A series of azastilbene derivatives, characterized by the presence of the 1,2,4-oxadiazole-5-one system as a linker of the two aromatic rings of stilbenes, have been prepared as novel potential inhibitors of p38 MAPK. Biological assays indicated that some of the synthesized compounds are endowed with good inhibitory activity towards the kinase. Molecular modeling data support the biological results showing that the designed compounds possess a reasonable binding mode in the ATP binding pocket of p38α kinase with a good binding affinity. The stilbene scaffold is a basic element for several biologically active natural and synthetic compounds which have demonstrated promising activity in the area of medicinal chemistry, targeting a wide variety of intracellular pathways ,4,5,6,7.cis double bond into an inactive form has been also achieved by the exploitation of heterocyclic rings in place of the ethene bridge [cis-locked analogs provide some interesting advantages such as the prevention of cis to trans isomerization, increased specificity since the trans conformation might be recognized by other cellular targets, and the presence of heterocyclic units that might improve the therapeutic potential of the synthesized potential drugs [A key structural factor for the biological activity is the presence of the double bond in a Z configuration: the olefinic bond forces the two aromatic rings to stay within an appropriate distance and gives the molecule the right dihedral angle to maximize the interaction with the target. The Z stilbene double bond can easily isomerize under the influence of heat, light, and protic media; therefore, many attempts have been addressed to modify the olefinic bridge in order to stabilize the configuration and to increase the biological effects of the compound ,23. In te bridge ,25,26. Tal drugs ,23.1)) [2)) [3)) [4)) [5)) [6)) [7)) [8)) [9)) [10)) [11a and 11b) [12)) [13)) [14)) and (15)) [16)) were prepared [Three-, four-, five-, and six-membered rings have been exploited to replace the olefinic moiety. Among the five-membered rings, 2-cyclopenten-1-ones (1)) , 2(5H)-f1)) [2)) , 1,3-oxa2)) [3))  and 4,5-3)) [4)) , diarylo4)) [5)) , furazan5)) [6)) , imizado6)) [7)) , pyrazol7)) [8)) , triazol8)) [9)) , 2,3-dih)) [10)) , 4,5-disand 11b) , and aryb) [12))  have bee)) [13)) , benzenend (15))  were alsnd (15)) ,38. Amonprepared  Figure ) [2)) [3cis-locked stilbene-based compounds with vicinal 4-fluorophenyl/4-pyridine rings were designed, synthesized, and evaluated as inhibitors of p38α mitogen-activated protein kinase (MAPK). The mitogen-activated protein kinases (MAPKs) are essential regulators for signal transduction pathways, involved in the control of cell functions including proliferation, gene expression, differentiation, mitosis, cell survival, and apoptosis [Further optimization of the structural diversity of stilbenes to increase their potency and biological activity has been explored. In this context, several poptosis . The p38poptosis  of severpoptosis ,43,44, bpoptosis , lung [4poptosis , cardiacpoptosis ,48, red poptosis , and fetpoptosis . Consequpoptosis ,53,54,551)) [2)) [3)) [4)) [5)) [6)) [Several selective stilbene-based p38 inhibitors, where the vicinal 4-fluorophenyl/pyridinyl motif is inserted into different ring scaffolds, have been developed, such as five-membered pyrazoles ((1)) ,57, isox1)) [2)) ,59, tria2)) [3)) ,61,62,633)) [4)) ,65, pyri4)) [5)) , and chr5)) [6)) . Many ofcis-restricted azastilbenes . Additional ligand-p38R interactions can be detected such as hydrogen bonding toward the core system from Lys53 and Tyr35 π-π stacking with the phenyl system. The binding mode of SB 203,580 shows that the ligand is not interfering with “hydrophobic region II” ,66. ThusAccording to these considerations, our design has been addressed to the construction of a new series of novel potential inhibitors of MAPK3, in which two vicinal aromatic systems at C-3 and N-2 are connected to a 1,2,4-oxadiazole-5-one system. The heterocyclic ring assures the required correct spatial assessment of two vicinal aryl groups, while the stabilizing hydrogen bond interactions to the hinge region and Lys53 of the enzyme pocket are mimed by the nitrogen atom of the pyridinyl unit at C-3 and by the oxygen atom of the carbonyl group, respectively . Moreove3a–l possess a reasonable binding mode in the ATP binding pocket of p38α kinase.The biological assays indicated that some of the synthesized compounds are endowed with a good inhibitory activity towards the p38α kinase. The synthesized 3,4-diaryl-1,2,4-oxadiazol-5-ones 3a–l exploits a recently reported route [1a–e to isocyanates as dipolarophiles. Isocyanates 2a–d were reacted at room temperature in dry acetone and in a nitrogen atmosphere with nitrones 1a–e, obtained by the reaction of the corresponding aldehydes with N-methyl or N-aryl hydroxylamine [3a–l in good yields  ed route  based onxylamine ,71,72,73d yields ; Table 11H-NMR spectra of the compounds show the presence of the diagnostic methine proton at C-3, whose resonance, as previously reported [3a–l. Furthermore, the methane 13C resonates as a doublet at 79–80 ppm, values lower than those of the 13C possible which are expected at higher fields (85–90 ppm).The structure of the obtained cycloadducts has been assigned on the basis of spectroscopic data . The 1H-reported , is in t2 from the molecular ion, is only amenable to cycloadducts 3a–l.As further support to the assigned structure, the presence in the MS spectra of the diagnostic peak at M-44, resulting from the loss of CO3a–l have been obtained as exclusive cycloadducts with no traces of the alternative cycloadducts , do not lead to the expected biological activity.The obtained results  highligh50 in the range of 0.10–5.1 µM. These values are perfectly in agreement with that obtained from the reference compound SB203580 (IC50 = 0.3 µM), thus highlighting the importance of a 4-fluorophenyl/pyridine motif in the molecular binding with the ATP pocket of the p38α kinase. In these compounds, the pyridine unit at C-3 in the oxazolidinone ring is able to exert the critical determinant hydrogen-bonding interaction in the hinge region of the enzyme. The most active compounds, 3e (0.08 mM) and 3f (1.1 mM), are characterized, besides the presence of the 4-fluoro-phenyl/pyridinyl motif, by an additional hydrophobic unit at N-2: the comparison with compounds 3l, which possesses a Me moiety , indicating that a consistent lipophilic interaction to HRII region of the enzyme could be the reason for the improved binding affinity of these derivatives. The substitution of the fluorine atom of 3e with a nitro group, CF3 or Cl, did not lead to anincrease of the inhibition of p38α for compounds 3a, 3c, and 3d.Compounds witha 4-fluoro-phenyl/pyridinyl motif  are shown to be endowed with good inhibitory activity, with an ICA possible binding mode for 3e, chosen as a model compound is shown in The possible binding mode of 3e suggests that several suitably substituted 1,2,4-oxadiazol-5-ones can mimic the binding mode of the known imidazole-based SB 203,580 inhibitor, i.e., the inhibitor hydrogen bonds to the hinge region and the phenyl ring can interact with the hydrophobic pocket. Moreover, the C=C double bond of the core ring system of the inhibitors witha 4-fluoro-phenyl/pyridinyl motif can be replaced with the simple C-N bond of the oxadiazole system and still obtain good inhibitory capacity.1H-NMR at 300 and 500 MHz, 13C-NMR at 75 and 125 MHz) were recorded with Varian instruments and are reported in ppm relative to TMS. Merck silica gel 60-F254 precoated aluminum plates have been used for thin-layer chromatographic separations. Flash chromatography was performed on Merck silica gel  (200–400 mesh). Preparative separations were carried out by a MPLC Büchi C-601 using Merck silica gel 0.040–0.063 mm.Solvents and reagents are commercial. HRMS were determined with an SQ Quantum XLS Triple Quadrupole GC-MS/MS. NMR spectra (Compounds were synthesized according to a recent report . A solut2-Phenyl-4-(4-trifluoromethyl)-3-(pyridin-4-yl)-1,2,4-oxadiazolidin-5-one (3c). Eluent cyclohexane/ethyl acetate 3:1; yellow sticky oil (yield 68%); ν max/cm−1: 1730 (C=O). 1H-NMR : δ 8.14 , 7.51 , 7.37–7.30 , 7.16. , 6.91 , 5.87 . 13C-NMR : δ 153.3, 150.0, 149.8, 146.5, 142.4, 133.8, 132.1, 127.1, 126.5, 125.3, 124.2, 124.1, 116.6, 89.4. HRMS-EI (m/z) calcd for C20H14F3N3O2 385.3462, found 385, 3470.2-Phenyl-4-(4-chlorophenyl)-3-(pyridin-4-yl)-1,2,4-oxadiazolidin-5-one (3d). Eluent cyclohexane/ethyl acetate 3: 1; yellow sticky oil (yield 58%). ν max/cm−1: 1730 (C=O). 1H-NMR : δ 8.10 , 7.48 , 7.40–7.30 , 7.25–6.97 , 5.73 . 13C-NMR : δ 153.3, 150.0, 149.8, 146.5, 137.2, 133.3, 129.0, 127.1, 126.6, 126.5, 124.2, 89.4. HRMS-EI (m/z) calcd for C22H19ClN2O3 C19H14ClN3O2 351.5607, found 351.5612.2-Benzyl-4-(4-fluorophenyl)-3-(pyridin-4-yl)-1,2,4-oxadiazolidin-5-one (3e). Eluent cyclohexane/ethyl acetate 3: 1; yellow sticky oil (yield 71%).ν max/cm−1: 1730 (C=O). 1H-NMR : δ 8.55 , 7.80 , 7.35–7.33 , 7.27–7.20 , 6,05 , 3.86 . 13C-NMR : δ 162.9, 153.3, 149.8, 137.7, 134.7, 128.5, 127.9, 127.0, 124.2, 123.2, 115.7, 86.9, 59.9. HRMS-EI (m/z) calcd for C20H16FN3O2 344.1330, found 344.1341.2-Benzyl-4-(4-nitrophenyl)-3-(pyridin-4-yl)-1,2,4-oxadiazolidin-5-one (3f). Eluent cyclohexane/ethyl acetate 4: 1; yellow sticky oil (yield 81%). ν max/cm−1: 1730 (C=O).1H-NMR :δ 8.30 , 7.45–7.40 , 7.35–7.20 , 6.04 , 3.95 . 13C-NMR : δ 153.3, 149.8, 146.5, 145.2, 143.5, 137.7, 131.1, 128.5, 127.9, 127.0, 124.2, 124.1, 86.9, 59.9. HRMS-EI (m/z) calcd for C20H16N4O4 376.1275, found 376.1282.2-Benzyl-4-(4-fluorophenyl)-3-phenyl-1,2,4-oxadiazolidin-5-one (3g). Eluent cyclohexane/ethyl acetate 4: 1; yellow sticky oil (yield 68%). νmax/cm−1: 1730 (C=O). 1H-NMR : δ7.55 , 7.45 , 7.35–7.20 , 5.90 . 13C-NMR : δ162.9, 153.3, 139.2, 137.5, 134.7, 128.5, 127.9, 127.0, 126.9, 123.2, 115.7, 86.9, 59.9.HRMS-EI (M7z) calcd for C21H17FN2O2 347.1163, found347.1170.2-Benzyl-4-(4-nitrophenyl)-3-phenyl-1,2,4-oxadiazolidin-5-one (3h).Eluent cyclohexane/ethyl acetate 4 1; yellow sticky oil (yield 70%). νmax/cm−1: 1730 (C=O). 1H-NMR : δ8.25 , 7.45 , 7.40 , 7.35–7.25 , 6.10 , 3.80 . 13C-NMR : δ 153.3, 145.2, 143.5, 139.2, 137.7, 131.1, 128.5, 127.9, 127.0, 126.7, 124.1, 86.9, 59.9. HRMS-EI (m/z) calcd for C21H16N3O4 335.1063, found 335.1070.2-Benzyl-4-(4-fluorophenyl)-3-methyl-1,2,4-oxadiazolidin-5-one (3i). Eluent cyclohexane/ethyl acetate 4: 1; yellow sticky oil (yield 70%). νmax/cm−1: 1730 (C=O).1H-NMR : δ 7.8 , 7.3–7.8 , 4.95 , 3.85 , 1.30 . 13C-NMR : δ 162.9, 153.0, 137.7, 134.7, 128.5, 127.9, 127.0, 123.2, 115.7, 78.7, 60.0, 19.6. HRMS-EI (m/z) calcd for C16H15 FN2O2 285.0503, found 285.0510.2-Phenyl-4-(4-fluorophenyl)-3-methyl-1,2,4-oxadiazolidin-5-one (3l). Eluent cyclohexane/ethyl acetate 4: 1; yellow sticky oil (yield 70%). νmax/cm−1: 1730 (C=O). 1H-NMR : δ7.70 , 7.35–7.20 , 5.0 , 1.30 . 13C-NMR : δ 162.9, 153.3, 150.0, 134.7, 128.5, 127.1, 126.5, 123.2, 116.6, 115.7, 81.2, 19.2.HRMS-EI (m/z) calcd for C15H13 FN2O2 271.0243, found 271.0250.−4 to 10−8 M.Results are given as IC50values (µM): the IC50 values were measured by testing four concentrations of compounds at least 6-fold.The p38 MAP kinase in vitro assays to determine the biological activity of the synthesized compounds were performed according to the published procedure : test cocis locked C-N bond.We have synthesized a series of azastilbenes where an isoxazolidine unit is the linker of the two vicinal 4-fluorophenyl/4-pyridine rings, with the C=C double bond of stilbene replaced by a The synthesized compounds have been evaluated as p38α inhibitors. The inhibition data show that the two best inhibitors, 3e and 3f, strongly reduce the activity of p38α . These data suggest that the core ring system of the reported 4-fluoro-phenyl/pyridinyl inhibitors can be replaced with the structurally simple C-N bond of the 5-membered ring and still obtain good inhibitory capacity.


Sanguisorba officinalis L. Chromatographic separation was conducted with gradient elution mode by using a DiamonsilTM C18 column  with the mobile phase of 0.1% acetic acid water (A) and methanol (B). The drift tube temperature of ELSD was set at 70 °C and the nitrogen cumulative flow rate was 1.6 L/min. The method was fully validated to be linear over a wide concentration range (R2 ≥ 0.9991). The precisions (RSD) were less than 3.0% and the recoveries were between 97.7% and 101.4% for all compounds. The results indicated that this method is accurate and effective for the determination of 23 functional components in Sanguisorba officinalis L. and could also be successfully applied to study the influence of processing method on those functional components in Sanguisorba officinalis L.A novel analytical method involving high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for simultaneous determination of 11 phenolic acids and 12 triterpenes in  Sanguisorba officinalis L.  has a long history of medical application. It was first to be seen in Shennong Herbal Medicine Classic, the first great pharmacy book in China. The dried roots of S. officinalis (Rosaceae) and Sanguisorba officinalis var longifolia have been officially listed in the Chinese Pharmacopoeia (2020 edition) -urs-12,18(19)-dien-28-oic acid β-D-glucopyranosyl ester (15), 3β-[(α-L-arabinopyranosyl)oxy]-urs-12,19(29)-dien-28-oic acid β-D-glucopyranosyl ester (16), 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-D-glucopyranosyl ester (17), pomonic acid (18), euscaphic acid (19), 2α, 3α-duhydroxyursa-12,19(29)-dien-28-oic acid β-D-glucopyranosyl ester (20), 1β-hydroxyeuscaphic acid (21), 3β,19α-dihydroxy-12-ursen-28-acid (22), and 2α-hydroxyoleanolic acid (23).The chemical constituents isolated from ycosides , phenoliycosides ,16. The terpenes  include S. officinalis, and there is a lack of simultaneous determination method for various triterpenes and phenolic acids. Using gallic acid as an indicator of quality control alone does not fully account for the quality of S. officinalis and the achievement of better control over its quality. These studies also show that the triterpenes in S. officinalis are more likely to be important components of its pharmacodynamic and physiological activity.The Chinese Pharmacopoeia only selected gallic acid as a quality control indicator for S. officinalis have strong physiological activity -urs-12,18(19)-dien-28-oic acid β-D-glucopyranosyl ester (88.975 min), 3β-[(α-L-arabinopyranosyl)oxy]-urs-12,19(29)-dien-28-oic acid β-D-glucopyranosyl ester (94.293 min), 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-D-glucopyranosyl ester (98.958 min), pomonic acid (101.456 min), euscaphic acid (103.725 min), 2α, 3α-duhydroxyursa-12,19(29)-dien-28-oic acid β-D-glucopyranosyl ester (105.710 min), 1β-hydroxyeuscaphic acid (108.168 min), 3β,19α-dihydroxy-12-ursen-28-acid (111.653 min), and 2α-hydroxyoleanolic acid (121.715 min).Y =a X + b, where Y and X are the logarithms of the peak area and concentration, respectively. As shown in R2 ≥ 0.9991) within the test ranges. Limits of quantitation ranged from 5.24 to 18.51 μg/mL. These results demonstrated that the HPLC-ELSD method is sensitive and precise for the quantitative determination of 23 analytes in S. officinalis.The calibration curves were calculated by double logarithmic transformation of the area and the concentration of the reference solution injected. Linearity was evaluated by five different concentrations of standard solutions. For calibration, the standard solutions with five different concentrations were analyzed. A plot of peak area versus sample size by ELSD was not linear, but the plot of peak area versus sample size in double logarithm was linear, which conformed to the mechanism of the ELSD. Their regression equations were calculated in the form of The experimental results of precision were 2.0%, 1.4%, 1.5%, 1.0%, 2.1%, 1.1%, 1.1%, 1.6%, 2.3%, 1.7%, 1.6%, 1.9%, 2.4%, 2.3%, 2.4%, 1.1%, 1.5%, 1.2%, 2.0%, 1.9%, 1.0%, 1.3%, 2.6%, 2.1%, respectively, which were all acceptable.The result of repeatability shown as follows. The concentration was calculated by external standard method, and the RSD of 23 compounds were 1.5%, 1.3%, 2.1%, 0.49%, 0.56%, 1.4%, 0.48%, 1.7%, 0.30%, 1.1%, 1.5%, 1.4%, 2.0%, 0.47%, 0.78%, 0.19%, 0.36%, 1.4%, 0.52%, 2.4%, 0.29%, 0.41%, 2.0%, respectively, which met the requirement.S. officinalis extract remained stable at least 12 h.The RE among measurements ranged from −2.8% to 2.9% ., which The result was shown in S. officinalis from 24 provinces was calculated. The source and collection time were shown in S. officinalis were shown in S. officinalis samples from different regions was listed in Based on the experimental analysis above, the content of 11 phenolic acids and 12 triterpenoids in 32 batches of All medicinal materials are purchased in the local Chinese medicine market in the 24 provinces mentioned below.S. officinalis, and the content of gallic acid should not be less than 1.0%. From the experimental results, the content of gallic acid recorded in the Chinese Pharmacopoeia (2020 edition) was different in 32 batches of S. officinalis, but all the results met the quality control requirements recorded in the pharmacopoeia.In Chinese Pharmacopoeia (2020 edition), gallic acid was used as a quantitative index in the medicinal materials and pieces of S. officinalis more reasonably and comprehensively, the content of phenolic acids and triterpenoids in S. officinalis was determined by HPLC. According to the content results, the contents of triterpenoids and phenolic acids in 32 batches are different, and some compounds cannot even be detected in S. officinalis. Among them, the content of ziyuglycoside I, catechin, and gallic acid in S. officinalis from Hebei was significantly higher than those in Gansu and Shanxi; the content of gallic acid and catechin in Shandong, Heilongjiang, and Shandong was higher; and the content of triterpenes in Inner Mongolia, Jiangxi, and Liaoning was also higher. The differences in the contents of triterpenoids and phenolic acids in S. officinalis are affected by many factors such as planting region, growing environment, and harvest time.Under the current experimental conditions, in order to evaluate the quality of S. officinalis from different regions were tested. The results of the test are helpful to evaluate the quality and to control the internal quality of S. officinalis.In this experiment, different batches of S. officinalis and processed S. officinalis are shown in The comparison of 23 compounds in unprocessed S. officinalis were well separated with symmetrical peaks and no tailing. The gradient elution method with different proportions was explored. The important parameter of ELSD conditions is the drift tube temperature which was evaluated at different drift tube temperatures from 60 °C to 80 °C. Good peak symmetries and efficiencies were achieved with a drift tube temperature at 70 °C. After examining a series of column temperatures, the final column temperature was determined to be 35 °C.The mobile phase combinations investigated included methanol–water, acetonitrile–water, methanol–0.1% acetic acid water, and acetonitrile–0.1% acetic acid water. The result showed that when methanol–0.1% acetic acid water was the mobile phase, 23 compounds in R2 ≥ 0.9991). The precisions were less than 3.0% and the recoveries were between 97.7% and 101.4% for all compounds. The results indicated that this method is accurate and effective for the determination of 23 functional components in Sanguisorba officinalis L.Linear of the method over a wide concentration range oxy]-urs-12,18(19)-dien-28-oic acid β-d-glucopyranosyl ester, 3β-[(α-L-arabinopyranosyl)oxy]-urs-12,19(29)-dien-28-oic acid β-D-glucopyranosyl ester, pomonic acid, 1β-hydroxyeuscaphic acid, and, 1β-hydroxyeuscaphic acid were decreased. Especially, compound (1), (3), (9), (11), (15), (16), and (21) were decreased significantly in batches from all three provinces. The content of methyl-6-O-galloyl-β-D-glucopyranoside, ethyl gallate, ellagic acid, and rosamultin was increased. Among them, (4) and (10) were increased significantly in batches from all three provinces. Sanguiin H-4 and 2α, 3α-dihydroxyursa-12,19(29)-dien-28-oic acid β-D-glucopyranosyl ester remain unchanged. Except for Heilongjiang, the content of caffeic acid in two batches of S. officinalis in Henan, Anhui decreased significantly. Syringic acid increased in batches from Heilongjiang, Anhui. 2-oxo-3,19-dihydroxy-olean-12-en-28-oic acid β-D-glucopyranosyl ester decreased in Heilongjiang and Anhui. Euscaphic acid and 3β,19α-dihydroxy-12-ursen-28-acid increased in Heilongjiang and Henan, but euscaphic acid decreased in batch from Anhui. In the batch from Heilongjiang, ziyuglycoside I and 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-D-glucopyranosyl ester increased, but decreased in Henan and Anhui, respectively.According to the result, compared with the unprocessed group, some compounds in S. officinalis according to the method in the Chinese pharmacopoeia  and applied the established content determination method to determine the processed and unprocessed S. officinalis to compare the content changes of each compound. Which provides a theoretical basis for the different effects of unprocessed and processed S. officinalis. The changes in the content of these compounds may provide some ideas for the study of revealing the specific pharmacological effects of the active ingredients of S. officinalis.We processed the The purity of these 23 analytes refined in our laboratory (identified by NMR and MS) were all more than 98%. As reference standards, the NMR and MS data about the isolated compounds were shown in m/z: 171 [M+H]+, 1H-NMR  δ 7.05 , 13C-NMR  δ 121.6 (C-1), 109.9 , 146 (C-3), 139.2 (C-4), 170 (C-7).gallic acid (1): ESI-MS m/z: 634 [M-H]-, 1H-NMR  δ 4.19 , 5.36 , 5.95 , 7.36 , 3.60 , 3.52 , 6.77 , 13C-NMR  δ 91.3 (C-1), 74.4 (C-2), 79.1 (C-3), 67.8 (C-4), 76.5 (C-5), 61.7 (C-6), 115.3 (C-1′), 126.3 (C-2′), 108.0 (C-3′), 145.8 (C-4′), 137.4 (C-5′), 146.0 (C-6′), 170.0 (C-7′), 115.4 (C-1″), 126.9 (C-2″), 107.6 (C-3″), 145.8 (C-4″), 137.5 (C-5″), 146.7 (C-6″), 171.4 (C-7″),δ 120.3 (C-1″‘), 110.5 , 146.7 , 140.6(C-4″‘), 166.4 (C-7″‘).sanguiin H-4 (2): ESI-MS m/z: 291 [M+H]+, 1H-NMR  δ 6.86 , 6.78 , 6.73 , 5.95 , 5.88 , 4.58 , 3.98 , 2.85 , 2.53 , 13C-NMR  δ 81.5 (C-2), 67.4 (C-3), 27.1 (C-4), 155.5 (C-5), 94.9 (C-6), 156.2 (C-7), 94.1 (C-8), 156.4 (C-9), 99.4 (C-10), 130.8 (C-1′), 113.9 (C-2′), 144.8 (C-3′), 144.8 (C-4′), 114.7 (C-5′), 118.7 (C-6′).catechin (3): ESI-MS β-D-glucopyranoside (4): ESI-MS m/z: 347 [M+H]+, 1H-NMR  δ 7.19 , 4.96 , 4.65 , 4.18 , 3.98 , 13C-NMR , δ 105.5 (C-1), 74.7 (C-2), 75.2 (C-3), 71.2 (C-4), 78.1 (C-5), 64.5(C-6), 56.4 (C-7), 167.1 (C-8), 121.0 (C-9), 110.0 , 149.4 , 135.2 (C-12).methyl-6-O-galloyl-m/z: 197 [M-H]-, 1H-NMR  δ 7.19 , 7.17 , 3.85 , 3.88 , 13C-NMR  δ 121.5 (C-1), 106.1 (C-2), 149.2 (C-3), 140.6 (C-4), 146.3 (C-5), 111.9 (C-6), 168.9 (C-7), 52.4 (3-OMe), 56.7 (7-OMe).3, 4-dihydroxy-5-methoxybenzyl ester (8): ESI-MS m/z: 673 [M+Na]+, 1H-NMR  δ 6.30 , 5.53 , 3.36 , 2.92 , 1.66 , 1.38 , 1.24 . 1.20 , 1.08 , 1.06 , 1.05 .rosamultin (13): ESI-MS m/z: 789 [M+H]+, 1H-NMR  δ 6.34 , 5.61 , 4.82 , 3.38 , 2.99 , 1.75 , 1.45 , 1.32 , 1.24 , 1.12 , 1.02 , 0.98 .ziyuglycoside I (14): ESI-MS β-[(α-l-arabinopyranosyl)oxy]-urs-12,18(19)-dien-28-oic acid β-d-glucopyranosyl ester (15): ESI-MS m/z: 771 [M+Na]+, 1H-NMR  δ 6.33 , 5.69 , 4.77 , 3.35 , 1.80 . 1.36 , 1.26 , 1.12 , 1.03 , 0.94 , 0.88 .3β-[(α-l-arabinopyranosyl)oxy]-urs-12,19(29)-dien-28-oic acid β-D-glucopyranosyl ester (16): ESI-MS m/z: 771 [M+Na]+, 1H-NMR  δ 6.31 , 5.53 , 5.19 , 5.04 , 4.77 , 3.83 , 3.37 , 1.27 , 1.27 , 1.12 , 1.05 , 0.95 , 0.88 .3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-D-glucopyranosyl ester (17): ESI-MS m/z: 657 [M+Na]+, 1H-NMR  δ 6.34 , 5.59 , 3.45 , 2.96 , 1.71 , 1.43 , 1.24 , 1.24 , 1.08 , 1.06 , 0.98 .3m/z: 473 [M+H]+, 1H-NMR  δ 5.52 , 4.35 , 3.06 , 1.73 , 1.21 , 1.13 , 1.11 , 1.06 , 1.00 , 0.91 .pomonic acid (18): ESI-MS α, 3α-dihydroxyursa-12,19(29)-dien-28-oic acid β-D-glucopyranosyl ester (20): ESI-MS m/z: 655 [M+Na]+, 1H-NMR  δ 6.29 , 5.50 , 4.29 , 3.78 , 3.76 , 1.25 , 1.13 , 1.11 , 1.04, 0.98 , 0.88 .2β-hydroxyeuscaphic acid (21): ESI-MS m/z: 527 [M+Na]+, 1H-NMR  δ 5.56 , 4.13 , 4.16 , 3.86 , 3.03 , 1.66 , 1.41 , 1.26 , 1.25 , 1.24 , 1.11 , 0.92 .1β,19α-dihydroxy-12- ursen -28-acid (22): ESI-MS m/z: 473.4 [M+H]+, 1H-NMR  δ 5.61 , 3.43 , 3.05 , 1.73 , 1.23 , 1.13 , 1.11 , 1.11 , 1.02 , 0.91 .3α-hydroxyoleanolic acid (23): ESI-MS m/z: 456 [M]+, 1H-NMR  δ 5.45 , 3.37 , 3.27 , 1.26 , 1.23 , 1.06 , 1.00 , 0.97 , 0.94 , 0.93 .2m/z 169.1→125.1 for gallic acid (1), m/z 633.1→300.9 for sanguiin H-4 (2), m/z 289.2→109.2 for catechin (3), m/z 179.0→135 for caffeic acid (5), m/z 197.0→122.8 for syringic acid (6), m/z 197.0→124.1 for ethyl gallate (7), m/z 197.1→182.2 for 3, 4-dihydroxy-5-methoxybenzyl ester (8), m/z 193.1→134 for ferulic acid (9), m/z 301→145 for ellagic acid (10), m/z 328.7→314 for 3, 3-dihydroxy ellagic acid (11), m/z 673.4→511.4 for rosamultin (13), m/z 784.5→437.4 for ziyuglycoside I (14), m/z 771.5→609.1 for 3β-[(α-L-arabinopyranosyl)oxy]-urs-12,18(19)-dien-28-oic acid β-D-glucopyranosyl ester (15), m/z 652.5→455.4 for 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-D-glucopyranosyl ester (17), m/z 655.4→493.0 for 2α, 3α-dihydroxyursa-12,19(29)-dien-28-oic acid β-D-glucopyranosyl ester (20), m/z 505.2→423.2 for 1β-hydroxyeuscaphic acid (21). Through the LC/MS/MS data shows that the HPLC-ELSD method we established is useful and effective.The LC/MS/MS data in the articles published in our laboratory was provided here for the discussion, as follows ,30: m/z S. officinalis (1.0 g), it was extracted by hot reflux with 10 mL 70% ethanol solution  3 times at 80 °C, 1 h each, and then filtrated. The combined filtrate was evaporated to steam, and the residue was dissolved in methanol to get a concentration equivalent to 0.005 g/mL of the S. officinalis extract.After crushing the dried root of Reference standards (1–23) were weighed accurately and dissolved by methanol in 10 mL volumetric bottle to yield a nominal concentration . The concentrations of mixed reference solution of analytes (1–23) were 51.3, 52.5, 60.1, 52.5, 75.0, 55.3, 52.6, 52.4, 105.0, 55.2, 52.6, 126.3, 90.1, 135.0, 117.5, 92.0, 94.0, 106.0, 74.0, 80.0, 80.0, 45.0, 60.0 μg/mL, respectively. The working standard solutions were prepared from the concentrated stock solution by further appropriate dilution in the methanol. All the solutions were stored at 4 °C and filtered through the 0.45 μm nylon membrane prior to analysis.TM C18 column  with the flow rate of 1.0 mL/min at 35 °C. The mobile phase was 0.1% acetic acid water (A) and methanol (B) with a gradient elution of 10–25% B at 0–20 min, 25–45% B at 20–50 min, 45–55% B at 50–55 min, 55–65% B at 55–70 min, 65–70% B at 70–90 min, 70–85% B at 90–115 min, 85–95% B at 115–125 min, 95–10% B at 125–130 min. The drift tube temperature of ELSD was 70 °C and nitrogen cumulative flow rate was 1.6 L/min.The analysis was performed on an Agilent series 1260 HPLC instrument coupled with an Agilent 1290 ELSD . The system was equipped with Diamonsil For calibration, the premixed working solution with at least five different concentrations (performed in triplicate) was analyzed, and the linear calibration curves were calculated by plotting the logarithm of concentration injected for each analysis. The mixed solution of the 23 reference compounds was further diluted to a series of concentrations by methanol for the gain of limit of quantification (LOQ). The LOQ under the present chromatographic conditions were determined at the signal-to-noise ratios of each analyte of about 10, respectively.The precision of the quantitative method was investigated by determining the 23 analytes in six replicates within one day. The RSD value of the peak area was adopted to evaluate precision.Repeatability was confirmed with six independent analytical sample solutions prepared according to the methods described above and variations expressed by RSD.To assess the stability of the developed method, the sample was stored at room temperature and injected into the HPLC apparatus at 0, 2, 4, 6, 8, 12 h after preparation, respectively. Calculate the concentration of the 23 analytes at each time point and compare with the value of 0 h, to obtain RE (relative error).S. officinalis powder with known content, each of ~0.5 g, were weighted accurately, put in conical bottle. Moreover, reference substances (1–23) were added precisely. The test sample solutions were prepared as Six parts of S. officinalis powder from 24 provinces were weighed to a total of 1.0 g. The test sample solutions were prepared as the S. officinalis was calculated with HPLC chromatographic conditions in Thirty-two batches of S. officinalis powder from the same origin was divided into unprocessed group and processed group. The unprocessed group precisely weighed 1.0 g, S. officinalis of processed group was heated until the surface was black and the inside was brown, taken out and left to cool at room temperature, and precisely weighed to 1.0 g after processing. Both groups were prepared as S. officinalis was developed based on HPLC-ELSD. This is the first report of simultaneous determination of 11 phenolic acids and 12 triterpenoids in S. officinalis extract, so this method could be used to evaluate the quality of S. officinalis. At the same time, it provides a basis for the establishment of the characteristic and fingerprint of traditional Chinese medicine of S. officinalis. The method is of great significance for the quality control of S. officinalis resources and guiding its application.In this study, a sensitive and reliable quantitative method for simultaneous analysis of phenolic acids and triterpenoids from α, 3α-dihydroxyursa-12,19(29)-dien-28-oic acid β-D-glucopyranosyl ester, processed of S. officinalis has effect on all other compounds. Therefore, processed has a great impact on the 23 functional components of S. officinalis.Except for sanguiin H-4 and 2


Euphorbia usambarica is a traditional medicine used for gynecologic, endocrine, and urogenital illnesses in East Africa; however, its constituents and bioactivities have not been investigated. A variety of compounds isolated from Euphorbia species have been shown to have activity against latent HIV-1, the major source of HIV-1 persistence despite antiretroviral therapy. We performed bioactivity-guided isolation to identify 15 new diterpenoids  along with 16 known compounds from E. usambarica with HIV-1 latency reversal activity. Euphordraculoate C (1) exhibits a rare 6/6/3-fused ring system with a 2-methyl-2-cyclopentenone moiety. Usambariphanes A (2) and B (3) display an unusual lactone ring constructed between C-17 and C-2 in the jatrophane structure. 4β-Crotignoid K (14) revealed a 250-fold improvement in latency reversal activity compared to crotignoid K (13), identifying that configuration at the C-4 of tigliane diterpenoids is critical to HIV-1 latency reversal activity. The primary mechanism of the active diterpenoids 12–14 and 21 for the HIV-1 latency reversal activity was activation of PKC, while lignans 26 and 27 that did not increase CD69 expression, suggesting a non-PKC mechanism. Accordingly, natural constituents from E. usambarica have the potential to contribute to the development of HIV-1 eradication strategies. Antiretroviral therapy (ART) durably blocks HIV-1 transcription by targeting viral enzymes; however, these drugs do not result in viral eradication due to the presence of replication-competent proviruses that are stably integrated into the genomes of a small population of long-lived memory T cells, known as the latent reservoir ,3,4.Euphorbia is one of the largest genera in Euphorbiaceae + , indicating 14 unsaturated degrees. The 1H NMR spectrum of 1 revealed six methyls, an oxygenated methine, two unsaturated methines, and a monosubstituted aromatic group , 7.58, 7.46 (2H); δC 166.2, 133.2, 130.4, 129.9 (2C), 128.6 (2C)] and one acetoxy (OAc)  could be identified in 1. Based on the COSY and HSQC spectra of 1, a series of COSY correlations between an olefinic methine /a methine /methylene , together with the allylic four-bond coupling between H-1 and a methyl group . Key HMBC correlations from H-1 and H-16 to an olefinic quaternary carbon C-2 (δC 141.3) and a ketone carbon C-3 (δC 206.8), and H-4 to C-3, indicated the presence of an α-methyl-α,β-unsaturated cyclopentanone moiety. Moreover, a series of COSY correlations between a methine , an olefinic methine , and an allylic coupled methyl group , together with the key HMBC correlations from H-7 to a carbonyl carbon C-5 (δC 163.4) and an oxygenated quaternary carbon C-14 (δC 85.8), H-8 to an olefinic quaternary carbon C-6 (δC 127.8), and H3-17 to C-5, C-6, and C-7, indicated the presence of an α-methyl-α,β-unsaturated-δ-lactone moiety. A gem-dimethylcyclopropane moiety could be identified by the key HMBC correlations from two methyl groups  to a methine C-9 , a quaternary carbon C-10 (δC 24.9), an oxygenated quaternary carbon C-11 (δC 63.4) and each other, and H-9 to C-10 and C-11. In addition, the 1H–1H COSY cross peak between H-8/H-9, a methyl group /a methine /an oxygenated methine , as well as the HMBC correlations from H-7 to C-14, H-8 to C-11 and C-13, H-9 to C-11 and C-14, H-12 to C-10 and C-11, and C-13 to C-14, demonstrated the presence of a six-membered ring fusion with the gem-dimethyl-cyclopropane moiety at C-9 and C-11, and the α-methyl-α,β-unsaturated-δ-lactone moiety at C-8 and C-14. The HMBC correlations from H-4 to C-14 and H-8 to C-15 indicated that the α-methyl-α,β-unsaturated cyclopentanone moiety was linked to C-14. The OAc and OBz groups should be connected to C-11 and C-12, respectively, based on HMBC correlations . The NMR spectra of 2 revealed clearly four OAc , one OBz (δH 7.93 (2H), 7.52, 7.39 (2H), δC 164.5, 133.5, 129.7, 129.6, 128.8), one propionate group , four methyls , a trans-disubstituted C=C , and a lactone carbonyl carbon . Further, comparing the 1D NMR data of 2 (10) + . The 1D NMR data , 1.20 (3H), δC 176.4, 34.2, 18.8, 18.5] instead of propanoyl. The isobutyryl group was connected to C-7 in 3 based on the HMBC correlation from H-7 (δH 5.38) to the OiBu carbonyl carbon (δC 176.4). The NOESY correlations of 3 revealed the same relative configuration as that of 2. The structure of 3 was established and named as usambariphane B.Compound NMR data  of 3 wer4 was purified as a colorless crystal. The molecular formula was identified as C40H52O16 by HR-ESIMS m/z 789.3346 [M + H]+ . Based on the comparison of the 1H and 13C NMR data (4 with those of usambariphane B (2), the skeleton of 4 was suggested to be a C22 dihomojatrophane with a double bond at ∆11,12  and a lactone moiety . A δ-lactone ring was constructed at C-5 and C-6 supporting by the 1H–1H COSY cross peak between H2-17 and H2-21, as well as the HMBC correlations from H-5 to C-17 and C-22, H2-17 to C-5, C-6, and C-22, and H2-21 to C-6 and C-22. Moreover, the 1D NMR data of 4 were highly close to those of euphosorophane D  at C-7 according to an HMBC correlation from H-7 (δH 5.40) to δC 174.6. The NOESY cross-peaks of 4 demonstrated the same relative orientations to those of euphosorophane D [4 was established and named as usambariphane C.Compound NMR data  for 4 wiophane D  except fophane D . Therefo5 was purified as a colorless crystal. The molecular formula was identified as C40H50O16 by HR-ESIMS m/z 787.3193 [M + H]+ . The inspection of 1D , a lactone moiety , and a ketone unit δC 211.4 (C-14). A δ-lactone ring was constructed at C-5 and C-6 supporting by the 1H–1H COSY cross peak between H2-17 and H2-21, and the HMBC correlations from H-5 to C-17 and C-22, H2-17 to C-5, C-6, and C-22, and H2-21 to C-6 and C-22. The ketone unit in 5 was located at C-14 based on the HMBC correlations from H-1, H-12, H-13, and H3-20 to C-14, respectively. An OH group was connected to C-15 by the confirmation of HMBC correlations from 15-OH to C-4, C-14, and C-15. Moreover, four OAc , one OBz , and one OPr  moieties were identified clearly by the examination of the NMR spectra. The HMBC correlations from H-3 to δC 169.1, H-7 to δC 173.4, H-8 to δC 170.0, and H-9 to δC 169.9, indicated the OPr was located at C-7, and three OAc were linked to C-3, C-8, and C-9, respectively. The location of the OBz at C-6 was confirmed by the NOESY correlations between the benzoyl proton signal δH 7.88 with H-5, H-8, and H-12. The last OAc was connected to C-2 based on the NOE cross-peak between the acetyl proton signal δH 2.26 with H3-16. The relative configuration of 5 was evaluated by the NOESY spectrum and comparison with a similar structure terracinolide J + . The 1D , 1.22 (3H), δC 175.2, 34.5, 19.0, 18.1] was presented at C-7 as confirmed by the HMBC correlation from H-7 (δH 6.39) to δC 175.2. The NOESY correlations of 6 revealed the same relative configuration as that of 5. The structure of 6 was established and named as usambariphane E.Compound . The 1D  and 2D N7 was obtained as a colorless crystal. The molecular formula was identified as C39H52O15 by HR-ESIMS m/z 761.3383 [M + H]+ . The 1D , δC 170.1, 21.0; δH 2.12 (3H), δC 170.9, 22.5; δH 2.06 (3H), δC 172.1, 20.9; δH 1.70 (3H), δC 172.2, 20.4], one OiBu , one OBz , four methyls , a trans-disubstituted C=C , and an exocyclic methylene . Further, the skeleton of 7 was established as a jatrophane type diterpenoid with two double bonds at ∆6,17 and ∆11,12 based on the series 1H–1H COSY correlations of H-3/H-4/H-5 and H-11/H-12/H-13/H-14 and H3-20, as well as the HMBC correlations from H-1 to C-2 and C-16, H-3 to C-1, C-2, C-4 and C-15, and H3-16 to C-1, C-2 and C-3, H-5 to C-3, C-4, C-6, C-15, C-17, H-7 to C-6 and C-9, H-8 to C-6 and C-10, H-9 to C-8 and C-11, H-11 to C-10 and C-13, H-12 to C-10, H-14 to C-1, C-4, C-12, C-13, and C-15, H2-17 to C-5, C-6, and C-7, H3-18 and H3-19 to C-9, C-10 and C-11, H3-20 to C-12, C-13, and C-14. The presence of the 3-OH group was deduced by the 1H–1H COSY cross-peak between H-3 and 3-OH, and the HMBC correlations from 3-OH to C-2, C-3, and C-4. Another OH group was located at C-8 by the COSY cross-peak between H-8 and 8-OH, and the HMBC correlations from 8-OH to C-7 and C-8. The third OH group was connected to C-15 by the confirmation of the HMBC correlations from 15-OH to C-1, C-4, and C-15. The HMBC correlations of H-1/δC 170.1 (OAc), H-5/δC 165.4 (OBz), H-7/δC 175.1 (OiBu), H-9/δC 172.1 (OAc), and H-14/δC 172.2 (OAc), demonstrated the locations of the acyl groups, and of necessity, the last OAc was located at C-2. The relative configuration of 7 was deduced by the NOESY spectrum. The H-4 and 15-OH in 7 can be assigned as α- and β-oriented, respectively, according to the comparison of the NMR data with those of known jatrophane-type diterpenoids + , indicating 18 degrees of molecular unsaturation. The 1D , δC 169.7, 20.7; δH 2.00 (3H), δC 169.9, 20.8], an OiBu , an OBz , a nicotinate group , four methyls , a trans-disubstituted C=C , ketone unit , and an exocyclic methylene . The skeleton of 8 was established as a jatrophane-type diterpenoid with two double bonds at ∆6,17 and ∆11,12, and the ketone unit at C-14 based on the series 1H–1H COSY correlations of H-3 (δH 4.76)/H-4 (δH 3.32)/H-5 (δH 5.67) and H-11/H-12/H-13 (δH 3.75)/H3-20; and the HMBC correlations from H2-1 (δH 2.95 and 2.27) to C-4 (δC 47.9), C-14, and C-15 (δC 89.0), H-3 to C-1(δC 51.4), C-2 (δC 92.0) and C-15, H-4 to C-15, H3-16 to C-1, C-2 and C-3 (δC 79.1), H-5 to C-3, C-6 (δC 144.9), and C-7 (δC 68.5), H-8 (δH 5.18) to C-6, C-7, C-9 (δC 80.6), and C-10 (δC 41.1), H-9 (δH 4.96) to C-10 and C-11, H-11 to C-9, C-10, C-13 (δC 44.4), C-18, and C-19, H-12 to C-10, H-13 to C-11, C-12, and C-14, H2-17 to C-5, C-6, and C-7, H3-18 and H3-19 to C-9 and C-10, H3-20 to C-12, C-13, and C-14. An OH group was located at C-3 based on a 1H–1H COSY cross-peak between H-3 and 3-OH (δH 3.57), and the HMBC correlations from 3-OH to C-3 and C-4. Another OH group was connected to C-15 by the confirmation of the HMBC correlations from 15-OH (δH 4.34) to C-1, C-4, and C-15. Furthermore, the HMBC correlations of H-5/δC 164.7 (OBz), H-7/δC 175.8 (OiBu), H-8/δC 169.9 (OAc), H-9/δC 169.7 (OAc), demonstrated the locations of these acyl groups, and thereby the ONic was located at C-2. According to the NOESY cross-peaks of H-1a/H-4, H-1a/H-13, H-3/H-4, H-4/H-7, H-4/H-13, H-5/H-8, H-5/15-OH, H-8/H3-19, H-9/H3-19, H3-19/H3-20, 3-OH/15-OH, 3-OH/H3-16, and comparison of the NMR data with those of -2,3,5,7,8,9,15-heptahydroxyjatropha-6(17),11-diene-14-one-2,3,8,9-tetraacetate-5-benzoate-7-(2-methylpropionate) [8 as depicted on 8 was established and named as usambariphane G.Compound . The 1D  and 2D N9 was obtained as a white amorphous powder with a molecular formula of C39H50O16 determined based on HR-ESIMS m/z 775.3172 [M + H]+ . The 1D , δC 172.0, 20.7; δH 2.19 (3H), δC 169.6, 22.9; δH 2.16 (3H), δC 170.4, 21.4; δH 2.15 (3H), δC 171.2, 21.7; δH 2.14 (3H), δC 171.0, 20.9], one OBz , four methyls , and a trans-disubstituted C=C , and a lactone carbonyl carbon . Further, comparing the NMR data of 9 with those of isoterracinolide A (10) + . The 1D , δC 173.9, 21.3], one OBz , four methyls , an oxygenated methylene , an oxygenated methine , two unsaturated methines , and a ketone unit . The interpretation of HMBC correlations suggested the skeleton of 14 was a tigliane-type diterpenoid + . The 1D , δC 174.1, 21.2], one OBz , five methyls , two oxygenated methines , two unsaturated methines , and a ketone unit . The interpretation of HMBC correlations demonstrated that 15 was a tigliane-type diterpenoid with an α-methyl-α,β-unsaturated cyclopentanone ring fused between C-4 and C-10, with two OH (δH 5.92 and 5.95) connected to C-5 and C-9 respectively, one OBz connected to C-12, and one OAc connected to C-13 and 20-methyl group. The NMR data of 15 was highly close to those of euphodendriane A + . The 1D , δC 26.9, CH3-17), three methyls , three methylenes , a methine , a set of trisubstituted aromatic ring , δC 125.2, CH-11; δH 8.17 , δC 133.4, CH-12; δC 135.9, C-13; δH 8.57 , δC 128.3, CH-14], two ketone units , and two quaternary carbons . The HMBC correlations of 16 from H2-1 to C-3, C-9, and C-20, H2-2 to C-3 and C-4, H-5 to C-1, C-4, C-9, and C-10, H2-6 to C-7, C-8, and C-10, H-12 to C-15, H-14 to C-7 and C-15, H3-17 to C-13, H3-18 and H3-19 to C-3, C-4, and C-5, and H3-20 to C-5 and C-10, suggested that 16 was an abietane-type diterpenoid + . The inspection of 1D  by the confirmation of the 1H–1H COSY cross-peaks between H2-1/H2-2/H-3 as well as the HMBC correlations from H2-1 to C-3 and H3-18 to C-3. The H-3 [δH 3.37 ] was identified as α-oriented by the NOESY correlations of H-3/H-5/H3-18 and the comparison of the proton signals for 17 with those of the similar compound 3β-hydroxy-abieta-8,11,13-trien-7-one + . The 1D , δC 9.0, CH3-17; δH 1.17 (3H), δC 25.9, CH3-18; δH 1.09 (3H), δC 22.4, CH3-19; δH 0.95 (3H), δC 15.0, CH3-20], four methylenes , four methine , six quaternary carbons , and two carbonyl carbons . The structure of 19 was suggested an abietane-type diterpenoid with a ketone carbon at C-3, an epoxy ring fused at C-8 and C-14, a double bond at ∆11,12, and an α-methyl-α,β-unsaturated δ-lactone ring formed as D ring according to the analysis of the COSY cross-peaks of H2-1/H2-2, H2-5/H2-6/H2-7, and H-9/H-11, as well as the HMBC correlations from H2-1 to C-3, C-5, and C-10, H2-2 to C-3 and C-10, H-5 to C-4, C-18, C-19, and C-20, H2-7 to C-5 and C-8, H-9 to C-1, C-5, C-8, C-12, C-14, and C-20, H-11 to C-8, C-9, C-12, and C-13, H-14 to C-7, C-8, C-12, and C-13, H3-17 to C-13, C-15, and C-16, H3-18 and H3-19 to C-3 and C-4, together with H3-20 to C-1, C-9, and C-10. Comparison of the 13C NMR data with those of the related compounds jolkinolide A + . The 1H and 13C NMR data  based on the inspection of the 1H–1H COSY cross-peaks between H2-1/H2-2/H-3 together with the HMBC correlations from H-3 to C-1, C-4, C-18, and C-19. Saturated methylene  and an oxygenated methine  were assigned to be at C-11 and C-12, respectively, according to the analysis of the 1H–1H COSY cross-peaks between H-9/H2-11/H-12 together with the HMBC correlations from H2-11 to C-8, C-10, and C-13, as well as H-12 to C-13 and C-15. The structure was found to be highly similar to the NMR features of gelomulide A + , m/z 515.2046 [M + Na]+ .2)Usambaricinophane A ; the 1H and 13C NMR spectroscopic data, see m/z 789.3327 [M + H]+ , m/z 811.3169 [M + Na]+ .3)Usambaricinophane B ; the 1H and 13C NMR spectroscopic data, see m/z 803.3488 [M + H]+ , m/z 825.3333 [M + Na]+ .4)Usambaricinophane C ; the 1H and 13C NMR spectroscopic data, see m/z 789.3346 [M + H]+  m/z 811.3171 [M + Na]+ .5)Usambaricinophane D ; the 1H and 13C NMR spectroscopic data, see m/z 787.3193 [M + H]+ , m/z 809.3011 [M + Na]+ .6)Usambaricinophane E ; the 1H and 13C NMR spectroscopic data, see m/z 801.3356 [M + H]+  m/z 823.3170 [M + Na]+ .7)Usambaricinophane F ; the 1H and 13C NMR spectroscopic data, see m/z 761.3383 [M + H]+ , m/z 783.3215 [M + Na]+ .8)Usambaricinophane G ; the 1H and 13C NMR spectroscopic data, see m/z 764.3230 [M + H]+ , m/z 786.3098 [M + Na]+ .9)Isoterracinolide C ; the 1H and 13C NMR spectroscopic data, see m/z 775.31721 [M + H]+ , m/z 797.3011 [M + Na]+ .14)4β-Crotignoid K ; the 1H and 13C NMR spectroscopic data, see m/z 495.2385 [M + H]+ , m/z 517.2202 [M + Na]+ .15)Euphodendriane B ; the 1H and 13C NMR spectroscopic data, see m/z 495.2396 [M + H]+ , m/z 517.2209 [M + Na]+ .16)16-Nor-abieta-8,11,13-trien-3,7,15-trione ; the 1H and 13C NMR spectroscopic data, see m/z 299.1648 [M + H]+ , m/z 321.1466 [M + Na]+ .17)16-Nor-3β-hydroxy-abieta-8,11,13-trien-7,15-dione ; the 1H and 13C NMR spectroscopic data, see m/z 301.1803 [M + H]+ , m/z 323.1623 [M + Na]+ .19)ent-8β,14β-Epoxyabieta-3-one-11,13(15)-dien-16,12-olide ; the 1H and 13C NMR spectroscopic data, see m/z 329.1753 [M + H]+ , m/z 351.1572 [M + Na]+ .20)ent-8β,14β-Epoxyabieta-3α-hydroxy-13(15)-en-16,12-olide ; the 1H and 13C NMR spectroscopic data, see m/z 333.2067 [M + H]+ , m/z 355.1886 [M + Na]+ .www.hivreagentprogram.org) and cultured in RPMI-based media supplemented with 10% fetal calf serum, 1% penicillin, and 1% streptomycin.The HIV-1-infected Jurkat T cell line (J-Lat 10.6) was obtained in January 2019 from the NIH/ATCC HIV-1 Reagent Program (www.thermofisher.com) per 105 cells for 30 min at 4 °C. Simultaneously, J-lat cells were also stained with antibodies against CD69 conjugated to an APC fluorophore . Cells were then washed and re-suspended in 1× PBS prior to flow cytometry acquisition evaluating for cellular viability, green fluorescent protein (GFP) expression, and CD69 expression. Flow cytometry was performed with a BD FACSCelesta or FACSCanoto flow cytometer with FACSDiva acquisition software  prior to analysis with FlowJo .After in vitro culture, J-lat cells were washed with phosphate-buffered saline (1× PBS) prior to staining with 0.1 µL fixable viability dye Live/Dead Aqua , at a concentration of 10 mM, and diluted with PBS, and tested with J-lat 10.6 cells at concentrations of 100, 10, and 1 μM. J-lat 10.6 cells were tested with compounds at a concentration of 2.5 × 10Statistical significance was analyzed using software from GraphPad Prism Version 7.0c . The mean values and standard deviations for all replicate J-lat results were calculated and used to create β-crotignoid K (14) revealed a very higher effect improvement compared to crotignoid K (13), indicating that configuration at the C-4 of tigliane diterpenoids is critical to HIV-1 latency reversal activity. (+)-Syringaresinol (26) and dimeric coniferyl acetate (27) showed no exhibition of CD69 expression, suggesting a non-PKC mechanism of latency reversal. Our results provide insights into the stereochemistry importance of bioactive diterpenoids and suggest that isolated compounds from E. usambarica can further research and development into therapeutic strategies for HIV-1 management, particularly as reactivators of latent HIV-1.In this study, 4


Bacillus cereus toxin cereulide (1) poses a significant safety risk in the food industry, causing emesis and nausea after consumption of contaminated foods. Analogously to cereulide, the structures of various isocereulides, namely, isocereulides A–G, have been recently reported and could also be identified in B. cereus-contaminated food samples. The HPLC fractionation of B. cereus extracts allows us to isolate additional isocereulides. By applying MSn sequencing, post-hydrolytic dipeptide, amino acid and α-hydroxy acid analyses using UPLC-ESI-TOF-MS to purify the analytes, seven new isocereulides H–N (2–8) could be elucidated in their chemical structures. The structure elucidation was supported by one-dimensional and two-dimensional NMR spectra of the isocereulides H (2), K (5), L and N (6 + 8) and M (7). The toxicity of 2–8 was investigated in a HEp-2 cell assay to determine their respective 50% effective concentration (EC50). Thus, 2–8 exhibited EC50 values ranging from a 0.4- to 1.4-fold value compared to cereulide (1). Missing structure-activity correlations indicate the necessity to determine the toxic potential of all naturally present isocereulides as single compounds to be able to perform a thorough toxicity evaluation of B. cereus-contaminated foods in the future.The emetic  Bacillus cereus is commonly categorized as a food-borne pathogen, causing gastrointestinal diseases and emesis arising from the production of various enterotoxins and the emetic toxin cereulide (1) +, [M + K]+ and [M + NH4]+ + . Guided cidation . FurtherStructure elucidation of isocereulides H–N (2–8). Following the recently reported structures for isocereulides A–G [+ pseudomolecular ions of the newly detected isocereulides H–N (2–8) were found to vary by 14–28 Da from the [M + Na]+ adduct of cereulide  (2 was detected as having an m/z of 1161.6522 ([M + Na]+), resulting in the elemental composition of C56H94N6O18Na. The observed mass shift of −14 Da indicated an overall loss of one methylene group compared to 1′s structure  and led toward the possible exemplary exchange of one Ala by one Gly, one Val by one 2-Abu moiety, one O-Leu by one O-Val, or one O-Val by one 2-hydroxybutanoic acid unit. Via UPLC-ESI–-TOF-MS analysis, after alkaline hydrolysis, the released dipeptides of dodecadepsipeptides 1–8 were investigated to gain a first insight into their amino acid and α-hydroxy acid composition. Analyzing the alkaline hydrolysate of 2, d-O-Leu-d-Ala , L-O-Val-L-Val  and one other dipeptide  were observed  .Exemplarobserved b in a ra–-TOF-MSe analysis of the candidate dipeptide released from 2  revealed an MSe fragmentation pattern with fragment ions at an m/z of 144.1020  and an m/z of 74.0243 , respectively. These observed m/z exhibited the expected pattern for O-Leu-Gly, providing the mother mass and the fragments formed after decarboxylation and cleavage of the peptide bond . The collected data on the released dipeptides of isocereulides H–N (2–8) are summarized in The eligible reference dipeptides for all isocereulide samples were synthesized through solid-phase peptide synthesis  and comp-Leu-Gly c and l-O-Ile-Gly d, respecl-O-Leu-Gly and d-O-Leu-Gly under given conditions demanded a subsequent stereospecific free amino acid and α-hydroxy acid UPLC-TOF-ESI–-MS analysis after acidic hydrolysis of alkaline hydrolysates. All hydrolyzed samples, including cereulide (1) and all respective enantiopure amino acids and α-hydroxy acids as references, were derivatized for specifying their stereochemistry. The obtained data from the analysis of isoindole-amino acid derivatives and MTPA-α-hydroxy acid esters from 2 . Fragmentation patterns differed only in the following MS4 scan, exhibiting key fragments (1: m/z 807.5 → 736.4/622.3. 2: m/z 793.5 → 734.4/622.3), thereby revealing the elimination of one Gly followed by one O-Leu moiety for 2. The accordance in the fragment ions of the respective MS5 spectrum  confirmed the preservation of the basic amino acid and α-hydroxy acid sequence of 1. Consequently, the structure of the cyclic isocereulide H (2) was identified as [2] ] a with it3–8) were elucidated  was identified as [2]. For isocereulide I (3), the differing dipeptide was identified as l-O-Val-l-2-Abu, resulting in the chemical structure [2]. Interestingly, the exchange of one amino acid by one non-proteinogenic amino acid has not been reported in a naturally occurring isocereulide so far. The structure of isocereulide K (5) was identified as [2], matching the originally reported chemical structure of isocereulide A  for 6 and d-O-Leu-d-Ala-l-O-Val-l-Val-d-O-Leu-d-Ser-l-O-Val-l-Val-d-O-Ile-d-Ala-l-O-Val-l-Val for 8. Due to the isobaric properties of d-O-Leu and d-O-Ile, the position of the d-O-Ile-d-Ala moiety in 8 could not be set unequivocally; therefore, it is interchangeable with the present d-O-Leu-d-Ala moiety. The same applies to isocereulide M (7), where the structure was proposed as d-O-Leu-d-Ala-l-O-Val-l-Val-d-O-Leu-Gly-l-O-Val-l-Val-d-O-Ile-d-Ala-l-O-Val-l-Val. The data on alkaline hydrolysis, acidic hydrolysis and MSn sequencing of all analyzed isocereulides are summarized in the This way, the structures of isocereulides I–N  were determined from pre-fractionated mixtures or their purified reference substances, with all values ranging from 347.4 to 361.1 Å2  values for the K361.1 Å2 . Due to 50 of cereulide (1) and isocereulides H–N (2–8) via HEp-2 cell assayDetermination of EC. The testing principle of the HEp-2 cell assay is based on the uncoupling of ATP synthesis, effectuated by supplemented ionophores, resulting in a facilitated ion influx into the mitochondria, thus disrupting the membrane potential. As a result, the 50% effective concentration (EC50) gives the amount of toxin required to inactivate half of the viable cells. The obtained EC50 for cereulide (1) and the structurally similar reference toxin valinomycin exhibited a discrepancy in respect to each other, displaying that 1 spawned a 13.7-times higher toxic effect than valinomycin at an equally applied concentration (data not shown). This result agrees well with literature findings, where 1 is described to hold an approx. 15-fold increased toxicity compared to valinomycin : δ (ppm) 0.83–0.94 , 0.84–1.00 , 1.00–1.19 ,1.65 , 1.70 , 1.81–2.02 , 2.02–2.19 , 2.36–2.61 , 4.13–4.24 , 4.44–4.54 , 4.60–4.68 , 4.73–4.84 , 5.29–5.34 , 5.38–5.46 , 5.55–5.62 , 5.62–5.70 , 8.60–8.68 , 8.86–8.91 , 8.97–9.06 , 9.08–9.14 , 9.25–9.30 . 13C-NMR : δ (ppm) 17.2 [C(5a)], 17.5 [C(5a)], 17.7 [C(8b/8c/11b/11c)], 18.0 , 19.18 , 19.23 [C(8b/8c/11b/11c)], 19.3 , 19.4 [C(8b/8c/11b/11c)], 19.7 [C(8b/8c/11b/11c)], 19.8 , 21.7 , 23.6 , 23.7 [C(2d)], 25.1 , 30.5 , 30.7 [C(8a)], 31.3 [C(8a)], 31.4 [C(11a)], 31.6 [C(8a)], 41.6 , 41.8 [C(2a)], 42.1 [C(14)], 49.9 [C(5)], 50.0 [C(5)], 59.4 , 59.8 [C(11)], 73.56 [C(2)], 73.63 [C(2)], 74.1 [C(2)], 79.1 [C(8)], 79.4 [C(8)], 79.5 [C(8)], 169.9 [C(15)], 171.0 , 171.6 [C(12)], 171.8 [C(3)], 172.08 [C(12)], 172.13 [C(12)], 172.17 , 172.9 [C(6)], 173.1 [C(6)].Isocereulide K (5)1H-NMR : δ (ppm) 0.82–1.06 , 1.06–1.24 , 1.65–1.77 , 1.89–2.05 , 2.05–2.19 , 2.42–2.54 , 2.55–2.65 , 4.69–4.77 , 4.80–4.86 , 5.41–5.46 , 5.48–5.52 , 5.56–5.60 , 5.62–5.70 , 8.87–8.93 , 8.93–8.98 , 9.05–9.20 , 9.15–9.20 , 9.21–9.29 .Isocereulide L (6) and Isocereulide N (8)1H-NMR : δ (ppm) 0.76–0.91 , 0.91–1.01 , 1.01–1.22 , 1.22–1.40 , 1.43–1.58 , 1.58–1.73 , 1.83–2.02 , 2.02–2.18 , 2.27–2.68 , 4.43–4.63 , 4.63–4.92 , 5.33–5.44 , 5.51–5.59 , 5.59–5.68 , 5.75 , 8.79–9.44 .Isocereulide M (7)1H-NMR : δ (ppm) 0.83–0.93 , 0.93–1.00 , 1.00–1.21 , 1.21–1.31 , 1.31–1.40 , 1.47–1.56 , 1.67 , 1.74 , 1.83–2.03 , 2.03–2.20 , 2.32–2.64 , 4.02 , 4.51 , 4.61 , 4.73–4.87 , 5.30 , 5.41–5.46 , 5.62 , 5.66 , 5.67 , 8.88 , 9.02 , 9.08 , 9.12 , 9.16 .


O-α-d-glucopyranosyl-6-O-hexadecanoyl-l-ascorbic acid , showed inhibitory effects on degranulation in vitro and on the passive cutaneous anaphylaxis (PCA) reaction in mice. In this study, several palmitoyl derivatives of ascorbic acid were synthesized and a structure–activity relationship study was performed to discover more potent ascorbic acid derivatives with degranulation inhibitory activity. 6-Deoxy-2-O-methyl-6-(N-hexadecanoyl)amino-l-ascorbic acid , in which a methyl group was introduced into the hydroxyl group at the C-2 position of ascorbic acid and in which the hydroxyl group at the C-6 position was substituted with an N-palmitoyl group, exhibited much higher inhibitory activity for degranulation in vitro than did 6-sPalm-AA-2G. 2-Me-6-N-Palm-AA strongly inhibit the PCA reaction in mice at lower doses than those of 6-sPalm-AA-2G. These findings suggest that 2-Me-6-N-Palm-AA may be a promising therapeutic candidate for allergic diseases.Allergy is an excessive immune response to a specific antigen. Type I allergies, such as hay fever and food allergies, have increased significantly in recent years and have become a worldwide problem. We previously reported that an ascorbic acid derivative having palmitoyl and glucosyl groups, 2- Type I allergies are an exaggerated response by the immune system centered on mast cells, and atopic dermatitis, atopic bronchial asthma, allergic rhinitis, and anaphylactic shock are typical diseases caused by type I allergies. IgE antibodies bound to high-affinity IgE receptors on the surface of mast cells and basophils in tissues and blood form a crosslinked structure when they interact with antigens, triggering a degranulation reaction ,2. This O-α-d-Glucopyranosyl-l-ascorbic acid  derivatives, having straight-acyl chains or branched-acyl chains of varying lengths − calcd for C23H39O7, 427.2701; found, 427.2692. 6-Palm-AA  was dissolved in DMF (60 mL), and K2CO3  and iodomethane  were added, and the reaction was carried out at room temperature for 45 min. After the reaction, the solution was separated with EtOAc and 2-M NaCl. The EtOAc layer was washed with 2-M NaCl, dehydrated with Na2SO4, concentrated, and subjected to Wakogel C-200 column chromatography. It was further recrystallized by n-hexane-EtOAc to purify 3-Me-6-Palm-AA . 1H NMR : δ 0.89 , 1.29 , 1.61 , 2.35 , 4.01 , 4.14 , 4.17 , 4.21 , 4.69 . 13C NMR : δ 13.0, 22.3, 24.5, 28.7, 28.97, 29.05, 29.2, 29.28, 29.33 (x 3), 29.4 (x 2), 31.6, 33.4, 58.3 (C-1″), 64.3 (C-6), 66.5 (C-5), 75.6 (C-4), 119.9 (C-2), 150.6 (C-3), 171.3, 173.7 (C-1). HRMS (m/z): [M-H]− calcd for C23H39O7, 427.2701; found, 427.2684.An alkyl group was selectively introduced into the hydroxyl group at the C-3 position of 6-Palm-AA, referring to the method of Nihro et al. . 6-Palm-2CO3  and iodomethane  were added, and the reaction was carried out at room temperature for 30 min. After the reaction, the solution was separated with EtOAc and 2-M NaCl. The EtOAc layer was washed with 2-M NaCl, dehydrated with Na2SO4, and concentrated to give crude 3-Me-6-Palm-AA (oil). The 3-Me-6-Palm-AA was dissolved in DMSO (10 mL), and K2CO3  and iodomethane  were added, and the reaction was carried out at room temperature for 30 min. The reaction mixture was separated with EtOAc and 2-M NaCl. The EtOAc layer was washed with 2-M NaCl, dehydrated with Na2SO4, concentrated, and subjected to Wakogel C-200 column chromatography. Recrystallization with n-hexane-EtOAc gave 2,3-Me-6-Palm-AA . 1H NMR : δ 0.88 , 1.29 , 1.61 , 2.35 , 3.78 , 4.03 , 4.13 , 4.16 , 4.22 , 4.75 . 13C NMR : δ 13.0, 22.3, 24.5, 28.7, 28.97, 29.05, 29.2, 29.28, 29.33 (x 3), 29.4 (x 2), 31.6, 33.4, 58.7 (C-1″), 59.7 (C-1′) 64.1 (C-6), 66.4 (C-5), 75.5 (C-4), 122.9 (C-2), 158.1 (C-3), 170.4, 173.6 (C-1). HRMS (m/z): [M-H]− calcd for C24H41O7, 441.2858; found, 441.2865.6-Palm-AA  was dissolved in DMSO (10 mL), and K2CO3  and chloromethyl methyl ether  were added, and the reaction was carried out at room temperature for 30 min. After the reaction, the solution was separated with EtOAc and 2-M NaCl. The EtOAc layer was washed with 2-M NaCl, dehydrated with Na2SO4, and concentrated to give 6-O-hexadecanoyl-3-O-methoxymethyl-l-ascorbic acid . 6-O-Hexadecanoyl-3-O-methoxymethyl-l-ascorbic acid  was dissolved in DMSO (20 mL), and K2CO3  and iodoethane  were added. The reaction was carried out at 50 °C for 30 min. After the reaction, the solution was separated with EtOAc and 2-M NaCl. The EtOAc layer was washed with 2-M NaCl, dehydrated with Na2SO4, and concentrated. The resulting 2-O-ethyl-6-O-hexadecanoyl-3-O-methoxymethyl-l-ascorbic acid  was dissolved in 60% formic acid–30% EtOH–10% H2O (30 mL) and reacted at 80 °C for 30 min. After the reaction, the mixture was evaporated and then separated with EtOAc and 2-M NaCl. The EtOAc layer was washed with 2-M NaCl, dehydrated with Na2SO4, concentrated, and subjected to Wakogel C-200 column chromatography. 2-Et-6-Palm-AA  was purified by recrystallization with n-hexane-EtOH. 1H NMR : δ 0.88 , 1.26 , 1.31 , 1.61 , 2.36 , 4.02 , 4.09 , 4.17 , 4.24 , 4.77 . 13C NMR : δ 13.0, 14.0 (C-2′), 22.3, 24.5, 28.8, 28.97, 29.05, 29.2, 29.29, 29.33 (x 3), 29.4 (x 2), 31.6, 33.4, 64.2 (C-6), 66.4 (C-5), 67.3 (C-1′), 75.7 (C-4), 120.4 (C-2), 159.7 (C-3), 171.3, 173.7 (C-1). HRMS (m/z): [M-H]− calcd for C24H41O7, 441.2858; found, 441.2839.6-Palm-AA  was dissolved in DMF (20 mL), and K2CO3  and iodoethane  were added, and the reaction was carried out at room temperature for 3 h. After the reaction, the solution was separated with EtOAc and 2-M NaCl. The EtOAc layer was washed with 2-M NaCl, dehydrated with Na2SO4, concentrated, and subjected to Wakogel C-200 column chromatography. 3-Et-6-Palm-AA  was purified by recrystallization with n-hexane-EtOAc. 1H NMR : δ 0.90 , 1.30 , 1.36 , 1.62 , 2.36 , 4.02 , 4.16 , 4.23 , 4.54 , 4.70 . 13C NMR : δ 13.0, 14.4 (C-2″), 22.3, 24.6, 28.8, 29.0, 29.1, 29.2, 29.4 (x 6), 31.7, 33.4, 64.3 (C-6), 66.6 (C-5), 67.2 (C-1″), 75.7 (C-4), 119.3 (C-2), 149.8 (C-3), 171.4, 173.7 (C-1). HRMS (m/z): [M-H]− calcd for C24H41O7, 441.2858; found, 441.2858.6-Palm-AA  was dissolved in DMSO (10 mL), and K2CO3  and iodoethane  were added, and the reaction was carried out at 30 °C for 2.5 h. After the reaction, the solution was separated with EtOAc and 2-M NaCl. The EtOAc layer was washed with 2-M NaCl, dehydrated with Na2SO4, concentrated, and dried to give crude 3-Et-6-Palm-AA (oil). 3-Et-6-Palm-AA was dissolved in DMSO (20 mL), and K2CO3  and iodoethane  were added, and the reaction was carried out at 30 °C for 30 min. After the reaction, the solution was separated with EtOAc and 2-M NaCl. The EtOAc layer was washed with 2-M NaCl, dehydrated with Na2SO4, concentrated, and subjected to Wakogel C-200 column chromatography. 2,3-Et-6-Palm-AA  was purified by recrystallization with n-hexane-EtOAc. 1H NMR : δ 0.88 , 1.28 , 1.30 , 1.37 , 1.61 , 2.35 , 4.03 , 4.06 , 4.14 , 4.22 , 4.52 , 4.74 . 13C NMR : δ 13.0, 14.06 (C-2′), 14.07 (C-2″), 22.3, 24.5, 28.7, 28.97, 29.05, 29.2, 29.28, 29.33 (x 3), 29.4 (x 2), 31.6, 33.4, 64.1 (C-6), 66.5 (C-5), 67.9 (C-1′), 68.1 (C-1″), 75.6 (C-4), 121.2 (C-2), 157.5 (C-3), 170.7, 173.6 (C-1). HRMS (m/z): [M-H]− calcd for C26H45O7, 469.3171; found, 469.3184.6-Palm-AA  was dissolved in DMSO (20 mL), and K2SO4 (16.7 mL) for 24 h at room temperature. The reaction mixture was added to ice-water (100 mL) and filtered. The residue was extracted with diethyl ether (140 mL) and washed twice with 2-N HCl (70 mL) containing 30% isopropanol. The diethyl ether layer was dehydrated with Na2SO4, concentrated, washed twice with n-hexane (30 mL), and filtered under reduced pressure to afford 2-P-6-Palm-AA . 1H NMR : δ 0.90 , 1.30 , 1.62 , 2.37 , 4.13 , 4.18 , 4.26 , 4.84 . 13C NMR : δ 13.0, 22.3, 24.5, 28.8, 28.98, 29.05, 29.2, 29.29, 29.33 (x 3), 29.35 (x 2), 31.6, 33.4, 64.1 (C-6), 66.5 (C-5), 75.8 (C-4), 114.0 (C-2), 158.6 (C-3), 169.4, 173.7 (C-1). HRMS (m/z): [M-H]− calcd for C22H38O10P, 493.2208; found, 493.2218.2-P-6-Palm-AA was synthesized according to the method of Suzuki et al. . AA-2P·32O  was reacted with palmitic acid  in H2SO4 (50 mL) for 24 h at room temperature. The reaction mixture was added to ice-water (100 mL) and filtered. The residue was extracted with diethyl ether (140 mL) and washed twice with 2-N HCl (70 mL) containing 30% isopropanol. The extract was concentrated, washed twice with n-hexane (30 mL), and filtered under reduced pressure to afford 2-S-6-Palm-AA . 1H NMR : δ 0.90 , 1.31 , 1.62 , 2.37 , 4.08 , 4.18 , 4.25 , 4.73 . 13C NMR : δ 13.0, 22.3, 24.6, 28.8, 28.98, 29.05, 29.2, 29.29, 29.33 (x 3), 29.4 (x 2), 31.6, 33.4, 64.4 (C-6), 66.6 (C-5), 75.8 (C-4), 118.7 (C-2), 152.6 (C-3), 171.7, 173.7 (C-1). MS (m/z): [M+H]+ calcd for C22H39O10S, 495.2258; found, 495.2607.2-S-6-Palm-AA was synthesized according to the method of Suzuki et al. . AA-2S·2O-Methyl-l-ascorbic acid (2-Me-AA) was synthesized by referring to the method of Kato et al. − calcd for C23H40NO6, 426.2861; found, 426.2866.2-o et al. . AscorbiAntigen-mediated degranulation assays were performed as described in our previous report . BrieflyCalcium ionophore-mediated degranulation assays were performed as described in our previous report . Briefly6 cells/6.0 mL/dish and cultured under 5% CO2 at 37 °C for 24 h. The cell layer was washed with medium, 5.0 mL of the sample (80 μM) was added, and then, the cells were incubated for 20 min. The supernatant was collected in a centrifuge tube, and the cell layer was washed twice with DPBS (−). The cells were detached using 0.05% trypsin–0.02% EDTA and collected in a centrifuge tube, along with the supernatant, and then centrifuged . The supernatant was removed, and ice-cold DPBS (−) (5 mL) was added. The number of cells was counted. The cell layer after centrifugation  was stored at −80 °C. The cell layer was added to 80% AcCN–50-mM ammonium acetate (200 μL) and sonicated for extraction . After extraction, the supernatant after centrifugation  was analyzed by HPLC. Separation for 2-Me-6-Palm-AA and 2-Me-6-N-Palm-AA was achieved by the isocratic elution of an Inertsil Ph-3 column  kept at 40 °C with 70% MeOH/H2O containing 0.5% formic acid at a flow rate of 0.7 mL/min. The absorbance at 240 nm was monitored. Separation for AA, 2-Me-AA, and 6-Amino-2-Me-AA was achieved by the isocratic elution of an Inertsil Amide column  kept at 40 °C with 80% AcCN–50-mM ammonium acetate at a flow rate of 0.7 mL/min. The absorbance at 260 nm was monitored.RBL-2H3 cells were seeded in 60-mm dishes at a density of 3 × 102O = 20/5/0.5/74.5 (v/v/v/v). After 3.5 h, the mice received an intravenous injection of saline containing DNP-HSA and Evan’s blue. After 30 min, each ear was removed and dissolved in 1-N KOH solution. The extravasated Evan’s blue dye was extracted with an acetone—0.3-M phosphoric acid (13:5) solution and centrifuged. Absorbance was measured (620 nm), and the percentage of the inhibitory effect on the PCA reaction was calculated.The PCA reaction inhibitory activities were evaluated as described in our previous report . An IgE-t-test. Multiple data comparisons were performed by analysis of variance, followed by Dunnett’s test (* p < 0.05 and ** p < 0.01).In vitro results were expressed as the means and SD, and in vivo results were expressed as the means and SE. Comparison of the two means was performed by the O-methyl-6-(N-hexadecanoyl)amino-l-ascorbic acid , that has higher antiallergic activity than that of 6-sPalm-AA-2G, an ascorbic acid derivative that was previously found to have antiallergic activity. The structure–activity relationship study of the 6-Palm-AA derivatives showed that retention of the hydroxyl group at the C-3 position of ascorbic acid is important for expression of the activity and that relatively small molecules such as a methyl group are suitable for substitution of the hydroxyl group at the C-2 position. Furthermore, the introduction of a palmitoyl group via an amid bond was found to result in greater degranulation inhibition and greater PCA reaction inhibition. The results indicate that 2-Me-6-N-Palm-AA is an effective drug candidate for the treatment of type I allergies.In this study, we succeeded in creating a new ascorbic acid derivative, 6-deoxy-2-


In parallel, the π electron-donor or electron-withdrawing character of the functional groups could modify the Highest Ocuppied Molecular Orbital (HOMO)–Lowest Unocuppied Molecular Orbital (LUMO) energy gap. Reactions of M(OAc)2·2H2O (M = Zn(II) and Cd(II); OAc = acetate) with 1,3-benzodioxole-5-carboxylic acid  and 4-acetylpyridine (4-Acpy) or isonicotinamide (Isn) resulted in the formation of four complexes. The elucidation of their crystal structure showed the formation of one paddle-wheel [Zn(μ-Pip)2(4-Acpy)]2 (1); a mixture of one dimer and two monomers [Zn(µ-Pip)(Pip)(Isn)2]2·2[Zn(Pip)2(HPip)(Isn)]·2MeOH (2); and two dimers [Cd(μ-Pip)(Pip)(4-Acpy)2]2 (3) and [Cd(μ-Pip)(Pip)(Isn)2]2·MeOH (4). They exhibit bridged , bridged, chelated and monodentated , or simultaneously bridged and chelated  coordination modes. Zn(II) centers accommodate coordination numbers 5 and 6, whereas Cd(II) presents coordination number 7. We have related their photophysical properties and fluorescence quantum yields with their geometric variations and interactions supported by TD-DFT calculations.Aggregation between discrete molecules is an essential factor to prevent aggregation-caused quenching (ACQ). Indeed, functional groups capable of generating strong hydrogen bonds are likely to assemble and cause ACQ and photoinduced electron transfer processes. Thus, it is possible to compare absorption and emission properties by incorporating two ligands with a different  Conversely, Cd(II) is likely to construct double bridged [Cd2(CO2)2] cores with four N-donor ligands, being the heptacoordinated environment is completed by chelate carboxylates and by designing a [Cd2(CO2)4]−2+2 motif. This core is commonly hindered for Zn(II) complexes  paddle-wheel and three dimeric [Cd2(CO2)4]−2+2 double bridged complexes were synthesized and their crystal structures were elucidated. The UV-Vis absorption measurements identified 4-Acpy as a promising ligand to avoid ACQ by minimizing intermolecular interactions. Fluorescence experiments confirmed the minimization of PET processes through complexation by enhanced fluorescence of the complexes with respect to the free ligands. Likewise, the work performed using Hg(II), HPip and a range of pyridine derivative ligands amine) resulted in the formation of monomeric, dimeric and polymeric or oligomeric metal complexes 2 (1); a mixture of one dimer and two monomers [Zn(µ-Pip)(Pip)(Isn)2]2·2[Zn(Pip)2(HPip)(Isn)]·2MeOH (2); and two dimeric complexes [Cd(μ-Pip)(Pip)(4-Acpy)2]2 (3) and [Cd(μ-Pip)(Pip)(Isn)2]2·MeOH (4). As a continuation of this work, in this paper, we have studied the reaction between M(OAc)Besides, their molecular and supramolecular structures have been analyzed and their photophysical properties have been studied. These results have been supported by TD-DFT calculations. 1–4 were prepared via a combination of M(OAc)2·2H2O (M = Zn(II) and Cd(II); OAc = acetate) salts and 1,3-benzodioxole-5-carboxylic acid  with dPy = 4-acetylpyridine (4-Acpy) or isonicotinamide (Isn) in MeOH as solvent at room temperature (RT). The M:Pip:dPy molar ratio used in the synthesis was 1:2:4 for complexes 1 and 3; 1:2:1 for complex 2; and 1:2:2 for complex 4. The corresponding crystals suitable for X-ray crystallographic analysis were grown via slow evaporation of their mother liquors  or via recrystallization in MeOH (4).Complexes 1H, and 13C{1H} NMR spectroscopies, and single crystal X-ray diffraction method. In addition, their UV-Vis and fluorescence spectra were recorded and their molar absorptivity (ε) and quantum yield  appear at 1556 (1), 1534 and 1502 (2), 1539 and 1506 (3), or at 1539 and 1502 cm−1 (4), whereas bands for υs(COO) arise at 1385 (1), 1385 (2), 1384 (3) and at 1385 cm−1 (4).The calculated Δ values (νas(COO)-νs(COO)) )2]2 (1). It crystallizes in the triclinic P-1 space group and has a dimeric paddle-wheel structure (4N] core belonging to the four Pip ligands and a 4-Acpy linker (2-η1:η1) coordination mode. The bond lengths and angles are within the range of analogous paddle-wheel complexes [[Zn(µ-Pip)tructure a. Each Zy linker b and exhy linker  20] wit wit2, 3.395 Å) and O dioxole atom of Pip  Å) (ac plane.These paddle-wheel units are assembled in groups of four by π⋯π interactions between Pip (Cg(2)) and 4-Acpy (Cg(3)) ligands in a 4-Acpy-Pip-Pip-4-Acpy sequence, stacking them through the ¯01 direclong the  and C-H⋯98(2) Å) c. All of2]2·2[Zn(Pip)2(HPip)(Isn)]·2MeOH (2). It crystallizes in the triclinic P-1 space group and comprises one dimeric and two monomeric structural units with two occluded MeOH molecules in the unit cell  of 58.41° for Zn(1A) and 58.81° for Zn(1B) 2·MeOH (4). They crystallize in the triclinic P-1 and monoclinic P2/c space groups, respectively, and contain two crystallographically independent molecules in the unit cell (molecules A and B). Both complexes have a dimeric structure with four Pip ligands and four 4-Acpy (3) (4) (Pip)(4-Acpy)Acpy (3) a,b or fo (3) (4) a,b, in wgeometry . The bongeometry .2-η2:η1), and the remaining two Pip linkers complete the coordination sphere by a bidentate chelate (μ1-η2) coordination mode. Both dimers (3 and 4) present intramolecular π⋯π interactions between the aromatic rings of 4-Acpy and Isn, respectively  a,b.4 are held together by the amide⋯amide pattern engaged in a head-to-head disposition which orders the dimers in chains (A⋯A and B⋯B) parallel to the [anti also partake in a double N-H⋯O and m-C-H⋯O associating the chains between A⋯B dimers (The dimeric units in l to the  directioB dimers a–g. PlanB dimers h.S parameter (2(CO2)2] units and the dimeric paddle-wheel [Zn2(CO2)4], bearing tetrahedral and octahedral geometries in [Zn2(CO2)2], or square pyramidal environments in [Zn2(CO2)4]. In turn, the most common motif in Cd(II) is the double bridged [Cd2(CO2)2], completed by two chelate ligands to form the [Cd2(CO2)2]−2+2 core and bearing coordination number 7  and the dimeric [Zn2(CO2)4]. The two display the same ligand disposition and distorted octahedral geometry, which seems to be stabilized by strong intermolecular N-H⋯O (dimer) and intramolecular O-H⋯O (monomer) interactions. In the monomeric unit, the µ1-η2 coordination mode combined with the intramolecular interaction between the HPip and Pip ligands force geometric constraints that accommodate the metal octahedral geometry ). By the same token, this deviation ) [2-η1:η1 and µ1-η1- coordination modes of the Pip ligands. Cd(II) dimers (3 and 4) present the common [Cd2(CO2)2]−2+2 core usually found bearing coordination number 7. Both dimers have similar S values (2.215 and 2.448 (3) or 1.995 and 2.015 (4))  a= 251 nm . In addiDFT calculations. The geometric optimization of complexes 1–4 has been performed in a MeOH solution using the Polarizable Continuum Model (PCM). The resulting complexes containing 4-Acpy exhibited an improvement of the geometric evaluator S from 0.240 to 0.181 (1) and from 2.215 and 2.448 to 2.048 and 1.889 (3). In contrast, complexes 2 and 4 presented significant differences between the X-ray diffraction data and the geometry in MeOH solution. The monomeric array in compound 2 is already stabilized by strong O-H⋯O intramolecular interactions between HPip and Pip ligands and suffers minor geometric changes with S values varying from 2.769 to 2.311. However, the dimer in 2, which is stacked in chains by strong amide⋯amide intermolecular interactions, presents significant differences between when they are in a solid state compared to being in solution. The S values change from 3.801, which corresponds to an octahedral geometry, to 8.571, acquiring a trigonal prismatic geometry  allows for a structural reorganization without such a marked difference in the S values, which shift from 2.015 and 1.995 to 3.367 and 2.288. Since MeOH has a solvent polarity parameter 2 (1). To a MeOH solution (15 mL) of Zn(OAc)2·2H2O , the 4-Acpy  was added drop wise, and the mixture was stirred for 5 min. Then, a MeOH solution (35 mL) of HPip  was added drop wise. The resulting solution was stirred for 5 h 30 min until a yellowish powder precipitated. The suspension was cooled down for 15 min and filtered off. The solid was washed twice with cold MeOH (5 mL) and dried under vacuum. Suitable colorless crystals were obtained via slow evaporation of the mother liquors for 6 days., 2912(w) [νal(C-H)], 1703(m) [ν(C=O)], 1638(m) , 1597(m) , 1556(m) [νas(COO)], 1502(m), 1437(s), 1385(s) [νs(COO)], 1355(m) , 1107(m), 1078(w), 1025(s) [δip(C-H)], 930(w), 919(m), 883(m), 807(m), 771(s) [δoop(C-H)], 730(m), 677(m), 588(m). 1H NMR : δ = 8.71 4-Acpy, 7.72 4-Acpy, 7.43 , 7.27 , 6.80 , 5.95 , 2.39 . 13C{1H} NMR : δ = 198.1 [OC-CH3], 171.4 [O2C-C], 150.8 [N-CH], 149.8 [O2C-C-(CH)2-C], 147.1 [O2C-C-CH-C], 143.1 [N-(CH)2-C], 128.9 [O2C-C], 124.7 [N-CH-CH], 121.8 [O2C-C-CH-CH], 109.5 [O2C-C-CH-C], 107.7 [O2C-C-CH-CH], 101.6 [O-CH2-O], 27.0 [-CH3]. UV-Vis in MeOH; wavelength : 203(4.93); 258(4.40); 282(4.20); 292(4.30).Yield: 365 mg (62%) (respect to Zn(OAc)2]2·2[Zn(Pip)2(HPip)(Isn)2]·2MeOH (2). To a MeOH solution (2 mL) of Zn(OAc)2·2H2O , a MeOH solution (6 mL) of Isn ligand  was added drop wise and the mixture was stirred for 5 min. Subsequently, a MeOH solution (16 mL) of HPip  was added drop wise. The solution was stirred for 4 h 30 min. Then, the reaction was concentrated until it reached half of the original volume, before being cooled down in an ice bath until a white powder precipitated. The resulting solid was filtered off, washed twice with cold Et2O (5 mL) and dried under vacuum. Suitable colorless crystals were obtained via slow evaporation of the mother liquors for 3 days.MeOH, 3318(w) [νas(N-H)], 3172(w) [νs(N-H)], 3111(w)–3071 (w) [νar(C-H)], 2904(w)–2789(w) [νal(C-H)], 1708(m) [ν(C=O)Isn], 1678(m) [ν(COOH)], 1629(m) , 1607(w) , 1553(m), 1534(m) [νas(COO)], 1502(m) [νas(COO)], 1435(s), 1417(s), 1385(s) [νs(COO)], 1353(s), 1300(m) , 1147(w), 1113(m), 1075(w), 1063(w), 1035(s) [δip(C-H)], 944(m), 928(m), 876(w), 861(m), 831(m), 804(m), 773(s) [δoop(C-H)], 720(m), 681 (m), 641(m), 580(m), 531(m). 1H NMR : δ = 8.66 Isn, 8.18 , 7.72 , 7.66 , 7.47 , 7.30 , 6.86 , 6.00 , 4.03 , 3.10 . 13C{1H} NMR : δ = 171.2 [O2C-C], 166.3 [OC-NH2], 150.2 [O2C-C-(CH)2-C], 149.6 [N-CH], 146.9 [O2C-C-CH-C], 141.6 [N-(CH)2-C], 128.9 [O2C-C], 124.6 [N-CH-CH], 121.6 [O2C-C-CH-CH], 109.4 [O2C-C-CH-C], 107.5 [O2C-C-CH-CH], 101.4 [O-CH2-O]. UV-Vis in MeOH; wavelength : 204(4.59); 225(3.24); 251(4.32); 276(4.05); 285(4.13); 292(4.26).Yield: 208 mg (83%) (respect to Zn(OAc)2]2 (3). To a MeOH solution (20 mL) of Cd(OAc)2·2H2O , a MeOH solution (30 mL) of HPip  and 4-Acpy  was added drop wise. The resulting solution was stirred for 19 h, concentrated to half of the original volume under vacuum, and kept in the fridge for 2 days until a white precipitate was formed. The powder was filtered, washed twice with cold MeOH (10 mL) and dried under vacuum. Suitable colorless crystals were obtained via slow evaporation of the mother liquors for 8 days., 3034(w) [νar(C-H)], 2905(m)–2779(w) [νal(C-H)], 1693(m) [ν(C=O)], 1632(w) , 1603(w) , 1539(m) [νas(COO)], 1506(m) [νas(COO)], 1436(m), 1415(m), 1384(s) [νs(COO)], 1360(s) , 1254(m), 1170(w), 1113(m) [ν(C-O-C)], 1035(m) [δip(C-H)], 1014(m), 923(m), 822(m) [δoop(C-H)], 806(m) [δoop(C-H)], 775(s) [δoop(C-H)], 682(m), 588(m), 530(s). 1H NMR : δ = 8.82 4-Acpy, 7.82 4-Acpy, 7.55 , 7.40 , 6.92 , 6.07 , 2.63 . 13C{1H} NMR : δ = 198.5 [OC -CH3], 172.3 [O2C-C], 151.2 [N-CH], 149.9 [O2C-C-(CH)2-C], 147.3 [O2C-C-CH-C], 143.1 [N-(CH)2-C], 129.5 [O2C-C], 125.1 [N-CH-CH], 121.9 [O2C-C-CH-CH], 110.0 [O2C-C-CH-C], 107.9 [O2C-C-CH-CH], 101.8 [O-CH2-O], 27.3 [-CH3]. UV-Vis in MeOH; wavelength : 203(4.95); 257(4.51); 291(4.45).Yield: 141 mg (55%) (respect to Cd(OAc)2(Isn)2]2·MeOH (4). To a MeOH solution (2 mL) of Cd(OAc)2·2H2O , a solution (6 mL) of Isn ligand  was added drop wise and the mixture was stirred for 5 min. Subsequently, a MeOH solution (16 mL) of HPip  was added drop wise. After 5 min of stirring, a white powder precipitated. The reaction was kept under stirring conditions for 5 h. Then, it was cooled down in an ice bath and the resulting solid was filtered off, washed twice with cold Et2O (5 mL) and dried under vacuum. Suitable colorless crystals were obtained via recrystallization in MeOH., 3316(w) [ν(N-H)], 3171(m) [ν(N-H)], 3069(w) [νar(C-H)], 2972(w)–2900(w) [νal(C-H)], 1704(m) [ν(C=O)Isn], 1626(m) , 1611(w) , 1553(sh.), 1539(s) [νas(COO)], 1502(m) [νas(COO)], 1434(s), 1415(m), 1385(s) [νs(COO)], 1349(m) , 1147(w), 1108(m), 1066(w), 1039(s) [δip(C-H)], 1015(m), 942(w), 921(m), 885(w), 855(m), 825(m), 806(m), 777(s) [δoop(C-H)], 723(m), 684(m), 641(s), 581(m), 530(m). 1H NMR : δ = 8.71 Isn, 8.23 Isn, 7.76 , 7.72 Isn, 7.54 , 7.39 , 6.91 , 6.06 . 13C{1H} NMR : δ = 172.1 [O2C-C], 166.6 [OCNH2], 150.5 [O2C-C-(CH)2-C], 149.7 [N-CH], 147.1 [O2C-C-CH-C], 141.6 [N-(CH)2-C], 141.1 [O2C-C], 129.2 [N-CH-CH], 124.9 [O2C-C], 121.8 [O2C-C-CH-CH], 109.8 [O2C-C-CH-C], 107.7 [O2C-C-CH-CH], 101.6 [O-CH2-O]. UV-Vis in MeOH; wavelength : 203(4.94); 256(4.41); 285(4.24).Yield: 325 mg (72%) (respect to Cd(OAc)1, 2 and 4) and yellow (3) prism-like specimens were used for the X-ray crystallographic analysis. The X-ray intensity data were measured on a D8 Venture system  equipped with a multilayer monochromator and a Mo microfocus (λ = 0.71073 Å). For 1–4, the frames were integrated with the Bruker SAINT software package  using a narrow-frame algorithm. For 1, the integration of the data yielded 8,790 independent reflections  and 7,734 (87.99%) were greater than 2σ(|F|2). The root-mean-square (RMS) deviation in the largest hole was 0.092 e-/Å3. For 2, the integration of the data yielded 18,771 independent reflections  and 10,867 (57.89%) were greater than 2σ(|F|2). The RMS deviation in the largest hole was 0.127 e−/Å3. For 3, the integration of the data yielded 12,559 independent reflections  and 9725 (77.43%) were greater than 2σ(|F|2). The RMS deviation in the largest hole was −1.028 e−/Å3. For 4, the integration of the data yielded 18,818 independent reflections  and 12,418 (65.99%) were greater than 2σ(|F|2). The RMS deviation in the largest hole was 0.123 e−/Å3.Colorless (1–4 were solved and refined using the Bruker SHELXTL Software Package (version-2018/3) [1–4 are reported in 1), 2124895 (2), 2124894 (3) and 2124892 (4). The molecular graphics were generated with Mercury 4.2.0 software [The structures of -2018/3) . The finsoftware  using thsoftware . The colMethodology and computational details. All of the calculations were performed using Gaussian09 software version D.01 [1–4 were completed using density functional theory (DFT) and time-dependent DFT (TD-DFT), respectively, using ωB97X-D [2 were optimized separately. The frequencies were also computed for each optimized structure to ensure that the geometries corresponded to an energy minimum. The HOMO and LUMO energetic levels were firstly examined and then the energy gaps calculated. For 1–4, the first 80 vertical absorptions from the ground state to the excited states were calculated and only the most probable transitions, those with higher oscillator strength (f) values, were selected for the electronic analysis. The shift in the theoretical absorption spectra, with respect to the experimental profile, is within the range of typical TD-DFT calculations (~0.3 eV) and are caused by computing the absorptions as vertical transitions [ion D.01 . The geo ωB97X-D ,41 funct ωB97X-D . The MeO ωB97X-D ,44. Sincnsitions .The methodology for the generation of the MOs was the same as has been previously reported  S.I.: F. The ana


Saccharomonospora piscinae that was isolated from dried fishpond sediment of Kouhu township, in the south of Taiwan, led to the isolation of three new compounds, saccharpiscinols A–C , and three new natural products, namely (2S)-5,7,3′,4′-tetrahydroxy-6,8-dimethylflavanone (4), methyl-4-hydroxy-2-methoxy-6-methylbenzoate (5), and (±)-7-acetyl-4,8-dihydroxy-6-methyl-1-tetralone (6). Compounds 4–6 were reported before as synthesized products, herein, they are reported from nature for the first time. The structures of the new compounds were unambiguously elucidated on the basis of extensive spectroscopic data analysis  and comparison with literature data. The effect of some isolates on the inhibition of NO production in lipopolysaccharide-activated RAW 264.7 murine macrophages was evaluated. Saccharpiscinol A showed inhibitory activities against LPS-induced NO production.Phytochemical investigation and chromatographic separation of extracts from the actinobacteria strain  T, which was isolated from a fishpond sediment sample collected from the southern area of Taiwan and had a unique morphology , two aromatic singlet protons at δH 6.52  and 6.49 , four mutually-coupling aliphatic protons  in ring C, two methoxyl signals at δH 3.81, 3.87 , and two active hydrogens at δH 5.60  and 5.74 . The 1H-1H COSY experiment of 1 revealed two spin systems of H-2/CH2–3/CH2–4 and H-5′/H-6′, which further confirmed the presence of resonances attributed to aliphatic protons belonging to the C ring, and aromatic resonances attributed to protons belonging to the B rings of flavan moieties. The 13C NMR data  showed 17 carbon resonances, comprising 12 olefinic carbons, two methylene carbons (δC 24.7 and 30.0), two methoxyl carbons (δC 56.1 and 56.5), and an oxygenated methane carbon (δC 77.2). Analysis of the 1H and 13C NMR data of 1  exhibited a very close structural resemblance to (2S)-7-hydroxy-6-methoxyflavan  groups in (2S)-7-hydroxy-6-methoxyflavan by a methoxy group [δH 3.87 ] at C-4′ and an OH group [δH 5.60 ] at C-3′ in 1. In the HMBC experiment, the cross-peaks from OH-7 to C-7/C-8, from OH-3′ to C-2′, from the C-6-methyoxyl protons to C-6, and from OMe-4′ to C-4′, placed the methoxyl groups at C-6 and 4′, and the hydroxy groups at C-7 and 3′, respectively ), indicating (S)-configuration at C-2 in comparison with the reported data of natural flavan analogues  in 2 replaced a 4-phenol moiety  in the C-2 position in (−)-4′-hydroxy-7-methoxy-8-methylflavan +, implying a molecular formula of C16H16O4. This was confirmed by the HREI-mass spectrum . The UV absorptions (232 and 283 nm) were similar to those of (−)-4′-hydroxy-7-methoxy-8-methylflavan D value of 3 with (2S)-2′,4′-dihydroxy-7-methoxy-8-methylflavan + peak at m/z 316.0945  in its HR-EI-MS. The UV absorptions  confirmed the presence of a flavonoid nucleus  in the B ring which were confirmed by HMBC correlations -5,7,3′,4′-tetrahydroxy-6,8-dimethylflavanone. This is the first time that compound 4 has been isolated from a natural source, although it had previously been synthesized + peak at m/z 196.0733 in its HREIMS spectrum . There are 10 carbon signals in the 13C NMR spectrum, including one ester C=O at δC 168.0, one methyl group at δC 19.7, two methoxyls at δC 51.7 and 55.9, and six aromatic carbon atoms with one deshielded signal at δC 159.6. The 1H and 13C NMR spectra of 5 are quite similar to those of methyl-2,4-dihydroxy-6-methyl benzoate, which suggested that compound 5 has a benzenoid structure. Comparing the 1H and 13C NMR spectra of compound 5 with those of the similar analogue, methyl-2,4-dihydroxy-6-methyl benzoate, it was presumed that a hydroxyl group  at C-2 of methyl-2,4-dihydroxy-6-methyl benzoate was substituted with a methoxy moiety in 8 from the signals at , which were confirmed by HMBC correlations. The HMBC correlations of OMe-2/C-2′ and Me-6/C-6, 1 and 5 determined the positions of methoxyl and methyl groups, which was confirmed by the correlations between OMe-2/H-3 and Me-6/H-5 in the NOESY spectrum  on an Inertsil ODS-3 column, 5 μm, 250 mm × 4.6 mm i.d.  using gradient of (A) water containing 0.1% phosphoric acid and (B) acetonitrile: 0–50 min, 90–50% A; 50–80 min, 50% A-100% B; flow 1 mL/min. Chromatograms were recorded at wavelength 237 nm. , we evaluated the inhibitory effects of saccharpiscinols A–C  and (2S)-5,7,3′,4′-tetrahydroxy-6,8-dimethylflavanone (4) on the production of NO induced by LPS -induced nitric oxide (NO) generation. The high cell viability (>80%) indicated that the inhibitory activity of LPS-induced nitrite production by compounds 1 and 2  did not result from its cytotoxicity. Compounds 3 and 4  also showed inhibition of the NO production of macrophages, but the low cell viability (<80%) suggested the possibility of cytotoxicity.Due to the small quantity of isolated compounds (d by LPS . They sh254 precoated plates (Merck). Column chromatography (CC): silica gel 60  and Spherical C18 100A Reversed Phase Silica Gel (RP-18) (particle size: 20–40 μm) (Silicycle). HPLC: Spherical C18 column  (Waters); LDC-Analytical-III apparatus. UV Spectra: Jasco UV-240 spectrophotometer; λmax (log ε) in nm. Optical rotation: Jasco DIP-370 polarimeter; in CHCl3. IR Spectra: Perkin-Elmer-2000 FT-IR spectrophotometer; ν in cm−1. 1H-, 13C- and 2D-NMR spectra: Varian-Mercury-400 and Varian-Unity-Plus-400 spectrometers; δ in ppm relative to Me4Si, J in Hz. ESI and HRESIMS: Bruker APEX-II mass spectrometer; in m/z.TLC: silica gel 60 FSaccharomonospora piscinae (06168H-1T) was used throughout this study, and deposited at the Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI). This actinobacteria was identified by M. T.Saccharomonospora piscinae, (06168H-1T), was isolated from dried fishpond sediment from the Kouhu township, in the south of Taiwan. The sample of fishpond sediment was dried at room temperature for 7 days. The sample suspension (100 µL) was plated on modified Sabouraud glucose agar  and incubated at 30 °C for 4 weeks. The spores or mycelia suspension were harvested with 20% (v/v) glycerol and stored at −20 °C.The actinobacteria, Saccharomonospora piscinae, (06168H-1T), was maintained on Sabouraud glucose agar (SGA) and the strain was cultured on potato dextrose agar slants at 30 °C for 7 days, and then the spores were harvested by sterile water. The spores (5 × 105) were seeded into 500 mL shake flasks containing 50 mL CM+YM  and cultivated with shaking (150 rpm) at 30 °C for 5 days. After the mycelium enrichment step, an inoculum mixing 100 mL mycelium broth and 100 mL CM+YM medium was inoculated into plastic boxes (25 cm × 30 cm) containing 1.3 kg sterile rice and cultivated at 30 °C for producing rice, and the abovementioned CM+YM medium was added to maintain the growth. After 21 days of cultivation, the rice was harvested, and used as a sample for further extraction.The actinobacteria, v/v, gradient; from 10:1 to 0:1 to generate seven fractions (Frs. 1–7). Fr. 2 was isolated by CC on silica gel eluted with hexane-EtOAc (from 5:1 to 1:1) to afford four subfractions (Fr. 2.1–2.4). Subfraction Fr. 2.3 was further purified by semi-preparative HPLC  to obtain 6 (1.2 mg). Subfraction Fr. 2.4 was further separated by HPLC  to obtain 5 (1.51 mg). Compound 3 (10.4 mg) was preferentially precipitated from Fr. 3. Fr. 5 was separated into 13 subfractions (Fr. 5–1—Fr. 5–13) by silica gel column chromatography eluted with a step gradient of petroleum ether–EtOAc . Fr. 5–2 was subjected to analytical RP-HPLC (85% MeOH–H2O) to obtain 4 (3.2 mg). Fr. 5–5 was also purified by analytical RP-HPLC (77.5% MeOH–H2O) to afford compounds 1 (3.2 mg) and 2 (10.4 mg).The rice fermentation was extracted five times with an equal volume of EtOAc. The combined EtOAc layers were evaporated to dryness under reduced pressure to give an EtOAc extract (12 g) which was subjected to silica gel column chromatography (CC) (petroleum ether-EtOAc 1): oil; [α]25 D −24 ; UV (MeOH): 230 (4.14), 283.0 (3.85) nm; IR (Neat): 3400 (OH), 1609, 1510 (aromatic C=C) cm−1; 1H NMR : δH 6.97 , 6.89 , 6.82 , 6.52 , 6.49 , 5.60 , 5.47 , 4.88 , 3.87 , 3.81 , 2.85 , 2.66 , 2.12 , 2.04 ; 13C NMR : δc 149.2 (C-9), 146.2 (C-4′), 145.6 (C-3′), 144.8 (C-7), 140.8 (C-6), 134.7 (C-1′), 112.2 (C-2′), 112.5 (C-10), 117.8 (C-6′), 111.4 (C-5), 110.5 (C-5′), 103.5 (C-8), 77.2 (C-2), 56.5 (OMe-6), 56.1 (OMe-6), 24.7 (C-4), 30.0 (C-3); HREIMS m/z 302.1153 [M]+ .Saccharpiscinol A (2): oil; [α]25 D = −15 ; UV (MeOH): 230 (4.14), 283.0 (3.85) nm; IR (Neat): 3400 (OH), 1609, 1510 (aromatic C=C) cm−1; 1H NMR : δH 6.84 , 6.57 , 6.43 , 4.92 , 3.95 , 3.85 , 2.87 , 2.70 , 2.15 , 2.10 , 1.93 ; 13C NMR : δc 156.7 (C-7), 153.2 (C-9), 148.8 (C-3′ and 5′), 139.3 (C-4′), 133.7 (C-1′), 126.1 (C-5), 114.2 (C-10), 113.8 (C-8), 105.5 (C-2′ and 6′), 103.1 (C-6), 77.2 (C-2), 61.1 (OMe-4′), 55.8 (OMe-7), 30.0 (C-3), 24.6 (C-4), 8.7 (Me-8); HREIMS m/z 316.1310 [M]+ .Saccharpiscinol B (3): oil; [α]25 D = −21.0 ; UV (MeOH): 232 (4.08), 283 (3.69) nm; IR (Neat): 3428 (OH), 1626 (aromatic C=C) cm−1; 1H NMR : δH 7.02 , 6.98 , 6.97 , 6.50 , 6.44 , 6.40 , 6.37 , 5.25 , 5.11 , 3.73 , 2.93 , 2.79 , 2.14–2.28 ; 13C NMR : δc 158.4 (C-7), 156.1 (C-2′), 155.6 (C-4′), 153.8 (C-9), 129.9 (C-5), 127.7 (C-6′), 108.3 (C-6), 117.7 (C-1′), 113.8 (C-10), 107.2 (C-5′), 104.1 (C-3′), 101.6 (C-8), 78.3 (C-2), 55.5 (OMe-7), 28.5 (C-3), 24.7 (C-4); EIMS (70 eV) m/z (%): 272 , 150 (42), 137 (90), 101 (20); HREIMS m/z 272.1047 [M]+ .Saccharpiscinol C (S)-5,7,3′,4′-Tetrahydroxy-6,8-dimethylflavanone (4): oil; [α]25 D = −22 ; UV (MeOH): 231 (4.44), 291 (4.35) nm; IR (Neat): 3394 (OH), 1721 (conjugated C=O), 1619, 1453 (aromatic C=C) cm−1; 1H NMR : δH 6.93, 6.80 , 6.79 , 5.24 , 3.02 , 2.71 , 2.00 , 1.99 ; 13C NMR : δc 198.4 (C-4), 164.1 (C-7), 160.2 (C-5), 159.3 (C-9), 146.7 (C-4′), 146.5 (C-3′), 132.2 (C-1′), 119.0 (C-2′), 116.2 (C-5′), 114.6 (C-6′), 104.7 (C-10), 104.1 (C-6), 103.3 (C-8), 80.0 (C-2), 44.2 (C-3), 8.2 (C-6), 7.4 (Me-8); EIMS (70 eV) m/z (%): 316 [M]+(100), 286 (12), 255 (10), 207 (13), 194 (20), 181 (68), 152 (52), 136 (17), 55 (10); HREIMS m/z 316.0945 [M]+ .The compound, (25): oil; + .The compound, (±)-7-Acetyl-4,8-dihydroxy-6-methyl-1-tetralone (6): gum; UV (MeOH): 221.0 (4.15), 253.0 (3.69), 281.0 (3.56) nm; IR (Neat): 3370 (OH), 1701 (ester C=O), 1604, 1454 (aromatic C=C) cm−1; 1H NMR : δH 8.57 , 6.34  6.29 , 3.76 , 3.72 , 2.16 ; 13C NMR : δc 168.0 (C-7), 159.2 (C-4), 158.4 (C-2), 137.8 (C-6), 116.0 (C-1), 109.1 (C-5), 97.1 (C-3), 55.9 (OCH3–2), 51.7 (OCH3–7), 19.7 (CH3–6); EIMS (70 eV) m/z (%): 196 , 165 (100), 122 (10); HREIMS m/z 196.0733 [M]+ .Methyl-4-hydroxy-2-methoxy-6-methylbenzoate  for 20 h. The concentration of NO in culture supernatants was determined as nitrite, a major stable product of NO, by a Griess reagent assay [Mouse macrophage cell line (RAW 264.7) was obtained from the Bioresource Collection and Research Center (BCRC 60001) and cultured at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (Gibco), 4.5 g/L glucose, 4 mM glutamine, penicillin (100 units/mL), and streptomycin (100 μg/mL) in a humidified atmosphere in a 5% COnt assay , and celnt assay .Under the support of the Ministry of Economic Affairs, the Bioresource Collection and Research Center at the Food Industry Research and Development Institute has been dedicated to the research work on the collection, separation, and preservation of bio-resource research in the past few years, and has constructed a complete indigenous strains resource bank in Taiwan. The applicant analyzed the active constituents from it, and obtained more than sixty active new compounds isolated from red yeast rice, endophytes, actinobacteria, and mushrooms, among which many new compounds have anti-cancer and anti-inflammatory effects ,21,22,23Streptomyces, there are also many rare species of actinobacteria, and there are many new species. Based on the concept of “new species and new compounds”, it is expected that special compounds can be found from these new strains. In recent years, studies have also found that these new species of actinobacteria can produce many active secondary metabolites. In order to further explore the efficacy of different strains of actinobacteria and expand the application range of actinomycetes, therefore, this project uses one new species of actinobacteria that has not been studied in the past. This species is cultured, extracted, purified and identified with high-level and highly active anti-inflammatory compounds with solid rice, in order to improve the research of actinobacteria at the country level, and develop health products related to actinobacteria. In summary, we have isolated and characterized three undescribed flavan derivatives, saccharpiscinols A–C, from an actinobacteria strain Saccharomonospora piscinae that was isolated from dried fishpond sediment from the Kouhu township, in the south of Taiwan. The absolute configurations of saccharpiscinols A–C were determined by comparing their optical activities with related derivatives. Saccharpiscinols A and B showed inhibitory activities against LPS-induced NO production. The discovery of flavan derivatives from actinobacteria pointed toward the potential use of endophytic or associated Saccharomonospora piscinae as alternative producers of flavan derivatives. The current results may encourage further investigations on the chemistry and bioactivity of flavan metabolites. These results also suggest that Saccharomonospora has distinct and diverse metabolites that arise under different fermentation conditions and soil-derived collections. It may therefore be possible to find more new bioactive natural products by searching Saccharomonospora species under special eco-environments. For the sake of better understanding the distribution of flavan acid analogs, the actinobacteria of the tilted research material and other special strains are worth examining for the presence of these secondary metabolites.Actinobacteria have the ability to produce a variety of physiologically active products, so they play a very important role in the food and pharmaceutical industries. Over the years, our team has also separated and collected actinomycetes resources from all over Taiwan and various environments. In addition to common