The chemical structures of these compounds were elucidated on the basis of physicochemical evidence including Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). Compounds 1–5 were obtained as complex compounds with disulfane or trisulfane and tetrahydro-2H-difurofuran-5(5aH)-one. In addition, the hypothetical biosynthetic pathways of these compounds were suggested.Five new cyclic organosulfur compounds, foliogarlic disulfanes A Allium plants (Allieae), such as garlic, onion, and chives, have been cultivated as not only foodstuffs but also medicinal plants in the worldwide from ancient. For example, the extract of Allium plants, such as garlic, has shown anticancer, antidiabetic, and antibacterial effects. In addition, the National Cancer Institute in the United States had focused on Allium species as expecting cancer prevention  have been reported by Nohara et al. (Allium fistulosum (green onion and welsh onion)  , A2 (2), and A3 (3) and foliogarlic trisulfane A1(4) and A2 (5), from the leaves of A. sativum and the biosynthetic pathways.Theisen, . Allium a et al. , 2014. TA. sativum (15.0 kg) were mixed with water. Then, acetone was added into the mixture to be 80% acetone solution. The solution was concentrated after standing for 4 days (96 h) at room temperature. The acetone extract was portioned between ethyl acetate (EtOAc) and water. The organic fraction was evaporated in vacuo and obtained EtOAc fraction as syrup . The EtOAc fraction was also subjected with the normal and reversed-phase column chromatography and high-performance liquid chromatography (HPLC) to give foliogarlic disulfanes A1 , A2 , and A3  and foliogarlic trisulfanes A1  and A2  (The fresh leaves of .00008%) .1 (1) was obtained as yellow oil and showed positive optical rotation (+160.9). In the Electrospray Ionization MS (ESIMS) measurement of 1, a pseudomolecular ion peak [M + Na]+ was observed at m/z 343.0282, and the molecular formula was determined as C12H16O6S2 on the basis of the High Resolution ESIMS (HRESIMS) peak and the 13C NMR data. The 13C NMR spectra of 1 showed signals corresponding to a secondary methyl group at δC 9.1 (6-CH3), a methine at δC 48.1 (C-6), a diastereotopic oxygen-bearing methylene at δC 74.98 (C-3); an oxygen-bearing methine at δC 75.03 (C-2); two methines neighboring the electron-withdrawing atom at δC 90.4 (C-3a) and δC 103.2 (C-7); an oxygen-bearing quaternary carbon at δC 79.6 (C-5a); a two-oxygen–bearing quaternary carbon at δC 119.1 (C-8a); and a lactone carbonyl carbon at δC 175.4 (C-5) -one skeleton. In addition, 1-propenyl disulfane structure at the side chain was confirmed by High Resolution MS (HRMS) and NMR (Nuclear Magnetic Resonance) data. Next, the NOESY spectrum of 1 showed key correlations between H-3a and H-6; and H-6 and H-7 -one skeleton were superimposable on those of known compound, kujounin A3, except for 1-propenyl disulfane moiety -3,5a-dihydroxy-6-methyl-7-tetrahydro-2H-difurofuran-5(5aH)-one.Foliogarlic disulfanes A.4 (C-5) . The corand H-3a . The het and H-7 . The res2 (2) and A3 (3) were isolated as yellow oil with positive specific rotations  were observed at m/z 343. The molecular formulas (2 and 3: C12H16O6S2) were determined on the basis of HRESIMS peaks at  and the 13C NMR data. The 1H and 13C NMR spectrum of 2 and 3 showed signals corresponding to a secondary methyl group, a methine, a diastereotopic oxygen-bearing methylene, and an oxygen-bearing methine  and A3 (3) were determined as -3,5a-dihydroxy-6-methyl-7-tetrahydro-2H-difurofuran-5(5aH)-one and -3,5a-dihydroxy-6-methyl-7-tetrahydro-2H-difurofuran-5(5aH)-one, respectively.Foliogarlic disulfanes A methine . On the nd 6-CH3 . The NOEnd 6-CH3 . In addi1 (4) and A2 (5) were isolated as yellow oil with positive specific rotations  were observed at m/z 375. The molecular formulas (4 and 5: C12H16O6S3) were determined on the basis of HRESIMS peaks at , and the 13C NMR data. On the basis of the detailed analysis of the 1H and 13C NMR, 2D-NMR  spectrum of 4 and 5, the relative structures of tetrahydro-2H-difurofuran-5(5aH)-one skeleton on 4 and 5 were found to be the same as those of 1 and 3, respectively -3,5a-dihydroxy-6-methyl-7-tetrahydro-2H-difurofuran-5(5aH)-one and -3,5a-dihydroxy-6-methyl-7-tetrahydro-2H-difurofuran-5(5aH)-one, respectively.Foliogarlic trisulfanes A1  and A2 , (b), and (c) by hydrolysis and is reconstructed to disulfane (d) and trisulfane (e) -one skeleton is formed from semidehydroascorbate by cyclization and sulfane formation with the intermediates d and e. Consequently, compounds 1–5 were presumed to be obtained  Jacob, . Finallyobtained .1–3 and foliogarlic trisulfanes 4 and 5, were isolated from the leaves of A. sativum. These compounds 1–5 have a tetrahydro-2H-difurofuran-5(5aH)-one skeleton with methyl group at 6-position and 2-propenyl disulfane or 2-propenyl trisulfane group at 7-position. Particularly, foliogarlic trisulfanes 4 and 5 with a trisulfane moiety are a rare compound derived from medicinal plants. The biological effects of these cyclic organosulfur compounds should be studied further.Five new organosulfur compounds, foliogarlic disulfanes l = 5 cm); IR spectra, JASCO  FT/IR-4600 Fourier Transform Infrared Spectrometer; ESIMS, Agilent Technologies  Quadrupole LC/MS 6130; HRESIMS, SHIMADZU LCMS-IT-TOF; 1H NMR spectra, JEOL  JNM-LA 500 (500 MHz) spectrometer; 13C-NMR spectra, JEOL JNM-LA 500 (125 MHz) spectrometer; NOESY spectra, JNM-ECA 600 (600 MHz) spectrometer; HPLC, a Shimadzu  SPD-20AVP UV-VIS detector. YMC-triart C18 (250 × 4.6 mm i.d. and 250 × 10 mm i.d.) and YMC-triart PFP (250 × 4.6 mm i.d. and 250 × 10 mm i.d.) columns were used for analytical and preparative purposes. The following experimental materials were used for chromatography: normal-phase silica gel column chromatography, silica gel BW-200 ; reversed-phase silica gel column chromatography, Cosmosil 140C18-OPN [Nacalai Tesque ], TLC, precoated TLC plates with silica gel 60F254  (ordinary phase), and silica gel RP-18 F254S  (reversed phase); reversed-phase HPTLC, precoated TLC plates with silica gel RP-18 WF254S. Detection was achieved by spraying with 1% Ce (SO4) 2–10% aqueous H2SO4 followed by heating.The following instruments were used to obtain physical data: specific rotations, a Horiba  SEPA-300 digital polarimeter  farmers' market  in April 2017. The plants were identified by the authors (H.M. and S.N.).Fresh leaves of A. sativum (15.2 kg) were chopped and mixed with water, and then acetone was added to the mixture to be 80% acetone solution. The mixture was soaked for 4 days (96 h) at room temperature. Evaporation of the filtrate under reduced pressure provided acetone extract . The extract was partitioned between EtOAc and H2O  to obtain EtOAc fraction  and aqueous phase. The EtOAc-soluble fraction (41.98 g) was subjected to normal phase silica gel column chromatography  to give nine fractions {Fr.1 , Fr.2 (715.5 mg), Fr.3 , Fr.4 , Fr.5 , Fr.6 , Fr.7 , Fr.8 (617.7 mg), Fr.9 }. r. 5  was further separated by reversed-phase silica gel column chromatography  to give 13 fractions {Fr.5-1 (52.3 mg), Fr.5-2 (21.0 mg), Fr.5-3 (31.0 mg), Fr.5-4 (19.1 mg), Fr.5-5 (84.4 mg), Fr.5-6 (281.7 mg), Fr.5-7 (43.5 mg), Fr.5-8 (26.3 mg), Fr.5-9 (67.9 mg), Fr.5-10 (482.9 mg), Fr.5-11 , Fr.5-12 (178.7 mg), Fr.5-13 (16.9 mg)}. Fr.5-5 (84.4 mg) was purified by HPLC {mobile phase: MeOH-H2O  [YMC-triart PFP (250 × 10 mm i.d.)]} to give 1 (6.0 mg) and 2 (16.3 mg). Fr.5-6 (281.7 mg) was purified by HPLC {mobile phase: MeOH-H2O  [YMC-triart C18 (250 × 10 mm i.d.)]} to give 1 (14.6 mg), 2 (16.0 mg), 4 (24.2 mg), and 5 (12.3 mg). Fr.5-6-5 (37.1 mg) was purified by HPLC {mobile phase: MeOH-H2O  [YMC-triart C18 (250 × 10 mm i.d.)]} to give 3 (14.7 mg) and 4 (4.5 mg).The fresh leaves of 12H16O6S2Na (M+Na)+: 343.0281, found: 343.0282; IR(ATR): 3,400, 2,975, 1,782 cm−1; 1H NMR (CD3OD), 13C NMR : given in Yellow oil; 12H16O6S2Na (M+Na)+: 343.0281, found: 343.0277; IR(ATR): 3,400, 2,970, 1,785 cm−1; 1H NMR , 13C NMR : given in Yellow oil; 12H16O6S2Na (M+Na)+: 343.0281, found: 343.0282; IR(ATR): 3,400, 2,981, 1,785 cm−1; 1H NMR , 13C NMR : given in Yellow oil; 12H16O6S3Na (M+Na)+: 375.0001, found: 374.9998; IR(ATR): 3,400, 2,975, 1,780 cm−1; 1H NMR , 13C NMR : given in Yellow oil; 12H16O6S3Na (M+Na)+: 375.0001, found: 375.0003; IR(ATR): 3,400, 2,931, 1,789 cm−1; 1H NMR (CD3OD), 13C NMR (CD3OD): given in Yellow oil; All datasets generated for this study are included in the article/MF: isolation of constituents and Structure elucidation of new compounds. SeiN: Structure elucidation of new compounds and overall supervision in this study. HH and DN: isolation of constituents. SouN: Preparation of plant extracts and overall supervision in this study. TY: Structure elucidation of new compounds. HM: overall supervision in this study.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.


Acanthopanax sessiliflorus (AS). Acanthopanax species have been used in traditional medicine as tonics, sedatives, and antispasmodics. An activity-guided isolation of the fermented bark of A. sessiliflorus (FAS) yielded several phytochemicals: acanthoside D (1), acanthoside B (2), daucosterol (3), protocatechuic acid (4), chlorogenic acid methyl ester (5), ciwujiatone (6), syringaresinol (7), farnesol (8), 3,4-dicaffeoylquinic acid (9), and falcarindiol (10). HPLC analysis showed that content of lignan glycoside (1) was decreased and 4 and 7 were increased after fermentation. Anti-inflammatory activities on FAS showed the decrease of nitric oxide (NO) production, and inhibitory activities of iNOS and COX-2, proinflammatory cytokines (IL-6 and tumor necrosis factor-α), and collagenase. The aglycone, syringaresinol (7), which was increased through fermentation showed enhanced activity than 1. Thus, FAS may have the potential to treat inflammatory disorders, such as arthritis.The fermentation was carried out on the bark of  Acanthopanax sessiliflorus, of the family Araliaceae, is abundant in Korea, China, Russia, and Japan. More than 30 Acanthopanax species grow in East Asia; more than 18 are known in Korea [Acanthopanax species have been used in traditional medicine to make tonics, sedatives, and antispasmodics and to treat rheumatoid arthritis, diabetes, bacterial infections, cancer, and hypertension [Acanthopanax species [rtension –4. Vario species –8. The m species , 10.α, prostaglandin and nitric oxide (NO), which is synthesized by inducible NO synthase (iNOS), are important chemical mediators of inflammation [Various molecules are involved in the induction and maintenance of inflammatory responses. In addition to major cytokines, such as interleukin- (IL-) 1, IL-6, and tumor necrosis factor- (TNF-) ammation . Prostagammation .α), and collagenase in lipopolysaccharide- (LPS-) treated RAW 264.7 macrophage cells were evaluated on the biotransformation product of A. sessiliflorus.Recent studies have been conducted to enhance the physiological activity of materials by fermentation using microorganisms such as fungi, yeast, lactic acid bacteria, and mushrooms. The benefits include nonsecondary pollution, a mild reaction, and the low cost of biotransformation. Microorganisms have many bioconversion elements. As a part of our previous related work , inhibitA. sessiliflorus was collected from Pyeongtaek, Republic of Korea, and certified by Dr. Kim . In addition, a voucher specimen (201804-AS) was deposited at the herbarium of the College of Pharmacy, Chung-Ang University.In April 2018, the bark of Lactobacillus plantarum subsp. argentoratensis was selected for this study, on the basis of a previous study [L. plantarum subsp. argentoratensis was purchased from the Korean Agricultural Culture Collection , inoculated into MRS broth, and grown at 37°C and 200 rpm for 48 h. Then, 1% (v/v) L. plantarum subsp. argentoratensis was inoculated into MRS medium with 1% (w/v) A. sessiliflorus extract, and fermentation was performed at 37°C and 200 rpm for 5 days. After fermentation, centrifugation was performed for 20 min at 3000 rpm, and the supernatant was evaporated to obtain the fermented A. sessiliflorus (FAS) extract.us study . L. planμm; GE Healthcare Bio-Science AB, Uppsala, Sweden), MCI-gel CHP 20P , and ODS-B gel . Thin-layer chromatography (TLC) was performed using a precoated silica gel 60 F254 plate  and chloroform : methanol  and chloroform : methanol : water (80 : 20 : 2 or 6 : 4:1). The spots were detected by application of a FeCl3 spray and 10% H2SO4 or anisaldehyde-H2SO4, followed by heat and UV irradiation (254 nm). The chemical structures were elucidated by using several types of instrumental analysis. 1D-nuclear magnetic resonance (NMR) experiments, such as 1H-(600 MHz) and 13C-(150 MHz) NMR, were performed using an JNM-ECZ600R  at the Center for Research facilities at Chung-Ang University.Chromatography was performed using the Sephadex LH-20 column (10–24 A. sessiliflorus (3.2 kg) were extracted four times with 70% ethanol at room temperature for 3 days. The ethanol was removed under vacuum, and 143.03 g of the extract (AS) was obtained. Sixty grams of the extract was fermented by using L. plantarum subsp. Argentoratensis; the rest was stored at −20°C. The FAS extract was subjected to Sephadex LH-20 column chromatography and eluted with MeOH : H2O gradient (from 0 : 10 to 10 : 0); six subfractions (fr-1 to 6) were obtained. Repeated column chromatography of fr-2 using an MCI gel column with a gradient solvent system of MeOH : H2O (from 0 : 10 to 10 : 0) yielded 1 , 2 , and 3 . fr-3 yielded 4 , 5 , 6 , 7 , and 8 . fr-4 yielded 9  and 10 .The barks of 2O) and B (acetonitrile (ACN); The compounds in the FAS extract were compared with those in the AS extract by using high-pressure liquid chromatography (HPLC). The mobile phase consisted of solvent A (0.2% acetic acid in H2), and the cells were counted by using a hemocytometer.Murine macrophage RAW 264.7 cells were purchased from the Korean Cell Line Bank . The cells were grown in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (Welgene), 100 IU/mL penicillin G, and 100 mg/mL streptomycin  at 37°C in a humidified atmosphere  was added to each well. After incubation for 4 h, the supernatant was aspirated. The formazan crystals in each well were dissolved in 100 μL dimethyl sulfoxide, and the absorbance at 540 nm was measured by using an enzyme-linked immunosorbent assay (ELISA) plate reader . Relative cell viability was evaluated from the quantity of MTT converted to the insoluble formazan salt compared with distilled water as the control. The cell viability was calculated as given in the following:The cytotoxicity was measured by the mitochondrial-dependent reduction of 3--2,5-diphenyltetrazolium-bromide  to formazan. RAW 264.7 cells were seeded in 96-well plates at a density of 1 × 105 cells/well) were seeded in 96-well plates and incubated for 16 h at 37°C in a humidified atmosphere (approximately 5% CO2). The cells were incubated in a serum-free medium containing the sample and 1 μg/mL LPS . After incubation for an additional 20 h, the NO content was evaluated by using the Griess assay. The supernatant was obtained, Griess reagent  was added, and the absorbance at 540 nm was recorded. NG-monomethyl-L-arginine monoacetate salt (L-NMMA) was used as the positive control. The inhibition of NO production was calculated from equation  were seeded in 6-well plates and preincubated for 16 h; subsequently, the samples were treated with LPS (1 μg/mL) for 24 h to induce the production of cytokines. The supernatant was evaluated using an ELISA kit  in accordance with the manufacturer's instructions. The cytokine concentrations were quantified by measuring the absorbance at 450 nm.The concentrations of IL-6 and TNF-6 cells/mL) were preincubated for 16 h and then treated with LPS (1 μg/mL). After incubation for 24 h, the cells were harvested and washed twice with phosphate-buffered saline. The cell lysates were prepared in RIPA buffer , and 1 mM/L EDTA; Thermo Fisher Scientific, MA, USA) for 30 min on ice. The cell lysates were centrifuged at 13,000 × g for 15 min at 4 °C, and 30 μg of the cell lysate was separated by to 7.5% SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred onto a PVDF membrane . Nonspecific binding to the membrane was blocked by incubation with blocking buffer (Thermo Fisher Scientific) for 60 min at room temperature. Then, the membrane was incubated with anti-mouse iNOS  and anti-mouse COX-2  overnight at 4°C. After washing, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody  for 60 min at room temperature. The bands were visualized by using the LAS-4000 luminescent image analyzer  and ECL detection reagent .RAW 264.7 cells (1 × 10μg) and 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (0.5 mg), as a substrate of collagenase, were added to 0.1 M Tris buffer (pH 7.4) in the presence or absence of samples to a total volume of 1.7 mL. The mixture was incubated at 37°C for 30 min, and 1 mL of 25 mM citric acid solution was added to terminate the enzyme reaction. Ethyl acetate (5 mL) was added, and the absorbance of the organic layer was measured by using UV spectrophotometry at 320 nm to calculate the inhibitory activity. The collagenase inhibitory activity was calculated from equation , followed by the Student–Newman–Keuls (S–N–K) test; statistical analyses were computed using Statistical Package for the Social Sciences  software package. Values were considered significantly different if the 1, 2, 6, 7), phenolic acids , and others . The isolated compounds were identified as acanthoside D (1) [2) [3) [4) [5) [6) [7) [8) [9) [10) [1H and 13C NMR spectra compared with references. 1H and 13C NMR spectra of the compounds (1–10) were included in Supplementary Materials as We isolated 10 compounds from FAS, namely, lignans (de D (1) , acantho (1) [2) , daucost [2) [3) , protoca [3) [4) , chlorog [4) [5) , ciwujia [5) [6) , syringa [6) [7) , farneso [7) [8) , 24, 3,4 [8) [9) , and fal[9) [10)  through 1): brown powder, 1H-NMR : δ 6.61 , 4.84 , 4.63 , 4.16 , 3.78 , 3.71 , 3.54 , 3.36 , 3.17-3.04 , 3.10-3.07 ; 13C-NMR : δ 153.1, 137.7 , 134.2 , 104.7 , 103.2 , 85.6 , 77.6 , 76.9 , 74.6 , 71.9 , 70.3 , 61.3 , 56.9 , 54.1 .Acanthoside D (2): brown powder, 1H-NMR : δ 6.61 , 6.55 , 4.83 , 4.63 , 4.57 , 4.13 , 3.75 , 3.71 , 3.70 , 3.54 , 3.60 , 3.16 , 3.10 , 3.01 , 2.99 ; 13C-NMR : δ 153.1 , 148.3 , 137.8 (C-1′), 135.2 (C-4″), 134.1 (C-4′), 131.9 (C-1″), 104.6 , 104.1 , 103.2 (C-1‴), 85.9 (C-6), 85.6 (C-2), 77.6 (C-5‴), 76.8 (C-3‴), 74.5 (C-2‴), 71.8 (C-8), 71.7 (C-4), 70.3 (C-4‴), 60.6 (C-6‴), 56.9 , 56.5 , 54.2 (C-1), 54.1 (C-5).Acanthoside B (3): white powder, 13C-NMR : δ 140.7 (C-5), 121.8 (C-6), 102.4 (C-1′), 78.5 (C-3), 78.4 (C-3′), 77.9 (C-5′), 75.2 (C-2′), 71.5 (C-4′), 62.7 (C-6′), 56.7 (C-14), 56.1 (C-17), 50.2 (C-9), 45.9 (C-24), 42.3 (C-13), 39.8 (C-4), 39.2 (C-12), 37.3 (C-1), 36.8 (C-10), 36.2 (C-20), 34.0 (C-22), 32.0 (C-7), 31.9 (C-8), 30.1 (C-2), 29.3 (C-25), 28.4 (C-16), 26.2 (C-23), 24.4 (C-15), 23.2 (C-28), 21.1 (C-11), 19.8 (C-27), 19.3 (C-19), 19.0 (C-26), 18.9 (C-21), 12.0 (C-29), 11.8 (C-18).Daucosterol (4): brown needle, 1H-NMR : δ 7.29 , 7.25 , 6.75 ; 13C-NMR : δ 167.9 (C-7), 150.3 (C-4), 145.2 (C-3), 122.5 (C-1), 122.1 (C-6), 117.0 (C-2), 115.6 (C-5).Protocatechuic acid (5): brown powder, 1H-NMR : δ 7.32 , 6.99 , 6.92 , 6.73 , 6.07 , 4.96 , 3.83 , 3.50 , 2.05 , 1.80 , 1.71 ; 13C-NMR : δ 174.1 (C-7), 166.0 (C-9′), 148.8 (C-4′), 145.9 (C-3′), 145.7 (C-7′), 126.0 (C-1′), 122.0 (C-6′), 116.3 (C-2′), 114.9 (C-5′), 114.4 (C-8′), 73.5 (C-1), 71.5 (C-5), 69.7 (C-4), 52.4 (C–CH3), 37.6 (C-6), 35.5 (C-2).Chlorogenic acid methyl ester (6): yellowish powder, 1H-NMR : δ 7.25 , 6.56 , 4.49 , 4.13 , 4.02, 3.78 , 3.40 , 3.46 , 2.43 ; 13C-NMR : δ 198.8 (C-7), 148.2 , 148.2 , 141.9 (C-4), 135.1 (C-4′), 132.4 (C-1′), 127.4 (C-1), 106.9 , 104.4 , 83.6 (C-7′), 70.3 (C-9), 60.3 (C-9′), 56.6 (C-3), 56.6 (C-5), 56.5 , 56.0 (C-8′), 49.0 (C-8).Ciwujiatone (7): light brown powder, 1H-NMR : δ 6.57 , 4.71 , 4.27 , 3.89 , 3.88 , 3.08 ; 13C-NMR : δ 147.2 , 134.3 (C-4′), 132.2 (C-1′), 102.7 , 86.2 , 71.9 , 56.5 , 54.4 .Syringaresinol (8): yellowish oil, 1H-NMR : δ 5.39 , 5.13 , 4.13 , 2.10-1.95 , 1.65 , 1.58 ; 13C-NMR : δ 139.9 (C-3), 135.4 (C-7), 131.4 (C-11), 124.4 (C-10), 123.9 (C-6), 123.3 (C-2), 59.4 (C-1), 39.8 (C-8), 39.6 (C-4), 26.8 (C-9), 26.4 (C-5), 25.8 (C-12), 17.8 (C-15), 16.4 (C-13), 16.1 (C-14).Farnesol (9): brown powder, 1H-NMR : δ 7.57 , 7.49 , 7.00 , 6.88 , 6.73 , 6.27, 6.17, 5.61 , 5.09 , 3.36 , 2.50-2.02 ; 13C-NMR : δ174.0 (C-7), 167.4 (C-9′), 167.1 (C-9″), 148.3 , 146.5(C-7′), 146.4 (C-7″), 145.3 (C-3′), 145.3 (C-3″), 126.4 (C-1′), 126.3 (C-1″), 122.0 (C-6′), 122.0 (C-6″), 115.2 , 113.9 , 113.4 (C-8′), 113.4 (C-8″), 74.6 (C-1), 74.5 (C-4), 68.6 (C-5), 67.8 (C-3), 37.2 (C-2), 37.0 (C-6).3,4-dicaffeoylquinic acid (10): brown oil, 1H-NMR : δ 5.92 , 5.60 , 5.50 , 5.25 , 5.20 , 4.93 , 2.10 , 1.37 , 1.25  0.87 ; 13C-NMR : δ 135.9 (C-2), 134.8 (C-10), 127.7 (C-9), 117.4 (C-1), 79.9 (C-7), 78.3 (C-4), 70.4 (C-5), 68.8 (C-6), 63.6 (C-3), 58.7 (C-8), 31.9 (C-15), 31.0 (C-13), 29.4 (C-14), 29.2 (C-12), 27.8 (C-11), 22.7 (C-16), 14.2 (C-17).Falcarindiol (1) was lower and syringaresinol (7) and protocatechuic acid (4) were higher in FAS than in AS  at the experimental doses . These r1–10 isolated from FAS, the inhibitory effects on NO production in RAW 264.7 cells were evaluated. FAS (IC50 = 12.31 ± 0.92 μg/mL) had strong inhibitory effects on NO production compared with AS (IC50 = 26.56 ± 1.28 μg/mL) and effects similar to those of the positive control, L-NMMA (IC50 = 10.16 ± 0.85 μg/mL). Most of the isolated compounds had strong inhibitory effects on NO production (7 (IC50 = 26.56 ± 1.28 μM), 8 (IC50 = 40.63 ± 3.38 μM), 9 (IC50 = 7.95 ± 2.03 μM), and 10 (IC50 = 21.87 ± 1.02 μM), more strongly inhibited NO production than the positive control, L-NMMA (IC50 = 19.68 ± 5.38 μM). In particular, the aglycone form (7) showed better activity than the glycoside (1).To assess the anti-inflammatory activity of AS, FAS, and compounds oduction . Some co7–10) that showed strong inhibitory activity in the NO production assay were selected for the test of iNOS and COX-2; in addition, compound 1 was selected for comparison with its aglycone, 7. The levels of iNOS and COX-2 were significantly increased in the LPS-stimulated cells compared with the control cells  showed better activity than the glycoside (1). These results indicated that the extracts and compounds inhibited NO production through a decrease in iNOS and COX-2 expression in the LPS-stimulated RAW 264.7 cells.To further characterize the mechanisms underlying the inhibition of the LPS-induced production of NO by the extracts and compounds, western blotting assays were performed. Four compounds (ol cells . Treatmeα) production was analyzed by using ELISA. RAW 264.7 macrophage cells were exposed to LPS, and the cytokine levels in these cells were measured for their evaluation of the inhibitory effects of AS, FAS, and the isolated compounds. In the LPS-treated group (control), IL-6 and TNF-α levels were detected to be 548.12 pg/mL and 777.89 pg/mL, respectively. In the AS and FAS treated groups, IL-6 and TNF-α levels were decreased  on cytokine production (IL-6 and TNF-α) were measured. In the LPS-treated group (control), IL-6 and TNF-α levels were 262.07 pg/mL and 302.88 pg/mL, respectively. IL-6 and TNF-α levels were decreased by treatment with the selected compounds. And the 9, 10, and the aglycone 7 showed better activity than the glycoside  on the collagenase reaction were measured. Compounds 9 (90.38%) and 10 (52.88%) showed potent inhibitory activities, and 1, 7, and 8 exhibited moderate activities. The aglycone form (7) (23.08%) showed better activity than the glycoside form (1) (15.38%).The inhibition of collagenase by AS, FAS, and the isolated compounds was evaluated. FAS (79%) more strongly inhibited collagenase when compared with AS 33%; . The effin vitro and in vivo [L. plantarum subsp. argentoratensis has tannase and can decompose tannin; thus, it has been used for the initial degradation of complex tannins [Vigna sinensis L., cowpea, increased phenolic compound content and enhanced antioxidant activity [7), has been reported to more strongly inhibit proinflammatory molecules  compared with acanthoside D, the glycoside form of lignan (1) [7) was better than that of acanthoside D (1). The HPLC analysis showed that the content of syringaresinol, the aglycone (7), increased during fermentation and the content of acanthoside D, the glycoside (1), decreased during fermentation. Protocatechuic acid (4) content also increased by bioconversion, and these compounds had good inhibitory effects on inflammatory-related factors. These results indicated that the variations due to bioconversion led to significant differences in the anti-inflammatory effects of the FAS and AS.There are many reports that the linkage of glycosyl groups of the aglycone can alter their bioactivities , 28. The in vivo , 31. Thi tannins . The feractivity . In anotactivity . Syringagnan (1) . In the A. sessiliflorus extract via fermentation was performed. The inhibitions of the production of NO and inflammatory molecules by AS, FAS, and the isolated compounds were measured in vitro. The results showed that the content of the compounds was different in FAS and AS. The increased compounds, syringaresinol (7) and protocatechuic acid (4), showed better activity than acanthoside D (1). Moreover, the anti-inflammatory activities of FAS were stronger than those of AS. These results indicated that FAS and the isolated compounds may be a potential treatment for inflammatory disorders such as arthritis.The biotransformation of the


Of the synthesized compounds, 2--N-acetamide (21a) achieved the highest in vitro cytotoxic activity against H1299, with an IC50 value of 12.5 nM in situ, and 0.47 and 0.14 nM against EGFR and HER2, respectively, values comparable to the IC50 of the approved drug imatinib (1). Our synthesized compounds were promising, demonstrating high selectivity and affinity for EGFR/HER2, especially the hinge region forming a hydrophobic pocket, which was mediated by hydrogen bonding as well as hydrophobic and electrostatic interactions, as indicated by molecular modeling. Moreover, the designed compounds showed good affinity for T790M EGFR, one of the main mutants resulting in acquired drug resistance. Furthermore, both pharmacokinetic and physicochemical properties of the designed compounds were within the appropriate range for human usage as predicted by the in Silico ADME study. The designed compound (21a) might serve as an encouraging lead compound for the discovery of promising anti-lung cancer agents targeting EGFR/HER2.Dual targeting of epidermal growth factor receptor (EGFR) and human EGFR-related receptor 2 (HER2) is a proven approach for the treatment of lung cancer. With the aim of discovering effective dual EGFR/HER2 inhibitors targeting non-small cell lung cancer cell line H1299, three series of thienotriazine and acetamide derivatives were designed, synthesized, and biologically evaluated. The synthesized compounds displayed IC Lung cancer continues to be one of the major causes of cancer-related mortality, and non-small cell lung cancer (NSCLC) cases account for approximately 90% of all lung cancer . Treatme2), imatinib (1), lapatinib (4), and erlotinib (3) have been approved by the US Food and Drug Administration (FDA) for treatment of EGFR-activating mutation-positive NSCLC patients (2) or imatinib (1) . Inut 25 nM . This su4) and neratinib (6) shows that the upper right warhead, or the hydrophobic tail, is essential for potency of kinase inhibition, while the lower left warhead, the hydrophobic head, is crucial for the dual EGFR/HER2 inhibitory activity. Therefore, to design dual TKIs, we kept the 4-aminoaniline with modified substitution at the meta position as the upper right warhead. On the other hand, the lower right warhead serves as an electrophilic moiety for covalent bond formation in the majority of second- and third-generation agents, and owing to the limited clinical benefit and toxicity, the left warhead was modified in the current study to cyclopentyl and/or cyclohexyl (series A and B) in an attempt to abolish the cytotoxicity. In addition, the hinge region of TKIs, which is occupied by the adenine ring of ATP, was modified to be occupied by thienotriazine in series A and B, and tetrahydrobenzothiophene in series C. Modification of the core scaffold was intended to diminish the cellular resistance common with quinazoline and improve the potency based on our previous publication. Another new modification in this study is a change in the position of the upper warhead from position 4 of quinoline and quinazoline in the majority of approved TKIs to position 2 of tetrahydrobenzothiophene, keeping the hydrophobic tail to mimic the dual EGFR/HER2 inhibitors. The cyano group, which is characteristic of neratinib (6), was kept at position 3 to mimic N3 of quinazoline-based TKIs. In this way, various regions of ATP-binding sites could be targeted by such substitution patterns on the mutant EGFR protein kinase domain to design molecules that are differentially selective. The design strategies are displayed in Moreover, it was observed that the structure of the majority of clinically approved TKIs is characterized by a hydrophobic head and hydrophobic tail linked to a central heteroaromatic system and NH spacer, as shown in 11–13) were synthesized as reported in our earlier research . Sc. Sc11–13ldehydes  with chlcompound . Then, c1H NMR, and EI-MS). Mass spectra showed the expected molecular ion peaks. IR spectra of the synthesized compounds  collectively showed secondary amine signals at around 3300–3500 cm−1, which indicates coupling between 2-chloro-N-acetamide (19) and heteroaryl amines  with the disappearance of signal corresponding to NH2. 1H NMR spectra of compounds (14–17a) showed multiple peaks of aromatic protons in the range of 7.80 to 8.70 δ (ppm) and a single peak for CH proton in the range of 7.4 to 8.4 δ (ppm). The spectrum of compound (15a) showed a single peak corresponding to OH proton at 10.15 δ (ppm), and the spectrum of compound (16a) showed a single sharp peak at 4.06 δ (ppm) corresponding to the OCH3 group of anisaldehyde. A single peak for CH proton appeared at 7.85 δ (ppm) and 7.64 δ (ppm) for compounds (15b) and (17b), respectively. The 1H NMR spectrum of compound (15b) showed a single peak corresponding to OH proton at 8.25 δ (ppm). The 1H NMR spectrum of compound (21a) showed the appearance of single peak at 11.88 δ (ppm) and multiple peaks at 6.95 δ (ppm) corresponding to NH proton and three aromatic protons of heteroarylamine, respectively. The NH proton of the coupling reaction appeared as a single peak at 3.65 δ (ppm). Another single peak belonging to proton of NH of thiophene appeared at 5.49 δ (ppm). Finally, a single sharp peak accounting for CH2 appeared at 3.08 δ (ppm). The 1H NMR spectrum of compound (21b) showed the appearance of single peaks at 11.69 and 6.58 δ (ppm), corresponding to NH proton and single aromatic proton of the heteroaryl amine, respectively. A single peak for NH proton of thiophene appeared at 5.01 δ (ppm) and a sharp peak of CH2 appeared at 3.65 δ (ppm). The spectrum also showed two single sharp peaks for the two methyl groups of the heteroarylamine at 2.42 and 2.54 δ (ppm).The structures of the synthesized compounds were confirmed by microanalyses and spectral data  were measured using sulfo-rhodamine B (SRB) assay thienotriazin-4-ylamino)benzene-1,3-diamine (13a) [A mixture of 3-  (0.01 mone (13a) .H-cyclopenta[4:5]thienotriazin-4-ylamino)-N-benzylidenebenzene-1,3-diamine (14a)3-: 371 , 361 (1.33%), 353 (1.21%), 343 (7.82%), 316 (11.55%), 108 (100%). IR (cm−1): 752 (C–S), 1261 (C–N), 1624 (C=N), 3429 (N–H). 1H NMR : δ (ppm) 1.29 , 3.08 , 3.27 , 7.08 , 7.71 , 7.80 , 11.81 . Elemental analysis calculated for C21H17N5S: C, 67.90; H, 4.61; N, 18.85. Found: C, 68.09; H, 4.66; N, 19.08.Mass spectrum: H-cyclopenta[4:5]thienotriazin-4-ylamino)-N-(2-hydroxybenzylidene)benzene-1,3-diamine (15a)3-: 387 , 375 (2.45%), 347 (6.59%), 345 (9.14%), 212 (85.96%), 119 (100%). IR (cm−1): 756 (C–S), 1149 (C–O), 1276 (C–N), 1616 (C=N), 3221 (N–H), 3352 (OH). 1H NMR : δ (ppm) 1.14 , 2.12 , 2.73 , 4.54 , 7.84 , 7.90 , 8.52 , 10.15 . Elemental analysis calculated for C21H17N5OS: C, 65.10; H, 4.42; N, 18.08. Found: C, 65.34; H, 4.47; N, 18.39.Mass spectrum: H-cyclopenta[4:5]thienotriazin-4-ylamino)-N-(3-nitrobenzylidene)benzene-1,3-diamine (16a)3-: 401 , 389 (79.4%), 362 (1.80%), 322 (1.20%), 293 (2.40%), 261 (1.50%), 249 (100%). IR (cm−1): 690 (C–S), 1246 (C–O–C), 1261 (C–N), 1678 (C=N), 3444 (N–H). 1H NMR : δ (ppm): 1.15 , 3.65 , 3.70 , 4.06 , 7.30 , 7.85 , 8.70 , 10.20 . Elemental analysis calculated for C22H19N5OS: C, 65.81; H, 4.77; N, 17.44. Found: C, 66.04; H, 4.84; N, 17.68.Mass spectrum: H-cyclopenta[4:5]thienotriazin-4-ylamino)-N-(4-methoxybenzylidene)benzene-1,3- diamine (17a)3-: 416 , 407 (2.47%), 403 (2.56%), 376 (4.56%), 222 (12.24%), 108 (100%). IR (cm−1): 732 (C–S), 1350 (NO2), 1527 (NO2), 1612 (C=N), 3363 (N–H). 1H NMR : δ (ppm): 1.80 , 2.62 , 2.64 , 4.80 , 7.28 , 7.31 , 7.34 , 8.50 . Elemental analysis calculated for C21H16N6O2S: C, 60.56; H, 3.87; N, 20.18. Found: C, 60.73; H, 3.91; N, 20.43.Mass spectrum: General procedure for synthesis of compounds (15b) and (17b)H-cyclohexa[4:5]thienotriazin-4ylamino)benzene-1,3-diamine (13b) [A mixture of 3-  (0.01 mone (13b) .H-cyclohexa[4:5]thienotriazin-4-ylamino)-N-(2-hydroxybenzylidene)benzene-1,3-diamine (15b)3-: 401 , 390 (3.80%), 375 (2.10%), 361 (6.49%), 346 (100%), 303 (5.30%). IR (cm−1): 756 (C–S), 1678 (C=N), 3228 (N–H), 3363 (OH). 1H NMR : δ (ppm): 1.13 , 3.05 , 3.10 , 3.60 , 7.13 , 7.81 , 7.85 , 8.25 . Elemental analysis calculated for C22H19N5OS: C, 65.81; H, 4.77; N, 17.44. Found C, 65.58; H, 4.39; N, 17.76.Mass spectrum: H-cyclohexa[4:5]thienotriazin-4-ylamino)-N-(3-nitrobenzylidene)benzene-1,3- diamine (17b)3-: 430 , 384 (8.60%), 354 (10.40%), 296 (6.30%), 103 (100%). IR (cm−1): 732 (C–S), 1350 (NO2), 1527 (NO2), 3360 (N–H). 1H NMR : δ (ppm): 1.14 , 3.04 , 3.11 , 3.20 , 7.31 , 7.37 , 7.45 , 7.64 .Mass spectrum: N-acetamide (19)2-Chloro-11) thiophene-3-carbonitrile (11)  (0.10 mo11) .General procedure for synthesis of compounds 20a,b) (0.009 mol) pyridin-3-ylamino)-N-acetamide (21a)2-(1m/z (%): 354 , 353 (4.83%), 352 (6.96%), 320 (57.61%), 291 (38.64%), 178 (100%). IR (cm−1): 789 (C–S), 1689 (C=O), 2224 (C≡N), 3214 (N–H), 3338 (N–H), 3443 (N–H). 1H NMR : δ (ppm): 1.20 , 3.06 , 3.08 , 3.65 , 5.49 , 6.95 , 11.88 . Elemental analysis calculated for C17H16N6OS: C, 57.94; H, 4.58; N, 23.85. Found: C, 58.21; H, 4.42; N, 24.18.Mass spectrum: N--2-acetamide (21b)m/z (%) 382 , 381 (1.08%), 380 (3.65%), 348 (1.44%), 320 (13.61%), 143 (100%). IR (cm−1): 778 (C–S), 1689 (C=O), 2224 (C≡N), 3285 (N–H), 3388 (N–H), 3444 (N–H). 1H NMR : δ (ppm): 1.18 , 1.75 , 2.42 , 2.54 , 3.02 , 3.65 , 5.01 , 6.58 , 11.69 . Elemental analysis calculated for C19H20N6OS: C, 59.98; H, 5.30; N, 22.09. Found: C, 60.29; H, 5.43; N, 22.41.Mass spectrum: N--2-acetamide (23)22) triazine scaffold and 3-cyanotetrahydrobenzothiophene occupied the ATP-binding site, showing the same drug–receptor interactions as two approved drugs: lapatinib (4) and neratinib (6). Additional binding sites, especially in hydrophobic pockets, were provided by our structural modification. The introduction of new functional groups with positions different to those commonly found in approved drugs could allow the designed compounds to interact with alternative binding sites, which could solve the problem of acquired drug resistance. Our new scaffolds might also serve as promising lead compounds for the discovery of new drugs to overcome acquired resistance in NSCLC patients.New chemical compounds belonging to tetrahydrobenzothienotriazine, dihydrocyclopentathienotriazine, and 3-cyanotetrahydrobenzothiophene bearing various heterocyclic systems at positions 4 and 2 were designed, synthesized, structurally elucidated, and biologically evaluated as anti-lung cancer agents. The synthesized compounds exhibited potent in situ cytotoxic activity toward human lung carcinoma cell line H1299 compared to gefitinib (


The SARs here highlighted along with their rationalization by in silico docking experiments into both target enzymes provide further insights into this class of inhibitors for their development as potential DML antidiabetic candidates.Diabetes mellitus (DM) is a complex disease which currently affects more than 460 million people and is one of the leading cause of death worldwide. Its development implies numerous metabolic dysfunctions and the onset of hyperglycaemia-induced chronic complications. Multiple ligands can be rationally designed for the treatment of multifactorial diseases, such as DM, with the precise aim of simultaneously controlling multiple pathogenic mechanisms related to the disease and providing a more effective and safer therapeutic treatment compared to combinations of selective drugs. Starting from our previous findings that highlighted the possibility to target both aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two enzymes strictly implicated in the development of DM and its complications, we synthesised 3-(5-arylidene-4-oxothiazolidin-3-yl)propanoic acids and analogous 2-butenoic acid derivatives, with the aim of balancing the effectiveness of dual AR/PTP1B inhibitors which we had identified as designed multiple ligands (DMLs). Out of the tested compounds,  In the last decade, the design and discovery of multi-target-directed ligands has emerged as a modern promising strategy to develop new drugs aimed to the treatment of multifactorial diseases, such as diabetes, cancer, infectious diseases, cardiovascular and neurodegenerative disorders. Therapies based on a single-target-directed agent may be insufficient for the management of these complex pathologies, thus combinations of different drugs generally represent therapeutic regimens necessary in order to achieve a higher efficacy. However, combination therapies may involve pharmacokinetics, toxicity or patient compliance problems.Therefore, recently multi-targeted ligands have attracted great interest in medicinal chemistry as a valuable alternative to combinations of single-targeted drugs. In particular, designed multiple ligands (DMLs) are compounds rationally designed to modulate two or more selected targets simultaneously and, thus, to achieve pharmacological effects favourable for the efficient management of multifactorial pathologies. Growing evidence indicates that DMLs may be endowed with enhanced efficacy and improved safety compared to drug combinations, especially in long-term therapies ,2,3,4.A drug design strategy frequently used to obtain DMLs starts from the knowledge of the distinct pharmacophores of agents active on each of the individual targets and consists in the integration of these pharmacophores into a single structure to obtain hybrid molecules. Hybridization should be carried out in such a way that the pharmacophoric elements inserted in a resulting DML maintain the ability to interact with specific sites of the different selected targets, thus producing simultaneously the desired effects. In order to obtain molecules endowed with appropriate drug-like properties, it is often preferred to partially overlap the different pharmacophores, by using or merging them. However, by applying the different possible approaches for the design of DMLs, it is quite likely to achieve lead compounds endowed with high potency towards only one of the desired targets, with less effect on the others; therefore, it is often necessary to carry out a balancing process, using structural modifications of the lead compound, aimed to modulate and balance the activity towards the selected targets until to reach an optimal ratio ,4.Recently we have reported an initial investigation on 4-thiazolidinone derivatives active as dual inhibitors of aldose reductase and protein tyrosine phosphatase 1B, in the context of our search for new candidates for the treatment of type 2 diabetes mellitus and its complications . DiabeteOn these bases, in the context of a search for new DMLs as potential antidiabetic agents, we selected as targets aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two enzymes differently involved in the development of DM and its complications. AR (E.C. 1.1.1.21) is an aldo-keto reductase capable to catalyse the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of a variety of aldehydes, including aldoses. Under hyperglycaemic conditions, such as in DM, an increased flux of glucose through the polyol pathway occurs. AR catalyses the first and rate-limiting step of this metabolic pathway, by reducing glucose to sorbitol which is then oxidized to fructose by sorbitol dehydrogenase. The increased glucose metabolism through the polyol pathway causes osmotic imbalance, decreases antioxidant cellular defence and promotes glycation and inflammatory events, thus leading to tissue damage which is responsible for the development of diabetic chronic complications ,11. AR iNumerous evidences have undoubtedly demonstrated that PTP1B (E.C. 3.1.3.48) is involved in the development of insulin-resistance, which is a characteristic condition in type 2 DM (T2DM). This protein tyrosine phosphatase acts as a major negative regulator of insulin signalling, by dephosphorylating specific phosphotyrosine residues of the activated insulin receptor and insulin receptor substrate proteins and thus attenuating insulin signalling pathways. PTP1B has been shown to be overexpressed in T2DM and in other pathologies associated with insulin resistance, such as obesity ,15,16. TOn these bases, both PTP1B and AR can be assumed as attractive molecular targets for the design of DMLs capable to control simultaneously different cellular mechanisms underlying the development of both T2DM and its complications. However, this research is still in its infancy and, so far, only few dual ALR2/PTP1B inhibitors have been reported, among which some natural compounds such as berberine ,24. Although these two enzymes belong to different families of proteins, the knowledge of their active sites and the structure-activity relationships (SARs) of inhibitors of each of these targets highlights several shared structural features that enable the design of dual inhibitors by merging the respective pharmacophores. In particular, our previous studies on different 4-thiazolidinone derivatives, which were investigated as AR or PTP1B inhibitors , evidenc1, 2, 2e) and the corresponding 2,4-thiazolidinedione counterpart 1e acetic acid derivatives, which are endowed with excellent AR inhibitory activity and 4-[(5-arylidene-4-oxothiazolidin-3-yl)methyl]benzoic acids, which we identified as potent PTP1B inhibitors, to obtain a series of new 4-thiazolidinone derivatives 1, 2,  that werrpart 1e , which e1–2, we designed and synthesised 3-(5-arylidene-4-oxothiazolidin-3-yl)propanoic acids 3a–f and 4a–f and 4--2-butenoic acids 5a–e methylidene]-2,4-dioxothiazolidin-3-yl}propanoic acid showed a moderate capability to inhibit human PTP1B. Therefore, this compound was assumed as a starting point for hit-to-lead optimization.5a–e was also suggested by our previous investigations on both AR and PTP1B inhibitors thiazolidin-3-yl}propanoic acid (3a). Yield 66% (409.6 mg); m.p. 177–180 °C; 1H-NMR (DMSO-d6): δ 2.59 ; 3.84 ; 7.09 ; 7.13 ; 7.19–7.23 ; 7.37 ; 7.44 , 7.54 ; 7.90 ; 12.45 . 13C-NMR (DMSO-d6): δ 32.0 (CH2), 38.0 (CH2), 119.5 (CH), 119.9 (CH), 120.8 (C), 122.8 (C), 124.8 (CH), 125.4 (CH), 130.8 (CH), 131.6 (CH), 132.7 (CH), 135.4 (CH), 156.3 (C), 158.2 (C), 165.8 (C), 167.4 (C), 172.4 (C). Anal. (C19H15NO5S) calcd: C 61.78; H 4.09; N 3.97; found: 61.92; H 4.03; N 3.99.3-{(5Z)-thiazolidin-3-yl}propanoic acid (3b). Yield 60% (372.3 mg); m.p. 198–202 °C; 1H-NMR (DMSO-d6): δ 2.60 ; 3.86 ; 7.11 ; 7.24 ; 7.45 ; 7.64 ; 7.90 ; 12.45 . 13C-NMR (DMSO-d6): δ 32.1 (CH2), 38.0 (CH2), 118.9 (CH), 120.1 (CH), 120.4 (C), 125.2 (CH), 128.2 (C), 130.9 (CH), 132.9 (CH), 133.0 (CH), 155.7 (C), 159.6 (C), 166.0 (C), 167.6 (C), 172.4 (C). Anal. (C19H15NO5S) calcd: C 61.78; H 4.09; N 3.97; found: C 61.97; H 3.98; N 4.01.3-{(5Z)-[5-(3-Benzyloxyphenyl)methylidene]-2,4-dioxothiazolidin-3-yl}propanoic acid (3c). Yield 25% (161.0 mg); m.p. 142–145 °C; 1H-NMR (DMSO-d6): δ 2.60 ; 3.86 ; 5.17 ; 7.14–7.20 ; 7.24 ; 7.34 ; 7.40 ; 7.44–7.48 ; 7.88 ; 12.45 . 13C-NMR (DMSO-d6): δ 32.0 (CH2), 38.0 (CH2), 70.0 (CH2), 116.7 (CH), 118.0 (C), 122.3 (C), 122.9 (CH), 128.3 (CH), 128.5 (CH), 129.1 (CH), 131.1 (CH), 133.3 (CH), 134.9 (CH), 137.2 (C), 159.3 (C), 165.9 (C), 167.6 (C), 172.4 (C). Anal. (C20H17NO5S) calcd: C 62.65; H 4.47; N 3.65; found: C 62.83; H 4.51; N 3.48.3-{(5Z)-[5-(4-Benzyloxyphenyl)methylidene]-2,4-dioxothiazolidin-3-yl}propanoic acid (3d). Yield 61% (392.9 mg); m.p. 195–199 °C; 1H-NMR (DMSO-d6): δ 2.58 ; 3.85 ; 5.19 ; 7.18 ; 7.34 ; 7.40 ; 7.46 ; 7.59 ; 7.88 ; 12.42 . 13C-NMR (DMSO-d6): δ 32.2 (CH2), 38.0 (CH2), 70.1 (CH2), 116.3 (CH), 118.7 (C), 126.2 (C), 128.4 (CH), 128.6 (CH), 129.1 (CH), 132.8 (CH), 133.4 (CH), 134.9 (CH), 137.0 (C), 160.8 (C), 166.1 (C), 167.7 (C), 172.6 (C). Anal. (C20H17NO5S) calcd: C 62.65; H 4.47; N 3.65; found: C 62.78; H 4.56; N 3.55.3-methylidene}thiazolidin-3-yl]propanoic acid (3e). Yield 81% (540.8 mg); m.p. 130–133 °C; 1H-NMR (DMSO-d6): δ 2.60 ; 3.06 ; 3.86 ; 4.25 ; 7.07 ; 7.16–7.24 ; 7.3–7.35 ; 7.44 ; 7.89 ; 12.45 . 13C-NMR (DMSO-d6): δ 32.0 (CH2), 35.4 (CH2), 38.0 (CH2), 68.9 (CH2), 116.6 (CH), 117.7 (C), 122.2 (C), 122.4 (CH), 126.9 (CH), 128.9 (CH), 129.5 (CH), 131.1 (CH), 133.4 (CH), 134.9 (CH), 138.8 (C), 159.4 (C), 165.9 (C), 167.6 (C), 172.4 (C). Anal. (C21H19NO5S) calcd: C 63.46; H 4.82; N 3.52; found: C 63.66; H 4.89; N 3.483-methylidene}thiazolidin-3-yl]propanoic acid (3f). Yield 73% (487.4 mg); m.p. 160–163 °C; 1H-NMR (DMSO-d6): δ 2.59 ; 3.06 ; 3.85 ; 4.28 ; 7.11 ; 7.23 ; 7.30–7.34 ; 7.57 ; 7.87 ; 12.44 . 13C-NMR (DMSO-d6): δ 32.1 (CH2), 35.3 (CH2), 37.9 (CH2), 69.0 (CH2), 116.0 (CH), 118.5 (C), 126.0 (C), 126.9 (CH), 128.9 (CH), 129.5 (CH), 132.8 (CH), 133.5 (CH), 138.6 (C), 160.9 (C), 166.1 (C), 167.7 (C), 172.4 (C). Anal. (C21H19NO5S) calcd: C 63.46; H 4.82; N 3.52; found: C 63.55; H 4.80; N 3.54.2O (35 mL) CS2  was added dropwise, in such a manner that the temperature of the reaction did not exceed 25 °C and the mixture was stirred at room temperature for 24 h. A solution of sodium bromoacetate, obtained by solubilizing bromoacetic acid  and NaOH  in H2O (45 mL), was added and the mixture was stirred at room temperature for a further 24 h. The mixture was acidified with HCl 6M until pH 3–4 and refluxed for 24 h. The mixture was then cooled and poured into H2O, providing a pale yellow precipitate. Then, if necessary, pH was adjusted again to 3–4 by the addition of HCl 6M and the solid was filtered off. The aqueous solution was extracted with ethyl acetate; the organic phase was washed with H2O, dried with anhydrous Na2SO4 and evaporated under reduced pressure, providing a further amount of 3-(4-oxo-2-thioxothiazolidin-3-yl)propanoic acid (7). The solid residue and the precipitate were collected and washed with ethyl ether to give pure compound 7. Yield 35% (897.2 mg); mp 159–162 °C; 1H-NMR (CDCl3): δ 2.77 , 4.01  4.31 . Anal. (C6H7NO3S2) calcd: C 35.11; H 3.44; N 6.82; found: C 34.96, H 3.34, N 6.92.To a solution of β-alanine  and NaOH  in H7)  glacial acetic acid (10 mL), sodium acetate  and the appropriate aldehyde (1.22 mmol) was refluxed for 3–4 h. The mixture was cooled and poured into H2O, providing a precipitate which was filtered off, washed with H2O and recrystallized from methanol to give pure 3-[(5-arylidene-4-oxo-2-thioxothiazolidin-3-yl)]propanoic acids 4.A mixture of 3-(4-oxo-2-thioxothiazolidin-3-yl)propanoic acid (3-{(5Z)[4-Oxo-5-(3-phenoxyphenyl)methylidene-2-thioxothiazolidin-3-yl]}propanoic acid (4a). Yield 46% (216.3 mg); m.p. 174–175 °C; 1H-NMR (DMSO-d6): δ 2.57 ; 4.15 ; 7.01–7.03 ; 7.08 ; 7.18 ; 7.27 ; 7.38–7.41 ; 7.49 ; 7.63 . 13C-NMR (DMSO-d6): δ 31.9 (CH2), 41.1 (NCH2), 120.0 (CH), 120.4 (CH), 120.5 (C), 121.8 (CH), 124.3 (CH), 125.5 (CH), 126.5 (CH), 131.4 (CH), 132.4 (CH), 133.3 (CH), 135.7 (C), 156.7 (C), 158.8 (C), 167.7 (C), 173.1 (C), 194.0 (C). Anal. (C19H15NO4S2) calcd: C 59.20; H 3.92; N 3.63; found: C 59.06; H 4.01; N 3.60.3-{(5Z)[4-Oxo-5-(4-phenoxyphenyl)methylidene-2-thioxothiazolidin-3-yl]}propanoic acid (4b). Yield 46% (216.3 mg); m.p. 218–219 °C; 1H-NMR (DMSO-d6): δ 2.62 ; 4.22 ; 7.11 ; 7.24 ; 7.45 ; 7.64 ; 7.76 . 13C-NMR (DMSO-d6): δ 31.7 (CH2), 40.8 (NCH2), 119.2 (CH), 120.8 (CH), 121.4 (C), 125.7 (CH), 128.4 (CH), 131.2 (C), 133.4 (CH), 133.9 (CH), 155.8 (C), 160.3 (C), 167.6 (C), 172.7 (C), 194.0 (C). Anal. (C19H15NO4S2) calcd: C 59.20; H 3.92; N 3.63; found: C 59.25; H 4.00; N 3.57.3-{(5Z)-5-[(3-Benzyloxyphenyl)methylidene]-4-oxo-2-thioxothiazolidin-3-yl}propanoic acid (4c). Yield 62% (302.2 mg); m.p. 207–209 °C; 1H-NMR (DMSO-d6): δ 2.62 ; 4.21 ; 5.16 ; 7.15–7.19 ; 7.33 ; 7.39 ; 7.44–7.47 ; 7.73 . 13C-NMR (DMSO-d6): δ 31.8 (CH2), 41.0 (CH2), 70.4 (CH2), 117.3 (CH), 119.0 (C), 123.8 (CH), 124.0 (C), 128.7 (CH), 129.1 (CH), 129.6 (CH), 131.8 (CH), 133.9 (CH), 135.2 (CH), 137.5 (C), 159.7 (C), 167.7 (C), 172.9 (C), 194.2 (C). Anal. (C20H17NO4S2) calcd: C 60.13; H 4.29; N 3.51; found: C 59.97; H 4.18; N 3.61.3-{(5Z)-5-[(4-Benzyloxyphenyl)methylidene]-4-oxo-2-thioxothiazolidin-3-yl}propanoic acid (4d). Yield 49% (238.8 mg); m.p. 170–173 °C; 1H-NMR (DMSO-d6): δ 2.61 ; 4.21 ; 5.20 ; 7.19 ; 7.34–7.48 ; 7.62 ; 7.78 . 13C-NMR (DMSO-d6): δ 31.5 (CH2), 40.6 (CH2), 70.2 (CH2), 116.5 (CH), 119.8 (C), 126.3 (C), 128.4 (CH), 128.6 (CH), 129.1 (CH), 133.5 (CH), 133.7 (CH), 137.0 (C), 161.2 (C), 167.3 (C), 172.4 (C), 193.7 (C). Anal. (C20H17NO4S2) calcd: C 60.13; H 4.29; N 3.51; found: C 60.17; H 4.35; N 3.45.3-[(5Z)-4-Oxo-{5-[3-(2-phenylethoxy)phenyl]methylidene}-2-thioxothiazolidin-3-yl]propanoic acid (4e). Yield 26% (131.2 mg); m.p. 136–138 °C; 1H-NMR (DMSO-d6): δ 2.63 ; 3.05 ; 4.20–4.26 ; 7.08 ; 7.15–7.18 ; 7.22 ; 7.29–7.34 ; 7.45 ; 7.76 . 13C-NMR (DMSO-d6): δ 31.5 (CH2), 35.5 (CH2), 40.7 (CH2), 69.1 (CH2), 117.0 (CH), 118.3 (C), 123.2 (C), 123.4 (CH), 127.1 (CH), 129.1 (CH), 129.7 (CH), 131.4 (CH), 133.7 (CH), 135.0 (CH), 138.9 (C), 159.6 (C), 167.3 (C), 172.5 (C), 193.9 (C). Anal. (C21H19NO4S2) calcd: C 61.00; H 4.63; N 3.39; found: C 61.18; H 4.74; N 3.25.3-[(5Z)-4-Oxo-{5-[4-(2-phenylethoxy)phenyl]methylidene}-2-thioxothiazolidin-3-yl]propanoic acid (4f). Yield 26% (131.2 mg); m.p. 171–174 °C; 1H-NMR (DMSO-d6): δ 2.63 ; 3.05 ; 4.23 ; 4.29 ; 7.12 ; 7.23 ; 7.30–7.34 ; 7.59 ; 7.77 ; 12.51 . 13C-NMR (DMSO-d6): δ 31.4 (CH2), 35.3 (CH2), 40.6 (CH2), 69.1 (CH2), 116.2 (CH), 119.6 (C), 126.1 (C), 126.9 (CH), 128.9 (CH), 129.5 (CH), 133.5 (CH), 133.8 (CH), 138.6 (C), 161.3 (C), 167.3 (C), 172.3 (C), 193.7 (C). Anal. (C21H19NO4S2) calcd: C 61.00; H 4.63; N 3.39; found: C 60.92; H 4.58; N 3.51.6 (2.5 mmol), methyl 4-bromocrotonate  and potassium carbonate  in anhydrous acetone (50 mL) was refluxed for 24 h. After cooling, the inorganic salts were filtered off and the solvent was evaporated under reduced pressure. The solid residue was washed with H2O and recrystallized from ethanol providing pure 4--2-butenoic acid methyl esters. A mixture of 5-arylidene-2,4-thiazolidinedione 2O and the precipitate was filtered off. The crude solid was washed with H2O and recrystallized from methanol to provide pure 2-butenoic acids 5. Chemical-physical and spectroscopic data of compounds 5a–c were reported in }-2-butenoic acid (5d). Yield 26% (220.6 mg); m.p. 170–172 °C; 1H-NMR (DMSO-d6): δ 4.47 ; 5.90 ; 6.83 ; 7.62–7.72 ; 8.03–8.15 ; 8.57 , 12.52 . 13C-NMR (DMSO-d6): δ 42.4 (CH2), 123.7 (CH), 124.1 (CH), 125.6 (CH), 126.2 (CH), 127.1 (CH), 127.5 (CH), 128.1 (CH), 129.5 (CH), 130.7 (CH), 130.8 (C), 131.5 (C), 133.8 (C), 141.2 (CH), 165.4 (C), 167.0 (C), 167.9 (C), 171.3 (C). Anal. (C18H13NO4S) calcd: C 63.71; H 3.86; N 4.13; found: C 63.59; H 3.76; N 4.22.4-{(5Z)-}-2-butenoic acid (5e). Yield 70% (593.9 mg); m.p. 235–238 °C; 1H-NMR (DMSO-d6): δ 4.45 ; 5.87 ; 6.81 ; 7.59–7.65 ; 7.71 ; 7.97 ; 8.04–8.08 ; 8.22 ; 12.49 . 13C-NMR (DMSO-d6): δ 42.4 (CH2), 122.1 (C), 123.6 (CH), 126.5 (CH), 127.8 (CH), 128.3 (CH), 128.7 (CH), 129.4 (CH), 129.6 (CH), 131.1 (CH), 131.8 (CH), 133.3 (C), 133.7 (C), 133.9 (C), 141.2 (CH), 165.8 (C), 167.0 (C), 167.7 (C). Anal. (C18H13NO4S) calcd: C 63.71; H 3.86; N 4.133; found: C 63.78; H 3.94; N 4.07.E. coli cells and purified to electrophoretic homogeneity as previously described [w/v) glycerol. AR activity was determined at 37 °C as previously described [The human recombinant AR was expressed in BL21(DE3)pLysS escribed . The purescribed  evaluati50 (concentration of compound required to determine a 50% inhibition of enzyme activity) values was performed in the above described assay conditions using 2 mM L-idose as substrate. All compounds tested as aldose reductase inhibitors were dissolved in DMSO and added to the assay mixture containing 8 mU of purified AR. The reaction was started by addition of the substrate. The DMSO concentration in all the assays was kept constant at 0.5% (v/v) in order to avoid effects on AR activity [The determination of ICactivity . 50 values were determined by nonlinear regression analysis using Prism GraphPad 6.0 fitting experimental data to the following equation:The ICiv/v0, represents the ratio between the activity measured in the presence of the inhibitor and the activity measured in the absence of inhibitor; Max and Min represent the expected maximal and minimal value of the activity and were fixed at 1 and zero, respectively. Slope, which describes the steepness of curve in the transition region, was fixed at −1. For each compound, at least five different concentrations of inhibitor in the triplicate assay were analyzed.In the equation, 4a, 4e and 4f was performed by measuring reaction rates with different L-idose concentrations s (S) in the absence and in the presence of different inhibitors concentrations. Data were analyzed by Linewever-Burk plots. The apparent dissociation constants i’K (for the ESI complex) and iK (for the EI complex) were determined from secondary plots of 1/maxV and KM/maxV as a function of the inhibitor concentration, respectively.The kinetic analysis of compounds 4a, 4e and 4f. In these conditions less than 15% of the enzyme activity was measured in the presence of 2 mM L-idose as substrate. The mixture was then extensively dialysed on Amicon ultrafiltration membrane (cut off 10 KDa) against 10 mM sodium phosphate buffer, pH 7.0. After dialysis, the enzyme activity was again measured as above and compared to that of a mixture in the absence of inhibitor and treated in the same conditions. In order to evaluate the reversibility of the inhibitory action, 8 mU of purified AR were assayed in the presence of 5 μM of compounds pNPP) as substrate. After an appropriate interval time, the reaction was stopped diluting assay solution with 2 mL of KOH 0.1 M. The amount of p-nitrophenol released was determined measuring the absorbance of the solution at 400 nm using a spectrophotometer and a 1-cm optical pathlength (εmM of p-nitrophenol is 18).PTP1B activity was determined as follow. An aliquot of human recombinant PTP1B was diluted in the assay buffer containing 0.075 M β-β-dimethylglutarate pH 7.0, 1 mM EDTA, 0.1 mM DTT and p-nitrophenyl phosphate , shows in the The ICpNPP. After 15 min, the reaction was stopped by adding 1 mL of KOH 0.2 M. The amount of pNP released was calculating measuring the absorbance at 400 nm of solutions using a spectrophotometer. Data obtained were normalized respect to control tests, which was carried out diluting the enzyme with DMSO, the solvent used to dissolve all compounds. Data reported in the figure represent the mean value +/− S.E.M. (n = 3).An aliquot of the enzyme was incubated for 60 min at 37 °C in the presence of saturating amount of PTP1B inhibitor. After this time, an aliquot of the enzyme was withdrawn and diluted 400 folds in the assay buffer containing 2.5 mM of 4e and 4f. Briefly, the main kinetic parameters, Km and Vmax, were calculated measuring the initial hydrolysis rate of PTP1B in the presence of increasing pNPP concentration (seven different concentrations in the 0.5–25 mM range). All assays were carried out in duplicate. Data obtained were fitted using the Michaelis-Menten equation. To evaluate the action mechanism of inhibitors, we studied the dependence of Km and Vmax from inhibitors concentration, using three different inhibitor concentrations, at least. Data obtained were then analysed by double reciprocal plot (Lineweaver-Burk plot). Kinetic analyses were carried out to dissect action mechanism of compound 2 at 37 °C. Differentiation of C2C12 was induced incubating murine myoblasts in DMEM supplemented with 2% horse serum. C2C12 cells were plated and grown until they reach 70% confluence. Then, cells were incubated in the presence of 2% horse serum for 96 h. Every 48 h, the medium was replaced with fresh medium. After 96 h, the degree of differentiation was evaluated analysing cells with a contrast phase microscope.Human liver cells (HepG2) and murine myoblasts (C2C12) were purchased by Sigma-Aldrich, which obtained them from the European Collection of Authenticated Cell Cultures (ECACC). HepG2 and C2C12 cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 2 mM glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin , in a humidified atmosphere and with 5% CO4e and 4f for 48 h at 37 °C. After this time, cells were washed with PBS and then incubated for 1 h with 0.5 mg/mL of MTT dissolved in complete medium. Then cells were washed and lysed with 300 μL of DMSO. The absorbance of solutions were evaluated using a microplate readers at 595 nm. All data were normalized respect to control experiment. Data showed in the figures represent the mean values ± S.E.M. (n = 4).HepG2 and C2C12 cells were incubated in the presence of increasing concentrations of compounds 4f  for 90 min. After this time, cells were washed with PBS and then stimulated or not with 10 nM insulin for 30 min. Then, cells were lysed and cellular proteins separated on a 12% SDS-PAGE. After electrophoresis, cellular proteins were transferred on a PVDF membrane by western blot technique and then probed against antibodies able to specifically recognize insulin receptor (I2033 Sigma Aldrich) or phosphorylated form of IR . Sample loading was evaluated probing PVDF membrane with anti-actin antibodies (ABT1485 Sigma Aldrich). HepG2 and differentiated C2C12 cells were starved for 24 h and then incubated in the presence of compound Crystal structure selection and preparation for PTP1B (PDB 1Q6T ) and theShape-based virtual screening was performed with ROCS   using de4a, 4e and 4f as well as 5a and 5d as dual AR/PTP1B inhibitors provided further insights into the possibility to develop 4-thiazolidinone derivatives as new potential multitarget antidiabetic candidates. Taking into account that the partial inhibition of selected targets, in particular enzymes, may be therapeutically more effective than the total control of a single target [1e and 2e, appear to be promising as lead compounds, whose inhibitory activity towards both target enzymes might be further improved. In particular, compound 4f exhibited the most interesting profile, with balanced AR/PTP1B inhibitory effectiveness at low micromolar concentrations, along with promising insulin-sensitizing activity in cultured cells. The IC50 values of 4f against both human PTP1B and human AR resulted in being in the low micromolar range, whereas those of the corresponding acetic acid analogue of series 2 differ of two orders of magnitude [The appreciable in vitro activity of compounds e target ,3, these4a, 4e and 4f as well as 2-butenoic acid derivatives 5a and 5d exhibited good capability to inhibit both human PTP1B and AR. The data reported here allowed us to extend the SARs of this class of DMLs, which were rationalized by in silico docking experiments into both target enzymes. Therefore, these findings can be of interest for the design of new 4-thiazolidinone derivatives endowed with improved pharmacological profiles. The SARs that emerged from our enzyme inhibition data, also considering our previous findings, highlighted that the AR inhibitory potency of the investigated 4-thiazolidinones is mainly influenced by the nature of the substituents in positions 2 and 3 of the heterocyclic scaffold and only in a lesser extent by the nature of the 5-arylidene moiety. On the other hand, this latter structural portion appeared to be critical to achieving good PTP1B inhibition, considering that a more extended 5-arylidene portion, in particular a (2-phenylethoxy)benzylidene moiety, showed to be more beneficial. In addition, a thiocarbonyl group in position 2 of the thiazolidinone scaffold generally proved to be advantageous to achieve gain in potency towards both target enzymes. The elongation of the carboxylic chain on N-3 in several cases improved inhibitory effectiveness towards PTP1B; propanoic acid derivatives


Dictamnus dasycarpus (Cortex Dictamni) were reported to be rich in anti-inflammation activity constituents, limonoids. Then, the investigation of anti-inflammation therapeutic limonoids from this plant was developed in the present study. Through the combination of various chromatographies isolation, six new limonoids, named dictamlimonol A (1), dictamlimonoside B (2), and dictamlimonols C–F (3–6), along with seven known ones (7–13), were obtained. Their structures were ascertained based on the extensive spectroscopic methods and ECD data analysis. Among them, compound 1 was the first 7,19-epoxy limonoid found in natural products. The anti-inflammatory effects of all limonoids were evaluated in lipopolysaccharide (LPS)-treated RAW 264.7 cell lines. Compounds 5, 7–11, and 13 were found to inhibit LPS-induced nitric oxide (NO) production. Moreover, dictamlimonol D (5), fraxinellone (11), and dasylactone A (13) were found to reduce the LPS-induced expressions of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), inducible nitric oxide synthase (iNOS), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and cyclooxygenase-2 (COX-2) at the protein levels in a dose-dependent manner. These findings support that the administration of Cortex Dictamni may be beneficial for inflammation.The root barks of perennial herb  Limonoids are highly oxygenated tetranorterpenoids derived from a precursor with a 4,4,8-trimethyl-17-furanyl steroid skeleton  are main resources of limonoids. The limonoids obtained from them could be subdivided as limonoid aglycones, degraded limonoids, and limonoid glucosides  have been used to treat inflammation, scabies, rheumatic pain, jaundice, and other symptoms  production in RAW 264.7 cell lines induced by lipopolysaccharide (LPS) were evaluated. Furthermore, the anti-inflammatory mechanism of activity compounds was studied by using Western blot assay.This study aims to clarify anti-inflammation limonoids in Cortex Dictamni was extracted by 70% ethanol–water and then partitioned in EtOAc-H2O to afford EtOAc and H2O layer extract, respectively. The H2O layer extract was eluted with H2O and 95% EtOH, successively. After subjecting to D101 macroporous resin column chromatography (CC), the silica gel, ODS, Sephadex LH-20 CC, and preparative high-performance liquid chromatography (pHPLC) were used to isolate the 95% EtOH eluate from D101 macroporous resin CC. As a result, six new limonoids, dictamlimonol A (1), dictamlimonoside B (2), and dictamlimonols C–F (3–6), as well as seven known ones, limonin (7)        Guo, , limonin1) was obtained as an optically active [[α]D25 −100.0, MeOH] white powder. The molecular formula, C26H30O9, of 1 was established by negative-ion ESI-Q-Orbitrap MS analysis [m/z 485.17969 [M–H]−; calcd for C26H29O9, 485.18061]. Its 1H, 13C NMR (5D5N), 1H 1H COZY, HSQC, and HMBC spectra indicated the presence of one furan ring at δH 6.60 , 7.68 , 7.77 , and four methyl groups at δH 0.91, 1.24, 1.40, 1.48 . According to the chemical shifts and the correlations observed in its 1H 1H COZY spectrum, “–O–CH–CH2-,” “–CH2-CH2-,” and “–CH=CH–O–” moieties were deduced. The planar structure of it was determined based on the key HMBC correlations from H-1 to C-3–5; H2-2 to C-3, C-10; H-5 to C-6; H-7 to C-6, C-9, C-14; H-15 to C-16; H-17 to C-14, C-20–22; H3-18 to C-12–14, C-17; H2-19 to C-1, C-5, C-7, C-10; H3-28 to C-4, C-5, C-29; H3-29 to C-4, C-5, C-28; H3-30 to C-7–9, C-14, which was a derivative of limonin (7). The relative configurations of C-13 and C-17 of 1 should be the same as those of 7 on the basis of biogenetic consideration, which indicated both 13-methyl and 17-furan ring presented α-orientations. Meanwhile, the NOE correlations between δH 1.40 (H3-18) and δH 2.89 (H-9), 4.72 (H-15); δH 2.89 (H-9); and δH 1.24 (H3-28), 4.61 (H-1), 4.72 (H-7); δH 1.24 (H3-28); and δH 4.61 (H-1) observed in its NOESY spectrum suggested that H-1, H-7, H-9, H-15, and H3-28 were α-oriented; on the other hand, δH 1.48 (H3-29) and δH 3.40 (H-5); δH 3.40 (H-5) and δH 4.62 (Hb-19); and δH 4.62 (Hb-19) and δH 0.91 (H3-30)  indicated that H-5, H2-19, H3-29, and H3-30 were β-oriented. Finally, the absolute configuration of 1 was clarified by comparison of experimental and calculated ECD data, which was recorded at the B3LYP/SVP level with the CPCM model  was consistent with the experimental data closely. The result indicated that the absolute configuration of 1 was 1S,5S,7R,8S,9R,10R,13S,14R,15S,17S. Thus, its structure was finally established and named dictamlimonol A. It was the first 7,19-epoxy limonoid found in natural products.Dictamlimonol A  was a white powder with negative optical rotation [[α]D25 −72.1, MeOH]. Its molecular formula was determined to be C20H26O9 [m/z 455.15375 [M + COOH]−, calcd for C21H27O11, 455.15479] by negative-ion ESI-Q-Orbitrap MS. d-glucose was detected from its acid hydrolysis product by HPLC analysis  spectra showed signals of two methyl groups at δH 0.84, 2.25 , two methylene groups at δH , , two oxygenated methylene groups at δH 4.11 , 4.97 , one furan ring at δH 6.44 , 7.55 , one β-d-glucopyranosyl at δH 4.53 , together with one α, β-unsaturated ketone group at δC 131.9 (C-14), 147.1 (C-8), 171.8 (C-16). The 1H 1H COZY spectrum of 2 suggested the presence of three partial structures written in bold lines  (H 0.77 (H3-18) and δH 6.52 (H-22), 7.72 (H-21), as well as biogenetic law suggested that 13-methyl and 17-furan ring were α-oriented. The correlation between δH 0.77 (H3-18) and δH 1.80 (H-11α) indicated that both 13-methyl and the proton with signal at δH 1.80 were in axial bond. Moreover, the coupling constant of H-9 was about 4 Hz, which suggested that the proton was presented in equatorial bond (one α-H in compound 2). Furthermore, the aglycone of it, 9β-hydroxyfraxinellone (12), was obtained when it was hydrolyzed with β-glucosidase. The calculated ECD spectrum of 2   between rum of 2  matched 3) was isolated as white powder with negative optical rotation . The molecular formula, C16H20O5, of 3 was established by negative-ion ESI-Q-Orbitrap MS [m/z 337.12790 [M + COOH]−, calcd for C17H21O7, 337.12818]. Comparing its 1H and 13C NMR spectroscopic data (3) with those of 2 indicated that they possessed the same substituent groups . At the same time, there were one more oxygenated methine proton at δH 4.90  and one more methoxy at δH 3.41  in 3. The long-range correlations from H-9 to C-8, C-14; H-15 to C-8, C-13, C-14, C-16; H-17 to C-14, C-20–22; H3-18 to C-12–14, C-17; H3-30 to C-8, C-9, C-14; 9-OCH3 to C-9 were observed in its HMBC spectrum, and its planar structure was elucidated. Moreover, the NOE correlations (H 0.95 (H3-18) and δH 4.90 (H-15), 6.40 (H-22), 7.45 (H-21) revealed that 13-methyl, 17-furan ring, as well as H-15 presented α-orientation, and the correlations between δH 0.95 (H3-18) and δH 1.73 (H-11α); δH 1.98 (H-11β); and δH 3.41 (9-OCH3), along with the coupling constant of H-9 (ca. J = 4 Hz), suggested that 9-OCH3 presented β-orientation. The calculated ECD spectrum of 3  was elucidated.Dictamlimonol C  was a white powder with negative optical rotation [[α]D25 −68.0, MeOH]. Its molecular formula, C17H22O5, was determined by its quasi-molecular ion peak at m/z 351.14261 [M + COOH]−  in the negative ESI-Q-Orbitrap MS experiment, which was 14 amu greater than that of 3. The 1H and 13C NMR (3) signals of 4 were similar to those of 3 except for C-9. The 9-methoxy in the structure of 3 has changed into an ethoxy in 4 at δH 1.25 , 3.47, 3.70 , which was consistent with the mass spectrometry data as well. In addition, in its HMBC spectra, the long-range correlation from H2-1' to C-9 was observed, which verified that the ethoxyl connected to the C-9 position. Meanwhile, the optical rotation, NOE correaltions  was deduced.Dictamlimonol D  was a white powder, too. It also had negative optical rotation [[α]D25 −9.1, MeOH]. The ESI-Q-Orbitrap MS experiment results indicated that the molecular fomula of 5 was the same as that of 3, C16H20O5 . The same planar structure of 5 and 3 was revealed by the 1H, 13C NMR (3), 1H 1H COZY, HSQC, and HMBC spectra. Meanwhile, the chemical shifts of C-9, 11, 12, 14, and 30 were different between these two compounds, which suggested they might be the C-9 epimers. In the NOESY spectrum of 5, NOE correlations (H 1.14 (H3-18) and δH 1.76 (H-11α), 4.98 (H-15), 6.41 (H-22), δH 1.47 (H-12β) and δH 3.79 (H-9), 5.44 (H-17). As a result, the relative configuration of 9-OCH3 was deduced to be α. Finally, the absolute configuration of dictamlimonol E (5) was determined to be 9R,13R,15S,17R by ECD spectra  was a white powder with negative optical rotation [[α]D25 −18.5, MeOH]. Its molecular formula, C15H18O5, was deduced by the ion peak at m/z 323.11221 [M + COOH]− . The aglycone of 6 was suggested to be similar to those of 3–5 by the comparison of their 1H, 13C NMR spectra. The differences were as follows: the substituent of C-9 disappeared; Δ8,14 had changed into Δ8,9; and there was one more oxygenated quaternary carbon at δC 75.4 (C-14) in 6. The presence of “–C=CH–CH2-CH2-” and “–CH=CH–O–” moieties was determined by the observation of its 1H 1H COZY spectrum. Moreover, the long-range correlations from H-9 to C-8, C-14; H-15 to C-13, C-14, C-16; H-17 to C-16, C-20–22; H3-18 to C-12–14, C-17; H3-30 to C-8, C-9, C-14 were observed in the HMBC spectrum. As a result, the planar structure of 6 was deduced. The NOE correlations . According to the relative stability, the relative configuration of 6 was speculated. The absolute configuration, 13S,14S,15S,17S, was clarified by comparing the experimental ECD spectrum (Dictamlimonol F (elations  between spectrum  with tha7–13) were determined.Meanwhile, comparing the NMR data reported in the literatures, the structures of seven known compounds . Although the specimen was limited, carbonyl substitution at position 23 showed enhanced activity (13 > 11). In compounds 3–6, α type alkoxy group substitution showed stronger inhibitory effects on NO production than β type (5 > 3 and 4).The structure–activity relationships on NO production in LPS-stimulated RAW 264.7 cells were summarized as follows: In compounds 5, 11, and 13 by determining the levels of these five proteins in LPS-stimulated RAW 264.7 cells.LPS can stimulate the acute inflammatory response by increasing expression of tumor necrosis factor (TNF-α) and interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and cyclooxygenase-2 (COX-2), successively  had been found to inhibit LPS-induced NO production and reduce the LPS-induced expressions of iNOS and COX-2 at the mRNA and protein levels in a dose-dependent manner by regulating NF-κB in RAW 264.7 macrophage-like cells , as well as the known compound, dasylactone A (13), in inflammation were characterized here firstly, which would provide new candidate drugs for inflammation-related diseases.Among the anti-inflammatory active compounds, fraxinellone (11) is the main constituent in Cortex Dictamni, and the content of it is about 0.15% in Cortex Dictamni, we can predict that the anti-inflammatory activity of the plant is mainly derived from limonoids, especially fraxinellone. Further mechanism and clinical studies for it is necessary.Since fraxinellone . IR spectra were recorded on a Varian 640-IR FT-IR spectrophotometer . Negative-ion mode ESI-Q-Orbitrap-MS was obtained on a Thermo ESI-Q-Orbitrap MS mass spectrometer connected with the UltiMate 3000 UHPLC instrument via ESI interface .Optical rotations were measured on a Rudolph Autopol® IV automatic polarimeter (l = 50 mm) . NMR spectra were determined on a Bruker 500-MHz NMR spectrometer  at 500 MHz for 18-MS-II , and Cosmosil PBr  were used to separate the constituents.CC was performed on macroporous resin D101 , silica gel , and ODS . pHPLC columns, Cosmosil 5CCortex Dictamni was purchased from the medicine market in Anguo city, Heibei province, China, and identified by Dr. Li Tianxiang . The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM.Cortex Dictamni (9.0 kg) was refluxed with 70% EtOH-H2O. The 70% EtOH extract (1725.3 g) was partitioned in an EtOAc-H2O mixture . The H2O layer (1253.8 g) was subjected to D101 macroporous resin CC (H2O → 95% EtOH). Then, H2O (1032.9 g) and 95% EtOH (123.4 g) eluates were obtained.3 → CHCl3-MeOH  → CHCl3-MeOH-H2O  → MeOH], and 12 fractions (Fr. 1–Fr. 12) were afforded. Fraction 7 (4.0 g) was isolated by ODS CC [MeOH-H2O ] to give nine fractions (Fr. 7-1–Fr. 7-9). Fraction 7-6 (923.7 mg) was separated by Sephadex LH-20 CC (MeOH), and three fractions (Fr. 7-6-1–Fr. 7-6-3) were obtained. Fraction 7-6-2 (526.5 mg) was purified by pHPLC  to gain dictamlimonoside B .The 95% EtOH eluate (90.0 g) was subjected to silica gel CC , and nine fractions (Fr. E-7-1–Fr. E-7-9) were obtained. Fraction E-7-2 (278.5 mg) was purified by pHPLC  to afford dictamlimonol D . Fraction E-8 (37.0 g) was centrifuged after dissolving in hexane, and two fractions (Fr. E-8-1–Fr. E-8-2) were gained. Fraction E-8-1 (70.0 mg) was isolated by pHPLC  to obtain dictamlimonol A  and limonin . Fraction E-8-2 (35.16 g) was subjected to silica gel CC [PE-EtOAc ], and nine fractions (Fr. E-8-2-1–Fr. 8-2-9) were afforded. Fraction E-8-2-5 (4.4 g) was separated by pHPLC  to provide nine fractions (Fr. E-8-2-5-1–Fr. E-8-2-5-9). Fraction E-8-2-5-1 (1396.6 mg) was subjected to pHPLC , and eight fractions (Fr. E-8-2-5-1-1–Fr. E-8-2-5-1-8) were given. Fraction E-8-2-5-1-3 (190.9 mg) was purified by pHPLC  to afford dictamlimonol F  and 9β-hydroxyfraxinellone . Fraction E-8-2-5-1-4 (81.0 mg) was isolated by pHPLC  to gain dictamlimonol E . Fraction E-8-2-5-1-6 (81.2 mg) was further subjected to pHPLC , and dictamlimonol C  was afforded. Fraction E-8-2-6 (7.1 g) was separated by silica gel CC [PE-EtOAc  → MeOH] to afford eight fractions (Fr. E-8-2-6-1–Fr. E-8-2-6-8). Among them, fraction E-8-2-6-8 was identified as obacunon . Fraction E-8-2-6-4 (3483.7 mg) was isolated by pHPLC , and 10 fractions (Fr. E-8-2-6-4-1–Fr. E-8-2-6-4-10) were obtained. Fraction E-8-2-6-4-1 (778.2 mg) was subjected to pHPLC  to provide six fractions (Fr. E-8-2-6-4-1-1–Fr. E-8-2-6-4-1-6). Fraction E-8-2-6-4-1-6 (70.5 mg) was purified by pHPLC  to gain dasylactone A . Fraction E-8-2-6-4-2 (693.5 mg) was isolated by pHPLC  to afford five fractions (Fr. E-8-2-6-4-2-1–Fr. E-8-2-6-4-2-5). Fraction E-8-2-6-4-2-5 (50.8 mg) was further purified by pHPLC , and 7α-obacunyl acetate  was obtained. Fraction E-8-2-7 (3.1 g) was separated by pHPLC  to obtain seven fractions (Fr. E-8-2-7-1–Fr. E-8-2-7-7). Fraction E-8-2-7-3 (80.6 mg) was purified by pHPLC  to gain limonin diosphenol .The EtOAc layer  was isolated by silica gel CC [hexane-EtOAc  → MeOH] to afford 10 fractions (Fr. E-1–Fr. E-10). Fraction E-4 (25.2 g) was separated by silica gel CC [hexane-EtOAc ] to gain fraxinellone (1). White powder; [α]D25 −100.0 ; UV λmax (MeOH) nm (log ε): 204 (4.05), 280 (3.35); CD  mdeg (λnm): −18.3 (223), −14.8 (223), −15.3 (228), +2.7 (271), −4.1 (307); IR νmax (KBr) cm−1: 3,515, 3,243, 2,985, 2,951, 1,746, 1,708, 1,652, 1,458, 1,390, 1,368, 1,281, 1,220, 1,176, 1,131, 1,105, 1,058, 1,023, 960, 898, 884, 828, 796; 1H NMR , 13C NMR ; see m/z 485.17969 [M – H]−  (Dictamlimonol A (5.18061) .2). White powder; [α]D25 −72.1 ; UV λmax (MeOH) nm (log ε): 213 (4.20); CD  mdeg (λnm): −17.5 (204), −12.4 (216), −21.8 (231), +13.0 (259); IR νmax (KBr) cm−1: 3,359, 2,931, 2,876, 1,755, 1,678, 1,504, 1,448, 1,406, 1,378, 1,344, 1,289, 1,226, 1,209, 1,161, 1,077, 1,048; 1H NMR , 13C NMR ; see m/z 455.15375 [M + COOH]−  (Dictamlimonoside B (5.15479) .3). White powder; [α]D25 −74.6 ; UV λmax (MeOH) nm (log ε): 206 (4.10); CD  mdeg (λnm): −15.4 (200), +4.9 (222), −4.7 (240), +1.8 (268), −0.2 (355); IR νmax (KBr) cm−1: 3,445, 3,152, 2,973, 2,941, 2,863, 2,825, 1,748, 1,653, 1,505, 1,458, 1,381, 1,350, 1,327, 1,278, 1,192, 1,162, 1,133, 1,109, 1,070, 1,025, 994, 907, 874, 800, 764, 732; 1H NMR , 13C NMR ; see m/z 337.12790 [M + COOH]−  (Dictamlimonol C (7.12818) .4). White powder; [α]D25 −68.0 ; UV λmax (MeOH) nm (log ε): 206 (4.10), 239 ; CD  mdeg (λnm): −11.3 (201), +0.8 (223), −1.1 (240), +0.8 (262), −0.2 (350); IR νmax (KBr) cm−1: 3,383, 3,147, 2,973, 2,941, 2,869, 1,750, 1,653, 1,506, 1,457, 1,380, 1,345, 1,325, 1,278, 1,265, 1,198, 1,162, 1,064, 1,024, 1,001, 911, 875, 800, 765, 731; 1H NMR , 13C NMR ; see m/z 351.14261 [M + COOH]−  (Dictamlimonol D (1.14383) .5). White powder; [α]D25 −9.1 ; UV λmax (MeOH) nm (log ε): 208 (3.94), 253 ; CD  mdeg (λnm): −17.3 (195), +11.3 (224), +4.1 (251), −1.1 (347); IR νmax (KBr) cm−1: 3,481, 3,148, 2,943, 2,876, 2,823, 1,749, 1,653, 1,506, 1,456, 1,380, 1,353, 1,282, 1,222, 1,163, 1,093, 1,023, 996, 911, 877, 802, 765, 731; 1H NMR , 13C NMR ; see m/z 337.12818 [M + COOH]−  (Dictamlimonol E (7.12818) .6). White powder; [α]D25 −18.5 ; UV λmax (MeOH) nm (log ε): 203 (4.01), 249 ; CD  mdeg (λnm): +23.9 (207), −21.4 (234), +1.7 (266); IR νmax (KBr) cm−1: 3,447, 3,149, 2,972, 2,944, 2,889, 1,745, 1,668, 1,550, 1,504, 1,450, 1,384, 1,305, 1,278, 1,160, 1,143, 1,023, 992, 970, 875, 806, 771, 758, 719; 1H NMR , 13C NMR ; see m/z 323.11221 [M + COOH]−  (Dictamlimonol F (3.11253) .2 (10.0 mg) was hydrolyzed with β-glucosidase (10.0 mg) in H2O (1.0 ml) at 37°C for 5 h. After cooling, the reaction mixture was partitioned with EtOAc. The EtOAc layer was separated by silica gel CC [Hexane-EtOAc ] to yield 9β-hydroxyfraxinellone .Compound d-glucose  from 2 was identified by comparison of its retention time and optical rotation with that of the authentic sample.Acid hydrolysis reaction was performed by using a similar method to our previously reported one  for 18 h. Each culture medium (50 μl) was mixed with an equal volume of Griess reagent after incubation. An ELISA plate reader was used to determine the nitrite level (a major stable product of NO) at 540 nm, and the concentrations were calculated by referring to a NaNO2 standard calibration curve.After pretreating compounds 5, 11, and 13 by determining the levels of these five proteins  in LPS-stimulated RAW 264.7 cells as previously reported  and Tukey's Studentized range test were used for the evaluation of the significant differences between means and post hoc, respectively.Values were statistically analyzed by using SPSS 17.0 software. 1), dictamlimonoside B (2), and dictamlimonols C–F (3–6), along with 7 known ones (7–13) were isolated from Cortex Dictamni by various chromatographies and identified by spectroscopies and chemical reactions. Among them, compound 1 was a first 7,19-epoxy limonoid found in natural products. Activity evaluation research showed several kinds of limonoids reduce expression of TNF-α, IL-6, iNOS, NF-κB, and COX-2 in LPS-stimulated RAW 264.7 cells. These findings support the idea that the administration of Cortex Dictamni may be beneficial for inflammation.In this study, 13 limonoids including 6 new ones, named dictamlimonol A (The datasets generated for this study are available on request to the corresponding author.YiZ and TW designed the research and wrote the manuscript. JR and YC performed the activity research. FS, SY, YinZ, and JY contributed to the isolation, purification, and characterization of all compounds. HW and YG performed the ECD calculation. HY perfected the language. All authors discussed, edited, and approved the final 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.


O-glucosyl-rhamnosyl-glucoside, quercetin-3-O-rhamnosyl-rhamnosyl-glucoside, kaempferol-3-O-glucosyl-rhamnosyl-glucoside and kaempferol-O-rhamnosyl-rhamnosyl-glucoside. The purities of all isolated flavonol triglycosides were above 95% based on HPLC, and the production yield of total flavonol glycosides from dry tea was 0.487%. Our study provides a preparation method of flavonol triglycosides from tea leaves, with relatively low cost of time and solvent but high production yield.Flavonol glycosides are important components of tea leaves, contributing to the bioactivities as well as bitterness and astringency of tea. However, the standards of many flavonol triglycosides are still not available, which restricts both sensory and bioactivity studies on flavonol glycosides. In the present study, we established a simultaneous preparation method of seven flavonol triglycoside individuals from tea leaves, which consisted of two steps: polyamide column enrichment and preparative HPLC isolation. The structures of seven flavonol triglycoside isolates were identified by mass and UV absorption spectra, four of which were further characterized by nuclear magnetic resonance spectra, namely, quercetin-3- O-glycosylation can enhance certain types of biological benefits such as anti-HIV activity, antirotavirus activity, antistress activity, antiobesity activity, and antiallergic activity − of quercetin and kaempferol, respectively, and the decrements of 162 m/z, 146 m/z and 308 m/z between fragments were attributed to the losses of the moieties of hexose, rhamnose and the disaccharide of these two monosaccharides. The MS and MS/MS fragmentation data, as well as the UV λmax of isolated compounds, verified the basic molecular structures of flavonol triglycosides.In our previous studies ,14, we eycosides ,19,20,21ycosides  was used1H-NMR and 13C-NMR measurements. The 1H- and 13C-NMR data were shown as follows:In order to further validate the molecular and structural characterization, the fractions corresponding to the big Peaks 2, 4, 6, 7 were collected and freeze dried for 1H-NMR  δ 7.69 , 7.63 , 6.88 , 6.42 , 6.22 , 5.09 , 4.56 , 4.43 , 3.94 , 3.81 , 3.76 , 3.73 , 3.64 , 3.52 , 3.48 , 3.47 , 3.44 , 3.43 , 3.41 , 3.38 , 3.34 , 3.28 , 3.25 , 1.11 . 13C NMR  δ 179.39 [C-4], 166.06 [C-7], 162.97 [C-5], 159.38 [C-2], 158.57 [C-9], 149.78 [C-4′], 145.81 [C-3′], 135.70 [C-3], 123.52 [C-6′], 123.10 [C-1′], 117.79 [C-2′], 116.06[C-5′], 105.62 , 104.91 [C-10], 102.35 [C-1′′′], 99.98 [C-6], 94.97 [C-8], 83.09 [C-3′′′], 78.20 [C-5′′], 77.58 , 77.19 [C-3′′], 75.72 [C-2′′], 75.46 [C-2′′′′], 72.59 , 71.37 [C-2′′′], 70.85 [C-4′′′′], 69.38 [C-5′′′], 68.81 [C-6′′], 62.05 [C-6′′′′], 17.94 [C-6′′′].Peak 2: Q-glu-rha-glu: 1H-NMR  δ 7.69 , 7.64 , 6.89 , 6.40 , 6.22 , 5.09 , 4.98 , 4.51 , 3.95 , 3.81 , 3.74 , 3.72 , 3.68 , 3.63 , 3.52 , 3.47 , 3.43 , 3.41 , 3.37 , 3.34 , 3.34 , 3.27 , 1.17 , 1.13 . 13C NMR  δ 179.43 [C-4], 166.28 [C-7], 163.01 [C-5], 159.38 [C-2], 158.58 [C-9], 149.84 [C-4′], 145.81 [C-3′], 135.78 [C-3], 123.56 [C-6′], 123.12 [C-1′], 117.79 [C-2′], 116.10 [C-5′], 105.62 [C-10], 105.02 [C-1′′], 103.87 [C-1′′′′], 102.46 [C-1′’’], 100.09 [C-6], 95.01 [C-8], 79.56 [C-3′′′], 78.24 [C-5′′], 77.21 [C-3′′], 75.77 [C-2′′], 74.13 [C-4′′′′], 73.26 [C-2′′′′], 72.19 , 71.94 [C-3′′′′], 71.42 [C-2′′′], 69.99 , 68.73 [C-6′′], 17.93 .Peak 4: Q-glu-rha-rha: 1H-NMR  δ 8.07 , 6.90 , 6.42 , 6.22 , 5.11 , 4.55 , 4.39 , 3.93 , 3.81 , 3.73 , 3.64 , 3.57 , 3.51 , 3.48 , 3.47 , 3.44 , 3.43 , 3.41 , 3.38 , 3.34 , 3.28 , 3.25 , 1.11 . 13C NMR  δ 179.42 [C-4], 166.18 [C-7], 163.01 [C-5], 161.50 [C-4′], 159.48 [C-2], 158.64 [C-9], 135.59 [C-3], 132.46 , 122.78 [C-1′], 116.17 , 105.67 , 104.72 [C-10], 102.33 [C-1′′′], 100.06 [C-6], 95.06 [C-8], 83.19 [C-3′′′], 78.19 [C-5′′], 77.58 , 77.15 [C-3′′], 75.77 [C-2′′], 75.46 [C-2′′′′], 72.57 [C-4′′′], 71.54 [C-4′′], 71.30 [C-2′′′], 70.85 [C-4′′′′], 69.43 [C-5′′′], 68.95 [C-6′′], 62.07 [C-6′′′′], 17.97 [C-6′′′].Peak 6: K-glu-rha-glu: 1H-NMR  δ 8.07 , 6.90 , 6.40 , 6.22 , 5.11 , 4.98 , 4.51 , 3.94 , 3.81 , 3.74 , 3.72 , 3.68 , 3.63 , 3.52 , 3.47 , 3.43 , 3.41 , 3.37 , 3.34 , 3.27 , 1.17 , 1.13 . 13C NMR  δ 179.40 [C-4], 166.13 [C-7], 162.99 [C-5], 161.48 [C-4′], 159.48 [C-2], 158.58 [C-9], 135.62 [C-3], 132.41 , 122.77 [C-1′], 116.15 , 105.66 [C-10], 104.77 [C-1′′], 103.87 [C-1′′′′], 102.43 [C-1′′′], 100.07 [C-6], 95.05 [C-8], 79.40 [C-3′′′], 78.16 [C-5′′], 77.20 [C-3′′], 75.78 [C-2′′], 74.10 [C-4′′′′], 73.22 [C-2′′′′], 72.18 , 71.92 [C-3′′′′], 71.51 [C-2′′′], 70.01 , 68.81 [C-6′′], 17.94 .Peak 7: K-glu-rha-rha: 1H- and 13C-NMR data of Q-glu-rha-glu, Q-glu-rha-rha, K-glu-rha-glu and K-glu-rha-rha were consistent with the reported data , which were higher than the chemical shift values of protons on sugar moieties. For instance, 5.09 ppm , 4.98 ppm  and 4.51 ppm  were corresponding to the protons at the glycosidic linkages of Q-glu-rha-rha, which were higher than those of protons on sugar moieties ranging from 3.95 ppm to 1.13 ppm. The relatively higher chemical shift values of anomeric protons are attributed to the deshielding effect. The 13C-NMR spectra of four isolated flavonol triglycosides were in general agreement with the references [The inset of ted data ,17,20. Fferences .O-glucoside , quercetin-3-O-glucoside , kaempferol-3-O-glucoside  were purchased from Chengdu DeSiTe Biological Technology Co., Ltd. . HPLC grade methanol and acetonitrile were purchased from Sigma-Aldrich . Polyamide (particle size 74–174 µm) and formic acid  were bought from Aladdin . The Ultrapure water was prepared by an EASYPure II UV Ultra Pure Water System .Green tea (dry leaves) was purchased from the local tea market , the chemical composition of which was shown in g and 4 °C for 5 min, the supernatant was collected and made up to 30 mL with water.Green tea was ground into powder and sieved through 100 µm. Five grams of green tea powder was extracted with 40 mL boiling water at 100 °C and 100 rpm for 30 min. After centrifugation at 2292× Polyamide was soaked in methanol for 24 h, and then was packed in a column (1 cm × 6.4 cm). The column was pre-equilibrated with 2% NaOH solution, 2% HCl solution and water prior to use.In order to achieve the maximum uploading volume of tea extract, different volumes of tea extract  were, respectively, uploaded onto polyamide column. The effluent was collected and submitted to UPLC–DAD–MS/MS analysis.v/v) → 20 mL of methanol; Method B: 20 mL of water → 20 mL of 15% methanol solution (v/v) → 20 mL of 30% methanol solution (v/v) → 20 mL of 45% methanol solution (v/v) → 20 mL of 70% methanol solution (v/v) → 20 mL of methanol. Flow rate was 5 mL/min. Each fraction was collected for chemical analyses.Tea extract (2.5 mL) was uploaded onto the polyamide column. Two different elution methods were used for preparing flavonol glycoside-enriched fraction: Method A: 20 mL of water → 20 mL of 60% methanol solution (Tea extract (2.5 mL) was uploaded onto the polyamide column and subsequently eluted by Method B at different flow rates of 2.5 mL/min, 5 mL/min and 8.00 mL/min. Each fraction was collected for chemical analyses.v/v/v), mobile phase B: water/acetonitrile/acetic acid = 30.0 : 0.5 : 69.5 (v/v/v), gradient elution: maintaining 55% A and 45% B during the first 35 min, and then linearly increasing to 35% A and 65% B till 55 min, and increasing to 100% B till 70 min, flow rate 2 mL/min. The fractions corresponding to Peaks 1–7 were collected for chemical analyses.The flavonol glycoside-enriched fraction  was collected and concentrated by a rotary evaporator. The concentrate was reconstituted with 4 mL water and then filtered through a 0.22 µm membrane before injection into the preparative HPLC AKTA purifier UPC100 . The preparative HPLC conditions were: YMC-Pack ODS-A column , injection volume 0.5 mL, mobile phase A: acetonitrile/acetic acid/water = 3.0 : 0.5 : 96.5  according to the reported method [m/z to 1000 m/z. The MS conditions were set as follows: cone voltage 30 V, extractor voltage 3.0 V, capillary voltage 3 kV, ion source temperature 150 °C, desolvation temperature 350 °C, desolvation gas flow 400 L/h. External standards including M-glu, Q-glu, and K-glu were respectively used for quantifying myricetin glycosides, quercetin glycosides and kaempferol glycosides based on UPLC chromatogram.After centrifugation at 13,201× 18 (2) column , column temperature 28 °C, mobile phase A: acetonitrile/acetic acid/water = 3 : 0.5 : 96.5 (v/v/v), mobile phase B: acetonitrile/acetic acid/water = 30 : 0.5 : 69.5 (v/v/v), gradient elution: linearly increasing from 30% B to 85% B during the first 35 min, and then holding at 85% B for another 5 min, detection wavelength 280 nm.The concentrations of catechin compounds and alkaloids were analyzed by LC 20A HPLC  according to our previously published method . Briefly3OD for 1H- and 13C-NMR measurement. The spectra were recorded on a Bruker AVANCE III spectrometer at 500 MHz with a 5 mm DCH cryoprobe . Integration of the spectra was performed on Mestnova 9.0.1 .The fractions corresponding to Peaks 2, 4, 6, 7 on preparative HPLC were freeze dried for NMR measurement. Ten mg of the obtained freeze-dried compounds was suspended in 0.6 mL CDAll analyses were carried out in triplicate. The results of above tests were expressed as the mean value ± SD. Statistical significance was carried out on SAS 9.4 software , using Tukey test. The structures of compounds were drawn by ChemOffice 2010 v12 .The present study developed a two stepwise method for simultaneously preparing abundant flavonol triglycosides from tea leaves, comprising enrichment of flavonol glycosides by polyamide column and preparative HPLC isolation. Seven flavonol glycoside individuals were isolated and characterized based on MS, UV absorption and NMR spectra, namely, Q-gal-rha-glu, Q-glu-rha-glu, Q-gal-rha-rha, Q-glu-rha-rha, K-gal-rha-rha, K-glu-rha-glu and K-glu-rha-rha. The production yield of TFG was 0.487% in total.


Citrus junos and investigated their anti-inflammatory effects by assessing the expression of pro-inflammatory cytokines in lipopolysaccharide-stimulated RAW 264.7 mouse macrophages and HT-29 human colon epithelial cells. Our findings revealed that limonoids significantly downregulated the pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α, and nuclear transcription factor κB. In particular, sudachinoid-type compounds, methyl sudachinoid A and sudachinoid B, and ichangensin-type compound, 1-O-methyichangensin downregulated the expression of pro-inflammatory cytokines more potently than other limonoids, nomilin and limonin, which have been previously reported to exhibit anti-inflammatory activities in other cells; nomilin and limonin were therefore employed as positive controls in this study. Herein, we reveal that the anti-inflammatory activities of limonoids including a new compound methyl sudachinoid A from C. junos were mediated via the downregulation of pro-inflammatory cytokines and these limonoids can be employed as potential therapeutic phytochemicals for IBD.Limonoids, a dominant group of phytochemicals in the Rutaceae family, are known to exhibit several pharmacological activities. To identify natural products having efficacy against inflammatory bowel disease (IBD), we isolated 13 limonoids including a new compound, methyl sudachinoid A, from the seeds of  Inflammation is a biological response to stimuli, such as pathogen infection, which serves as a major obstacle in the maintenance of a high quality of life . InflammThe aberrant production of pro-inflammatory factors, such as chemokines e.g., IL-8), often results in chronic inflammation , often r, abrogatCitrus species from the Rutaceae family include coumarins, flavonoids, limonoids and carotenoids that have various pharmacologic activities , three methines attached to the oxygen proton signal , two methoxy groups [δH 3.43  and 3.18 ], two methine proton signals [δH 2.87  and 2.54 ], five methylene proton signals , and six methyl groups .Compound  extract . Based o13C-NMR spectrum of compound 1 contained resonances corresponding to three carbonyl carbon groups , one double bond that might be the conjugated enone [δc 148.7 (C-22) and 135.5 (C-20)], two acetal carbon signals [δc 108.1 (C-1) and 103.5 (C-23)], three oxygenated methane carbon signals , two methoxy groups [δc 56.0 (C-OCH3) and 48.2(C-OCH3)], and six methyl carbon signals . In the H-C multiple bond correlation (HMBC) spectrum, the correlation of δH 1.13 (H-18) with δc 69.3 (C-14) and 75.1 (C-17); δH 3.43 (OCH3) with δc 103.5 (C-23); δH 7.61 (H-22) with δc 103.5 (C-23), 135.5 (C-20), and 169.0 (C-21); δH 4.42 (H-15) with δc 166.9 (C-16); δH 1.19 (H-24) with δc 69.3 (C-14); δH 1.11 (H-19), 1.23 (H-2), and 3.18 (OCH3) with δc 108.1 (C-1); and δH 1.16 (H-25) and 1.03 (H-26) with δc 79.4 (C-4) confirmed the structure of compound 1. Further validation of the structure was achieved with the 1H-1H  correlation  spectroscopy  (COSY) correlations of δH 7.61 (H-22) with δH 6.08 (H-23) and δH 2.87 (H-9) with δH 1.71 (H-11)  . Based o2), ichangensin (3), 1-O-methyichangensin (4), nomilin (5), deacetylnomilin (6), methylnomilinate (7), methyldeacetylnomilinate (8), deacetylnomilinic acid-17-O-glucopyranoside (9), nomilinate A ring lactone (10), obacunone (11), limonin (12), and ichangin (13)   c 0.4 MeOH); 1H-NMR : δH 7.61, 6.08, 5.32, 4.42, 3.43, 3.18, 2.87, 2.74, 2.54, 2.20, 1.74, 1.71, 1.23, 1.23, 1.19, 1.16, 1.13, 1.11, 1.03; 13C-NMR : δC 208.4, 169.0, 166.9, 148.7, 135.5, 108.1, 103.5, 79.4, 75.1, 69.3, 56.0, 55.6, 52.5, 49.5, 48.9, 48.2, 40.5, 39.7, 36.6, 31.6, 26, 23.6, 18.9, 18.4, 17.6, 15.6, 14.5. High-resolution electrospray ionization mass spectrometry (HRESIMS) m/z 505.2444 [M+H]+ . The spectra are available as Methyl sudachinoid A  and cultured in Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% heat-inactivated fetal bovine serum (FBS) , 100 μg/mL streptomycin, and 100 IU/mL penicillin. The cells were incubated as monolayers for 72 h at 37 °C in a humidified environment containing 5% CO5 cells/well) and maintained in DMEM containing 10% FBS for 24 h. Then, cells were treated with different concentrations of limonoids for 1 h prior to stimulation with lipopolysaccharide  for 18 h. Cell viability was measured by an MTT assay, which involved the incubation of the cultured cells with MTT (0.05 mg/mL) at 37 °C for 4 h. After removal of the supernatants, the absorbance of the formazan solution was measured at 570 nm using a microplate reader.The cells were seeded in 96-well plates , IL-6 and TNF-α  levels in RAW 264.7 mouse macrophage cells, and the level of IL-8  in HT-29 cells using ELISA kits, as per the manufacturer’s protocol. Relative production (%) was calculated as ratio of the production levels of LPS treated and control group.RAW 264.7 and HT-29 cells were seeded in 96-well plates at a density of 1 × 106 cells/well in 6-well plates in culture medium for 24 h. Seeded cells were treated with compounds 1, 2, and 4 for 1 h, then stimulated with LPS (1 µg/mL). After 1 h, the cells were washed two times with cold phosphate-buffered saline (PBS) and whole cell lysates were extracted with protein extraction solution . The protein concentration was determined by the Bradford reagent and Western blot analysis was done as described previously [Mouse macrophage were seeded at the density of 1 × 10eviously . The prin = 3). Data analysis was carried out using one-way analysis of variance (ANOVA). p-values < 0.05 were considered to indicate significant differences.The data are presented as mean ± standard deviation (