Second, a comprehensive metabolomic analysis conducted by ultrahigh-performance liquid-chromatography/electrospray-ionization mass spectrometry (UHPLC-Qtof-MS) coupled with multivariate statistics was performed to fully characterize the chemical components and discover the potential biomarkers between frankincense and processed frankincense. In total, 81 metabolites, including the 18 separated compounds, were selected as potential biomarkers between frankincense and processed frankincense among 153 detected compounds for their VIP values of greater than one. The tirucallane-type compounds and components with 9,11-dehydro structures clearly occurred at high levels in the processed frankincense, while lupine-type compounds and those with 11-keto structures were significantly higher in frankincense. Then, a network pharmacology model was constructed to decipher the potential mechanisms of processing. Intestinal absorption properties prediction indicated the possibility of processing-related absorption enhancement. A systematic analysis of the constructed networks showed that the C-T network was constructed with 18 potential biomarkers and 69 targets. TNF and IL-1β were among the top-ranked and were linked by 8 and 7 pathways, which were mainly involved in inflammation. The arachidonic acid metabolism pathway exhibited the highest number of target connections. Finally, the prediction was validated experimentally by an intestinal permeability and efficacy assay. The experiments provided convincing evidence that processed frankincense harbored stronger inhibition effects toward TNF-α-, IL-1β- and arachidonic acid-induced platelet aggregation. The processing procedure leads to changes of the chemical metabolites, which triggers the enhancement of absorption and cure efficiency. The global change of the metabolites, absorption and pharmacological effects of processing were depicted in a systematic manner.In-depth research on processing can promote the globalization of processed herbs. The purpose of this study is to propose an improved strategy for processing effect investigation. Frankincense and processed frankincense were used as research subjects. First, high-speed countercurrent chromatography (HSCCC) and preparation high-performance liquid chromatography (PHPLC) techniques were used for major compounds isolation and minor compounds concentration. Processed frankincense was subjected to two stepwise solvent systems, namely,  Herbal medicine is attracting increasing attention and acceptance in the world due to its special contribution to the treatment of chronic diseases. The processing of herbal medicine, called Pao Zhi, is a unique part of traditional Chinese medicine (TCM) and has been widely used in the preparation of Chinese materia medica for thousands of years. It can alter the properties and functions of remedies, increase medical potencies, and reduce the toxicities and side effects of raw herbs . In the in vitro and in vivo bioavailability evaluations of the multitarget treatment strategy.Chemical studies of processing provided useful information for understanding chemical changes. However, the identification and evaluation of the changes of compounds during processing are hampered by the content differences among complex components in herbal medicines and the limitations of analytical methods. This generates a critical requirement for appropriate methods to reveal definitive information. Additionally, phytochemical analysis is strongly dependent on efficient separation methodologies that are scalable to the preparative scale due to the need for bioactivity evaluation. Metabolomics is an ‘omics’ approach that aims to comprehensively analyze all metabolites in a biological sample . HoweverBoswellia sacra Flück. (B. carteri Birdw), B. papyrifera (Del.) Hochst., or B. serrata Roxb, has been used as a traditional medicine in many countries , the gum resin of ountries . Noteworbarriers . China ibarriers . This reIn this paper, an improved strategy is put forward to investigate processing effects of frankincense. Principal compounds separation and minor concentrated metabolites enrichment from frankincense and processed frankincense were conducted separately by HSCCC and PHPLC techniques for larger lab-scale preparation and subsequent structural identification. A comprehensive analysis including lower-concentration metabolites was conducted by UHPLC-Qtof-MS to entirely characterize the chemical components and to discover the potential biomarkers between frankincense and processed frankincense. Then, a network pharmacology screening was developed to predict the effect of processing. Finally, the prediction was validated experimentally through an intestinal permeability and efficacy assay. The global changes of the metabolites and the intestinal absorption and pharmacological effects of processing were supposed to be depicted in a systematic manner.Boswellia papyrifera according to the literature . U937 cells were from the Pasture Institute of Iran. FBS, RPMI-1640 medium, fetal bovine serum and penicillin/streptomycin were obtained from Gibco . The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo . Lipopolysaccharide (LPS) and propylene glycol methyl ether acetate (PMA) were purchased from Sigma-Aldrich . The Assay Design Enzyme-Linked Immunosorbent Assay kit (ELISA) was obtained from Beijing 4A Biotech Co., Ltd. . 3-Acetyl-11-keto-β-boswellic acid (AKBA) was purchased from the National Institute for Control of Biological and Pharmaceutical Products of China . 11-Keto-β-boswellic acid (KBA), α-boswellic acid (α-BA), β-boswellic acid (β-BA), 3-acetyl-α-boswellic acid (α-ABA), and 3-acetyl-β-boswellic acid (β-ABA) were purchase from ChromaDex Int. . Tsugaric acid A and β-elemolic acid were from Extrasynthese . β-Elemonic acid was obtained from Chengdu DeSiTe Biological Technology Co., Ltd. .In total, five batches frankincense were purchased from Beijing Tongrentang Medicine Corporation, Ltd. , Beijing Ben Cao Fang Yuan Medicine Corporation, Ltd.  and Beijing Qiancao Traditional Chinese Medicine Corporation, Ltd. . Their plant species were identified to belong to terature . Rice vi211AE induction cooker  equipped with temperature control and time programming functions was used for processing. The processed frankincense was prepared by stir-frying with rice vinegar according to the procedure recorded in the Chinese Pharmacopoeia (2015 edition). Briefly, 200 g frankincense was put into a heated pot at the temperature 240°C, stir-fried quickly for 9 min and sprayed with 20 mL rice vinegar [this component has been investigated (The Al-Fstigated ] during stigated . All theThe processed sample powder (1 kg) was extracted with 30.0 L methanol under sonication for 30 min. After filtration and evaporation, the methanol extraction (540.05 g) was obtained with a yield of 54.05%.n-heptane:methyl acetate:acetonitrile:water  was thoroughly mixed in a separatory funnel and allowed to stand until three clear layers were formed at 25°C. Then, the upper phase, middle phase and lower phase were separated. The sample solution was prepared by dissolving 1.0 g of the methanol extraction in 5 mL of the middle phase.A three-phase solvent system for HSCCC separation composed of 18 column  using a mobile phase of methanol and water (containing 0.1% formic acid) at a flow rate of 1.0 mL/min. The gradient program  was 60:40 (t = 0 min), 60:40 (t = 30 min), 80:20 (t = 35 min), 80:20 (t = 45 min), and 85:15 (t = 60 min). The peak areas of the upper phase and the middle phase were recorded as Aupper and Alower, respectively. The partition coefficients (K) of KBA, AKBA, α-BA, β-BA, α-ABA, and β-ABA were obtained by the following equation: DK = Aupper/Alower.The upper phase (2 mL) and the middle phase (2 mL) were delivered in glass test tubes in which 10 mg of methanol extraction was added, mixed thoroughly, and allowed to stand until two clear layers were formed. Each phase solution (5 μL) was determined by an Agilent 1200 HPLC apparatus . HPLC separation was performed on an Agilent XDB-CTM P-900  as the stationary phase. The apparatus was then rotated at 800 rpm while the lower phase was pumped into the coil column at a flow rate of 3.0 mL/min. After hydrodynamic equilibrium was reached, the retention rate was determined, and the sample solution was injected into the column. During the separation process, the column temperature was controlled at 25°C. The effluent from the coil-column was monitored by an ÄKTA PurifierTM UV-900 , and fractions were collected by an ÄKTA PurifierTM Frac-920 .The head-to-tail mode was applied for HSCCC separation. The upper phase was filled into the multilayer coiled columns of the TBE-300 HSCCC apparatus  by an ÄKTA Purifier18 column . The elution was carried out in isocratic mode with different ratios of methanol and water containing 0.1% formic acid at 5 mL/min. The detection wavelengths were 210, 250, and 280 nm. The PHPLC fractions were collected and evaporated to dryness.The fractions were further purified by a preparative HPLC apparatus  equipped with a G13611A prep pump, G2260A prep automatic sampler, G1315D diode array detector, G1364B prep fraction collector and Agilent Zorbax SB-C1 H-NMR and 13C-NMR spectra. The NMR spectra were recorded on Bruker AM-400 spectrometers  using trimethylsilyl (TMS) as the internal reference.The target compounds obtained by HSCCC and PHPLC separation were identified by their UV, MS,18 column  using a mobile phase of methanol and water (containing 0.1% formic acid) in the positive mode and of methanol and water (containing 0.2% formic acid and 5 mM ammonium formate) in the negative mode, both at a flow rate of 0.5 mL/min. The gradient programs of the positive and negative modes  were both 70:30 (t = 0 min), 85:15 (t = 10 min), 90:10 (t = 15 min), 92:8 (t = 22 min), 95:5 (t = 25 min), and 98:2 (t = 40 min). The injection volume of each sample was 2 μL. Both positive and negative modes were performed. The ESI conditions were as follows: gas temperature, 300°C; drying gas, 8 L⋅min-1 and nebulizer, 20 psi. The TOF conditions were as follows: fragmentor, 200 V; skimmer, 60 V and OCTRFV, 200 V. Accurately weighed powder samples (50 mg) of frankincense or processed frankincense were placed in conical flasks separately and sonicated with 10 mL methanol for 30 min. The supernatant solution was filtered through a 0.22-μm filter membrane. The HSCCC fractions were evaporated to dryness and redissolved by methanol and then filtered through a 0.22-μm filter membrane. Each sample was prepared in triplicate and detected twice using UHPLC-Qtof-MS.High-resolution mass spectra were measured on an Agilent 6520 Accurate-Mass TOF LC/MS . UHPLC separation was performed on an Agilent XDB-CThe mass data acquired were imported to MassHunter Profinder (version B.06.00) and Mass Profiler Professional (version B.02.00) for peak detection and alignment. The retention time and m/z data for each peak were determined by the software. The parameters were set as follows: the full scan mode was employed in the mass range of 100–600 amu; mass tolerance, 0.1 Da and noise elimination level, 5.p-value for all analyses in this study was set to 0.05.Multivariate statistical analyses, including unsupervised principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA), were performed using the Simca-P (version 13.0)  statistical package to find the potential biomarkers. The significant The structural identification of potential biomarkers between frankincense and processed frankincense was performed according to the retention time, UV-visible spectra and mass characteristics of the reference substances and separated compounds. Meanwhile, the relationships between the separated compounds and potential biomarkers were considered. The possessed discriminatory power of potential biomarkers was investigated by network pharmacology.1. With respect to absorption predictions of frankincense and processed frankincense having problems with multiple compound attributes, a decision analysis method was proposed. A probability value (Pij) and weight index (WI)  value and Caco-2 permeability values, were used to predict the absorption properties of the potential biomarkers the possessed discriminatory power. Their absorption parameters were obtained in TCMSPdex (WI)  of compon is the number of potential biomarkers possessing discriminatory power in m number of different frankincense samples. ijc represents the concentration of compound j in i kinds of frankincense samples.Here, To obtain the target of the biomarkers of frankincense and processed frankincense, a similarity ensemble approach (SEA)  and PharP-values were set at 0.05 as the cut-off criterion. These analytical results were annotated by Pathview (2.The latest pathway data were extracted from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database  for KEGGPathview  in the RThe component-target (C-T) network was constructed to find the key targets. Then, the target-pathway (T-P) network was established to determine the relationships between the targets and pathways. Cytoscape 3.5.1 , an open2 were continuously bubbled through the incubation buffer. The inner solution of the everted gut-sac was collected using a syringe fitted with a needle. An accurately measured 3 mL aliquot of the inner solution of the everted gut-sac and 10 mL ethyl acetate was added. The mixture was extracted three times. The organic layer was collected, dried by nitrogen, redissolved in 100 μL methanol and tested by ultrahigh-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QqQ-MS) for compound bioavailability investigation. This study was approved by the Research Ethics Committee of Institute of Basic Theory of Chinese Medicine, China Academy of Chinese Medical Sciences.The powders of frankincense and processed frankincense (3 g) obtained using an 80-mesh sieve were individually mixed in 500 mL 1% CMC-Na and ground for 10 min to obtain suspensions. The rat everted gut-sac model was chosen . It was An Agilent 1260 UHPLC/6410 QqQ system  equipped with an electrospray ionization (ESI) source  was utilized for determination of the inner solution of the everted gut-sac. The negative ion mode was chosen. The nitrogen temperature was kept at 300°C, the flow rate at 11 L/min, nebulizer at 15 psi, and the capillary voltage at 4,000 V.18 column  (0.5 mL/min) using a mobile phase of water (containing 0.1% formic acid and 10 mM ammonium formate) and acetonitrile with gradient elution. The gradient program  was 85:15 (t = 0 min), 85:15 (t = 20 min), 98:2 (t = 35 min), and 98:2 (t = 40 min). Each sample was prepared in triplicate. The injection volume of each sample was 2 μL. Bioavailability was evaluated by the uptake rate of the compounds. Rate of uptake = Cinnersolutionofevertedgut-sac/Cfrankincenseorprocessedfrankincensesuspensions × 100%.UHPLC condition was carried out on an Agilent XDB-C2. Next, 10 μL CCK-8 was added to incubate for another 3 h. For the validation assay, cells were seeded into a 12-well cell culture plate at a density of 1 × 106 cells/well and incubated with 100 ng/mL PMA for 48 h in a 5% CO2 incubator. Then, they were washed twice, stimulated by LPS and treated with or without frankincense and processed frankincense extracts at 8 μg/mL and 16 μg/mL and validated by CCK-8 assay for 48 h. Cell supernatants were harvested. TNF-α and IL-1β production were detected using an ELISA kit. All data were expressed as the mean ± standard deviation (SD). The statistical significance was evaluated using SPSS version 22.0 .Human leukemic U937 cells were cultured in RPMI-1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin. They were placed into a 96-well cell culture plate for cell viability assay using CCK-8. For this, 100 ng/mL PMA was added. Then, the cells were washed with PBS, followed by incubation with methanol extract of frankincense or processed frankincense (2–100 μg/mL) for up to 48 h at 37°C in 5% COFor the arachidonic acid (AA)-induced platelet aggregation assay, male rats were anesthetized with chloral hydrate (300 mg/kg). The blood drawn from the abdominal aortas was anticoagulated with frankincense and processed frankincense extracts (16 μg/mL) and heparin (20 U/mL). All platelet aggregation studies were performed using a Chrono-log platelet aggregometer . This system measures the increase in impedance caused by platelet aggregates between two pairs of electrodes, enabling two simultaneous measurements for duplicate analysis. Single-use cuvettes containing a Teflon-coated stirrer (800 rpm) were filled with prewarmed 500 μL physiologic saline and 500 μL whole blood. After 10 min of incubation, tests were initiated by adding a stimulating agonist to the test cell. In our study, we used AA (0.5 mM) as the agonist. Aggregation was recorded for 6 min, and the results were given in the maximum aggregation and area under the curve (AUC). AUC (aggregation × 6 min) in units and were used for further analysis.n-hexane, ethanol, and water with a volume ratio of 6:5:1 for the separation of triterpenoid acids from frankincense. Considering the high separation capacity of the three-phase solvent system for various compounds  are required for effective HSCCC separation  and flow rate  of the mobile phase, along with the separation temperature , were the main factors influencing the separation. Under the conditions of the same flow rate (3 mL/min) and the same separation temperature (25°C), the rotating speed of 800 rpm was proven to realize the best separation. The flow rate of 3 mL/min exhibited the best retention rates of the stationary phase, and the separation temperature was settled at room temperature (25°C). All optimized conditions were applied for HSCCC separation of the methanol extracts.The HSCCC separation chromatogram of processed frankincense is shown in Figure Eighteen compounds were isolated from the methanol extract of processed frankincense.-, corresponding to a molecular formula of C30H46O3. Both showed the same signal at δ 5.0–6.0  results were as follows: δ 5.68 , 5.48 , 5.33 , 1.38 , 1.19 , 1.13 , 0.93 , 0.86 , and 0.8 . The13C-NMR  results were: δ 35.7 (C-1), 27.2 (C-2), 76.2 (C-3), 56.3 (C-4), 45.7 (C-5), 16.1 (C-6), 32.5 (C-7), 41.9 (C-8), 154.9 (C-9), 38.6 (C-10), 116.1 (C-11), 121.2 (C-12), 145.3 (C-13), 42.7 (C-14), 25.8 (C-15), 27.0 (C-16), 32.8 (C-17), 45.6 (C-18), 46.8 (C-19), 31.2 (C-20), 34.1 (C-21), 36.0 (C-22), 12.5 (C-23), 181.0 (C-24), 20.0 (C-25), 21.2 (C-26), 24.5 (C-27), 26.7 (C-28), 26.9 (C-29), and 27.2 (C-30). The compound 9 1H-NMR  results were as follows: δ 5.68 , 5.48 , 5.33 , 1.38 , 1.16 , 1.11 , 1.03 , 0.93 , 0.86 , and 0.8 . The 13C-NMR  results were: δ 35.3 (C-1), 26.4 (C-2), 75.3 (C-3), 54.3 (C-4), 43.8 (C-5), 16.3 (C-6), 34.7 (C-7), 41.2 (C-8), 156.3 (C-9), 36.3 (C-10), 119.2 (C-11), 123.3 (C-12), 143.2 (C-13), 41.9 (C-14), 27.3 (C-15), 28.3 (C-16), 34.2 (C-17), 58.1 (C-18), 38.2 (C-19), 38.9 (C-20), 28.6 (C-21), 42.7 (C-22), 13.2 (C-23), 179.3 (C-24), 18.2 (C-25), 22.1 (C-26), 24.8 (C-27), 26.9 (C-28), 15.4 (C-29), and 23.8 (C-30). Compound 9 gave a longer retention time in HPLC analysis than did compound 3. This result was agreed with the conclusion that β-configurations always give longer retention times than do α-configurations. Therefore, compound 3 was identified as 9,11-dehydro-α-bowellic acid, and compound 9 was identified as 9,11-dehydro-β-bowellic acid  results were as follows: δ 5.67 , δ 5.49 , δ 5.33 , δ 2.08 , 1.32 , 1.18 , 0.93 , 0.89 , and 0.81 . The 13C-NMR  results were: δ 35.9 (C-1), 25.9 (C-2), 76.5 (C-3), 53.0 (C-4), 45.9 (C-5), 15.8 (C-6), 32.5 (C-7), 41.9 (C-8), 154.9 (C-9), 38.4 (C-10), 116.1 (C-11), 121.2 (C-12), 145.3 (C-13), 42.7 (C-14), 25.8 (C-15), 27.0 (C-16), 32.8 (C-17), 45.6 (C-18), 46.8 (C-19), 31.2 (C-20), 34.1 (C-21), 36.0 (C-22), 12.7 (C-23), 181.0 (C-24), 20.0 (C-25), 21.2 (C-26), 24.5 (C-27), 26.7 (C-28), 26.9 (C-29), 25.3 (C-30), 170.2 (C-31), and 21.9 (C-32). The compound 10 1H-NMR  results were as follows: δ 5.67 , δ 5.49 , δ 5.33 , δ 2.08 , 1.30 , 1.25 , 1.18 , 0.98 , 0.93 , 0.89 , and 0.81 . The 13C-NMR  results were: δ 33.69 (C-1), 24.33 (C-2), 76.67 (C-3), 57.37 (C-4), 46.9 (C-5), 17.4 (C-6), 33.19 (C-7), 41.34 (C-8), 152.5 (C-9), 39.03 (C-10), 116.56 (C-11), 123.05 (C-12), 141.63 (C-13), 43.39 (C-14), 26.22 (C-15), 28.27 (C-16), 31.86 (C-17), 47.44 (C-18), 39.42 (C-19), 39.06 (C-20), 28.71 (C-21), 40.66 (C-22), 23.25 (C-23), 181.45 (C-24), 19.53 (C-25), 21.19 (C-26), 23.67 (C-27), 31.20 (C-28), 21.77 (C-29), 21.50 (C-30), 170.20 (C-31), and 21.50 (C-32), The α and β isomers were differentiated according to the number of angular methyl signals. The compound 4 was identified as 3-acetyl-9,11-dehydro-α-bowellic acid and compound 10 was identified as 3-acetyl-9,11-dehydro-β-bowellic acid  results were as follows: δ 5.23 , δ 4.41 , 2.04 , 1.75 , 1.64 , 1.01 , 0.93, 0.91 , and 0.82 . The 13C-NMR  results were: δ 35.2 (C-1), 22.2 (C-2), 82.1 (C-3), 38.4 (C-4), 51.4 (C-5), 17.8 (C-6), 27.5 (C-7), 135.1 (C-8), 134.8 (C-9), 37.5 (C-10), 20.8 (C-11), 28.4 (C-12), 44.8 (C-13), 50.2 (C-14), 30.2 (C-15), 27.9 (C-16), 48.2 (C-17), 16.4 (C-18), 17.5 (C-19), 47.6 (C-20), 84.8 (C-21), 33.5 (C-22), 26.3 (C-23), 132.4 (C-24), 133.1 (C-25), 20.8 (C-26), 18.6 (C-27), 22.4 (C-28), 20.4 (C-29), 25.1 (C-30), 171.2 (C-31), and 19.1 (C-32). The compound was identified as 3β-acetoxy-5α-lanosta-8,24-dien-21-acid  results were as follows: δ 5.25 , 5.10 , 3.50 , 1.75 , 1.70 , 1.53 , 0.97 , 0.93 , 0.90 , and 0.75 . The 13C-NMR  results were: δ 31.2 (C-1), 25.3 (C-2), 76.5 (C-3), 41.2 (C-4), 44.5 (C-5), 23.9 (C-6), 118.2 (C-7), 145.7 (C-8), 48.3 (C-9), 38.1 (C-10), 17.5 (C-11), 33.4 (C-12), 43.3 (C-13), 51.1 (C-14), 33.9 (C-15), 30.9 (C-16), 47.1 (C-17), 18.8 (C-18), 21.8 (C-19), 49.7 (C-20), 181.4 (C-21), 32.4 (C22), 27.1 (C-23), 123.6 (C-24), 132.2 (C-25), 17.7 (C-26), 25.7 (C-27), 27.8 (C-28), 21.8 (C-29), and 27.3 (C-30). The compound was identified as 3α-hydroxy-tirucall-7,24-dien-21-oic acid  results were as follows: δ 5.25 , δ 5.10 , δ 4.70 , 2.06 , 1.71 , 1.58 , 0.97 , 0.93 , 0.91 , 0.90 , and 0.75 . The 13C-NMR  results were: δ 37.4 (C-1), 26.4 (C-2), 78.9 (C-3), 44.5 (C-4), 52.4 (C-5), 22.4 (C-6), 116.5 (C-7), 146.3 (C-8), 48.1 (C-9), 35.1 (C-10), 22.1 (C-11), 30.2 (C-12), 46.2 (C-13), 51.2 (C-14), 34.9 (C-15), 28.9 (C-16), 49.7 (C-17), 17.5 (C-18), 14.8 (C-19), 49.2 (C-20), 182 (C-21), 33.2 (C-22), 28.4 (C-23), 124.8 (C-24), 132.8 (C-25), 24.9 (C-27), 23.7 (C-28), 24.2 (C-29), 26.2 (C-30), 171.2 (C-31), and 14.2 (C-32). The compound was identified as 3α-acetyloxy-tirucall-7,24-dien-21-oic acid  results were as follows: δ 4.68 , δ 4.56 , and δ 4.05 . The 13C-NMR  results were; δ 38.1 (C-1), 27.1 (C-2), 75.90 (C-3), 53.0 (C-4), 41.7 (C-5), 20.1 (C-6), 34.9 (C-7), 40.9 (C-8), 50.0 (C-9), 31.7 (C-10), 21.7 (C-11), 25.4 (C-12), 37.9 (C-13), 42.9 (C-14), 26.4 (C-15), 35.6 (C-16), 43.0 (C-17), 48.4 (C-18), 48.0 (C-19), 150.3 (C-20), 29.8 (C-21), 40.0 (C-22), 9.8 (C-23), 181.0 (C-24), 15.8 (C-25), 18.5 (C-26), 15.0 (C-27), 18.5 (C-28), 110.6 (C-29), and 21.7 (C-30). The compound was identified as lupeolic acid  results were as follows: δ 4.69  δ 4.57 , δ 1.68 , and δ 2.08 . The 13C-NMR  results were: δ 38.8 (C-1), 26.8 (C-2), 76.84 (C-3). 37.0 (C-4), 55.1 (C-5), 15.9 (C-6), 34.3 (C-7), 40.9 (C-8), 49.9 (C-9), 37.2 (C-10), 19.9 (C-11), 27.6 (C-12), 38.3 (C-13), 43.0 (C-14), 30.0 (C-15), 35.6 (C-16), 43.1 (C-17), 48.4 (C-18), 48.1 (C-19), 152.2 (C-20), 33.7 (C-21), 40.1 (C-22), 21.6 (C-23), 21.2 (C-24), 15.93(C-25), 16.1 (C-26), 14.5 (C-27), 18.2 (C-28), 109.5 (C-29), 21.7 (C-30), 182.1 (C-31), and 19.5 (C-32). The fragmentation rules and NMR spectral characteristics of frankincense metabolites may aid in discovery and identification. This compound was identified as lupeol acetate , mobile phase composition , gradient elution procedure, flow rate of the mobile phase  and column temperature  were optimized to obtain the proper separation conditions. This demonstrated that the Agilent XDB-C18  column achieved the best separation, which was proven suitable for the analyses of α-amyrin and β-amyrin.Chromatographic parameters such as the column type  sample was produced by mixing equal aliquots of each frankincense sample. The precision was evaluated by injecting the QC sample six times. The reproducibility was determined by analyzing the six replicates of the QC sample within 1 day. A system stability test was carried out by injecting a QC sample every five samples during the whole sample analysis procedure.The extracted ion chromatographic peaks from five ions including the retention time, signal intensity and mass accuracy were selected for method validation in negative ion mode. The relative standard deviations (RSDs) of the retention time for the precision, reproducibility and system stability were 0.14%∼0.32%, 0.16%∼0.87% and 0.14%∼0.43%. The RSDs of the signal intensity were 2.83%∼3.55%, 3.49%∼4.31% and 3.86%∼4.79%. The RSDs of the mass accuracy were 0.14%∼0.26%, 0.22%∼0.29% and 0.23%∼0.31%.To investigate the chemical changes that occurred after processing, five batches of frankincense and processed frankincense were analyzed. Holistic chemical profiling and discrimination methods have been developed using UHPLC-Qtof-MS together with chemometric methods such as PCA and OPLS-DA.The total methanol extracts of frankincense and processed frankincense were analyzed. Meanwhile, the frankincense and processed frankincense HSCCC fractions yields in the Section “HSCCC Separation of Processed Frankincense” were also analyzed. Typical UHPLC chromatograms of frankincense and processed frankincense HSCCC fractions are shown in three-dimensional chromatograms Figure . The X–YR2X = 0.851, Q2= 0.685), as shown in Figure PCA and OPLS-DA were utilized to classify the metabolic phenotypes and identify the differentiating metabolites from the HSCCC fractions of frankincense and processed frankincense. A PCA score plot for the first and second principal components was utilized to depict the general variation among the samples  database, which yielded 10 KEGG pathways , including AA metabolism, the IL-17 signaling pathway, serotonergic synapses, insulin resistance, the TNF signaling pathway, the NF-kappa B signaling pathway, etc. (Supplementary Figure Furthermore, the canonical pathways associated with the targets were extracted from the Kyoto Encyclopedia of Genes and Genomes (KEGGAs the result of absorption prediction, processing was believed to have a positive effect on frankincense absorption. To confirm this idea, there is an urgent need for validation. Gastrointestinal permeability is a fundamental parameter determining oral drug absorption in oral administration, and the rat everted gut-sac model is a classical model utilized to investigate intestinal absorption . In the The procedure of optimizing the MRM transitions for all target analytes was as follows. The precursor and product ions were determined by standard solutions in scan and product ion modes. The fragmentor energy and collision energy parameters were further optimized based on the abundance of precursor and product ions (as shown in Supplementary Table 50 = 0.75 μM, while AKBA did so at an IC50 = 2.7 μM (As shown in the T-P network, TNF and IL-1β were among the top-ranking compounds and were linked by 8 and 7 pathways Figure . LPS is = 2.7 μM . This meAdditionally, AA and its metabolites in AA metabolism are significant precursors of various bioactive molecules in the cardiovascular system . They haThe aim of this study was to establish a strategy for realizing the investigation of the effects of processing procedures by comparing the global metabolites, (as shown in Figure AL and ZL provided the concept and designed the study. ZN, CW, and YL conducted the experiments and wrote the manuscript. ZN, CW, YL, ZS, XM, and DL participated in the experiments. AL and ZL contributed to revising and proofreading the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Castanopsis species (Fagaceae), three new phenolic compounds, -[1′-oxyethyl]-3-hydroxy-dihydrofuran-2(3H)-one (1), -2-[2′-oxyethyl]-dihydroxybutanoic acid (2), and -3-hydroxymethyl-3,4-dihydro-5,6,7-trihydroxy-4-(4′-hydroxy-3′-methoxyphenyl)-1H-[2]-benzopyran-1-one (3) were isolated from the fresh leaves of Castanopsis fargesii. In addition, a known phenolic glycoside, gentisic acid 5-O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranoside (4) was also isolated and identified. Their structures were elucidated by means of spectroscopic methods including one- and two-dimensional NMR techniques.In the course of a phytochemical and chemotaxonomical investigation of  Castanopsis belongs to the Fagaceae family and is commonly found in the evergreen forests of East Asia. There are about 120 species of Castanopsis, however, the classical plant taxonomy of the species is very complicated and sometimes confusing − and [M + Na]+ ion peaks at m/z 459.1143  and 483.1118 , respectively. In the 1H- and 13C-NMR spectra  and four aromatic carbon signals at δC 109.1 and 145.2, along with an ester carbonyl signal at δC 166.4 suggested the presence of a galloyl group -3-hydroxy-dihydrofuran-2(3H)-one.Compound  spectra , two proC 166.4) . A secon2 was isolated as a yellow amorphous powder and gave a positive FeCl3 test (dark blue). The presence of a galloyl group was deduced from the 13C-NMR signals  and 13C-NMR data. The 13C-NMR and DEPT spectra showed signals attributable to a methyl (δC 16.9), a methylene (δC 34.2), an oxymethine (δC 71.4), an oxymethylene (δC 60.2), an oxy quaternary (δC 77.7), and a carboxyl (δC 175.4) carbons. The NMR data of 2 -butanoic acid moiety could be constructed by 1H-1H COSY correlations (C 71.4), H-3 with C-1, H-1′ with C-1, and H-2′ with C-2 (C 165.9) indicated that the galloyl group was attached to C-2′. Based on these results, the structure of 2 was determined to be 2-[2′--oxyethyl]-2,3-dihydroxybutanoic acid. Compound  signals  6]. The. The2 waata of 2  were simelations  of H-4 wwith C-2 . The HMB2 were established using the modified Mosher’s method − peak at m/z 347.0768 in the LC-MS/IT-TOF . Comparison of the 1H- and 13C-NMR data of 3 -3-[(β-d-glucopyranosyl)oxymethyl]-3,4-dihydro-5,6,7-trihydroxy-4-(4′-hydroxy-3′-methoxyphenyl)-1H-[2]-benzopyran-1-one -benzopyran-1-one.Compound ata of 3  and  . Compariis 3S,4S . Based o50 value > 10 μM).All isolates were subjected to a cytotoxicity assay in vitro against human lung epithelial A549, human hepatocellular carcinoma SMMC-7721 cell, human gastric carcinoma MGC-803 cell, liver hepatocellular HepG2 cell, and human breast adenocarcinoma MCF-7 tumour cell. Unfortunately, none of the isolates showed inhibitions of those tumour cells at the highest concentration tested  was deposited in the Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute of Botany, China.The leaves of 1H- and 13C-NMR spectra were measured in acetone at 27 °C, using an Avance 500 spectrometer . Coupling constants and chemical shifts were given in Hz and on a δ (ppm) scale, respectively. GC was performed on a 6890N instrument equipped with a FID detector  operated at 280 °C . LC-MS/IT-TOF was recorded on a LCMS-IT-TOF spectrometer . Semi-preparative HPLC was performed on an Agilent 1200 apparatus equipped with a UV detector and a Zorbax SB-C-18 (9.4 × 250 mm) column (Agilent). Column chromatography (CC) was performed using Sephadex LH-20 , MCI gel CHP 20P , Diaion HP20SS , Chromatorex ODS , and Toyopearl Butyl-650C  columns. TLC was performed on precoated Kieselgel 60 F254 plates  with toluene–HCO2Et–HCO2H  as the solvent, and spots were detected by spraying with a 2% ethanolic FeCl3.Optical rotations were measured with a 341 digital polarimeter . C. fargesii (5.20 kg) were cut into small pieces and extracted three times with EtOH/H2O  by maceration at room temperature for 7 days. The extracts were combined and concentrated under reduced pressure to give an aqueous solution. The solution was partitioned with Et2O four times to give the Et2O fraction (32.4 g). The aqueous layer was subjected to Sephadex LH-20 CC (8 cm i.d. × 40 cm) with 0%–100% MeOH–H2O  to give 9 fractions: frs 1 (15.6 g), 2 (84.5 g), 3 (26.6 g), 4 (27.0 g), 5 (130.0 g), 6 (12.9 g), 7 (3.3 g), 8 (2.3 g), and 9 (2.2 g). Fraction fr. 2 (84.5 g) was separated by MCI gel CHP 20PCC (6 cm i.d. × 40 cm) with MeOH–H2O  to yield seven fractions, and fraction fr. 2-2 (5.3 g) was further fractionated by Diaion HP20SS CC (4 cm i.d. × 30 cm) with H2O containing increasing proportions of MeOH  to give 4 (105 mg). The Et2O fraction was subjected to MCI gel CHP 20PCC (5 cm i.d. × 50 cm) with 0%–100% MeOH in H2O  to yield 10 fractions: frs E-1 (5.3 g), 2 (7.5 g), 3 (1.6 g), 4 (3.2 g), 5 (4.3 g), 6(1.5 g), 7 (2.3 g), 8 (1.0 g), 9 (9.9 g) and 10 (3.5 g). Fr. E-3 was fractionated by Toyopearl Butyl-650C CC (3 cm i.d. × 30 cm) with 0%–100% MeOH–H2O containing 0.1% CF3CO2H (TFA)  to give fr. E-31 (1.3 g) and fr. E-32 (122 mg). Fr. E-32 was further purified by Chromatorex ODS CC (3 cm i.d. × 30 cm) with 0%–80% MeOH in H2O  to give 3 (12 mg). Fraction E-4 was separated by Sephadex LH-20 CC (4 cm i.d. × 40 cm) with H2O containing increasing amounts of MeOH  to yield Fr. E-41 (250 mg), Fr. E-42 (150 mg), Fr. E-43 (296 mg) and Fr. E-44 (1.7 g). The Fr. E-42 and Fr. E-43 were further purified by semi-preparative HPLC  to give 1  and 2 , respectively.The fresh leaves of d-glucopyranosyl)oxyethyl]-3-hydroxy-dihydrofuran-2(3H)-one-− 459.1143  and [M + Na]+ 483.1118 . -3-Hydroxy-1'-hydroxyethyl-dihydrofuran-2(3H)-one (Hydrochloride of1): c = 0.15, MeOH); UV (MeOH) λmax nm (log ε): 265 (3.16); CD (MeOH) λmax (Δε) 267 (5.7), 251 (3.2), 211 (1.2). 1H-NMR  δ 4.36 , 4.25 , 2.11 , 2.33 , 1.21 , 4.12 , 1.21 ; LC-MS/IT-TOF m/z 169.0475 [M + Na]+ .-2-[2'-oxyethyl]-dihydroxybutanoic acid  (2): Yellow amorphous powder; c = 0.12, MeOH); UV (MeOH) λmax nm (log ε): 267 (4.26); CD (MeOH) λmax (Δε) 268 (10.6), 252 (4.6), 212 (1.4). 1H- and 13C-NMR data, see m/z 339.0672 [M + Na]+ .-3-Hydroxymethyl-3,4-dihydro-5,6,7-trihydroxy-4-(4'-hydroxy-3'-methoxyphenyl)-1H-[2]-benzopyran-1-one (3): Brown amorphous powder; c = 0.11, MeOH); UV (MeOH) λmax nm (log ε): 220 (4.26), 278 (2.35); CD (MeOH) λmax (Δε) 288 (11.4), 242 (6.5), 218 (2.7). 1H- and 13C-NMR data, see m/z 347.0768 [M − H]− .3I (30 mg) and K2CO3 (15 mg) were added to a solution of 2 (10 mg) in DMF (5 mL). After stirring for 24 h at room temperature (r.t.), the reaction mixture was suspended in H2O and extracted with CHCl3. The CHCl3 layer was vacuum dried to afford a residue (6.2 mg). Then, DMAP (3.8 mg), Et3N (4.0 μL), and (R)-(−)-MTPACl (3.0 μL) were added to a solution of the residue (3.1 mg) in CH2Cl2 (1.0 mL) and stirred for 4 h at r.t. The reaction mixture was dried under a stream of N2. Separation of the residue was done by a silica gel column  to afford the (S)-MTPA ester derivative (2.1 mg). The (R)-MTPA ester derivative (2.3 mg) was obtained according to the same procedure using (S)-(+) MTPACl. CHS)-MTPA Ester derivative of2: Colorless oil; 1H-NMR  δ 7.0512–7.3901 (7H), 4.8212 , 1.3648 , 2.2511 , 2.3004 , 4.2526 , 4.2812 , 3.3206–3.8516 (-OCH3 × 5). LC-MS/IT-TOF m/z 611.17147 [M + Na]+ .(3R)-MTPA Ester derivative of2: Colorless oil; 1H-NMR  δ 7.0510–7.3902 (7H), 4.8210 , 1.3603 , 2.2572 , 2.3088 , 4.2548 , 4.2858 , 3.3312–3.8716 (-OCH3 × 5). LC-MS/IT-TOF m/z 611.1710 [M + Na]+ .(32 (10.2 mg) was dissolved in acetone (1.0 mL) and treated with DMP (0.2 mL) and PPTS (6.5 mg) at r.t. After 4 h, Et3N (7.5 μL) was added and the mixture was concentrated by N2 blowing. The residue was separated on a silica gel column  to afford the 2,3-O-isopropylidene derivative (3.2 mg) of 2.Compound 2,3-O-Isopropylidene Derivative of2: Colorless oil; 1H-NMR  δ 6.98 , 4.36 , 1.21 , 2.11 , 2.23 , 4.25 , 4.29 , 1.27 , 1.31 ; LC-MS/IT-TOF m/z 379.1007 [M + Na]+ .1 (6 mg) was dissolved in MeOH (4.0 mL) and 1 M H2SO4 (2.0 mL) and refluxed for 2 h on a H2O bath. After the hydrolysate was cool, H2O (8.0 mL) was added, then extracted with EtOAc (3 × 10.0 mL). The EtOAc layer was vacuum dried and chromatographed on semi-preparative HPLC eluting with a gradient of MeOH–H2O  to afford 3-hydroxy-1′-hydrooxyethyl-dihydrofuran-2(3H)-one. The aqueous layer was neutralized with aqueous Ba(OH)2 and evaporated under reduced pressure to give a residue. The residue was dissolved in pyridine (100 μL), subsequent treated with 0.1 mL cysteine methyl ester hydrochloride  and warmed at 60 °C for 1 h, then the trimethysilylation reagent HMDS/TMCS  was added and warmed at 60 °C for 30 min. The reaction mixture was partitioned between water and hexane. The hexane extract was analyzed by GC [tR values of d-glucose and l-glucose were 13.25 min and 15.32 min, respectively. The thiazolidine derivatives of the samples were confirmed by comparison with authentic standards.Compound All isolates were tested for cytotoxicity in vitro against A549, SMMC-7721, MGC-803, HepG2, and MCF-7 tumour cells via the MTT assay ,17 with 1–3 and a known compound 4 from the leaves of C. fargesii. The 2,3-dihydroxy-2-(2-hydroxyethyl)-butanoic acid moiety in 1 and 2 is an unusual carboxylic acid in Nature, thus, these compounds might be recognized as chemotaxonomic markers. Our preliminary examination also suggested this plant contains triterpene HHDP esters, which are important chemotaxonomical markers of Castanopsis sp.; therefore, further phytochemical investigations of the leaves of C. fargesii are in progress.In this study, we separated and identified three new compounds


Illicium pachyphyllum fruits against two grain storage insects, Sitophilus zeamais and Tribolium castaneum, and to isolate any insecticidal constituents from the essential oil. The essential oil of I. pachyphyllum fruits was obtained by hydrodistillation and analyzed by GC-MS. A total of 36 components of the essential oil were identified, with the principal compounds in the essential oil being trans-ρ-mentha-1(7),8-dien-2-ol (24.56%), d-limonene (9.79%), caryophyllene oxide (9.32%), and cis-carveol (5.26%) followed by β-caryophyllene (4.63%) and bornyl acetate. Based on bioactivity-guided fractionation, the three active constituents were isolated and identified as trans-ρ-mentha-1(7),8-dien-2-ol, d-limonene and caryophyllene oxide. The essential oil of I. pachyphyllum fruits exhibited contact toxicity against S. zeamais and T. castaneum adults, with LD50 values of 17.33 μg/adult and 28.94 μg/adult, respectively. trans-p-Mentha-1(7),8-dien-2-ol  exhibited stronger acute toxicity against S. zeamais and T. castaneum adults than either caryophyllene oxide (LD50 = 34.09 μg/adult and 45.56 μg/adult) and d-limonene (LD50 = 29.86 μg/adult and 20.14 μg/adult). The essential oil of I. pachyphyllum possessed fumigant toxicity against S. zeamais and T. castaneum adults with LC50 values of 11.49 mg/L and 15.08 mg/L, respectively. trans-p-Mentha-1(7),8-dien-2-ol exhibited stronger fumigant toxicity against S. zeamais and T. castaneum adults, respectively, with LC50 values of 6.01 mg/L and 8.14 mg/L, than caryophyllene oxide (LC50 = 17.02 mg/L and 15.98 mg/L) and d-limonene (LC50 = 33.71 mg/L and 21.24 mg/L). The results indicate that the essential oil of I. pachyphyllum fruits and its constituent compounds have potential for development into natural insecticides or fumigants for the control of insects in stored grains. The aim of this research was to determine chemical composition and insecticidal activity of the essential oil of  Sitophilus and Tribolium species are two of the major pests of stored grains and grain products in the tropics and subtropics  instruments using CDCl3 or DMSO-d6 as the solvent with TMS as internal standard. The crude essential oil of Limonene  δ: 5.33–5.49 , 4.73 , 2.03–2.18 , 1.88–2.02 , 1.79–1.86 , 1.76 , 1.68 , 1.50 . 13C-NMR  δ: 149.8 (C-8), 133.5 (C-1), 120.9 (C-6), 109.2 (C-9), 40.9 (C-4), 30.6 (C-2), 30.5 (C-5), 27.8 (C-3), 23.7 (C-7), 21.2 (C-10). The data matched a previous report [Limonene , yellow trans-ρ-Mentha-1(7),8-dien-2-ol  δ: 4.85 , 4.77 , 4.72 , 4.37 , 2.50–2.59 , 2.45–2.50 , 2.21 , 1.97–2.02 , 1.86–1.89 , 1.82–1.86 , 1.73 , 1.48–1.56 , 1.29 . 13C-NMR  δ: 149.8 (C-1), 149.4 (C-8), 109.9 (C-7), 108.9 (C-9), 72.4 (C-2), 39.0 (C-3), 38.1 (C-4), 32.6 (C-5), 29.9 (C-6), 21.0 (C-10). The data matched a previous report [ien-2-ol , A colors report .Caryophyllene oxide  δ: 5.00 , 4.88 , 2.90 , 2.64 , 2.33–2.37 , 2.28 , 2.09–2.15 , 1.72–1.74 , 1.69 , 1.65–1.67 , 1.62 , 1.44 , 1.36–1.39 , 1.23 , 1.03 , 1.01 , 0.99 . 13C-NMR  δ: 151.8 (C-1), 112.8 (C-12), 63.8 (C-9), 59.9 (C-8), 50.7 (C-5), 48.9 (C-2), 39.7 (C-3), 39.1 (C-7), 34.0 (C-4), 30.2 (C-10), 29.9 (C-14), 29.8 (C-11), 27.2 (C-6), 21.6 (C-13), 17.0 (C-15). The data matched a previous report [ne oxide , colorles report .S. zeamais and T. castaneum adults was tested as described by Liu and Ho [n-hexane. A Whatman filter paper (diameter 2.0 cm) were each impregnated with 10 μL dilution, and then placed on the underside of the screw cap of a glass vial . The solvent was allowed to evaporate for 15 s before the cap was placed tightly on the glass vial, each of which contained 10 insects inside to form a sealed chamber. Fluon (ICI America Inc.) was used inside the glass vial to prevent insects from contacting the treated filter paper. Preliminary experiments demonstrated that 15 s was sufficient for the evaporation of solvents. n-Hexane was used as a control. Five replicates were carried out for all treatments and controls, and they were incubated for 24 h. The insects were then transferred to clean vials with some culture media and returned to the incubator and observed daily for determination of end-point mortality, which was reached after one week. The experiments were repeated in three times. The observed data were corrected for control mortality using Abbott’s formula and LC50 values were calculated by using Probit analysis [The fumigant activity of the essential oil and the pure compounds against u and Ho . Range-fanalysis . S. zeamais and T. castaneum adults was measured as described by Liu and Ho [n-hexane. Aliquots of 0.5 μL of the dilutions were applied topically to the dorsal thorax of the insects, using a Burkard Arnold microapplicator. Controls were determined using n-hexane. Five replicates were carried out for all treatments and controls. Both treated and control insects were then transferred to glass vials  with culture media and kept in incubators. Mortality of insects was observed daily until end-point mortality was reached one week after treatment. The experiments were repeated in three times. The observed data were corrected for control mortality using Abbott’s formula and the results from all replicates were subjected to probit analysis using the PriProbit Program V1.6.3 to determine LC50 values [The contact toxicity of the essential oil/pure compounds against u and Ho . Range-f0 values . PyrethrI. pachyphyllum fruits and its constituent compounds, trans-ρ-mentha-1(7),8-dien-2-ol, d-limonene, and caryophyllene oxide have potential for development into natural insecticides/fumigants for control of insects in stored grains.The study indicates that the essential oil of


Viburnum sargentii Koehne. The structures of the compounds, namely 9'-O-methylvibsanol (3), furcatoside A (4) and lareciresinol (5) were elucidated by analysis of spectroscopic data and comparison with values for previously known analogues. In addition, (+)-catechin (1), (+)-epicatechin (2) were also isolated. This work also examined the cytotoxic effects of three compounds 3-5 (25-100 µM) in MCF-7 and A549 cells after 24, 48 and 72 h of exposure. Our results showed that 9'-O-methylvibsanol (3) exhibited strong concentration-dependent anticancer effects according to the MTT assay and produced morphological changes consistent with apoptosis, as confirmed by Ho3342 staining analysis revealed that more apoptotic cells were observed after 9'-O-methylvibsanol (3) treatment.Compounds were isolated from a methanol extract of the dried stem barks of  V. sargentii Koehne was identified. The Viburnum genus (Caprifoliacea) comprises more than 200 species, shrubs or trees, mainly distributed in Southern America and in Asia and very abundant in the Chinese spontaneous flora -. 9'Furcatoside A (4). C32H42014, (300MHz CD3OD) 6.0 . 6.62 , 2.01, 2.2 , 0.95 , 3.3-4.8 (6H), 6.4/ 7.7 , 7.5 ; 91.5 (C-1), 140.7 (C-3), 115.3 (C-4), 36.4 (C-5), 28.7 (C-6), 38.0 (C-7), 80.7 (C-8), 4.67 (C-9), 71.6 (C-10), 70.0 (C-11), 172.8 (COO), 20.8 (COOCH3), 173.1 (C-1′), 44.3 (C-2′), 26.8 (C-3′), 22.7 (C-4′), 22.7 (C-5′), 101 .9 (C-1″), 71.3 (C-2″), 75.3 (C-3″), 71.8 (C-4″), 78.1 (C-5″), 62.7 (C-6″), 127.1 (C-1′′′), 131.3 , 116.9 , 161.4 (C-4′′′), 146.8 (C-7′′′), 115.2 (C-8′′′), 168.3 (C-9′′′), ESI-MS (negative ion mode): m/z 355.04 [M-H]-. (5Lariciresinol ): C20H24O6, (300MHz CD3OD) 2.42 , 2.54 , 2.72 , 2.95 , 3.77 , 3.88 , 3.90 , 3.93 , 3.99 , 4.75 , 4.99 , 6.62-6.86 ; 132.3 (C-1), 112.0 (C-2), 146.5 (C-3), 144.6 (C-4), 114.3 (C-5), 121.0 (C-6), 32.5 (C-7), 42.7 (C-8), 72.3 (C-9), 134.5 (C-1′) 108.6 (C-2′), 146.8 (C-3′), 145.9 (C-4′), 114.0 (C-5′), 118.6 (C-6′), 82.8 (C-7′), 52.5 (C-8′), 59.2 (C-9′), 55.1 , ESI-MS (negative ion mode): m/z 358.85 [M-H]-. 2 at 37 ºC. Compounds isolated from V. sargentii were dissolved in dimethyl sulfoxide (DMSO) and the final DMSO concentration in all cultures was 0.5%. Human cancer cell line, A549 (human lung adenocarcinoma), MCF-7 (human breast adenocarcinoma), used in this work was purchased from the Korean Cell Line Bank (KCLB). A549, MCF-7 cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/mL streptomycin, in a humidified incubator containing 5% CO5 cells/well in 96 well plates. After incubation for 24 h. at 37 ºC, cells were exposed to various concentrations of 9'-O-methylvibsanol (3), furcatoside A (4) and Lariciresinol (5) and incubated for 12-72 h. Doxorubicin (1–10 µM), Paclitaxel (25 nM–1 µM) were used as positive controls; negative control groups used same amount of DMSO. After incubation, the medium was removed and cells in each well were incubated with PBS contained 5 mg/mL MTT for 4 h. at 37 ºC in 5% CO2 incubator. MTT solution was then discarded and 100 μL of DMSO was added into each well to dissolve insoluble formazan crystals. Plates were then kept agitation for 30 min at room temperature for complete solubilization. The level of colored formazan derivative was analysed on a ELISA reader  at a wavelength of 570 nm. Results were expressed as the mean percentage of cell growth inhibition. The IC50 value was expressed as the concentration of compounds that inhibited the growth of cells by 50%. -2,5-diphenyltetrazolium bromide (MTT) assay is a common assay for cell cytotoxicity . Briefly by 50%. .4 cells/well) were incubated for 24 h in 12 well plates. After incubation, the cells were untreated or treated with 9'-O-methylvibsanol (3) of four different concentrations for 24–72 h. Then the medium was removed and cells in wells were washed once with PBS. They were observed by phase contrast inverted microscope  at 400 × magnification (Cells (5 × 10fication . DoxorubO-methylvibsanol (3). After 12, 24 and 72 h of incubation, cells were washed with PBS and fixed with methanol for 10 min at room temperature. Fixed cells were washed with PBS, and stained with 1 ug/mL benzimidazole Ho33342 solution for 30 min at incubation. The cells were washed twice more with PBS and analyzed with a fluorescence microscope  showed highly cytotoxicity of MCF-7, A549 cells with the lowest IC50 range (83.2 to 29.3 µM). Furthermore, the anti-tumor effects 9'-O-methylvibsanol (3) on MCF-7, A549 cells has not been reported previously. Consequently, we examined whether the apoptotic pathway is involved in the cell death caused by 9'-O-methylvibsanol (3) in MCF-7, A549 cells. Firstly, we found that the MCF-7, A549 cells treated with 9'-O-methylvibsanol (3) acquired apoptotic morphological features such as becoming round in shape, reduction in cell volume, destabilization of the plasma membrane, indicating an increasing progression toward cell death in a dose-dependent manner (O-methylvibsanol (3) inhibits the proliferation of MCF-7, A549 cells by inducing apoptosis, cells treated with compound were examined after Ho33342 staining. Results showed that MCF-7, A549 cells treated with 9'-O-methylvibsanol (3) displayed typical apoptotic phenomena with chromatin condensation and karyopyknosis  for its development as a cancer chemopreventive and/or anticarcinogenic agents.However, further studies are needed to determine the mechanism of apoptosis pathway. In general, depending on the cell, apoptosis can be initiated in two ways: by extrinsic pathway or by an intrinsic pathway. In the former, plasma membrane death receptors are involved and the apoptosis signal is provided by interaction between the ligand and the death receptor, which then activates caspase-8 and apoptotic cell death ,17. Howe


Among the known isolates, 5–8, 10–13, 16–20, and 25 were obtained from the genus for the first time; 4, 14, 15, and 21–24 were isolated from the species for the first time. Meanwhile, the NMR data of 16 was first reported here. Their structures were determined by means of chemical and spectroscopic methods. On the other hand, their inhibitory effects on sodium oleate-induced triglyceride (TG) overloading in HepG2 cells were evaluated. As a result, two new compounds (1 and 2), together with known isolates 7–11, 13, 14, 16–18, 20, 21, and 25 possessed significant inhibitory effects in the cells.Three new compounds, apetalumosides C Lepidium apetalum seed extract ;tragalin  + quasi-molecular ion at m/z 391.0739 . Twelve signals were displayed in its 13C-NMR  to δC 92.4 (C-1′), and δH 5.57  to δC 96.1 (C-1) observed in the HSQC spectrum indicated that there were two sugar units in 3. Combined with its MS and 1H-NMR spectrum (H 4.68 (H-1′)) and a set of 13C-NMR (δC 63.7 (C-6′), 72.1 (C-4′), 74.4 (C-2′), 80.0 (C-3′), 83.1 (C-5′), 92.4 (C-1′)) signals revealed the presence of 1-thio-β-d-glucopyranosyl  to give thymidine . Fraction 1-8-1 (253.1 mg) was purified by PHPLC [CH3CN–H2O  + 1% HOAc] to yield 3′,5′-dimethoxy-4-O-β-d-glucopyranosyl cinnamic acid . Fraction 1-8-3 (579.2 mg) was separated by PHPLC  + 1% HOAc), and adenosine  was gained. Fraction 1-8-4 (1.3 g) was isolated by PHPLC  + 1% HOAc) to yield seven fractions (Fr. 1-8-4-1–1-8-4-7). Fraction 1-8-4-5 (130.9 mg) was further purified by PHPLC  + 1% HOAc) to yield sinapoylglucose . Fraction 1-8-7 (573.6 mg) was separated by Sephadex LH-20 CC ) and PHPLC  + 1% HOAc) to give lariciresinol 4′-O-β-d-glucopyranoside . Fraction 1-8-8 (1.1 g) was prepared by PHPLC  + 1% HOAc), and -aegineoside  was yielded. Fraction 1-8-10 (917.9 mg) was purified by PHPLC  + 1% HOAc) to obtain astragalin  and isorhamnetin 3-O-β-d-glucopyranoside . Fraction 1-8-11 (1.3 g) was isolated by PHPLC ) to give 1(E),2(E)-di-O-sinapoyl β-d-glucopyranoside . Fraction 1-12 (8.0 g) was subjected to ODS CC ), and nine fractions (Fr. 1-12-1–1-12-9) were given. Fraction 1-12-8 (1.4 g) was further prepared by PHPLC  + 1% HOAc) to yield sinapoyl-9-sucrosecoside . Fraction 1-12-9 (2.1 g) was purified by PHPLC  + 1% HOAc) to obtain 1,2-disinapoylgentiobiose  and drabanemoroside . Fraction 1-13 (13.7 g) was isolated by PHPLC  → MeOH) to give twenty-one fractions (Fr. 1-13-1–1-13-21). Fraction 1-13-3 (606.1 mg) was purified by PHPLC  + 1% HOAc) to gain apetalumoside D . Fraction 1-13-4 (780.2 mg) was separated by PHPLC  + 1% HOAc) to yield l-tryptophan . Fraction 1-13-6 (543.7 mg) was further purified by PHPLC ) to obtain 4,9-di-O-β-d-glucosyl sinapoyl alcohol . Fraction 1-13-16 (217.6 mg) was isolated by PHPLC ), and isorhamnetin 3,4′-O-β-d-diglucoside  was yielded. Fraction 1-13-17 (369.6 mg) was prepared by PHPLC ) to gain apetalumoside C1 . Fraction 1-13-20 (779.2 mg) was separated by PHPLC ) to obtain quercetin 3-O-α-l-rhamnopyranosyl(1→2)-α-l-arabinopyranoside . Fraction 1-14 (8.0 g) was subjected to Sephadex LH-20 CC ), and seven fractions (Fr. 1-14-1–1-14-7) were given. Fraction 1-14-7 (477.8 mg) was separated by PHPLC  + 1% HOAc) to yield 2-O--2,4,6-trihydroxyphenylacetic acid 4-O-β-d-glucopyranoside . Fraction 1-15 (14.1 g) was isolated by PHPLC ), and eleven fractions (Fr. 1-15-1–1-15-11) were obtained. Fraction 1-15-1 (2.5 g) was further prepared by PHPLC ) to give quercetin 3-O-β-d-glucopyranosyl-7-O-β-d-gentiobioside . Fraction 1-15-5 (2.3 g) was subjected to Sephadex LH-20 CC ) and finally separated by PHPLC ) to yield kaempferol 3-O-β-d-glucopyranosyl-7-O-β-d-gentiobioside . Fraction 1-15-8 (294.4 mg) was purified by PHPLC ) to gain isorhamnetin 3-O-β-d-glucopyranosyl-7-O-β-d-gentiobioside .Fraction 1 (80 g) was subjected to silica gel CC ), and seven fractions (Fr. 2-1–2-7) were given. Fractions 2-4 (102.8 mg) and 2-5 (159.6 mg) were further purified by PHPLC ) to yield stachyose  and 1-thio-β-d-glucopyranosyl(1→1)-1-thio-α-d-glucopyranoside . Fraction 2-6 (102.8 mg) was separated by PHPLC  to gain TgSSTg .Meanwhile, fraction 2 (4.0 g) was isolated by PHPLC : Yellow powder; [α]c = 0.95, MeOH); IR υmax (KBr) cm−1: 3362, 2937, 1699, 1653, 1600, 1516, 1457, 1340, 1286, 1179, 1113, 1066, 827; UV λmax (MeOH) nm (log ε): 334 (4.19), 266 (4.09), 245 (4.20). 1H-  and 13C-NMR  spectroscopic data, see m/z 977.2555 [M − H]− .Apetalumoside D (2): White powder; [α]c = 0.94, MeOH); IR υmax (KBr) cm−1: 3399, 2922, 1616, 1519, 1463, 1336, 1222, 1113, 1025, 876, 825; UV λmax (MeOH) nm (log ε): 277 ; 242 (3.82). 1H-  and 13C-NMR  spectroscopic data, see m/z 593.1333 [M + Na]+ .d-glucopyranosyl(1→1)-1-thio-α-d-glucopyranoside1-Thio-β- (3): White powder. [α]c = 0.97, H2O); IR υmax (KBr) cm−1: 3368, 2888, 1636, 1411, 1356, 1273, 1097, 1042, 874; 1H-  and 13C-NMR  spectroscopic data, see m/z 391.0739 [M + H]+ .Sinapoyl-9-sucrosecoside (16): Pale yellow powders; The NMR data of 16 in DMSO-d6 is first reported. 1H-NMR  δ: 6.89 , 7.60 , 6.44 , 3.65 , 3.69 , H2-1′), 3.87 , 4.12 , 4.18 , , 4.55 ), 5.46 , 3.54 , 3.82 , 3.41 , 4.18 , , 3.92 , H2-6′′), 3.87 ; 13C-NMR  δ: 126.5 (C-1), 106.8 , 149.2 , 139.3 (C-4), 147.3 (C-7), 115.5 (C-8), 169.2 (C-9), 64.1 (C-1′), 105.1 (C-2′), 83.6 (C-3′), 76.0 (C-4′), 79.0 (C-5′), 65.1 (C-6′), 93.1 (C-1′′), 73.0 (C-2′′), 74.5 (C-3′′), 71.7 (C-4′′), 71.9 (C-5′′), 64.1 (C-6′′), 56.9 ; HRESI–TOF–MS: Negative-ion mode m/z 547.1680 [M − H]− .Acid Hydrolysis of1: the solution of compound 1 (2.0 mg) in 1 M HCl (1 mL) was treated by using the same method as described in reference [1 was heated under reflux for 3 h. The reaction mixture was then analyzed by CH3CN–H2O . As a result, d-glucose was detected from the aqueous phase of 1 by comparison of its retention time and optical rotation with that of the authentic sample, d-glucose (tR 8.8 min (positive)).mpound 1 .0 mg in Materials: HepG2 cells were purchased from Cell Resource Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College . Dulbecco’s modified Eagle’s medium (DMEM), penicillin and streptomycin were purchased from Thermo Scientific . Fetal Bovine Serum (FBS) was obtained from Mediatech . TG assay kits were purchased from Biosino Bio-Technology And Science Inc. . Sodium oleate (SO) and orlistat were obtained from Sigma-Aldrich Corporation .Cell culture: HepG2 cells were routinely cultured in DMEM-based medium as described before [d before . After cInduction and evaluation of TG overloading: TG overloading was induced as described before [d before . Brieflyp < 0.05.Statistical analyses were undertaken with SPSS v12.0 . The significance of the differences between the mean values was determined using an analysis of variance (ANOVA). The differences were considered statistically significant at 1–25) including three new ones, apetalumosides C1 (1), D (2), and 1-thio-β-d-glucopyranosyl(1→1)-1-thio-α-d-glucopyranoside (3), were obtained from the seeds of L. apetalum. Among the known isolates, 5–8, 10–13, 16–20, and 25 were obtained from the genus for the first time; 4, 14, 15, 21–24 were isolated from the species for the first time. Meanwhile, the NMR data of 16 was first reported here. Their structures were determined by means of chemical and spectroscopic methods. Moreover, their inhibitory effects on TG overloading were evaluated in HepG2 cells. The results showed that phenol compounds, including five flavonoids (7–11), five sinapic acid groups  and one lignin (20), together with two new compounds (1 and 2) as well as two other isolates (21 and 25) have significant TG-lowering effects, among of which, 10, 13 and 21 exhibited a level of activities almost comparable to that of orlistat. It is suggested that the above compounds contained in the L. apetalum might be part of the material basis involved in the lipid metabolism.Summed up, twenty-five compounds (


Pseudallescheria boydii F44-1. By adding amino acids to GPY culture medium, two new bisindole alkaloids, pseudboindoles A and B (1 and 2), together with 11 known indole alkaloids were isolated from the culture broth. Their structures were elucidated by comprehensive analysis of the NMR, MS, IR, and UV spectra. The 3,3′-cyclohexylidenebis(1H-indole) (3) showed cytotoxic activity against various cancer cell lines.The composition of the culture medium has great influence on the metabolite production of the marine fungus  Scedosporium apiospermum F41-1 produced 12 new indole alkaloids. Among them, scedapin C and scequinadoline D displayed significant antiviral activity against hepatitis C +  (−1) and benzene ring (1618 and 1456 cm−1). UV maxima at 222 and 282 nm also displayed the conjugated system containing a benzene ring. The 13C NMR and DEPT showed one methylene, six methines and three quaternary carbons , 7.34 , 7.21  and 7.12  , one methine group , one hydroxyl group , and a broad singlet  , H-7 to C-4a (δC 127.6), H-2 to C-3 (δC 112.4) . Consequently, the chemical structure of pseudboindole A (1) was illustrated as 1,3-di(1H-indol-3-yl)propan-2-ol (Pseudboindole A (1.14919) , which h carbons . The 1H .8, H-5) , indicatbrs, NH) . The 1H−/H-6/H-7  and the C 112.4)  were ind H-4/H-8 . The rempan-2-ol .2) was revealed to be C20H20N2OS by HR-(−)ESI-MS at m/z 335.12247 [M−H]− , 7.34 , 7.16  and 7.04  and 1H−1H COSY correlations of H-4/H-5/H-6/H-7 (1H−1H COSY correlations of H-1/H-2 (C 136.60 or 136.56), H-5 (H-7)/C-4a (δC 126.80 or 126.71), and H-2/C-4a/C-7a/C-3 (δC 118.67 or 118.40)  was connected to a sulfoxide group, which can enable the further analysis of the HMBC correlation of H3-12 to C-10 (δC 53.0). Consequently, the chain partial structure of −CHCH2CH2SOCH3 was established. The HMBC correlations of H-8/C-3 indicated that two 3-substitued indole parts were connected to C-8 (δC 33.2), which was confirmed by the NOESY correlation of H-4/H-8 (2) was unambiguously established, as shown in The molecular formula of pseudboindole B   indicate H-4/H-8 . Therefo3 was a brown amorphous powder. It had the molecular formula C22H22N2, which was established on the basis of the HR-(−)ESI-MS ion at m/z 313.17120 [M−H]−  /C-8 (δC 39.5), H-9 /C-8, H-10 /C-8, the two 3-substitued indole parts and the chain part of −CH2CH2CH2CH2CH2− were connected to C-8. The chemical structure of compound 3 was illustrated as 3,3′-cyclohexylidenebis(1H-indole), as seen in H-indole) (3) can be synthesized by the reaction of an indole with cyclohexanone, and showed rather potent enhancing activity (140%) on Am80-induced HL-60 cell differentiation  aniline (5) indol-1-yl)ethan-1-ol (12) + .Pseudboindole A (2). Brown amorphous powder. mp 113.9−114.8 °C. UV (MeOH) λmax (log ε): 282 (3.82), 222 (4.51). IR υmax 3420, 2980, 2926, 1659, 1419, 1332, 1197, 1094, 1050, 937, 881, 741 cm-1. 1H and 13C NMR data, see m/z 335.12247 [M−H]− .Pseudboindole B (H-indole) (3). Brown amorphous powder. mp 114.7−115.9 °C. UV (MeOH) λmax (log ε): 283 (3.88), 224 (4.63). IR υmax 3405, 2935, 1686, 1618, 1456, 1415, 1097, 1336, 1242, 1103, 1046, 1012, 817, 738 cm-1. 1H and 13C NMR data, see m/z 313.17120 [M−H]− .3,3′-cyclohexylidenebis-2,5-diphenyl-2H-tetrazolium bromide) assay. The tested human cancer cell lines were seeded in 96-well plates at a density of 3 × 107 cells/L, and the compounds were added at various concentrations (7.864−30.00 μM). After 72 h, MTT was added to the culture medium at a final concentration of 0.5 mg/mL, and the plates were incubated for 4 h at 37 °C. The supernatant was removed. The formazan crystals were dissolved in DMSO (150 μL) with gentle shaking at room temperature. The absorbance at 570 nm was recorded with a microplate reader , and the data were analyzed with the SPSS (version 13.0) [The in vitro cytotoxic activity of on 13.0) .1H NMR signals in the aromatic region of 6.50−8.50 ppm, two new bisindole compounds, pseudboindoles A and B (1 and 2), together with 11 known indole alkaloids (3−12) were efficiently isolated from the marine fungus Pseudallescheria boydii F44-1. The 3,3′-cyclohexylidenebis(1H-indole) (3) showed significant cytotoxic activity against various cancer cell lines. The result proves again that an amino acid-directed strategy is effective for inducing the marine fungi to produce diverse alkaloids. However, the specific quantitative relationship between amino acids and alkaloids and their biosynthesis pathways still need further study. After revealing these relationships, the application of this strategy will be more efficient.By tracking characteristic


Compared to cultures in the same medium without SAHA, compounds 1–4, 8, 9, 11, and 12 were solely observed under SAHA condition. The structures of these compounds were elucidated based on spectroscopic analysis, specific rotation analysis, ECD, and X-ray crystallographic analysis. (±)-3, (±)-4, and (±)-5 were further resolved into the corresponding optically pure enantiomers and their absolute configurations were determined for the first time. Compounds 11 and 12 showed selective antibacterial against Pseudomonas aeruginosa with a minimum inhibitory concentration (MIC) of 17.4 and 13.9 μM, respectively. Compound 10 exhibited better α-glucosidase inhibitory activity than the assay control acarbose with IC50 values of 117.3 and 255.3 μM, respectively.A chemical-epigenetic method was used to enhance the chemodiversity of a marine algicolous fungus. Apart from thirteen known compounds, (+)-brevianamide R ((+)- Aspergillus is a kind of productive fungal genus and has been reported to produce many new NPs  propanamide (1), (+)-brevianamide X ((+)-2) and (‒)-brevianamide X ((‒)-2), (±)-brevianamide R (3) +  , one sp3 methine , three sp3 methylenes , and two methyls  (H 2.79), H-8 (δH 4.64), and NH2  to carbonyl carbon (δC-9 173.7) along with the 1H–1H COSY (1H–1H COSY correlations from H-10 (δH 2.25) to H-12 (δH 0.86), and H-13 (δH 0.84) through H-11 (δH 1.91), demonstrated an isobutyl fragment. The remaining fragment, C4H2ON2, could be a pyrazin-2(1H)-one nucleus from the HMBC correlations from H-4 (δH 7.01) to C-3 (δC 155.2) and C-6 (δC 138.0). Furthermore, the key HMBC correlations from H-10 to C-5 (δC 120.8) and C-6, and H-7 to C-2 (δC 156.2) and C-3, allowed for the linkage of the isobutyl to C-6 and the 3-substituted propionamide moiety to C-3, respectively. Thus, structure 1 was established as 3-[6-(2-methylpropyl)-2-oxo-1H-pyrazin-3-yl] propenamide, which was only recorded in the Aurora Fine Chemicals with ID number K13.052.726 without any physical and chemical properties reported. Consequently, 1 was identified as a new naturally occurring compound.Compound 46.1213) . 1D (Tab46.1213)  and HSQC46.1213)  NMR specδH 0.84) . The HMBδH 0.84)  correlat–1H COSY  between 2 was isolated as light yellow crystal with the molecular formula of C21H23N3O4 from a HRESIMS peak at m/z 404.1570 [M + Na]+  , two sp3 methylenes , one sp3 methine , five sp2 methines , and one sp2 methylene , which demonstrated the presence of a monosubstituted double bond (1H–1H COSY (H 6.11) to H-22  and the HMBC (H 6.11) to C-20 (δC 40.5), C-23 (δC 28.1), and C-24 (δC 28.3), along with H-23 (δH 1.57) to C-20 and C-24, which demonstrated the presence of a 2-methylbut-3-en-2-yl fragment. The 1H–1H COSY correlations from H-13 (δH 7.37) to H-16 (δH 7.43), along with the HMBC from H-13 (δH 7.37) to C-12 (δC 127.4), C-11 (δC 104.5), and C-17 (δC 127.4), and H-16 (δH 7.43) to C-12 and C-17, which demonstrated the presence of a 2,3-bisubstituted indole ring. Moreover, the key HMBC from H-21, H-23, and H-24 (δH 1.54) to C-19 (δC 146.2) connected the 2-methylbut-3-en-2-yl group to C-19 of the indole ring. Chemical shifts of C-1 (δC 165.7) and C-4 (δC 161.3) indicated two amide carbonyl carbons. In addition, 1H–1H COSY correlations from H-6  to H-8 (δH 4.41) through H-7  and HMBC correlations from H-6 to C-4 and C-9 (δC 91.0), H-7 to C-9, H-8 (δH 4.41) to C-1 and C-9 and, H-10 (δH 7.29) to C-3 (δC 125.1) and C-4 were observed. This data indicated a 2-methylene- pyrrolo diketopiperazine nucleus. The key HMBC correlations from H-10 to C-11, C-12, and C-19 ) propanamide (1): a brown amorphous powder, mp 141–143 °C; UV (MeOH) λmax (log ε) 217 (3.75), 230 (3.79), 262 (3.15), 325 (3.66) nm; IR (KBr) νmax 3400, 3198, 2959, 2929, 2861, 1655, 1614, 1529, 1408, 1168, 575 cm−1; 1H and 13C NMR data .3-+; 1H and 13C NMR ; ECD  λmax (Δε) 212 (+26.2), 258 (–19.1), 342 (+11.8) nm. (–)-3: mp 140–142 °C; c 0.13, MeOH); ECD  λmax (Δε) 212 (–25.9), 258 (+16.7), 340.5 (–10.8) nm.(±)-Brevianamide R ( NMR see . (+)-3: 4): a colorless powder; ESI-MS m/z 388.2 [M + Na]+; 1H and 13C NMR ; ECD  λmax (Δε) 212 (+21.8), 258 (–14.3), 340 (+9.3) nm. (–)-4: mp 160–163 °C; c 0.2, MeOH); ECD  λmax (Δε) 213 (–19.3), 258 (+15.8), 340 (–9.7) nm.(±)-Brevianamide Q ( NMR see . (+)-4: 5): a light yellow powder; ESI-MS m/z 372.3 [M + Na]+; 1H and 13C NMR ; ECD  λmax (Δε) 212 (+24.3), 258 (–12.2), 330 (+6.3) nm. (–)-5: mp 131–132 °C; c 2.0, MeOH); ECD  λmax (Δε) 213 (–22.2), 258 (+11.0), 330 (–5.7) nm.(±)-Brevianamide V ( NMR see . (+)-5: 12): a white solid; UV (MeOH) λmax (log ε) 224 (3.42), 262 (2.95), 200 (1.36), 301 (1.23) nm; IR (KBr) νmax 3401, 2954, 1656, 1598, 1454, 1322, 1262, 1207, 1161, 1104, 1031, 998, 952, 843, 677 cm−1; 1H and 13C NMR  diorcinol-4-carboxylate (12a): a white solid; UV (MeOH) λmax (log ε) 222 (3.34), 228 (3.32), 279 (1.49) nm; IR (KBr) νmax 3440, 2950, 1731, 1587, 1463, 1324, 1268, 1193, 1158, 1093, 1064, 1028, 996, 935, 836 cm−1; 1H and 13C NMR   .2 was obtained as a light-yellow monoclinic crystal with molecular formula C11H17O2N3 from CH3CH2OH and CH2Cl2. Space group P-1, a = 9.8007 (9) Å, b = 10.4560(12) Å, c = 12.9125(14) Å, α = 122.466(3)°, β = 97.4200(10)°, γ = 106.988(2)°, V = 1125.2(2) Å3, Z = 2, Dcalcd = 1.259 mg/m3, μ = 0.089 mm-1, F(000) = 454, crystal size 0.34 × 0.21 × 0.13 mm, T = 298(2) K, 2.27° ≤ 2θ ≤ 25.02°, 5737 reflections collected, 3915 unique (R(int) = 0.0364). Final R indices R1 = 0.0310, wR2 = 0.0748 based on 3915 reflections with I > 2 sigma(I) (refinement on F2), 316 parameters, 0 restraint. Crystallographic data (excluding structure factors) for structure (±)-2 in this dissertation have been deposited in the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1837585 -1223-336033; e-mail, deposit@ccdc.cam.ac.uk).Racemic The calculations were performed by using the density functional theory (DFT) using Gaussian 03. The preliminary conformational distributions search was performed by HyperChem 7.5 software. All ground-state geometries were optimized at the B3LYP/6-31G(d) level. Conformers within a 2 kcal/mol energy threshold from the global minimum were selected to calculate the electronic transitions. The overall theoretical ECD spectra were obtained according to the Boltzmann weighting of each conformers. Solvent effects of methanol solution were evaluated at the same DFT level by using the SCRF/PCM method.2CO3 (20 mg) and MeI (0.03 mL), and the mixture was then stirred for 2 h [2O, 4 mL/min) to give dimethylether derivative 12a .To a solution of 12 (10 mg) in DMF (1 mL) were added K for 2 h . The reaB. subtilis ATCC6051, S. aureus ATCC6538, S. aureus ATCC25923, P. aeruginosa ATCC10145, E. aerogenes, and E. coli ATCC11775 and the pathogenic fungi were C. albicans ATCC10231, and C. glabrata ATCC2001. Ciprofloxacin and ketoconazole were used as positive controls against bacteria and fungi, respectively. All of the microbial strains used in the bioassays are preserved at the Key Laboratory of Chemistry for Natural Products of Guizhou Province, Chinese Academy of Sciences.The MICs of the compounds were tested using the broth microdilution method ,32. The 50 value of 255.3 μM.The inhibitory effects were assayed as described previously ,13,33. AAspergillus versicolor OUCMDZ-2738 under chemical-epigenetic modification, including a new naturally occurring diketopiperazine derivative (1) and two new compounds, (+)-brevianamide X ((+)-2) and (–)-brevianamide X ((–)-2). We also chirally separated the racemic brevianamides R (3), Q (4), and V (5) into the corresponding optically pure compounds and resolved their absolute configurations for the first time. We clearly determined the structure of the methyl diorcinol-4-carboxylate (12) by preparing 3,3′-dimethylether derivative (12a). Compounds 11 and 12 showed selective antibacterial activity against P. aeruginosa both with the MIC value of 17.4 and 13.9 μM, respectively.We identified sixteen compounds from the fermentation of


Moringa oleifera Lam. is a commonly used plant in herbal medicine and has various reported bioactivities such as antioxidant, antimicrobial, anticancer and antidiabetes. It is rich in nutrients and polyphenols. The plant also has been traditionally used for alleviating allergic conditions. This study was aimed to examine the anti-allergic activity of M. oleifera extracts and its isolated compounds.M. oleifera leaves, seeds and pods were extracted with 80% of ethanol. Individual compounds were isolated using a column chromatographic technique and elucidated based on the nuclear magnetic resonance (NMR) and electrospray ionisation mass spectrometry (ESIMS) spectral data. The anti-allergic activity of the extracts, isolated compounds and ketotifen fumarate as a positive control was evaluated using rat basophilic leukaemia (RBL-2H3) cells for early and late phases of allergic reactions. The early phase was determined based on the inhibition of beta-hexosaminidase and histamine release; while the late phase was based on the inhibition of interleukin (IL-4) and tumour necrosis factor (TNF-α) release.E)–undec-6-enoate (1) and 3,5,6-trihydroxy-2--4H-chromen-4-one (2) together with six known compounds; quercetin (3), kaempferol (4), β-sitosterol-3-O-glucoside (5), oleic acid (6), glucomoringin (7), 2,3,4-trihydroxybenzaldehyde (8) and stigmasterol (9) were isolated from M. oleifera extracts. All extracts and the isolated compounds inhibited mast cell degranulation by inhibiting beta-hexosaminidase and histamine release, as well as the release of IL-4 and TNF-α at varying levels compared with ketotifen fumarate.Two new compounds; ethyl-(M. oleifera and its isolated compounds potentially have an anti-allergic activity by inhibiting both early and late phases of allergic reactions.The study suggested that  Moringa oleifera Lam. (M. oleifera) is well known for its nutritional and medicinal values +. 1H NMR : δH 0.85 , 1.25 , 1.32 , 1.35 , 1.62 , 2.02 , 2.31 , 2.79 , 4.15 , 5.4 . 13C NMR : δc 14.3 , 22.8 (C-12), 24.9 (C-6), 29.1 (C-5), 29.7 (C-11), 31.9 (C-10), 35 , 60.1 (C-2), 130 , 174.3 (C-3).Elution of the hexane leaf fraction (18.91 g) with hexane-ethyl acetate  yielded compound 2. Reddish-brown liquid (3.9 mg), TLC: Rf 0.50 , HRESIMS (+ve mode) m/z: 176.9835 [M + 2H]2+ .1H NMR : δH 6.83 , 7.89 . 13C NMR : δc 113.3 (C-10), 114.3 (C-7), 122.1 (C-1′), 123.0 (C-6), 128.5 (C-3′), 128.5 (C-5′), 131.4 (C-8), 138.5 (C-3), 138.5 (C-4′), 147.5 (C-2′), 147.5 (C-6′), 161.8 (C-9), 162.9 (C-5), 168.9 (C-2), 173.8 (C-4).The ethyl acetate leaf fraction (3.38 g) was loaded onto Sephadex LH-20 using methanol-water  yielding four fractions. The third fraction was further eluted using Sephadex LH-20 with chloroform-dichloromethane-methanol  as a mobile phase giving four fractions. The last fraction was further eluted in silica gel column chromatography with ethyl acetate-chloroform  as a mobile phase yielding compound 3. Yellow powder (5.9 mg), m.p 312–314 °C. TLC: Rf 0.49 , HRESIMS (+ve mode) m/z: 325.0294 [M + Na]+. 1H NMR : δH 6.20 , 6.41 , 6.91 , 7.66 , 7.76 . 13C NMR : δc 93.3 (C-8), 98.2 (C-6), 103.3 (C-10), 114.9 (C-2′), 115.1 (C-5′), 120.6 (C-6′), 123.0 (C-1′), 136.1 (C-3), 145.1 (C-3′), 146.9 (C-2), 147.6 (C-4′), 157.1 (C-9), 161.3 (C-5), 164.5 (C-7), 176.2 (C-4).The ethyl acetate leaf fraction (3.38 g) was loaded onto Sephadex LH-20 using methanol-water  yielding four fractions. The fourth fraction was further eluted using Sephadex LH-20 with chloroform-dichloromethane-methanol  as a mobile phase yielding compound 4. Yellow powder (1.3 mg), m.p 276–279 °C. TLC: Rf 0.68 , HRESIMS (+ve mode) m/z: 309.0334 [M + Na]+. 1H NMR : δH 6.19 , 6.41 , 6.91 , 6.91 , 8.10 , 8.74 .13C NMR : δc 93.1 (C-8), 97.9 (C-6), 103.2 (C-10), 114.9 (C-3′), 114.9 (C-5′), 122.3 (C-1′), 129.3 (C-2′), 129.3 (C-6′), 135.7 (C-3), 146.7 (C-2), 156.9 (C-9), 159.2 (C-4′), 161.1 (C-5), 164.2 (C-7), 176.0 (C-4).The ethyl acetate leaf fraction (3.38 g) was loaded onto Sephadex LH-20 using methanol-water  yielding four fractions. The fourth fraction was further eluted using Sephadex LH-20 with chloroform-dichloromethane-methanol  as a mobile phase giving another six fractions. The fifth fraction was subjected to silica gel column chromatography using chloroform-methanol  as a mobile phase to obtain compound 5. White powder (8.5 mg), m.p 209–211 °C. TLC: Rf 0.67 , HRESIMS (+ve mode) m/z: 599.4190 [M + Na]+ . 1H NMR : δH 0.68 , 0.83 , 0.83 , 0.83 , 0.83 , 0.92 , 0.92 , 0.92 , 0.96 , 1.00 , 1.00 , 1.17 , 1.17 , 1.25 , 1.25 , 1.25 , 1.37 , 1.38 , 1.42 , 1.52 , 1.52 , 1.53 , 1.65 , 1.65 , 1.82 , 1.95 , 1.98 , 2.63 , 2.90 , 3.00 , 3.08 , 3.12 , 3.48 ), 3.48 ), 3.48 ), 3.68 ), 4.25 , 4.45 , 4.91 , 5.35 . 13C NMR : δc 12.2 (C-18), 12.3 (C-29), 19.1 (C-21), 19.4 (C-27), 19.6 (C-19), 20.2 (C-26), 23.1 (C-28), 24.3 (C-15), 25.9 (C-23), 28.3 (C-16), 29.1 (C-25), 29.7 (C-2), 31.8 (C-7), 31.8 (C-8), 31.8 (C-22), 36.0 (C-10), 36.0 (C-20), 36.7 (C-1), 37.3 (C-12), 38.8 (C-4), 42.3 (C-13), 45.6 (C-24), 50.0 (C-9), 55.9 (C-17), 56.6 (C-14), 61.6 (C-6′), 70.6 (C-4′), 73.9 (C-2′), 77.2 (C-5′), 77.3 (C-3), 77.3 (C-3′), 101.2 (C-1′), 121.7 (C-6), 140.9 (C-5).The acetone leaf fraction (0.95 g) was eluted in the silica gel column chromatography using ethyl acetate-chloroform  as a mobile phase to obtain seven fractions. The sixth fraction was separated using ethyl acetate  yielding four fractions. The fourth fraction was then purified using chloroform-methanol  to give compound 6. White amorphous liquid (24.5 mg), TLC: Rf 0.74 , HRESIMS (+ve mode) m/z: 603.4867 [2 M + K]+ . 1H NMR : δH 0.90 , 1.30 , 1.65 , 2.03 , 2.03 , 2.37 , 5.37 , 5.37 . 13C NMR : δc 14.1 (C-18), 22.7 (C-17), 24.7 (C-3), 27.2 (C-8), 27.2 (C-11), 29.0 (C-4), 29.0 (C-5), 29.1 (C-14), 29.1 (C-15), 29.4 (C-13), 29.5 (C-6), 29.7 (C-12), 29.8 (C-7), 31.9 (C-16), 34.3 (C-2), 129.8 (C-10), 130.0 (C-9), 179.8 (C-1).The ethyl acetate seed fraction (2.10 g) was eluted in the silica gel column chromatography using hexane-ethyl acetate  as a mobile phase yielding three fractions. The second fraction was further eluted using hexane-ethyl acetate  yielding compound 7. Yellow liquid (28.1 mg), TLC: Rf 0.36 , HRESIMS (+ve mode) m/z: 572.3460 [M + H]+ . 1H NMR : δH 1.23 , 3.20 , 3.49 , 3.64 , 3.83 , 3.86 , 4.03 , 4.10 , 4.23 , 4.34 , 4.47 , 4.64 , 5.44 , 7.02 , 7.09 , 7.27 , 7.30 . 13C NMR : δc 16.7 (C-6″), 21.2 (C-7), 43.0 (C-4′), 45.5 (C-3′), 47.1 (C-2′), 47.9 (C-6′), 59.8 (C-5′), 65.9 (C-1′), 69.2 (C-5″), 69.3 (C-2″), 70.6 (C-3″), 72.3 (C-4″), 98.4 (C-1″), 116.1 (C-6), 116.7 (C-2), 128.7 (C-3), 129.0 (C-5), 131.7 (C-4), 155.7 (C-1), 156.0 (N).The acetone seed fraction (2.21 g) was eluted in the silica gel column chromatography using chloroform-methanol  as a mobile phase yielding five fractions. The fourth fraction was eluted using the same mobile phase to obtain compound 8. Reddish brown liquid (1.8 mg), TLC: Rf 0.73 , HRESIMS (+ve mode) m/z193.0489[M + K] +. 1H NMR : δH 6.98 , 7.83 , 9.89 . 13C NMR : δc 116.0 (C-1), 116.0 (C-5), 130.2 (C-3), 132.4 (C-2), 132.4 (C-4), 161.2 (C-6), 190.8 (C-7).The hexane pod fraction (19.2 g) was eluted on the silica gel column chromatography using hexane-ethyl acetate  as a mobile phase yielding four fractions. The second fraction was further eluted using hexane-ethyl acetate  giving five fractions. The third fraction was purified using hexane-ethyl acetate  yielding compound 9. White crystal (28.1 mg), m.p 166–168 °C. TLC: Rf 0.39 , HR ESIMS (+ve mode) m/z: 451.3425 [M + K] + . 1H NMR : δH 0.70 , 0.82 , 0.83 , 0.87 , 0.95 , 0.95 , 1.02 , 1.05 , 1.08 , 1.12 , 1.18 , 1.28 , 1.37 , 1.46 , 1.48 , 1.49 , 1.52 , 1.53 , 1.55 , 1.58 , 1.71 , 1.85 , 1.88 , 1.98 , 2.25 , 2.32 , 3.55 , 5.04 , 5.16 , 5.38 . 13C NMR : δc 12.0 (C-18), 12.3 (C-29), 19.4 (C-19), 19.9 (C-27), 21.1 (C-11), 21.1 (C-26), 21.3 (C-22), 24.3 (C-15), 25.6 (C-28), 29.0 (C-16), 29.8 (C-2), 31.9 (C-7), 31.9 (C-8), 31.9 (C-25), 36.5 (C-10), 37.8 (C-1), 39.8 (C-12), 40.0 (C-20), 42.3 (C-13), 44.0 (C-4), 50.1 (C-9), 51.2 (C-24), 55.9 (C-17), 56.8 (C-14), 71.8 (C-3), 121.8 (C-6), 129.3 (C-23), 138.3 (C-22), 140.8 (C-5).The ethyl acetate pod fraction (3.0 g) was eluted in the silica gel column chromatography using chloroform-ethyl acetate  as a mobile phase eluting fourteen fractions. The second fraction was further subjected to silica gel column chromatography using hexane-ethyl acetate  to obtain compound 5 cells/mL. The cells were incubated for 24 h at 37 °C in a humidified incubator  prior to reconstitution with 100 μL of test sample. Then the cells were incubated for another 24 h. 10 μL of 3--2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL in PBS buffer) was added into the mixture and was incubated further for 4 h. Upon the appearance of purple formazan crystal, the medium was removed and 100 μL of DMSO was added. After shaking the plates for 10 min, the absorbance was immediately measured using a microplate reader at 570 nm. Percentage of cell viability was determined as using the formula below, whereby OD means optical density:RBL-2H3 cells from the Japanese Collection of Research Bioresources Cell Bank  in enriched media (100 μL) were seeded in 96 well plates with cell concentrations of 2.5 × 102 and 95% air. The medium was changed every two days and the cells were sub-cultured upon 80% confluency. Once the cells reached the third passage with stable morphology and growth, the cells were used for the bioassay experiment. Only cells from third to fifth passages were used in this study.RBL-2H3 cells were cultured in MEM containing 10% FBS and 1% penicillin-streptomycin at 37 °C with 5% CO5 / 400 μL) in enriched medium were seeded and allowed to adhere onto 24 well plates in the humidified incubator  for two hours. The cells were then sensitized with anti-DNP IgE  overnight at 37 °C in 5% CO2.RBL-2H3 cells  (PIPES), 40 mM NaOH, 0.1% BSA, pH 7.2) and were reconstituted with 160 μL Siraganian buffer. After 10 min of incubation at 37 °C in 5% CO2 and 95% air, the cells were treated with 20 μL of test samples  or 20 μL of ketotifen fumarate  and incubated for another 10 min. Cells degranulation was then stimulated by the addition of 20 μL DNP-BSA (10 mg/mL) allergen for 30 min at 37 °C in 5% CO2 and 95% air. Aliquot of the supernatant (50 μL) was then transferred into a 96 well plate and incubated with a substrate  in 1:1 ratio at 37 °C for 1 h. The reaction was quenched with the addition of 200 μL of stop solution . The absorbance was measured at 405 nm using a microplate reader. Cells that were exposed to allergen were used as negative control. While cells that did not exposed to allergen were used as normal. The positive control used was ketotifen fumarate. The percentage inhibition was determined using the equation below.The inhibitory effect of beta-hexosaminidase release from RBL-2H3 cells were measured using the method described by Shahari et al. . The sen2 and 95% air. Aliquot of the supernatant (50 μL) was then centrifuged at 1000 x g at 8 °C for 20 min and the concentration of histamine released was measured using a Histamine ELISA Kit . For late-phase allergic reaction assay, the cells were incubated with 40 μL of DNP-BSA (10 mg/mL) allergen for 4 h at 37 °C in 5% CO2 and 95% air. The supernatant (100 μL) was centrifuged at 1000 x g at 8 °C for 20 min and the concentrations of IL-4 and TNF-α were measured using the respective ELISA kits .The inhibitory effects on histamine and cytokines release from RBL-2H3 cells were measured using the previously described methods , 25. Then = 3). The IC50 values with 95% confidence intervals were determined using a GraphPad Prism 5 software. Statistical significance of the data  was analysed using one-way ANOVA with GraphPad Prism 5.The data obtained were reported as mean value ± standard error of mean (SEM) representing triplicate measurements (M. oleifera leaf extract yielded a new compound (compound 1). Compound 1 was obtained as white amorphous powder (34.2 mg) with HRESIMS molecular ionisation at m/z 235.1295 corresponding to [M + Na] +. Based on the value, chemical formula of the compound was deduced as C13H24O2 with a molecular weight of 212.33 g/mol. 1H and 13C NMR spectral data of the compound (Table 3J correlation measurement between the carbon at δ 24.9 (C-6) and deshielded methylene at δ 2.3 (H-4), and 3J correlation between the carbon at δ 24.9 (C-6) and olefin at δ 5.4 (H-8)  of olefin and terminal methyl at δ 0.85 (H-13) confirmed the position of olefin at C-8 and C-9. Although the olefin resonated as a multiplet, the compound was deduced to be in E conformation which was more stable compared to Z conformation. The spectroscopical data analysis suggest the identity of compound 1 as ethyl-(E)-undec-6-enoate.The elution of hexane fraction of compound . Compouncompound . Compounowder 34. mg with 2 was obtained as reddish-brown crystals (3.9 mg) from the ethyl acetate fraction of M. oleifera leaves with HRESIMS molecular ionisation at m/z 176.9835 that correspond to [M + 2H]2+. Based on the MS data, the compound was deduced to have a molecular weight of 350.24 g/mol with a molecular formula of C15H10O10. From the NMR spectral data  and δ 7.89 (H-8) were observed on the 1H NMR spectrum. The doublets peaks were ortho-positioned to each other with J coupling value of 8.6 at A ring of the flavanol. 3J correlation between the doublets proved the ortho-position of the hydrogen. As no other hydrogen signals were observed, it was deduced that all carbons in ring B were attached to hydroxyl groups. In addition, 3J correlation between δ7.89 (H-8) and δ 168.9 (C-2) confirmed the position of the hydrogen at ring A of the flavanol structure. Based on the spectral data obtained, compound 2 was concluded to be 3,5,6-trihydroxy-2--4H-chromen-4-one.Compound 3) [4) [O-glucoside (5) [6) [7) [8) [9) [8) was not included in the bioassay study as the compound was isolated in minute quantity.Other seven isolated compounds were identified as quercetin (3) , kaempfe3) [4) , 28, β-sside (5) , oleic a (5) [6) , glucomo [6) [7) , 2,3,4-t [7) [8)  and stig [8) [9) , 34 baseM. oleifera extracts and the isolated compounds at concentrations of 7.81, 15.62 and 31.25 μg/mL were found not cytotoxic towards the RBL-2H3 cells as presented in Table Cell viability assay of 50 value of 7.17 ± 1.69 μg/mL than ketotifen fumarate with IC50 value of 15.19 ± 1.10 μg/mL (Table H-chromen-4-one (2), quercetin (3), kaempferol (4) and β-sitosterol-3-O-glucoside (5) exhibited significantly higher inhibitory activity than ketotifen fumarate at varying concentrations of 15.62 and 31.25 μg/mL, and p-values  (IC50: 10.43 ± 1.51 μM) and quercetin (3) (IC50: 19.07 ± 7.19 μM) were found to be significantly more active than ketotifen fumarate (IC50: 35.70 ± 2.59 μM).All extracts Fig. a and isomL Table . Four ofn = 3) with significance values of *p < 0.05 and **p < 0.01 as compared to positive control.Data are presented as mean ± SEM (O-glucoside (5), showed significant inhibitory activity at varying concentrations -undec-6-enoate (1), quercetin (3) and kaempferol (4) significantly inhibited histamine release in a concentration dependent manner  and glucomoringin (7) showed significant inhibitory activity at higher concentrations (15.62 and 31.25 μg/mL)  and stigmasterol (9) significantly inhibited only at the highest concentration  (IC50: 7.77 ± 27.86 μM) was found to be more active than ketotifen fumarate (IC50: 16.38 ± 0.10 μM) in inhibiting histamine release.All extracts significantly inhibited histamine release Fig. b compareons Fig. b. M. oleM. oleifera leaf and pod extracts showed higher inhibitory activity with IC50 values of 2.32 ± 0.08 and 2.74 ± 0.17 μg/mL, respectively, than ketotifen fumarate (IC50: 3.08 ± 0.49 μg/mL) (Table H-chromen-4-one (2) and glucomoringin (7), exhibited higher inhibition percentage of IL-4 release at 31.25 μg/mL than ketotifen fumarate  (IC50: 7.33 ± 1.76 μM) and glucomoringin (7) (IC50: 7.59 ± 0.39 μM) showed the most comparable inhibitory activity to ketotifen fumarate (IC50: 7.24 ± 1.16 μM).Generally, all extracts Fig. a and theate Fig. a. Howeve50 values of 1.20 ± 0.29, 2.52 ± 0.33 and 2.52 ± 0.15 μg/mL, respectively, than ketotifen fumarate (IC50: 2.91 ± 0.33 μg/mL) (Table 7) (IC50: 3.86 ± 8.26 μM) and β-sitosterol-3-O-glucoside (5) (IC50: 5.26 ± 0.00 μM) were found to be even more active than ketotifen fumarate (IC50: 6.85 ± 0.77 μM)  Table b. HoweveMoringa oleifera is a commonly used plant that has high nutritional and medicinal values. However, scientific report on its anti-allergic property to support the traditional use is still inadequate. High content of flavonoids and flavanol glucosides in M. oleifera such as quercetin, astragalin, kaempferol and rutin could be associated with the anti-allergic potential of the plant [he plant .M. oleifera ethanol (80%) extracts of various plant parts. Ethyl-(E)-2-undec-6-enoate (1), 3,5,6-trihydroxy-2--4H-chromen-4-one (2), quercetin (3), kaempferol (4) and β-sitosterol-3-O-glucoside (5) were isolated from the leaves extract. Oleic acid (6) and glucomoringin (7) were isolated from the seeds extract; while 3,5,6-trihydroxy-2--4H-chromen-4-one (2), β-sitosterol-3-O-glucoside (5), 2,3,4-trihydroxybenzaldehyde (8) and stigmasterol (9) were isolated from the pod extract. Amongst the compounds isolated, ethyl-(E)-2-undec-6-enoate (1) and 3,5,6-trihydroxy-2--4H-chromen-4-one (2) were new compounds isolated in natural product chemistry, whereas 2,3,4-trihydroxybenzaldehyde (8) is the first to be reported of its presence in Moringaceae species.From this study, nine compounds were isolated from the ƐRI complex triggering the basophils to degranulate and release mediators such as beta-hexosaminidase and histamine [RBL-2H3 cells derived from rat basophils were used to determine the allergic responses in vitro. Basophils have similarities with mast cells based on the composition of the granules and reactions towards allergens . The masistamine . Histamiistamine .Early phase of an allergic reaction occurs minutes after allergen sensitisation, releasing histamine and other mediators . WhereasM. oleifera leaf, seed and pod extracts exhibited an anti-allergic activity by inhibiting the early and late phases of allergic reaction. The leaves extract particularly suppressed the release of beta-hexosaminidase, IL-4 and TNF-α more than the positive control, ketotifen fumarate. Nevertheless, it also showed better activity in the late phase of allergic reaction (Table H-chromen-4-one (2), quercetin (3), kaempferol (4) and β-sitosterol-3-O-glucoside (5) isolated from the leaves extract inhibited beta-hexosaminidase more actively than ketotifen fumarate. In the early phase of allergic reaction, quercetin (3) was found to be more potent than ketotifen fumarate in inhibiting histamine release. In the late phase, 3,5,6-trihydroxy-2--4H-chromen-4-one (2) and quercetin (3) inhibited release of TNF-α, while β-sitosterol-3-O-glucoside (5) inhibited both IL-4 and TNF-α release more actively than ketotifen fumarate. Flavonoids were reported to have anti-allergic properties by inhibiting histamine and IL-4 release [H-chromen-4-one (2), quercetin (3) and kaempferol (4), might be responsible for the anti-allergic property due to their ability to stabilise mast cell from degranulation and inhibit the release of IL-4 and TNF-α from IgE-mediated RBL-2H3 cells. Quercetin (3) and kaempferol (4) have been reported to inhibit mast cells from degranulation by suppressing the calcium influx from extracellular medium [3) and kaempferol (4) also inhibited PLCγ and PKC pathways of the IgE-mediated allergic reaction by reducing the expression of 5-lipoxygenase, cyclooxygenase-2 and the production of leukotriene B4 and prostaglandin E2 [3) and kaempferol (4) reduced mast-cell count of passive cutaneous anaphylaxis in mice [3) with two hydroxyl groups (3′4’-trihydroxy) at the flavanol ring B has higher activity than kaempferol (4) with one hydroxyl group (4′-hydroxy group). In contrast, the current study proposed that flavanol with saturated hydroxy group on ring B of 3,5,6-trihydroxy-2--4H-chromen4-one (2) decreased the inhibitory activity of the compound.In this study, on Table . Previou release . Three fr medium , 41. Ther medium , 43. In  in mice . QuercetM. oleifera pods had higher inhibitory activity against beta-hexosaminidase and at the late phase of allergic reaction than ketotifen fumarate. The activity could be associated with its isolated compounds; 3,5,6-trihydroxy-2--4H-chromen-4-one (2), β-sitosterol-3-O-glucoside (5) and stigmasterol (9). Stigmasterol (9) only inhibited TNF-α release in RBL-2H3 cells more significantly than ketotifen fumarate. This is supported by the fact that stigmasterol (9) did not inhibit beta-hexosaminidase release from A23187-induced RBL-2H3 cells [O-tetradecanoylphorbol-13-acetate (TPA). With an addition of a sugar moeity on the sterol, β-sitosterol-3-O-glucoside (5) inhibited beta-hexosaminidase, IL-4 and TNF-α release but did not show any significant activity on histamine release. β-sitosterol-3-O-glucoside (5) might directly inhibit beta-hexosaminidase activity instead of inhibiting mast cell degranulation. In previous studies, β-sitosterol-3-O-glucoside (5) showed only 7.0% reduction of beta-hexosaminidase release from A23187-induced RBL-2H3 cells [The findings from this study showed that the extract of H3 cells . HoweverH3 cells . It alsoH3 cells  and reduH3 cells .M. oleifera seeds extract inhibited the release of beta-hexosaminidase, histamine and TNF-α more actively than ketotifen fumarate. It could be attributed to the presence of the isolated compound, glucomoringin (7). To the best of the authors’ knowledge, this is the first report of glucomoringin (7) and its effect on mast cell stabilising activity and its potential use in inhibiting the late phase of allergic response.From this study, the E)-undec-6-enoate (1) and oleic acid (6) exhibited the weakest mast cell stabilizing activity. Oleic acid (6) was reported to weakly inhibit histamine and beta-hexosaminidase release by antigen- and A23187-induced RBL-2H3 cells [2+ that induced cell degranulation [Long chain-containing chemical structure as seen in ethyl- displayed the highest inhibitory activity on beta-hexosaminidase and TNF-α release. Meanwhile, quercetin (3) was the most potent against histamine release inhibition while, β-sitosterol-3-O-glucoside (5) exhibited the highest inhibition on IL-4 release. Overall, this study suggested the anti-allergic property of ethanol (80%) extract of M. oleifera leaves, seeds and pods, as well as the isolated compounds by stabilising mast cell from degranulation and inhibiting the early and late phases of allergic reaction in anti-DNP IgE sensitised RBL-2H3 cells induced by DNP-BSA. Two new compounds; ethyl-(E)-2-undec-6-enoate (1) and 3,5,6-trihydroxy-2--4H-chromen-4-one (2), were isolated from the leaves extract. However, only the latter compound was active against the beta-hexosaminidase and TNF-α release.The leaves extract exhibited the highest inhibition against the release of beta-hexosaminidase, IL-4 and TNF-α, while the seed extract inhibited the histamine release the most. Further study revealed that isolated compound such as glucomoringin (