the reaction was finished , the solvent was evaporated and the crude 1,2-distigmasterylcarbonoyl-3-palmitoyl-sn-glycerol (dStigC-PA) and 2,3-distigmasterylcarbonoyl-1-oleoyl-sn-glycerol (dStigC-OA) were purified by column chromatography .To a solution of the corresponding monoacylglycerol  was used as a visualization reagent. Visualization was determined using UV light (λ = 254 and 365 nm). Compounds were also detected by spraying the plates with an H2SO4/CH3OH mixture , followed by heating to 120–200 °C. Products were purified using chromatography on silica gel columns . NMR spectra  were recorded on a Jeol 400 MHz Year Hold Magnet spectrometer . Chemical shifts were referenced to the residual solvent signal . ATR-IR spectra were collected using a Nicolet iS10 spectrometer . Spectra were obtained from 4000 cm−1 to 520 cm−1 at 32 scans, with a spectral resolution of 4 cm−1 and a blank window for background. HRMS spectra were recorded using the ESI technique on an ESI-Q-TOF Premier XE spectrometer . Melting points  were determined on a Boetius apparatus .TLC analyses used to monitor the progress of reactions were performed on 0.2 mm silica gel 60 F254 plates  with a mixture of hexane and ethyl acetate or chloroform, methanol, and acetic acid in various ratios. Primuline spray  showed no cytotoxic activity against normal human cells when lipids were added to the cell cultures at 100 µg/mL and lower concentrations. The use of these compounds as functional food additives requires further studies on their thermal and oxidative stability, however, they may become a safe source of phytosterols for humans.


Herbidospora daliensis originating from Taiwan led to the isolation of five new compounds, for which we propose the name herbidosporadalins A–E (1–5), one isolated for the first time, herbidosporadalin F (6), together with two known compounds (7 & 8). Their structures were elucidated by spectroscopic analyses, including 1D- and 2D-NMR experiments with those of known analogues, and on the basis of HR-EI-MS mass spectrometry, their anti-inflammatory activities were also evaluated. Of these isolates, herbidosporadalin A (1), B (2), F (6) and G (8) showed NO inhibitory activity, with IC50 values of 11.8 ± 0.9, 7.1 ± 2.9, 17.8 ± 1.7, and 13.3 ± 6.5 μM, stronger than the positive control quercetin (IC50 = 36.8 ± 1.3 μM). To the best of our knowledge, this is the first report on 3,4-seco-friedelane metabolites  from the genus Herbidospora.Bioassay-guided fractionation of extracts derived from solid cultures of a  Actinobacteria are a very special group of microorganisms that mainly grow in soil and can also be found in oceans, streams, lake water or sediment, animals, plants, nodules, compost, hot springs, geothermal and other environments. Its morphology is as varied as that of fungi, and it has structures such as substrate hyphae, aerial hyphae, spore, spore chain and sporangium in its life history. However, it resembles prokaryotes-like bacteria in physiological structure, so it has been mistaken for fungi for a long time, and some people regard it as a microorganism between bacteria and fungi-boundary microorganisms. Actinobacteria themselves have the ability to produce a variety of physiologically active products . The 1H NMR spectrum indicated that 1 possessed a coumarin nucleus based on a pair of characteristic doublets at δH 7.75  and 6.35 , one singlet aromatic proton at δH 7.36 , two doublets benzofuran protons at δH 6.80  and 7.68 , two Me groups at δH 1.26  and 4.71 , two Me groups at δH 1.26  and one aliphatic methoxy group at δH 3.24 . The 13C NMR and DEPT spectra of 1 displayed 17 carbons, including one lactone carbonyl carbon at δC 210.6; two olefinic methines at δC 106.8 (C-3′) and 146.8 (C-2′); 8 aromatic carbons at δC 148.2 (C-7), 144.3 (C-4), 143.5 (C-8a), 131.9 (C-8), 126.0 (C-6), 116.5 (C-4a), 114.7 (C-3), and 113.5 (C-5); two methyls at δC 21.4 (C4″) & 20.6 (C-5″); one methoxy at δC 49.3 (OCH3-2″); an adjacent oxymethylene at δC 75.6 (C-1″); and an oxygenated quaternary carbon at δC 75.5 (C-3″). In the HMBC spectrum, the conjugated lactone carbonyl carbon  revealed correlations with H-3 (δH 6.35) and H-4 (δH 7.75), one oxygenated quaternary carbon C-8a (δC 143.5) showed correlations with H-4 (δH 7.75) & H-5 (δH 7.36), and the other oxygenated quaternary carbon  showed the correlations with H-2′ (δH 7.68), H-5 (δH 7.36) and H-3′ (δH 6.80). These 1D and 2D NMR spectroscopic data suggested the existence of an alkyl butyl substituent at C-8 and a methoxy group at C-2″. The deshielded methylene signal appeared at δH 4.37  and 4.71 , which was correlated to carbon  from the HSQC spectrum. The tertiary carbon  revealed interactions with (CH3)2-4″ and 5″ (δH 1.26), CH2-1″ (δH 4.37/4.71), and OCH3-2″ (δH 3.24), and an oxygenated quaternary carbon  revealed interactions with the Me group  and oxymethine  in the HMBC spectrum. These data and HMBC correlations showed unambiguously the structure of the alkylbuty unit as a 3-hydroxy-2-methoxy-3-methylbutoxy group. Furthermore, compound 1 showed dextrorotatory optical activity with [α]25D + 111.8 , and the absolute configuration of C-2″ was proposed as S-form after comparison with the (R)-heraclenol chromen-7-one and was named herbidosporadalin A.Compound NMR data  of 1 shoraclenol , except 2 was obtained as an optically inactive yellow oil. [α]25D ± 0 . The molecular formula was established as C15H16O5 by EIMS and HRESIMS analysis (276 [M]+). The UV absorption band at λmax 321 (4.45), 258 (3.94), 248 (3.91), and 222 (4.30) nm were characteristic of the coumarin skeleton and the IR (1732 cm−1), and aromatic moiety . The 1H NMR spectrum , 5.52 , 9.71 ] in 2 was substituted for an ethyl group  in murrayacarpin A. 13C NMR spectrum (C 18.4 (C-3′), 20.0 (C-4′), 52.8 (C-2′), 72.0 (C-1′), 203.8 (CHO-2′)].Compound spectrum  was simid the IR 32 cm−1, spectrum  also supH 5.52) is associated with C-7, 8, 9; δH 9.71 (s) is associated with C-2′, 3′, 4′; and it was determined that the branched chain of compound 2 contains a tert-butyl group, and the mass peak at m/z 205 (100) seen in the EI-MS spectrum was presumed to be the fragment of the molecular parent peak removed -CCHO(CH3)2-. Compound 2 is as follows: 7-methoxy-8-coumarin, designated as herbidosporadalin B. Tertiary butyl is very rare in nature, and it has been found in compounds isolated from Ginkgo biloba in the past. The structure of 1 is a quite interesting new skeleton, and its possible biosynthesis pathway is speculated, as shown in The associations of the HMBC signals are as follows: H-1′  data determined the molecular formula to be C22H20O3 ), indicating 13 degrees of unsaturation. The 13C NMR (−1), and aromatic  functionalities in the IR spectrum together with the 1H-NMR spectrum of 3 showed characteristics of a chromene ring, i.e., of the ABX-pattern H-atoms H-7 , H-8 , H-10 ), CH3-9 , of a pair of magnetically equivalent Me groups ). The base peak at m/z 317 ([M-Me]+) in the EI-MS and a set of signals at δC 27.7 (Me(6 × 2)), 77.3 (C(3′), 128.2 (C-11), 136.4 (C-12), 123.2 (C-7), 128.9 (C-8), 137.4 (C-9), and 123.1 (C-10) in the 13C-NMR spectrum provided further support for the presence of a geminally dimethyl-substituted chromene system. The remaining unsaturation degrees suggested the presence of a typical biphenyl unit that was supported by the remaining 12 aromatic C-atom signals in the 13C-NMR spectrum. The existence of two phenolic OH groups was confirmed by the signals of two exchangeable H-atoms in the 1H-NMR (δH 5.42 and 4.87) and two oxygenated aromatic C-atoms in the 13C-NMR spectrum (δC 144.8 (C-3) and 154.8 (C-10). The 1H-NMR spectrum showed a set of ds of ortho-coupled H-atoms at δH 7.48  and 6.88 ), typical for a para-substituted aryl moiety. Therefore, one OH group was assigned to C-10. A set of doublets of meta-coupled H-atoms at δH 7.06  and 7.40  belonged to a 1,2,3,5-tetrasubstituted benzene moiety (ring A). A HMBC experiment allowed positioning of the substituents at ring A. The H-10 signal at δH 7.57 showed a two-bond connectivity with C-11 (δC 128.2), and a three-bond connectivity with C-12 (δC 136.4), C-13 (δC 122.6) and C-11 (δC 128.2) in the HMBC plot (H 7.00 showed a two-bond connectivity with C-4 (δC 145.8), and a three-bond connectivity with C-14 (δC 138.5), C-1 (δC 112.4), and C-1′ (δC 134.5), which suggested that the second OH group was attached to C-3. Further support for the determination of the structure was provided by the signal of H-1 at δH 7.40, showing a two-bond connectivity with C-13 (δC 122.6), and a three-bond connectivity with C-3 (δC 112.9), C-14 (δC 138.5), and C-1′ (δC 134.5) in the HMBC plot. The 1H- and 13C-NMR (3) as 2-(4-hydroxyphenyl)-6,6,9-trimethyl-6H-benzo[c]chromen-4-ol.Compound  13C NMR  and DEPTMBC plot . H-3 at  13C-NMR  and HMBC 13C-NMR  establis4 was isolated as an amorphous powder, and its molecular formula was established as C16H16O6 by HREIMS. The UV spectrum exhibited bands at 233 sh, 286 nm, suggesting the presence of a benzophenone moiety. A band attributable to ketone (1729 cm−1) was observed in the IR spectrum. The 1H NMR data  7.22 , 6.84 ], two symmetrical aromatic protons [δH 6.08 ] in another benzene ring, the existence of two phenolic OH groups was confirmed by the signals of two exchangeable H-atoms in the 1H-NMR (δH 6.02 and 5.32) and two oxygenated aromatic C-atoms in the 13C-NMR spectrum (δC 150.5 (C-4′)) and 158.5 (C-4), and three methoxy groups , indicating the presence of a 4-hydroxy-3-methoxyphenyl moiety and a 4-hydroxy-2,6-dimethoxyphenyl.Compound NMR data  of 4 shoC 194.2 (C-7)]. The HMBC spectrum (H 7.59), H-6′ (δH 7.22)/C-7 (δC 194.2), OCH3-3′ (δH 3.93)/C-3′(δC 146.5), OCH3-2 & 6 (δH 3.63)/C-2 & 6 (δC 158.6), H-5′ (δH 6.84)/C-1′ (δC 131.1), C-3′ (δC 146.5), C-4′ (δC 150.5), H-6′ (δH 7.22)/C-2′ (δC 110.1), C-4′ (δC 150.5), C-7 (δC 194.2), H-2′ (δH 7.59)/C-4′ (δC 150.5), C-6′ (δC 126.2), C-7 (δC 194.2), H-3 (δH 6.08)/C-1 (δC 110.4), C-2 (δC 158.6), C-4 (δC 158.5), and C-5 (δC 92.0). This result was further supported by the key NOESY data (H 7.59 (H-2′)/OCH3-3′ (δH 3.93), δH 7.22 (H-6′)/6.84 (H-5′), and δH 6.08 (H-3)/OCH3-2 (δH 3.63). Thus, the biphenylpropanoid structure of 4 was elucidated as 4,4-dihydroxy-2,6,3′-trimethoxy-benzophenone, which was confirmed by DEPT, COSY, HSQC, NOESY . The 1H and 13C chemical shifts of the hydrogen and carbon atoms are given in The structure elucidation of three secotriterpenes compounds 5 was obtained as a light white solid with a 197–198 °C melting point, and the high resolution electron impact mass spectrometry (HR-EI-MS) data determined the molecular formula to be C32H54O2 ), indicating six degrees of unsaturation. The IR spectrum indicated the presence of carbonyl (1736 cm−1), and double bond (1633 cm−1) functionalities. The 1H NMR spectrum of 5  showed seven methyl singlets resonating at δH 1.15 , 0.99 , 0.97 , 0.97 , 0.96 , 0.92 , 0.86 , and one triplet at 1.23 , respectively, on the basis of comparison with those reported in the literature for closely related triterpenes.Compound 1H-NMR and 13C-NMR spectra  and 1.23 ] and the terminal double bond  of 5 replaced a hydroxyl group at C-3 and a single bond at C-4–C-23  of 3,4-seco-friendelan-3-oic acid.The  spectra  of 5 wer3-24 (δH 0.96) and these hydrogens with C-4 (δC 42.0), C-10 (δC 58.3) and C-6 (δC 41.5); C-10 (δC 58.3) with CH3-25 (δH 0.86); CH3-25 with C-8 (δC 53.0), C-9 (δC 38.6) and C-11 (δC 35.1); C-8 (δC 53.0) with CH3-26 (δH 0.97); CH3-26 (δH 0.97) with C-13 (δC 39.6), C-14 (δC 38.3) and C-15 (δC 32.2); C-13 and C-14 with CH3-27 (δH 0.99); CH3-27 (δH 0.99) and CH3-30 (δH 0.97) with C-19 (δC 35.2), C-20 (δC 28.1), and C-21 (δC 32.7). The relative configuration of 1 was determined on the basis of a NOESY experiment +]. Its IR spectrum showed carbonyl absorption at 1737 cm−1. The 1H and 13C NMR data . The main differences between them were that the oxymethine group [δH 4.07 (C-2)] at C-2 in 8 was replaced by a methylene group after observing HMBC correlations from CH2-2  to C-3 (δC 173.8) and C-1 (δC 21.0). Compound 6 was first isolated from a natural source, though it has since been synthesized + at m/z 290.1144 by HR-EI-MS , corresponding to 8 degrees of unsaturation. The IR spectrum exhibited absorptions of a strong intermolecular hydrogen bonding at 3416 cm−1 and a conjugated carbonyl group (1660 cm−1). The 1H and 13C NMR data /δC 162.1 (C-7), 157.4 (C-5), 156.0 (C-8a), 111.4 (C-6), 105.0 (C-4a), and 89.7 (C-8)], an olefinic methine [δC/δH 108.6/6.03 ], an olefinic methyl [δC/δH 20.9/2.34 ], a carbonyl carbon signal , and a carbon signal . Furthermore, the characteristic UV absorption signals at 291, 256, and 232 nm for the chromone skeleton and the above-mentioned NMR data indicated that compound 7 showed signals and coupling patterns similar to those of cnidimol A -7-methoxy-2-methylchromone  in 8 was substituted for a vinyl group  in 5. The 1H- and 13C-NMR  + .Herbidosporadalin A (2): yellowish oil; +, 175 (23); HREIMS m/z 276.0995 [M]+ .Herbidosporadalin B (3): oil; UV (MeOH): 287 (4.39), 265 (4.60), 212 (4.59) nm; IR (Neat): 3390 (OH), 1598, 1504 (aromatic ring) cm−1; 1H NMR : see 13C NMR : see m/z (%): 332 , 317 (100), 306 (23), 158 (12); HREIMS m/z 332.1429 [M]+ .Herbidosporadalin C (4): amorphous powder; mp. 177–179 °C; UV (MeOH): 310 (4.30), 281 (4.3), 230 (4.5) nm; IR (Neat): 3353 (OH), 1593 (CO) cm−1; 1H NMR : see 13C NMR : see m/z (%): 304, 287 (33), 181 (100), 123 (13); HREIMS m/z 304.0937 [M]+ .Herbidosporadalin D (5): amorphous solid; mp. 197–198 °C; + .Herbidosporadalin E (6): white needles; mp. 185–186 °C; + .Herbidosporadalin F (Z)-4′-acetoxy-3′-methylbut-2′-enyl]-7-methoxy-2-methylchromone (7): oil; UV (MeOH): 291 (3.97), 256 (4.27), 251 (sh) (4.27), 232 (4.33) nm; IR (Neat): 3416 (OH), 1660 (conjugated ketone group), 1626, 1493 (benzene) cm−1; 1H NMR : see 13C NMR : see m/z (%): 290 , 272 (35), 257 (58), 231(52), 189 (54); HREIMS m/z 290.1144 [M]+ .5-Hydroxy-6-+ .2-Hydroxyl-3,4-seco-friedelan-3-oic acid ethyl-ester  for 20 h. The concentration of NO in culture supernatants was determined as nitrite, a major stable product of NO, by Griess reagent assay [Mouse macrophage cell line (RAW 264.7) was obtained from the Bioresource Collection and Research Center (BCRC 60001) and cultured at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum , 4.5 g/L glucose, 4 mM glutamine, penicillin (100 units/mL), and streptomycin (100 μg/mL) in a humidified atmosphere in a 5% COnt assay , and celActinobacteria have great economic and biotechnological value and have long been recognized as the main microorganisms in the medical industry. To date, there are tens of thousands of antibiotics produced by microorganisms, of which more than 70% are derived from Actinobacteria [Actinobacteria have various structures and biological activities, including antibacterial, antifungal, antitumor, insecticidal and herbicidal, enzyme inhibition, immune regulation, etc. [Actinobacteria have great potential for the development of new medicines. As part of our investigations aimed at exploring structurally novel bioactive secondary metabolites from actinomycetes, chemical research on the fermentation extract of the H. daliensis led to the isolation of seven previously undescribed compounds, namely, herbidosporadalins A–G (1–7), and one compound isolated from nature for the first time, namely, herbidosporadalin H (8)  in RAW 264.7 cells. In addition, compounds 1, 2, 7, and 8 showed potent inhibition with IC50 values ≤ 17.8 μM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation, stronger than the positive control quercetin (IC50 = 36.8 ± 1.3 μM). To the best of our knowledge, this is the first report on 3,4-seco-friedelane metabolites (6–8) from the genus Herbidospora.bacteria . Secondaon, etc. , which iin H (8) . The strIn previous surveys, there have been many reports on the activity of actinomycete metabolites in the literature, but few reports of active natural products were evaluated. The current study reported the inhibitory activities of active natural products against nitric oxide production. Therefore, it is worth continuing to study the effect of such substances upon other various pro-inflammatory cytokines, such as interleukin (IL)-12p40, IL-6, and tumor necrosis factor (TNF)-α.


Poriae Cutis were separated by high-speed countercurrent chromatography (HSCCC) and identified using ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) and nuclear magnetic resonance (NMR). The in vitro anti-inflammatory activities of the purified triterpenoids on RAW 264.7 cells were also investigated. Triterpenoids, poricoic acid B, poricoic acid A, dehydrotrametenolic acid, and dehydroeburicoic acid were obtained; their levels of purity were 90%, 92%, 93%, and 96%, respectively. The results indicated that poricoic acid B had higher anti-inflammatory activity than those of poricoic acid A by inhibiting the generation of NO in lipopolysaccharide (LPS)-induced RAW 264.7 cells. However, dehydrotrametenolic acid and dehydroeburicoic acid had no anti-inflammatory activity. In addition, the production of cytokines  in cells treated with poricoic acid B decreased in a dose-dependent manner in the concentration range from 10 to 40 μg/mL. The results provide evidence for the use of Poriae Cutis as a natural anti-inflammatory agent in medicines and functional foods.In this study, triterpenoid compounds from  Poria cocos (Schw.) Wolf (“fuling” in Chinese) is a widely used medicinal fungus. According to the Chinese Pharmacopoeia, about one-tenth of traditional Chinese medicine preparations include P. cocos -. UV λmax: 242 nm. The 1H- and 13C-NMR spectra revealed H-11 (δ 5.21)/C-11 (δ 120.7), H-7 (δ 5.17)/C-7 (δ 119.0), C-9 (δ 138.3), C-8 (δ 142.3), H-18 (δ 0.99), H-19 (δ 0.93), H-26 (δ 1.50), H-27 (δ 1.58), H-29 (δ 1.68), H-30 (δ 1.50), H-16 (δ 4.54), H-28 , H-24 (δ 5.27), C-3 (δ 178.3), C-21 (δ 180.0). Based on the structural features of triterpene compounds and the data given in the literature, compound-1 was identified as poricoic acid B -. UV λmax: 242 nm. The 1H- and 13C-NMR spectra presented H-11 (δ 5.27)/C-11 (δ 119.5), H-7 (δ 5.21)/C-7 (δ 117.8), C-9 (δ 137.1), C-8 (δ 141.1), H-18 (δ 1.09), H-19 (δ 1.02), H-26 (δ 0.98), H-27 (δ 0.99), H-29 (δ 1.74), H-30 (δ 1.50), H-16 (δ 4.54), H-28 , C-3 (δ 176.9), C-21 (δ 178.5), C-24 (δ 155.3), C-25 (δ 33.6), C-31 (δ 106.7). Based on the structural features of triterpene compounds and the data given in the literature, compound-4 was identified as poricoic acid A -. UV λmax: 243 nm. The 1H- and 13C-NMR spectra indicated that H-11 (δ 5.37)/C-11 (δ 116.5), H-7 (δ 5.61)/C-7 (δ 121.0), C-9 (δ 146.4), C-8 (δ 142.7), H-18 (δ 1.01), H-19 (δ 1.07), H-26 (δ 1.67), H-27 (δ 1.62), H-28 (δ 1.22), H-29 (δ 1.13), H-30 (δ 1.06), H-3 (δ 3.46), H-24 (δ 5.33), C-3 (δ 77.9), C-21 (δ 178.9), C-24 (δ 124.8), C-25 (δ 131.5). Based on the structural features of triterpene compounds and the data given in the literature, compound-4 was identified as dehydrotrametenolic acid [Compound-3 : ESI-MS 4 (m/z): 467.3518 [M-H]-. UV λmax: 243 nm. The 1H- and 13C-NMR spectra showed that H-11 (δ 5.62)/C-11 (δ 116.5), H-7 (δ 5.61)/C-7 (δ 121.0), C-9 (δ 146.4), C-8 (δ 142.7), H-18 (δ 1.02), H-19 (δ 1.08), H-26 (δ 1.04), H-27 (δ 1.05), H-28 (δ 1.23), H-29 (δ 1.14), H-30 (δ 1.08), H-3 (δ 3.46), H-31 , C-3 (δ 77.9), C-21 (δ 178.9), C-24 (δ 160.8), C-31 (δ 106.8). Based on the structural features of triterpene compounds and the data given in the literature, compound-4 was identified as dehydroeburicoic acid [Compound-4 : ESI-MS Poriae Cutis were different and are shown in The cytotoxic effect of poricoic acid A, poricoic acid B, dehydrotrametenolic acid, and dehydroeburicoic acid on RAW 264.7 cells was evaluated by an MTT assay. It is generally acknowledged that when adding samples, if the cell viability is below 90%, it indicates cytotoxicity. If the cell viability is higher than this critical value, the sample is non-cytotoxic and can be used for further research . The viaPoria cocos showed weak cytotoxic activities on MGC-803 and HepG2 cell lines.In summary, the maximum concentrations of poricoic acid A, poricoic acid B, dehydrotrametenolic acid, and dehydroeburicoic acid without significant cell activity inhibition on RAW264.7 cells were 30, 40, 1, and 0.5 μg/mL, respectively. The difference may be related to their chemical structures. Poricoic acid A and poricoic acid B belong to 3,4-seco-lanostan-7,9(11)-diene type triterpenes, which were less cytotoxic to RAW264.7 cells; dehydrotrametenolic acid and dehydroeburicoic acid are both lanosta-7,9(11)-diene type triterpenes, which were reported to have a higher cytotoxicity [Poriae Cutis on NO production in LPS-induced RAW 264.7 cells in co-culture experiments are shown in p > 0.05), but the change became significant with a further increase in concentration. The NO production in cells treated with poricoic acid A significantly decreased to 47% at 30 μg/mL. Poricoic acid B dose-dependently decreased NO production at concentrations of 20, 30, and 40 μg/mL, and considerably suppressed NO production to 29%, 34%, and 68%, respectively. However, we found that the IC50 of poricoic acid A that inhibited NO production in LPS-activated cells was 18.12 μΜ (8.77 μg/mL) [Nitric oxide (NO) has been acknowledged as an important signaling molecule related to inflammatory responses . NaturalThe anti-inflammatory activities of four triterpenes  were assessed by an LPS-stimulated NO production model on RAW 264.7 cells. The results revealed that poricoic acid B exhibited a strong NO production inhibitory activity in a dose-dependent manner. Thus, the secretion of cytokines including tumor necrosis factor (TNF)- α, interleukin (IL)-1β, and interleukin (IL)-6 was measured with poricoic acid B in a medium of RAW 264.7 in co-culture experiments, which were quantified by an ELISA assay.5 pg/mL). The result indicated that LPS increased the release of pro-inflammatory cytokines [Cyclocarya paliurus showed similar inhibition of IL-6 and TNF-α [It has been reported that LPS-induced RAW 264.7 cells secrete pro-inflammatory factors TNF-α, IL-1β, and IL-6 during the inflammation process ,31. As sytokines , and pornd TNF-α .Poriae Cutis: two 3,4-seco-lanostan-7,9(11)-diene type triterpenes (poricoic acid B and poricoic acid A) and two lanosta-7,9(11)-diene type triterpenes (dehydrotrametenolic acid and dehydroeburicoic acid). The cytotoxicity of the 3,4-seco-lanostan-7,9(11)-diene type triterpenes was lower than that of the lanosta-7,9(11)-diene type triterpenes. The 3,4-seco-lanostan-7,9(11)-diene type triterpenes were found to inhibit the NO generation in LPS-induced RAW264.7 cells. However, the lanosta-7,9(11)-diene type triterpenes had no effect on the production of NO. In addition, poricoic acid B significantly inhibited the secretion of TNF-α, IL-1β, and IL-6 in LPS-induced RAW 264.7 cells. Our results indicated that 3,4-seco-lanostan-7,9(11)-diene type triterpenes exhibited more anti-inflammatory activities than lanosta-7,9(11)-diene type triterpenes, and provided experimental evidence that Poriae Cutis is a potential source of natural anti-inflammatory agents for use in pharmaceuticals and functional foods.In this study, four triterpenoids were isolated from


Patrinia scabiosaefolia is a medical and edible Chinese herb with high nutritional and medicinal value. The continuing study of its chemical constituents led to the discovery of nine unique iridoids and iridoid glycosides, including three new iridoids (1-3) and six previously unknown irioid glycosides (5-10), and one known compound (4). Among them, compound 1 was a deformed iridoid, while compounds 3, 5-7, and 10 formed a new ring in their skeletons which was uncommon in this genus. For compound 3, the new ring existed between C-3 and C-10, while a 1,3-dioxane appeared between C-7 and C-10 in compounds 5-7 and 10. Moreover, compound 10 was a bis-iridoid glycoside, which was the first reported in P. scabiosaefolia. And the sugar of irioid glycosides (5-10) was glucose at C-11, except in 9 which had a 5-deoxyglucose moiety. All their structures were confirmed based on the extensive spectroscopic analysis, including IR, UV, HR-ESI-MS, ECD, and 1D- and 2D-NMR experiments. Their cytotoxic activities against HL-60, A-549, SMMC-7721, MCF-7, SW480 were also tested. Nardostachys, Valeriana, and Patrinia, which can be found all over the country  and six novel irioid glycosides (5-10), and one known compound (4)  . Among t*GEL ODS-A-HG . Fractions were monitored by TLC, and spots were visualized by UV light and sprayed with 10% H2SO4 in EtOH, followed by heating.Optical rotation was obtained on a JASCO P-1020 digital polarimeter . UV spectra were measured by a Shimadzu UV-2401 PC spectrophotometer . IR spectra were obtained on a Bruker Tensor 27 infrared spectrophotometer  with KBr pellets. Mass spectra were performed on an API QSTAR time-of-flight spectrometer  and LC/MS-IT-TOF  spectrometer. NMR spectra were recorded on Bruker DRX-500 and Av III-800 instruments with TMS as the internal standard . The chemical shifts were given in δ (ppm) with reference to the solvent signal. Column chromatography was performed on silica gel , Lichroprep Rp-18 gel , MCI gel CHP-20P , Sephadex LH-20 , and YMCPatrinia scabiosaefolia were collected in October 2010 from Shucheng county, Anhui Province, People's Republic of China, and were stored in a cool and dry place at room temperature. The material was identified by Prof. Shou-Jin Liu in Anhui University of Chinese Medicine and a voucher specimen (Wan1295) was deposited in Anhui University of Chinese Medicine. The plants of P. scabiosaefolia are common in the local area and the collection was permitted. We also ensured that the local population of P. scabiosaefolia was not destroyed through the means of collection at different locations.The whole plants of P. scabiosaefolia were extracted with 95% ethanol (3 × 75 L) under room temperature and concentrated under reduced pressure. Then the residue (3 kg) was dissolved in water and partitioned successively with n-butanol to yield n-butanol extract (0.85 kg) after concentration. The n-butanol extract was subjected to silica gel column chromatography eluted with a gradient of CHCl3-MeOH  to obtain five fractions 1-5 by TLC plate analysis.The air-dried and powdered whole plants (29 kg) of 3-MeOH  to afford 4 subfractions (Fr.2-1 to Fr.2-4). Fr.2-1 (5.9 g) was performed by Rp-18 column chromatography  to afford 6 subfractions (Fr.2-1-1 to Fr.2-1-6). Fraction 2-1-2 (1.0 g) was applied to Sephadex LH-20 column chromatography  and then purified by silica gel column chromatography repeatedly to obtain 1 (1.0 mg). Fraction 2-2-3 (1.3 g) was submitted to silica gel column chromatography eluted with a gradient of EtOAc-MeOH  to afford A (180 mg) and B (240 mg), and then purified by semi-prep. HPLC  to obtain 3 (7.0 mg) and 4 (16.0 mg) respectively. Fraction 2-2-4 (2.1 g) was separated by the same methods as Fraction 2-2-3 and purified by semi-prep. HPLC  to afford 5 (1.0 mg) and 10 (16.0 mg).Fraction 2 (22 g) was separated by silica gel column chromatography eluted with a gradient system of CHCl3-MeOH  to afford 6 subfractions (Fr.3-1 to Fr.3-6). Fr.3-3 (4.3 g) was separated by Rp-18 column chromatography  to get 5 subfractions (Fr.3-3-1 to Fr.3-3-5). Fraction 3-3-3 (560 mg) was applied to Sephadex LH-20 column chromatography , and silica gel column chromatography eluted with CHCl3- MeOH  to obtain 2 (3.0 mg).Fraction 3 (15 g) was subjected to silica gel column chromatography eluted with a gradient system of CHCl2O, 30:70→ 100:0, v/v) to obtain 6 subfractions (Fr.4-1 to Fr.4-6). Fr.4-2 (3.4 g) was applied to silica gel column chromatography eluted with a gradient system of EtOAc-MeOH , then isolated by Sephadex LH-20 column chromatography , and further purified by semi-prep. HPLC  to afford 6 (3.0 mg) and 7 (10 mg). Fr.4-4 (5.6 g) was repeatedly separated by silica gel column chromatography and Sephadex LH-20 column chromatography , and finally purified by semi-prep. HPLC  to afford 8 (3.0 mg) and 9 (16.0 mg).Fraction 4 (28 g) was submitted to Rp-18 column chromatography (MeOH-H1)Patrirscabioin M ; UV (MeOH) λmax (log ε): 204 (3.42) nm; IR (KBr) νmax 3,428, 2,925, 2,855, 1,632, 1,384, 1,101 cm−1; 1H and 13C NMR data, see m/z 235 [M + Na]+; HREIMS m/z 235.0943 [M + Na]+ .2)Patrirscabioin N ; UV (MeOH) λmax (log ε): 248 (3.72) nm; 203 (3.69) nm; IR (KBr) νmax 3,425, 2,927, 2,874, 1,727, 1,625, 1,384, 1,161, 1,088 cm−1; 1H and 13C NMR data, see +; HREIMS m/z 293.1354 [M + Na]+ .3)Patrirscabioin O ; UV (MeOH) λmax (log ε): 203 (3.03) nm; IR (KBr) νmax 3,444, 2,926, 2,870, 1,725, 1,634, 1,383, 1,088 cm−1; 1H and 13C NMR data, see +; HREIMS m/z 295.1515 [M + Na]+ .5)Patrinoside B ; UV (MeOH) λmax (log ε): 218 (4.15) nm; IR (KBr) νmax 3,426, 2,929, 2,873, 1,727, 1,642, 1,076 cm−1; 1H and 13C NMR data, see m/z 593 [M + Na]+; HREIMS m/z 593.2931 [M + Na]+ .6)Patrinoside C ; UV (MeOH) λmax (log ε): 209 (4.16) nm, 217 (4.18) nm; IR (KBr) νmax 3,431, 2,926, 2,876, 1,727, 1,637, 1,384, 1,075 cm−1; 1H and 13C NMR data, see m/z 537 [M + Na]+; HREIMS m/z 537.2302 [M + Na]+ .7)Patrinoside D ; UV (MeOH) λmax (log ε): 209 (4.06) nm, 217 (4.06) nm; IR (KBr) νmax 3,429, 2,924, 2,855, 1,724, 1,635, 1,384, 1,075 cm−1; 1H and 13C NMR data, see m/z 551 [M + Na]+; HREIMS m/z 551.2467 [M + Na]+ .8)Patrinoside E ; UV (MeOH) λmax (log ε): 205 (3.95) nm, 214 (3.94) nm; IR (KBr) νmax 3,429, 2,925, 2,857, 1,725, 1,635, 1,384, 1,077 cm−1; 1H and 13C NMR data, see m/z 497 [M + Na]+; HREIMS m/z 497.1988 [M + Na]+ .9)Patrinoside F ; UV (MeOH) λmax (log ε): 207 (4.18) nm; IR (KBr) νmax 3,441, 2,925, 2,860, 1,727, 1,639, 1,121, 1,065 cm−1; 1H and 13C NMR data, see m/z 467 [M + Na]+; HREIMS m/z 467.1889 [M + Na]+ .10)Patrirscabiobisin D ; UV (MeOH) λmax (log ε): 209 (4.24) nm, 217 (4.25) nm; IR (KBr) νmax 3,426, 2,926, 1,724, 1641, 1,077 cm−1; 1H and 13C NMR data, see m/z 663 [M + Na]+; HREIMS m/z 663.2627 [M + Na]+ .2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3--5-(3-carboxymethoxyphenyl)-2- (4-sulfopheny)-2H-tetrazolium (MTS)   as positive controls. After the incubation, MTS (100 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS-50% DMF after removal of 100 μL medium. The optical density of the lysate was measured at 490 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by the Reed and Muench method . All the cells were cultured in RPMI-1640 or DMEM medium , supplemented with 10% fetal bovine serum (Hyclone) at 37°C in a humidified atmosphere with 5% COh method .The CHARMM force field and DFT/TDDFT calculations were performed with Discovery Studio 4.0 and Gaussian09 program package, respectively. Conflex conformational search generated low-energy conformers within a 20 kcal/mol energy window and were subjected to geometry optimization using DFT method without imposing any symmetry constraints at the B3LYP/6-31G(d) level. Frequency calculations were carried out using the same level to verify that the molecular structures were true minimum. The calculated ECD spectra were generated by the program SpecDis2 using a Gaussian band shape with 0.3eV exponential half-width from dipole-length dipolar and rotational strengths , δC 196.3 (d)] and a pair of double bond , two oxygenated methine groups , and one oxygenated methylene at δC 66.6 (t). These data were similar to those of 8,9-didehydro-7-hydroxydolichodial ] to C-10 (δC 66.6), C-8 (δC 48.6), and C-9 (δC 57.1), and OMe (δC 54.5) were observed, and the correlations of H-1/H-9 and H-9/H-5 in the ROESY spectrum -1,10-epoxy-7,10-dihydroxy-dolichodial, named Patriscabioin M.The absolute configurations of H-5 and H-7 were both  spectra . Hence, 2, a light yellow oil, had a molecular formula of C14H22O5 based on HRESIMS . Detailed comparisons of 1H-NMR and 13C-NMR  and δC 104.1 , a conjugated olefinic bond at δC 193.3 ; δC 164.1 , δC 125.6 , and one oxygenated methylene at δC 62.3 . These data were similar to patriscabioin I , except for the substituent at C-1 (2 it was n-butanol group , while it was methyl in patriscabioin I. The correlation from H-1 [4.92 ] to C-1' [δC 70.7 (t)] in HMBC implied the location of n-butanol group at C-1 -1-n-butoxy-7,10-dihydroxy-11-aldehyde-5,6-dihydrovaltrate hydrin, named patriscabioin N.Compound  13C-NMR  suggestet at C-1 . In compp at C-1 . The con3 was found to have a molecular formula of C14H24O5, determined by its positive HRESIMS data  and 13C-NMR spectrum  and δC 99.5 , with a n-butanol group ) located in C-1, which was confirmed by the correlation from H-1 to C-1' in HMBC  was the added 1 unsaturation, of which the former's degree was 2, and the latter was 3. Meanwhile, the NMR spectra exhibited the disappeared double bonds and the appearance of two methines, among which the chemical shift was δC 94.7 , but there was no other unsaturated group. Therefore, it was presumed that there was a ring that existed in the skeleton of compound 3. The new ring was formed by the ether bond between C-3 and C-10, which was further verified by the fragment of H-3—H-4—H-5—H-9—H-8—H-10 in COSY, and the correlation from H-3 to C-10 in HMBC -1-n-butoxy-3,10-epoxy-7-hydroxy-3,4,5,6-tetrahydrovaltrate hydrinm, named patriscabioin O.Compound spectrum , with 3  in HMBC . However in HMBC . Accordiin ROESY . There win ROESY . Hence, 5 was obtained as a slight yellow oil. Its molecular formula was assigned as C29H46O11 on the basis of positive-ion HR-ESI-MS at m/z 593.2931 [M + Na]+  and 13C-NMR data  and δC 93.0 , a trisubstituted olefinic bond at δH 6.35 , δC 139.8 , and δC117.3 , an oxymethine at δC 79.3 (C-7) and two oxygenated methylenes at δC 67.1  and δC 69.7 . The above data indicated the existence of 7,10,11-trihydroxy-3-en valtratehydrin. It was found that 5 had a 3-methylcrotonyl group at δC 166.4 (s), δC 116.1 (d), δC 161.2 (s), δC 27.6 (q), δC 20.6 (q) and a glucopyranose unit at δC 103.2 (d), δC 78.2 (d), δC 78.0 (d), δC 75.1(d), δC 71.7 (d), δC 62.8 (t) through further detailed analysis of its 1D-NMR and 2D-NMR. The 3-methylcrotonyl group and glucopyranose were arranged at C-1 and C-11 by the correlations from δH 5.90 (H-1) to δC 166.4 (C-1'), and δH 4.29 (H-1”) to δC 69.7 (C-11”) in HMBC spectrum  of H-1” could also be observed. Thus, eight carbons remained, including two methyl groups, four methylene groups, and two methine groups. The 1H-1H COSY correlations (2 (H-3”')—CH2(H-4”')—CH —CH3 (H-7”') and CH(H-2”')—CH3(2”'-Me). Combined with the correlations of H-2”'/2”'-Me, and H-1”'/ C-10, C-7 in HMBC, these revealed the presence of a 1,3-dioxane through acetalation between OH-7, OH-10 and C-1.”' It meant that 1,3-dioxane was formed by hemiacetal formation from hydroxyl groups of C-7 and C-10 and aldehyde of 2-methyl-heptaldehyde.Compound NMR data . Carefulspectrum . The typelations  showed t5 was similar to patriscabioins A-L and patriscabiobisins A and B . The ROESY cross peaks of H-1”'/H-8 and 2”'-Me/H-1”' suggested the α-orientation of H-1”' and 2”'-Me. Thus, the structure of 5 was identified as shown and named patrinoside B.The relative configuration of 6 had the molecular formula C25H38O11 based on its quasimolecular ion peak at m/z 537.2302 [M + Na]+  in HR-ESI-MS spectrum and 13C-NMR data  to C-2”' (δC 38.2), and H-4”' (δH 0.90) to C-2”' (δC 38.2), C-3”' (δC 18.2) in HMBC. Therefore, the structure of 6 was deduced as shown and named patrinoside C.Compound NMR data . Compari7 was identified as C26H40O11 from HR-ESI-MS at m/z 551.2467 [M + Na]+ . Comparing 1H-NMR and 13C-NMR data of 7 with those of 6, it was found that they shared similar NMR data, and the obvious distinction between them was the appearance of a methylene (δC 34.8) and the change in chemical shift of methyl signal of C-4' (δC 27.6→ 12.3). It indicated that a 3-methylcrotonyl group at C-1 in 6 was replaced by the 3,4-dimethylcrotonyl group in 7. This was ultimately confirmed by 1D and 2D NMR spectra. In HMBC spectrum, the correlations between H-2' (δH 5.68) and C-4' (δC 34.8) and C-5' (δC 12.3) provided solid evidence for the existence of the 3,4-dimethylcrotonyl group. Finally, the structure of 7 was elucidated as shown and named patrinoside D.The molecular formula of 8 was analyzed to have a molecular formula of C22H34O11 by HR-ESI-MS at m/z 497.1988 [M + Na]+ , whose molecular weight is 54 less than that of 7. Careful analysis of its NMR data found that it had the same aglycone as compound 7, as well as a glucose at C-11 and a 3,4-dimethylcrotonyl group at C-1, but lacked an oxymethine at δC 102.0, two methylenes and a methyl, which suggested that it did not have a 1,3-dioxaneformed at C-7 and C-10. This could be further verified by the chemical shifts at C-7 and C-10 which were shifted up field from δC 79.2 to 72.7 and form δC 67.1 to 62.1, respectively, compared with compound 7. Finally, the structure of compound 8 was characterized as -1-O--7,10-dihydroxy-11-β-D-glucose-5,6-dihydrovaltrate hydrin and named patrinoside E.Compound 9 was found to have a molecular formula of C21H32O10 by HR-ESI-MS at m/z 467.1889 [M + Na]+ . Careful analysis of its 1D and 2D NMR data led to finding that it was a 7,10,11-trihydroxy-3-envaltratehydrin iridoid with a 3-methylcrotonyl group at C-1 and a sugar. The 1H-1H COSY correlations (H 4.22)—CH —CH —CH —CH2 —CH2 . Combined with the correlations between H-5” (δH 1.35) and C-2” (δC 76.9) and C-3” (δC 72.2) in the HMBC, all these signals hinted the sugar was 5-deoxyglucose, which was linked to C-11 from the correlation of H-11 /C-1” (δC 103.8). The β-configuration was determined by the coupled constant of H-1” . Ultimately, the structure of compound 9 was confirmed as -1-O-(3-methylcrotonyl)-7,10-dihydroxy-11-(5-deoxy-β-D-glucofuranose)-5,6-dihydrovaltrate hydrin and named patrinoside F.Compound elations  showed a10 was formulated as C31H44O14 from HR-ESI-MS at m/z 663.2627 [M + Na]+  and 13C-NMR data (C 103.3 (d), 75.1 (d), 78.1 (d), 71.7 (d), 78.8 (d), 62.8 (t) and the anomeric proton signals at δH 4.27  suggested that compound 10 contained a glucose at C-11 by the correlation from the anomeric proton (δH 4.27) to C-11 (δC 69.6) in HMBC (C 93.3 (d), δC 139.9 (d), δC 117.2 (s), δC 77.9 (d), δC 66.9 (t), δC 69.6 (t), and δH 5.80 , combined with the 3-methylcrotonyl  at C-1, found one iridoid was patrinoside A , δC 151.7 (s), δC 134.1 (t), two methylene at δC 39.4 (t), and δC 63.5 (t), and four methynes at δC 37.5 (t), δC 73.2 (d), δC 46.1 (d), δC 42.1 (d) in 13C NMR, except for the absence of one methyl, one pair of double bonds, and an aldehyde group. However, it did include an oxygenated methylene, two methines, and an acetal at δC 102.3 (d). This suggested that the other iridoid was 7,10-dihydroxydolichodial. And two units were linked through the aldehyde group between the aldehyde group at C-1' of 7,10 -dihydroxydolichodial and hydroxyl groups at C-7 and C-10 of patrinoside A, which was further verified by the correlations of H-1'/C-7, C-10 in the HMBC spectrum. The α-orientations of H-1”', H-8”', and H-9”' were determined by the ROESY correlations of H-7”' with H-1”', H-8”,' and H-9”'. Additionally, these were further proven by the good agreement of ECD spectra between compound 10 and patriscabiobisin A and B which were similar to compound 10. Thus, the structure of compound 10 was characterized as shown, namely patriscabiobisin C.Compound NMR data . The car in HMBC . The rem1-10 were a series of 5,-dihydrovaltrate hydrins with characteristic substituents, such as 3-methylcrotonyl group or 3,4-dimethylcrotonyl group in the Valerianaceae family, belonging to iridoids derived from iridodial. Iridoial was enoxylated and then subjected to intramolecular hemiacetal formation to form iridoid. And iridoid suffered a series of chemical changes to achieve diverse compounds (1-10)  . Iridodid by ECD .5 which showed the cell inhibition of 102.42, 95.13, 73.07, and 80.93% against HL-60, SMMC-7721, MCF-7, and SW-480 respectively . Unfortunately, none of the compounds showed significant cytotoxicities at 40 μM, except for compound ectively .P. scabiosaefolia is a medical and edible Chinese herb with high nutritional value and a wide range of biological activities. Research showed plants in the Patrinia genus are rich in iridoids and terpenoids. In our ongoing study, we found a series of iridoids and iridoid glycosides, including three new iridoids (1-3) and six novel irioid glycosides (5-10), and one known compound (4). Among them, compound 1 was a deformed iridoid, compound 3 formed a cycle between C-3 and C-10, compounds 5-7 with a 1,3-dioxane between C-7 and C-10, and compound 10 was a bis-iridoid glycoside, which was the first reported in P. scabiosaefolia. Cytotoxicity assays found compound 5 showed good cell inhibition against HL-60, SMMC-7721, MCF-7, and SW-480. All these results enriched the study on the chemical constituents and activities of Patrinia genus. However, the activities of these compounds were not thorough, just the evaluation of the cytotoxicity assays, and the other activities and their mechanisms are worth further exploration.The original contributions presented in the study are included in the article/ZL, JH, and WK conceived the research subject. ZL, YN, LZ, LM, SC, and MW contributed to the development and writing of the manuscript. WK and ZL contributed in validating, reviewing, and supervising the project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The neural precursor cell expressed by developmentally downregulated gene 4-2 (NEDD4-2) is a ubiquitin E3 ligase that has a high affinity toward binding and ubiquitinating glutamate ionotropic receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type subunit 1 . Since dysregulation of GRIA1 surface expression is relevant to the responsiveness to AMPA receptor (AMPAR) antagonists (perampanel and GYKI 52466) in chronic epilepsy rats, it is likely that NEDD4-2 may be involved in the pathogenesis of intractable epilepsy. However, the role of NEDD4-2-mediated GRIA1 ubiquitination in refractory seizures to AMPAR antagonists is still unknown. In the present study, both AMPAR antagonists recovered the impaired GRIA1 ubiquitination by regulating protein phosphatase 2B (PP2B)-extracellular signal-regulated kinase 1/2 (ERK1/2)-serum and glucocorticoid-regulated kinase 1 (SGK1)-NEDD4-2 signaling pathway in responders (whose seizure activities are responsive to AMPAR), but not non-responders (whose seizure activities were uncontrolled by AMPAR antagonists). In addition, cyclosporin A  co-treatment improved the effects of AMPAR antagonists in non-responders, independent of AKT signaling pathway. Therefore, our findings suggest that dysregulation of PP2B-ERK1/2-SGK1-NEDD4-2-mediated GRIA1 ubiquitination may be responsible for refractory seizures and that this pathway may be a potential therapeutic target for improving the treatment of intractable epilepsy in response to AMPAR antagonists.  Epilepsy is a brain function disorder characterized by recurrent and unprovoked seizures. The prevalence of epilepsy in the general population is approximately 0.6−0.8% . InitialThe pilocarpine model (including LiCI-pilocarpine model) serves as a reliable animal model of intractable epilepsy. The profiles of spontaneous recurrent seizures in this model resemble those of human MTLE. This model shows limbic seizures that become secondarily generalized, evolving to SE, which lasts for several hours (acute period). The SE is followed by a latent “seizure-free” period about 15−30 days) and by a chronic period characterized by the presence of spontaneous recurrent seizures. The lesions of mesial temporal structures, including a well-known hippocampal sclerosis, in this model are also similar to those of human MTLE patients ,5. There−30 days The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) is one of the major subtypes of ionotropic glutamate receptors. AMPARs are comprised of combinations of glutamate ionotropic receptor AMPA type subunit 1 —GRIA4, which are assembled as homo- or heterotetramers . The reg+ channel (Nav)1.6 [+ channels Kv7/KCNQ [NEDD4-2 gene are identified through genomic mutation screening in patients with epilepsy [The neural precursor cell expressed by developmentally downregulated gene 4-2 (NEDD4-2) is a ubiquitin E3 ligase that has a high affinity toward binding and ubiquitinating membrane proteins . Some ne(Nav)1.6 , voltageKv7/KCNQ ,14,15 anKv7/KCNQ . NEDD4-2Kv7/KCNQ ,17. Therepilepsy ,19,20. Fepilepsy ,17,21,22On the other hand, multifactorial events are involved in the underlying mechanisms of pharmacoresistant epilepsy: (1) a reduced yield of AED concentration in the brain by hyper-activation of drug efflux transporter or sustained inflammatory conditions ,24, (2) Here, we demonstrate that the anti-convulsive effects of AMPAR antagonists are closely related to the regulation of GRIA1 ubiquitination via protein phosphatase 2B (PP2B)-extracellular signal-regulated kinase 1/2 (ERK1/2)-serum and glucocorticoid-regulated kinase 1 (SGK1)-NEDD4-2 signaling pathway. In addition, impairment of this signaling pathway resulted in refractory seizures to AMPAR antagonists, which was improved by cyclosporin A  co-treatment. Therefore, our findings suggest that the PP2B-ERK1/2-SGK1-NEDD4-2 pathway may be a potential therapeutic strategy to improve the treatment of intractable MTLE in response to AMPAR antagonists.Male Sprague Dawley (SD) rats (seven weeks old) were provided with a commercial diet and water ad libitum under controlled temperature, humidity and lighting conditions . Animal protocols were approved by the Institutional Animal Care and Use Committee of Hallym University . All reagents were purchased from Sigma-Aldrich , except where noted.Animals were intraperitoneally (i.p.) given LiCl (127 mg/kg) 24 h before pilocarpine treatment. On the next day, animals were treated with pilocarpine  20 min after atropine methylbromide (5 mg/kg i.p.). Two hours after SE on-set, animals were administered diazepam  as needed. Control animals received saline in place of pilocarpine. Animals were video-monitored 8 h a day for general behavior and occurrence of spontaneous seizures by four weeks after SE . We clas2O:O2). Some animals were also implanted with a brain infusion kit 1  to infuse with vehicle or cyclosporin A  into the right lateral ventricle . The CsA concentration did not affect spontaneous seizure activities in chronic epilepsy rats [Control and epilepsy rats were implanted with monopolar stainless steel electrodes  in the right hippocampus  under isoflurane anesthesia  over a seven-day period. Thereafter, perampanel or GYKI 52466 was daily administered by the aforementioned method. Non-responders were also connected with Alzet 1007D osmotic pump  containing vehicle or CsA (250 μM). The pump was placed in a subcutaneous pocket in the dorsal region. After recording (18 h after the last drug treatment), animals were used for Western blot.®, Roche Applied Science, Branford, CT, USA) and 1 mM sodium orthovanadate. Protein concentrations were calibrated by BCA protein assay  and equal amounts of total proteins were incubated with NEDD4-2, SGK1 or GluA1 antibody  containing a protease inhibitor cocktail , phosphatase inhibitor cocktail . Thereafter, total protein concentration was calibrated using a Micro BCA Protein Assay Kit . Western blot was performed by the standard protocol: Sample proteins (10 μg) were separated on a Bis-Tris sodium dodecyl sulfate-poly-acrylamide gel (SDS-PAGE) and transferred to membranes. Membranes were incubated with 2% bovine serum albumin (BSA) in Tris-buffered saline , and then reacted with primary antibodies  and phosphatase inhibitor cocktail  = 5.1, p = 0.024, Friedman test, n = 7), seizure duration  = 6.8, p = 0.022, repeated measures ANOVA, n = 7) and the seizure severity (χ2(1) = 5.6, p = 0.018 Friedman test, n = 7) over a one-week period , the total seizure duration was 538.3 ± 127 s (t(12) = 8.54, p < 0.001 vs. vehicle, Student t-test, n = 7), and the average seizure severity was 1.9 ± 0.4 over a one-week period , the total electroencephalographic (EEG) seizure duration and average seizure severity  were 12.6 ± 2.9, 945.8 ± 102 s and 3.7 ± 0.5 over a one-week period, respectively n = 7, A–C. In rk period A,B. The χ2(1) = 4.6, p = 0.033, Friedman test, n = 6), total seizure duration  = 5.9, p = 0.033, repeated measures ANOVA, n = 6), and seizure severity (χ2(1) = 4.7, p = 0.031, Friedman test, n = 6) in responders over a one-week period , the total seizure duration was 571.2 ± 94.1 s (t(11) = 6.82, p < 0.001 vs. vehicle, Student t-test, n = 6), and the average seizure severity was 2.5 ± 0.4 over a one-week period  = 14.2, p < 0.001 vs. control animals, Student t-test; t(12) = 13.3, p < 0.001 vs. control animals, Student t-test) and 0.65 times (t(12) = 12.9, p < 0.001 vs. control animals, Student t-test) the control level in the vehicle-treated epilepsy rats, respectively  = 35.8, p < 0.001 vs. vehicle, one-way ANOVA; F = 1.4, p = 0.28 vs. vehicle, one-way ANOVA; F = 8.3, p = 0.003 vs. vehicle, one-way ANOVA; F = 47.9, p < 0.001 vs. vehicle, one-way ANOVA; F = 0.01, p = 0.99 vs. vehicle, one-way ANOVA; t(12) = 11.2, p < 0.001 vs. control animals, Student t-test; t(12) = 13.1, p < 0.001 vs. control animals, Student t-test; F = 38.8, p < 0.001 vs. vehicle, one-way ANOVA; F = 20.7, p < 0.001 vs. vehicle, one-way ANOVA; In responders to perampanel and GYKI 52466, NEDD4-2 protein levels were increased to 0.78 and 0.77 times the control level, respectively  plays a key role in NEDD4-2 phosphorylation, and SGK1 and NEDD4-2 regulate one another in a reciprocal manner: SGK1-mediated NEDD4-2 phosphorylation increases SGK1 ubiquitination by NEDD4-2 . Therefot(12) = 7.5, p < 0.001 vs. control animals, Student t-test; t(12) = 14.9, p < 0.001 vs. control animals, Student t-test) and 0.43 times (t(12) = 18.5, p < 0.001 vs. control animals, Student t-test) the control level in epilepsy rats, respectively (t(12) = 24.3, p < 0.001 vs. control animals, Student t-test) and 0.34 times (t(12) = 25.8, p < 0.001 vs. control animals, Student t-test) the control level  = 29.7, p < 0.001 vs. vehicle, one-way ANOVA; F = 18.6, p < 0.001 vs. vehicle, one-way ANOVA; F = 36.5, p < 0.001 vs. vehicle, one-way ANOVA; F = 0.1, p = 0.92 vs. vehicle, one-way ANOVA; F = 1.7, p = 0.22 vs. vehicle, one-way ANOVA; In responders, both AMPAR antagonists restored SGK1 protein levels to control level (t(12) = 14.4, p < 0.001 vs. control animals, Student t-test; F = 19.6, p < 0.001 vs. vehicle, one-way ANOVA; Unlike NEDD4-2, SGK1 ubiquitination was reduced to 0.52 times the control level in the epileptic hippocampus (Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinases (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/AKT pathways phosphorylate SGK1 at S78 and S422 sites, respectively ,38,39,40t(12) = 11.6, p < 0.001 vs. control animals, Student t-test) and 0.52 times (t(12) = 10.9, p < 0.001 vs. control animals, Student t-test) the control level in epilepsy rats, respectively, without altering ERK1/2 protein level (t(12) = 10.0, p < 0.001 vs. control animals, Student t-test) and 1.57 times (t(12) = 8.7, p < 0.001 vs. control animals, Student t-test) the control level in epilepsy rats, respectively, while PDK1 protein level was unchanged (t(12) = 8.9, p < 0.001 vs. control animals, Student t-test) and 1.81 times (t(12) = 8.4, p < 0.001 vs. control animals, Student t-test) the control level in epilepsy rats, respectively, without changing AKT protein level  = 0.3, p = 0.78 vs. vehicle, one-way ANOVA; F = 26.9, p < 0.001 vs. vehicle, one-way ANOVA; F = 15.6, p < 0.001 vs. vehicle, one-way ANOVA; F = 50.5, p < 0.001 vs. vehicle, one-way ANOVA), PDK1 phosphorylation ratio  = 49.6, p < 0.001 vs. vehicle, one-way ANOVA), AKT phosphorylation level  = 52, p < 0.001 vs. vehicle, one-way ANOVA) and AKT phosphorylation ratio  = 45.4, p < 0.001 vs. vehicle, one-way ANOVA) to control level without affecting their protein levels  and protein phosphatase 2B (PP2B) inhibit ERK1/2 kinase activity by dephosphorylating threonine and tyrosine residues ,43. In ot(12) = 16.3, p < 0.001 vs. control animals, Student t-test; t(12) = 26, p < 0.001 vs. control animals, Student t-test) and 0.58 times (t(12) = 8, p < 0.001 vs. control animals, Student t-test) the control level in epilepsy rats, respectively (t(12) = 12.6, p < 0.001 vs. control animals, Student t-test; t(12) = 30.3, p < 0.001 vs. control animals, Student t-test) and 0.55 times (t(12) = 12.9, p < 0.001 vs. control animals, Student t-test) the control level in epilepsy rats, respectively  = 0.3, p = 0.73 vs. vehicle, one-way ANOVA), PP2A phosphorylation levels  = 0.1, p = 0.93 vs. vehicle, one-way ANOVA) or its phosphorylation ratio  = 0.11, p = 0.89 vs. vehicle, one-way ANOVA; F = 17.2, p < 0.001 vs. vehicle, one-way ANOVA), respectively, without altering its protein level  = 32.7, p < 0.001 vs. vehicle, one-way ANOVA; In responders, neither AMPAR antagonist affected PP2A protein levels  was co-treated with perampanel or GYKI 52466 in non-responders. In non-responders to perampanel, total seizure frequency was 12.4 ± 3.1, total seizure duration was 946.4 ± 153.1 s, and average seizure severity was 4 ± 0.4 over a one-week period n = 5, A–C. In nχ2(3) = 8.4, p = 0.038, Friedman test, n = 5), total seizure duration  = 4.2, p = 0.023, repeated measures ANOVA, n = 5), and seizure severity (χ2(3) = 9.7, p = 0.021, Friedman test, n = 5) in both perampanel- and GYKI 52466-treated groups over a one-week period  = 3.58, p = 0.02, paired Student t-test, n = 5; z = 2.03, p = 0.042, Wilcoxon signed rank test, n = 5; z = 2.04, p = 0.041, Wilcoxon signed rank test, n = 5; t(4) = 3.2, p = 0.03, paired Student t-test, n = 5; z = 2.02, p = 0.043 vs. vehicle, Wilcoxon signed rank test, n = 5; CsA co-treatment gradually decreased seizure frequency  = 4.73, p = 0.001, Student t-test) and GYKI 52466 (t(8) = 4.9, p = 0.001, Student t-test), respectively (t(8) = 4.67, p = 0.002, Student t-test) and GYKI 52466 (t(8) = 5.88, p < 0.001, Student t-test), respectively (t(8) = 6.8, p < 0.001, Student t-test) and GYKI 52466 (t(8) = 6.2, p < 0.001, Student t-test), respectively (t(8) = 4, p = 0.004, Student t-test) and GYKI 52466 (t(8) = 4.8, p = 0.001, Student t-test), respectively (t(8) = 5.9 p < 0.001, Student t-test) and GYKI 52466 (t(8) = 5.8, p < 0.001, Student t-test; t(8) = 6.7, p < 0.001, Student t-test) and GYKI 52466, respectively (t(8) = 5.1, p < 0.001, Student t-test; t(8) = 7, p < 0.001, Student t-test) and GYKI 52466 (t(8) = 4, p = 0.004, Student t-test), respectively  = 7.3, p < 0.001 vs. control animals, Student t-test; t(8) = 4.5, p = 0.002, Student t-test) and GYKI 52466 (t(8) = 4.7, p = 0.001, Student t-test), respectively (t(8) = 9.2, p < 0.001, Student t-test) and GYKI 52466 (t(8) = 5.7, p = 0.004, Student t-test), respectively (t(8) = 5.7, p < 0.001 vs. control animals, Student t-test; t(8) = 9, p < 0.001, Student t-test) and GYKI 52466 (t(8) = 11.5, p < 0.001, Student t-test), respectively  are less sensitive to AMPAR activation but very sensitive to AMPAR inhibition. Briefly, the direct AMPAR stimulation by AMPA treatment elevates the synchrony of neuronal activity in wild-type mice more than NEDD4-andi2 mice. In contrast, NEDD4-andi2 mice are very sensitive to 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline  with regard to spontaneous spike frequency, although average spontaneous spike amplitude and electrode burst activity do not differ after AMPA or NBQX treatment for either genotype [NEDD4-andi2 mice show a higher seizure susceptibility than kainic acid, which is recovered by the genetically reducing GRIA1 level [Although the pathogenesis of MTLE has been studied for decades, the underlying mechanisms of intractable MTLE are still elusive. Recently, the dysregulation of ubiquitination has been considered as a potential factor for the generation of refractory epilepsy, since ubiquitination is involved in the modulation of synaptic function ,48. Ubiqgenotype . ParadoxA1 level . FurtherA1 level . In addiA1 level . TherefoA1 level ,49, AMPAA1 level ,24,25 wo+ channels (ENaC), Nav1.6, KCNQ2/3 and KCNQ3/5, and facilitates their ubiquitination [The binding of NEDD4-2 to substrates is differently regulated by phosphorylation. In the unphosphorylated state, NEDD4-2 binds to epithelial Natination ,13,14,15tination ,17,36,50tination . Interestination . ConsistSGK1 is activated by phosphorylating S78 and S422 sites through MAPK-ERK1/2 and PI3K-PDK1-AKT pathways, respectively ,38,39,40In the present study, PP2A and PP2B expressions in chronic epilepsy rats were lower than those in control animals. Furthermore, their phosphorylation ratios were also reduced in epilepsy animals. Since phosphorylation inhibits protein phosphatase activities ,46, it iPP2A also leads to SGK1 inactivation . In the + and K+, but not Ca2+ [2+-permeable) AMPAR [2+-permeable AMPAR in the epileptic hippocampus results in a subsequent elevated Ca2+ influx followed by PP2B activation [2+-permeable AMPAR level. Furthermore, the hyper-activation of AKT-glycogen synthase kinase 3β (GSK3β)-Ca2+/cAMP response element-binding protein (CREB) pathway leads to increased Ca2+-permeable AMPAR in non-responders [2+-permeable AMPAR expression would be a fundamental reason for the lack of response to the AMPAR antagonists in the non-responders. However, the present data show that the lower efficacies of AMPAR antagonists to the PP2B-ERK1/2-SGK1-NEDD4-2-mediated GRIA1 ubiquitination and the inhibition of spontaneous seizure activities  AMPAR ,10,26. Ativation ,56,57. Ativation ,10,26. Tsponders ,26, suggon GRIA1 ,24,25) ion GRIA1 ,58,59. TOn the other hand, deubiquitination  also plays a role in AMPAR-mediated neurotransmission ,61. UbiqThe present study reveals that AMPAR antagonists ameliorated spontaneous seizure activity by affecting the PP2B-mediated ERK1/2-SGK1-NEDD4-2 signaling pathway, which is relevant to the enhanced GRIA1 ubiquitination. In addition, the dysregulation of this pathway was one of the causes of refractory seizures to AMPAR antagonists. Therefore, our findings suggest that PP2B-ERK1/2-SGK1-NEDD4-2 pathway may be one of the potential therapeutic targets for the treatment of intractable TLE.


For comparison purposes, the variation of the substitution pattern at the coumarin unit was analyzed with the natural product 6,7-dihydroxycoumarin (Esculetin) as well as 5,7-dihydroxycomarin in the case of the bithiophene dye. Crucial steps for extension of the conjugated system involved Sonogashira reaction yielding highly fluorescent molecules. Spectroscopic characterization showed that the extension of conjugation via the alkynyl bridge resulted in a strong red-shift of absorption and emission spectra (in solution) of approximately 73–79 nm and 52–89 nm, respectively, relative to 6,7-dimethoxy-4-methylcoumarin (λabs = 341 nm and λem = 410 nm). Theoretical density functional theory (DFT) calculations show that the Lowest Unoccupied Molecular Orbital (LUMO) is mostly centered in the cyanoacrylic anchor unit, corroborating the high intramolecular charge transfer (ICT) character of the electronic transition. Photovoltaic performance evaluation reveals that the thieno  thiophene unit present in dye 8 leads to the best sensitizer of the set, with a conversion efficiency (η = 2.00%), best VOC (367 mV) and second best Jsc (9.28 mA·cm−2), surpassed only by dye 9b (Jsc = 10.19 mA·cm−2). This high photocurrent value can be attributed to increased donor ability of the 5,7-dimethoxy unit when compared to the 6,7 equivalent (9b).A set of 3-ethynylaryl coumarin dyes with mono, bithiophenes and the fused variant, thieno [3,2- Following O’Regan and Grätzel’s seminal application  of an orAlthough the original ruthenium(II)-polypyridyl chromophores used in DSSCs, such as N3  and N719To be adequately applied in DSSCs, organic dyes must present a donor-π-acceptor (D-π-A) structure. The push-pull effect in these D-π-A dyes leads to efficient intramolecular charge transfer (ICT) from the donor to the acceptor unit through the π-bridge upon light absorption. Among the many classes of organic compounds used, coumarins are of particular interest due to their wide use as fluorescent sensors ,18,19, eo-halide phenylene spacers on dye structure and device performance.This allows for a decrease in the energy gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), making coumarins great candidates as new sensitizers for DSSCs. Hara et al. ,27,28 weRecently we have shown that the introduction of a linear ethynyl π-bridge into coumarin-based conjugated donor–acceptor systems resulted in redshifted absorption and emission spectra relatively to the styryl counterparts . Electrob] thiophene, as well as a benzotriazole unit containing a long alkyl chain  were then obtained through a high-yield Sonogashira coupling with ethynyltrimethylsilane, followed by the removal of the trimethylsilyl group, which at first was accomplished with tetrabutylammonium fluoride (TBAF). Since this method resulted in poor yields, partly due to the possible degradation of the unprotected ethynyl derivative, a method employing K2CO3 in MeOH described by Wang et al.  thiophene in the case of compound 4, and alkylation, double bromination + 303.1047; Found 303.1053.5,7-dimethoxy-3-((trimethylsilyl)ethynyl)coumarin (2b). Starting from 147.1 mg  of 3-bromo-5,7-dimethoxycoumarin (1b), 114.0 mg (72.9%) of 5,7-dimethoxy-3-((trimethylsilyl)ethynyl)coumarin (2b) were obtained. 1H-NMR  δ (ppm) 8.14 , 6.37 , 6.25 , 3.88 , 3.84 , 0.25 . HRMS-ESI(+) Calculated for C16H19O4Si [M + H]+ 303.1047; Found 303.1041.3 (0.06 eq), CuI (0.12 eq), Pd(PPh3)4 (0.15), aldehyde (1 eq.) and 5 mL of dry dioxane were added under a N2 atmosphere. After a few minutes ethynylcoumarin (3) (1 eq.) and dry (i-Pr)2NH (2 eq.) were added and the solution was stirred at 45°C overnight under a N2 atmosphere. Once the reaction was confirmed to be complete by TLC (hexane/AcOEt (7:3 v/v)), the solution was cooled to room temperature, the solvent was removed under reduced pressure and the solid residue dried in vacuo before being purified by flash chromatography with DCM/MeOH (99.8:0.02 v/v and 99.5:0.05 v/v) as eluent, affording a bright yellow solid in all cases.To a sealed tube, PPh5-ethynyl)thieno  tiophene-2-carbaldehyde (4). Starting from 104.6 mg  of 5-bromothieno  thiophene-2-carbaldehyde, 89.5 mg (51.5%) of 5-ethynyl)thieno  tiophene-2-carbaldehyde (4) were obtained. 1H-NMR  δ (ppm) 9.99 , 7.92 , 7.89 , 7.54 , 6.87 , 6.86 , 3.98 , 3.94 ; HRMS-ESI(+) Calculated for C20H13O5S2 [M + H]+ 397.0199; Found 397.0197.5′-ethynyl)--5-carbaldehyde (5a). Starting from 132.8 mg  of 5-bromo--5-carbaldehyde, 95.2 mg (46.3%) of 5′-ethynyl)--5-carbaldehyde (5a) were obtained. 1H-NMR  δ (ppm) 9.85 , 7.89 , 7.71 , 7.30 , 6.87 , 6.85 , 3.92 , 3.87 ; HRMS-ESI(+) Calculated for C22H15O5S2 [M + H]+ 423.0355; Found 423.0348.5′-ethynyl)--5-carbaldehyde (5b). Starting from 154.0 mg  of 5-bromo--5-carbaldehyde, 22.4 mg (14.1%) of 5′-ethynyl)--5-carbaldehyde (5b) were obtained. 1H-NMR  δ (ppm) 10.02 , 8.29 , 8.08 , 7.69–7.67 , 7.53 , 6.67 , 6.63 , 4.04 , 3.99 ; HRMS-ESI(+) Calculated for C22H15O5S2 [M + H]+ 423.0355; Found 423.0349.5-ethynyl)-thiophene-2-carbaldehyde (6). Starting from 64.8 mg  of 5-bromothiophene-2-carbaldehyde, 58.7 mg (50.8%) of 5-ethynyl)-thiophene-2-carbaldehyde (6) were obtained. 1H-NMR  δ (ppm) 9.87 , 7.95 , 7.71 , 7.41 , 6.89 , 6.87 , 3.94 , 3.89 ; HRMS-ESI(+) Calculated for C18H13O5S [M + H]+ 341.0478; Found 341.0478.2-decyl-7-ethynyl)-2H-benzo[d]triazole-4-carbaldehyde (7). Starting from 113 mg  of 7-dibromo-2-decyl-2H-benzo[d]triazole-4-carbaldehyde, 42.5 mg (50.8%) of 2-decyl-7-ethynyl)-2H-benzo[d]triazole-4-carbaldehyde (7) were obtained. 1H-NMR  δ (ppm) 10.50 , 8.11 , 7.99 , 7.81 , 6.96 , 6.93 , 4.90 , 3.99 , 3.94 , 2.26–2.18 , 1.43–1.30 , 0.90 ; HRMS-ESI(+) Calculated for C30H34N3O5 [M + H]+ 516.2493; Found 516.2509.v/v)), the solvent was evaporated under reduced pressure, the solid residue was washed 3–5 times with ACN, acidified with HCl (10%) and washed 3–5 times with distilled water. After each washing step the solvent used was centrifuged  to recover any lost product.To a round-bottom flask containing aldehyde (1 eq.), cyanoacetic acid (3 eq.), 5 mL of acetonitrile (ACN) and dry piperidine (2.7 eq.) were added and the resulting solution was stirred under reflux for 24 h. Once the reaction was confirmed to be complete by TLC ethynyl)thienothiophen-2-yl)acrylic acid (8). Starting from 15 mg  of 5-ethynyl)thienotiophene-2-carbaldehyde (4), 7.8 mg (44.5%) of 2-cyano-3-ethynyl)thienothiophen-2-yl)acrylic acid (8) were obtained. 1H-NMR  δ (ppm) 8.75 , 8.37 , 8.28 , 8.09 , 7.89 , 7.26 , 7.14 , 3.90 , 3.82 . HRMS-ESI(+) Calculated for C23H14NO6S2 [M + H]+ 464.0257; Found 464.0249.2-cyano-3-ethynyl)--5-yl)acrylic acid (9a). Starting from 95 mg  of 5′-ethynyl)--5-carbaldehyde (5a), 54.7 mg (49.7%) of 2-cyano-3-ethynyl)--5-yl)acrylic acid (9a) were obtained. 1H-NMR  δ (ppm) 8.57 , 8.38 , 8.08 , 7.73 , 7.70 , 7.53 , 7.37 , 7.15 , 4.02 , 3.92 . HRMS-ESI(+) Calculated for C25H16NO6S2 [M + H]+ 490.0414; Found 490.0408.2-cyano-3-ethynyl)--5-yl)acrylic acid (9b). Starting from 23.2 mg  of 5′-ethynyl)--5-carbaldehyde (5b), 14.1 mg (52.5%) of 2-cyano-3-ethynyl)--5-yl)acrylic acid (9b) were obtained. 1H-NMR  δ (ppm) 8.53 , 8.27 , 8.04 , 7.70 , 7.67 , 7.52 , 6.65 , 6.61 , 4.04 , 3.99 . HRMS-ESI(+) Calculated for C25H16NO6S2 [M + H]+ 490.0414; Found 490.0406.2-cyano-3-ethynyl)-thiophen-2-yl)acrylic acid (10). Starting from 28.1 mg  of 5-ethynyl)-thiophene-2-carbaldehyde (6), 17.7 mg (50.8%) of 2-cyano-3-ethynyl)-thiophen-2-yl)acrylic acid (10) were obtained. 1H-NMR  δ (ppm) 8.50 , 8.36 , 7.98 , 7.57 , 7.22 , 7.11 , 3.89 , 3.81 . HRMS-ESI(+) Calculated for C21H14NO6S [M + H]+ 408.0536; Found 408.0529.2-cyano-3-ethynyl)-2H-benzo[d]triazol-4-yl)acrylic acid (11). Starting from 51,7 mg  of 2-decyl-7-ethynyl)-2H-benzo[d]triazole-4-carbaldehyde (7), 8.9 mg (50.8%) of 2-cyano-3-ethynyl)-2H-benzo[d]triazol-4-yl)acrylic acid (11) were obtained. 1H-NMR  δ (ppm) 8.92 , 8.55 , 8.46 , 7.97 , 7.42 , 7.17 , 4.95 , 4.04 , 3.93 , 2.22–2.15 , 1.44–1.14 , 0.85 . HRMS-ESI(+) Calculated for C33H35N4O6 [M + H]+ 583.2551; Found 583.2542.The ground state molecular geometry was optimized using the density functional theory (DFT) by means of the Gaussian 09 program  , under C4/water solution (40 mM) at 70 °C for 30 min, washed with water and ethanol and sintered at 500 °C for 30 min. This procedure is essential to improve the adherence of the subsequently deposited nanocrystalline layers to the glass plates, as well as to serve as a ‘blocking-layer’, helping to block charge recombination between electrons in the FTO and holes in the I−/I3− redox couple. Afterwards, the TiO2 nanocrystalline layers were deposited on these pre-treated FTO plates by screen-printing the transparent titania paste  using a frame with polyester fibers with 43.80 mesh per cm2. This procedure, involving two steps (coating and drying at 125 °C), was repeated twice. The TiO2-coated plates were gradually heated up to 325 °C, then the temperature increased to 375 °C in 5 min, and afterwards to 500 °C. The plates were sintered at this temperature for 30 min, and finally cooled down to room temperature. A second treatment with the same TiCl4/water solution (40 mM) was performed, following the procedure described previously. This second TiCl4 treatment is also an optimization step that enhances the surface roughness for dye adsorption, thus positively affecting the photocurrent produced by the cell under illumination. Finally, a coating of reflective titania paste  was deposited by screen-printing and sintered at 500 °C. This layer of 150–200 nm sized anatase particles functions as a ‘photon-trapping’ layer that further improves the photocurrent. Each anode was cut into rectangular pieces (area: 2 cm × 1.5 cm) with a spot area of 0.196 cm2 and a thickness of 15 μm. The prepared anodes were soaked for 16 h in a 0.5 mM solution of the dye in dichloromethane:methanol:H2O (65:20:2), at room temperature in the dark. The excess dye was removed by rinsing the photoanodes with the same solvent as that employed for the dye solution.The detailed procedure has been described elsewhere . The con−/I3− (0.8 M LiI and 0.05 M I2), in an acetonitrile/valeronitrile  mixture. The electrolyte was introduced into the cell via backfilling under vacuum through the hole drilled in the back of the cathode. Finally, the hole was sealed with adhesive tape.Each counter-electrode consisted of an FTO-glass plate (area: 2 cm × 2 cm) in which a hole (1.0 mm diameter) was drilled. The perforated substrates were washed and cleaned with water and ethanol to remove any residual glass powder and organic contaminants. The transparent Pt catalyst  was deposited on the conductive face of the FTO-glass by doctor blade: one edge of the glass plate was covered with a strip of an adhesive tape (3 M Magic) both to control the thickness of the film and to mask an electric contact strip. The Pt paste was spread uniformly on the substrate by sliding a glass rod along the tape spacer. The adhesive tape strip was removed, and the glasses heated at 550 °C for 30 min. The photoanode and the Pt counter-electrode were assembled into a sandwich type arrangement and sealed (using a thermopress) with a hot melt gasket made of Surlyn ionomer . The electrolyte was prepared by dissolving the redox couple, IFor each compound, at least two cells were assembled under the same conditions, and the efficiencies were measured 5 times for each cell resulting in a minimum of 10 measurements per compound.2) . The thickness of the oxide film deposited on the photoanodes was measured using an Alpha-Step D600 Stylus Profiler .Current-Voltage curves were recorded with a digital Keithley SourceMeter multimeter (PVIV-1A)  connected to a PC. Simulated sunlight irradiation was provided by an Oriel solar simulator  and the superior donor ability of the 5,7-disubstituted coumarin are promising avenues for the synthesis of more efficient coumarin-based donors.In the current work, five new 3-ethynylaryldimethoxycoumarin-based chromophores were synthesized by previously reported methods, with the aim of investigating not only the effect of varying the heterocyclic π-bridge on the device performance, but also assess the difference in properties between the 6,7- and 5,7-substitution patterns on the coumarin moiety. Through absorption and fluorescence emission spectroscopy, both in solution and adsorbed onto porous TiOrin dyes ,30,31,32


Tyrosol (T) and hydroxytyrosol (HOT) and their glycosides are promising candidates for applications in functional food products or in complementary therapy. A series of phenylethanoid glycofuranosides (PEGFs) were synthesized to compare some of their biochemical and biological activities with T and HOT. The optimization of glycosylation promoted by environmentally benign basic zinc carbonate was performed to prepare HOT α-L-arabino-, β-D-apio-, and β-D-ribofuranosides. T and HOT β-D-fructofuranosides, prepared by enzymatic transfructosylation of T and HOT, were also included in the comparative study. The antioxidant capacity and DNA-protective potential of T, HOT, and PEGFs on plasmid DNA were determined using cell-free assays. The DNA-damaging potential of the studied compounds for human hepatoma HepG2 cells and their DNA-protective potential on HepG2 cells against hydrogen peroxide were evaluated using the comet assay. Experiments revealed a spectrum of different activities of the studied compounds. HOT and HOT β-D-fructofuranoside appear to be the best-performing scavengers and protectants of plasmid DNA and HepG2 cells. T and T β-D-fructofuranoside display almost zero or low scavenging/antioxidant activity and protective effects on plasmid DNA or HepG2 cells. The results imply that especially HOT β-D-fructofuranoside and β-D-apiofuranoside could be considered as prospective molecules for the subsequent design of supplements with potential in food and health protection. These inexpensive and environmentally benign Zn(II) promoters have been found to be efficient and selective for 1,2-trans-glucosylation of tyrosol and hydroxytyrosol with the phenolic hydroxyls protected by easily removable acetyl or t-butyldimethylsilyl groups. Various per-O-acetylated hexopyranosyl, pentopyranosyl, and rungiosyl bromides have been successfully used to determine the viability and scope of this method D20 = −74.9° ; , δ 7.11 , 7.08 , 7.06 , 5.05 , 5.01 , 4.95 , 4.38 , 4.19 , 4.05 , 3.93 , 3.72 , 2.89 , 2.27 , 2.26  2.09 , 2.07 ; 13C NMR , δ 170.7 (COCH3), 170.3 (COCH3), 169.8 (COCH3), 168.4 (COCH3), 168.3 (COCH3), 142.0 (CPh), 140.6 (CPh), 137.9 (CPh), 127.2 (CHPh), 123.9 (CHPh), 123.2 (CHPh), 105.6 (C-1), 81.4 (C-2), 80.4 (C-4), 77.1 (C-3), 67.8 (OCH2CH2), 63.3 (C-5), 35.4 (OCH2CH2), 20.9 (COCH3), 20.9 (COCH3), 20.8 (COCH3), 20.8 (COCH3), 20.7 (COCH3); HRMS (APCI): calcd. for C23H28O12 [M + H]+ = 497.16535, found 497.16529.2-ethyl 2,3,5-tri- CHCl3); , Entry 1O-benzoyl-2,3-di-O-acetyl-β-D-apiofuranoside (17). Colorless syrup; Rf = 0.48 ; [α]D20 = −53.9° ; , δ 8.10–8.07 , 7.58 , 7.46 , 7.05–7.02 , 5.43 , 5.00 , 4.83 , 4.74 , 4.17 , 3.91 , 3.67 , 2.88 , 2.22 , 2.21 , 2.09 , 2.01 ; 13C NMR , δ 169.8 (COCH3), 169.3 (COCH3), 168.4 (COCH3), 168.3 (COCH3), 166.0 (COPh), 142.0 (CPh), 140.7 (CPh), 133.3 (CHBz), 131.6 (CPh), 130.0 (2xCHBz), 129.8 (CBz), 128.5 (2xCHBz), 127.2 (CHPh), 123.9 (CHPh), 123.3 (CHPh), 105.7 (C-1), 84.1 (C-3), 76.5 (C-2), 72.7 (C-4), 68.4 (OCH2CH2), 63.9 (C-5), 35.5 (OCH2CH2), 21.2 (COCH3), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3); HRMS (ESI): calcd. for C28H30O12 [M + Na]+ = 581.16295, found 581.16339.2-ethyl 5- CHCl3); , Entry 3O-acetyl-β-D-ribofuranoside (18). Colorless syrup; Rf = 0.39 ; [α]D20 = −38.8° ; , δ 7.09 , 7.07 , 7.03 , 5.25 , 5.21 , 4.98 , 4.28–4.23 , 4.23 , 3.95 , 3.92 , 3.60 , 2.85 , 2.26 , 2.26 , 2.09 , 2.05 , 2.04 ; 13C NMR , δ 170.7 (COCH3), 169.8 (COCH3), 169.7 (COCH3), 168.4 (COCH3), 168.4 (COCH3), 142.0 (CPh), 140.7 (CPh), 137.5 (CPh), 127.2 (CHPh), 124.0 (CHPh), 123.3 (CHPh), 105.2 (C-1), 78.7 (C-4), 74.8 (C-2), 71.7 (C-3), 68.4 (OCH2), 64.8 (C-5), 35.4 (OCH2CH2), 20.9 (COCH3), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3), 20.6 (COCH3); HRMS (ESI): calcd. for C23H28O12 [M + Na]+ = 519.14730, found 519.14694.2-ethyl 2,3,5-tri- CHCl3); , Entry 6O-acetylated glycofuranoside (2 mmol) was dissolved in dry MeOH (30 mL) and cooled to 4 °C, and 0.5 M MeONa (3.4 mL) was added dropwise. The mixture was stirred at 4 °C until completion of the reaction was indicated by TLC (EtOAc:MeOH 6:1). The mixture was neutralized to pH 5 with Dowex® 50WX8 (H+ form). The resin was then filtered off, and the filtrate was evaporated to dryness at temperature not higher than 40 °C. The residue was purified by column chromatography on silica gel (EtOAc:MeOH 6:1) and concentrated.Per-6). Yield 44%. Colorless syrup; Rf = 0.48 ; [α]D20 = −21.8° ; 1H NMR , δ 6.72–6.69 , 6.68 , 6.57 , 4.90 , 3.98 , 3.92 , 3.89 , 3.84 , 3.77 , 3.67 , 3.61 , 2.77 ; 13C NMR , δ 146.1 (CPh), 144.6 (CPh), 131.8 (CPh), 121.2 (CHPh), 117.0 (CHPh), 116.3 (CHPh), 109.4 (C-1), 85.3 (C-4), 83.5 (C-2), 78.7 (C-3), 70.0 (OCH2), 63.0 (C-5), 36.5 (OCH2CH2); HRMS (ESI): calcd. for C13H18O7 [M + Na]+ = 309.09447; found 309.09447.2-ethyl α-L-arabinofuranoside (7). Yield 36%. Colorless syrup; Rf = 0.51 ; [α]D20 = −12.9° . 1H NMR , δ 6.67 , 6.66 , 6.53 , 4.92 , 3.87 , 3.84 , 3.80 , 3.73 , 3.57 , 3.55 , 3.51 , 2.71 ; 13C NMR , δ 146.1 (CPh), 144.6 (CPh), 131.8 (CPh), 121.2 (CHPh), 117.0 (CHPh), 116.3 (CHPh), 110.4 (C-1), 80.4 (C-3), 78.0 (C-2), 74.9 (C-4), 70.5 (OCH2), 65.5 (C-5), 36.6 (OCH2CH2); HRMS (ESI): calcd. for C13H18O7 [M + Na]+ = 309.09447; found 309.09457.2-ethyl β-D-apiofuranoside (8). Yield 31%. Colorless syrup; Rf = 0.50 ; [α]D20 = −12.8° . 1H NMR  δ 6.67 , 6.65 , 6.53 , 4.85 , 4.00 , 3.91 , 3.90–3.83 , 3.65 , 3.54 , 3.43 , 2.69 ; 13C NMR  δ 146.1 (CPh), 144.6 (CPh), 131.8 (CPh), 121.2 (CHPh), 117.1 (CHPh), 116.2 (CHPh), 108.6 (C-1), 84.8 (C-4), 76.3 (C-2), 72.9 (C-3), 70.0, (OCH2), 65.2 (C-5), 36.5 (OCH2CH2); HRMS (ESI): calcd. for C13H18O7 [M + Na]+ = 309.09447; found 309.09448.2-ethyl β-D-ribofuranoside 6] (1%). The mixtures were incubated at 50 °C for 20 min. Then, trichloroacetic acid  was added to each sample and the mixture centrifuged at 900× g for 10 min. Finally, 2.5 mL of the upper layer from supernatants was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% FeCl3 and the spectrophotometer GENESYS™ 10 Bio, Spectronic, was used to measure the absorbance at the wavelength of 700 nm for the samples and the standard solutions.Free aglycones rding to  and 76]1 and 2 asample is the sample’s absorbance or the absorbance of the standard and Acontrol is the absorbance of the control‘s reaction . Aglycones and PEGFs were evaluated for their ability to scavenge DPPH  radicals by the assay modified by  and by u2+ ions was used to detect the DNA-protective/DNA-damaging potential of aglycones and PEGFs as described 2 induced2:95% air. All media and supplements used for the maintenance of cells in culture were purchased from Gibco Life Technologies BRL . Human hepatoma HepG2 cells were used for cytotoxicity assessment and the investigation of potential genotoxic/protective action of aglycones and PEGFs. This cell line represents a useful and suitable tool for the detection of dietary genotoxic mutagens/carcinogens and various xenobiotics that could pose health risks to humans because of its functional drug-metabolizing abilities . HepG2 c6/plate, cultivated for 24 h and treated with T, HOT, and PEGFs (0–2000 µM). After the 48 h treatment, the cells were washed with phosphate buffered saline  and incubated with a medium containing MTT (1 mg/mL) for further 4 h. Then, the medium was removed and replaced by 200 μL of dimethyl sulfoxide . The absorbance was measured at 540 and 690 nm using an xMark™ microplate spectrophotometer . The viability of HepG2 cells was calculated by the formulaFor determination of cytotoxicity by MTT assay ,72, HepG2O2. The conventional comet assay procedure was performed in alkaline conditions, as suggested in [2O2 (300 μM in PBS on ice in the dark), while the slides not treated with H2O2 were kept for 5 min in cold PBS. All slides were washed in PBS and placed in a lysis solution consisting of 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl (pH = 10.0), and 1% Triton X-100 for 1 h at 4 °C. After lysis, the slides were transferred into an electrophoresis buffer  for unwinding (40 min at 4 °C) and subjected to electrophoresis  for 30 min at 4 °C. The slides were then neutralized in 0.4 M Tris-HCl (pH = 7.5), drained, and stained with EtBr (5 μg/mL). For the assessment of DNA-damaging/-protective effects of T, HOT, and PEGFs, the concentration range of 50–200 μM was selected for the 48 h treatment of HepG2 cells (their viability was above 70%), followed by 5 min of incubation with Hested in , and modested in . In brieAt least 100 EtBr-stained nucleoids/sample/three slides in one electrophoresis run were scored with a Carl Zeiss AxioImager.Z2 fluorescence microscope using a computerized image analysis Metafer 5 . As a parameter for the expression of DNA damage, the percentage of DNA in the tail was chosen. t-test. If data were normally distributed but did not pass the equality-of-variance test, differences were tested using unpaired t-test with Welch’s correction. If the data were non-normally distributed, a Mann–Whitney U test was used. All tests were two-tailed and performed at the significance level α = 0.05. The p < 0.05 was considered statistically significant for all analyses .For statistical analysis of the cell viability results (%) and the mean tail DNA (%), SigmaPlot 12.5 and Prism GraphPad 8.4.3 were used. The normality of the distribution was tested by the Shapiro–Wilk test, and the equality of the data variance was tested by Levene’s test. If normally distributed, differences were tested by unpaired trans-glycofuranosides were prepared by modifying the Koenigs–Knorr conditions, using the environmentally friendly basic zinc carbonate as a promoter. The new glycofuranosides, as well as previously enzymatically prepared T and HOT fructofuranosides, were compared with their aglycones (T and HOT) to determine a possible relationship between their structure, their antioxidant capacity, and their DNA-damaging and DNA-protective potential. The results showed that HOT glycofuranosides and HOT possess significant radical scavenging/antioxidant activities comparable to those of GA. The compounds protected plasmid DNA in a dose-dependent manner in the order HOT = HOTFRU > T > HOTAPI > TYBFRU. For glycofuranosides, the effectiveness of this protection can be explained by the different abilities of their primary hydroxyls to participate in radical stabilization together with ortho-diphenols of HOT aglycon. The compounds have no genotoxic effect on human HepG2 cells at the concentrations studied. HOT, HOTFRU, and HOTAPI were the best candidates for protection at the cellular level. However, it should be noted that in the concentration range of 5–500 μM, HOTFRU and HOTRIB were less toxic to cells than HOT, T, and other PEGFs. HOTFRU has been shown to have antioxidant potential comparable to that of HOT at lower toxicity.A series of new hydroxytyrosol 1,2-