D101 Macroporous Resin Research Articles

Pomegranate peel-derived punicalagin: Ultrasonic-assisted extraction, purification, and its α-glucosidase inhibitory mechanism

The pomegranate peel is a by-product of pomegranate fruit rich in polyphenols. In this study, pomegranate peel polyphenols were explored using LC-MS/MS, and punicalagin was the most abundant compound. The highest yield (505.89 ± 1.73 mg/g DW) of punicalagin was obtained by ultrasonic-assisted extraction (UAE) with the ethanol concentration of 53%, sample-to-liquid ratio of 1:25 w/v, ultrasonic power of 757 W, and extraction time of 25 min. Punicalagin was further purified by the macroporous resin D101 and prep-HPLC, reaching the purity of 92.15%. The purified punicalagin had the IC50 of 82 ± 0.02 µg/mL against α-glucosidase, similar to the punicalagin standard with IC50 of 58 ± 0.014 µg/mL, both exhibiting a mixed inhibitory mechanism. Molecular docking further revealed that a steric hindrance with the intermolecular energy of −7.99 kcal/mol was formed between punicalagin and α-glucosidase. Overall, pomegranate peel is a promising source of punicalagin to develop anti-diabetic functional foods.

Screening and identification of acetylcholinesterase inhibitors from Terminalia chebula fruits based on ultrafiltration and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry

Traditional Chinese medicines (TCMs) are one of the most important sources of numerous lead compounds in drug discovery. However, rapid screening and identification of bioactive compounds from complex medicinal plants remain a technical challenge. In this work, a method based on ultrafiltration (UF) and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-qTOF-MS/MS) was developed to rapidly screen and identify specific acetylcholinesterase (AChE) inhibitors from Terminalia chebula fruits. The water extract of T. chebula fruits was subjected to column chromatography on D101 macroporous resin with increasing proportions of ethanol as the eluent to produce five fractions. Fraction E2 (30% ethanol eluent) exhibited the strongest AChE inhibitory activity and was incubated with AChE. After incubation, ultrafiltration and dissociation, 17 compounds including eight gallotannins, seven ellagitannins and two other compounds in fraction E2 were screened out and their molecular structures were identified by MS/MS spectra. Subsequently, the commercially available standards of chebulagic acid, corilagin, chebulanin and ellagic acid were used to verify the compound identification, AChE inhibitory assay in vitro and molecular docking. The study revealed that chebulanin, chebulagic acid and corilagin were responsible for AChE inhibitory activities with IC50 values of 0.35 ± 0.02 mM, 0.42 ± 0.03 mM and 0.72 ± 0.03 mM, respectively. Chebulagic acid, corilagin, chebulanin, and ellagic acid could fit well into the binding pocket of the model with binding energy values ranging from −8.7 to −6.4 kcal/mol. In conclusion, the results suggested that the developed method could be a rapid, effective, convenient and high-throughput screening approach for AChE inhibitors from TCMs.

Optimal extraction, purification and antioxidant activity of total flavonoids from endophytic fungi of Conyza blinii H. Lév.

BackgroundFlavonoids are widely used in the market because of their antibacterial, antiviral, and antioxidant activities. But the production speed of flavonoids is limited by the growth of plants. CBL9 (Chaetomium cruentum) is a flavonoid-producing endophytic fungi from Conyza blinii H. Lév, which has potential to produce flavonoids.MethodsIn this study, we isolated total flavonoids from endophytic fungus CBL9 of Conyza blinii H. Lév using macroporous resin D101. The process was optimized by response surface and the best extraction process was obtained. The antioxidant activities of total flavonoids were analyzed in vitro.ResultsIt was found that the best parameters were 25 °C pH 2.80, 1.85 h, and the adsorption ratio reached (64.14 ± 0.04)%. A total of 60% ethanol was the best elution solvent. The elution ratio of total flavonoid reached to (81.54 ± 0.03)%, and the purity was 7.13%, which was increased by 14.55 times compared with the original fermentation broth. Moreover its purity could rise to 13.69% after precipitated by ethanol, which is very close to 14.10% prepared by ethyl acetate extraction. In the antioxidant research, the clearance ratio of L9F-M on DPPH, ABTS, •OH, •O2−, (96.44 ± 0.04)% and (75.33 ± 0.03)%, (73.79 ± 0.02)%, (31.14 ± 0.01)% at maximum mass concentration, was higher than L9F.ConclusionThe result indicated using macroporous resin in the extraction of total flavonoid from endophytic fungus is better than organic solvents with higher extraction ratio, safety and lower cost. In vitro testing indicated that the flavonoid extracted by macroporous resin have good antioxidant activity, providing more evidence for the production of flavonoid by biological fermentation method.

Co-immobilized Alcohol Dehydrogenase and Glucose Dehydrogenase with Resin Extraction for Continuous Production of Chiral Diaryl Alcohol.

Ni2+-functionalized porous ceramic/agarose composite beads (Ni-NTA Cerose) can be used as carrier materials to immobilize enzymes harboring a metal affinity tag. Here, a 6×His-tag fusion alcohol dehydrogenase Mu-S5 and glucose dehydrogenase from Bacillus megaterium (BmGDH) were co-immobilized on Ni-NTA Cerose to construct a packed bed reactor (PBR) for the continuous synthesis of the chiral intermediate (S)-(4-chlorophenyl)-(pyridin-2-yl) methanol ((S)-CPMA) NADPH recycling, and in situ product adsorption was achieved simultaneously by assembling a D101 macroporous resin column after the PBR. Using an optimum enzyme activity ratio of 2:1 (Mu-S5: BmGDH) and hydroxypropyl-β-cyclodextrin as co-solvent, a space-time yield of 1560 g/(L·d) could be achieved in the first three days at a flow rate of 5 mL/min and substrate concentration of 10 mM. With simplified selective adsorption and extraction procedures, (S)-CPMA was obtained in 84% isolated yield.

Natural Deep Eutectic Solvents for the Extraction of Bioactive Steroidal Saponins from Dioscoreae Nipponicae Rhizoma

In the present study, a simple and environmentally friendly extraction method based on natural deep eutectic solvents (NADESs) was established to extract four bioactive steroidal saponins from Dioscoreae Nipponicae Rhizoma (DNR). A total of twenty-one types of choline chloride, betaine, and L-proline based NADESs were tailored, and the NADES composed of 1:1 molar ratio of choline chloride and malonic acid showed the best extraction efficiency for the four steroidal saponins compared with other NADESs. Then, the extraction parameters for extraction of steroidal saponins by selected tailor-made NADES were optimized using response surface methodology and the optimal extraction conditions are extraction time, 23.5 min; liquid–solid ratio, 57.5 mL/g; and water content, 54%. The microstructure of the DNR powder before and after ultrasonic extraction by conventional solvents (water and methanol) and the selected NADES were observed using field emission scanning electron microscope. In addition, the four steroidal saponins were recovered from NADESs by D101 macroporous resin with a satisfactory recovery yield between 67.27% and 79.90%. The present research demonstrates that NADESs are a suitable green media for the extraction of the bioactive steroidal saponins from DNR, and have a great potential as possible alternatives to organic solvents for efficiently extracting bioactive compounds from natural products.

Bioactivity-Guided Isolation of Phytochemicals from Vaccinium dunalianum Wight and Their Antioxidant and Enzyme Inhibitory Activities

Vaccinium dunalianum Wight, usually processed as a traditional folk tea beverage, is widely distributed in the southwest of China. The present study aimed to investigate the antioxidant, α-glucosidase and pancreatic lipase inhibitory activities of V. dunalianum extract and isolate the bioactive components. In this study, the crude extract (CE) from the buds of V. dunalianum was prepared by the ultrasound-assisted extraction method in 70% methanol and then purified with macroporous resin D101 to obtain the purified extract (PM). Five fractions (Fr. A–E) were further obtained by MPLC column (RP-C18). Bioactivity assays revealed that Fr. B with 40% methanol and Fr. D with 80% methanol had better antioxidant with 0.48 ± 0.03 and 0.62 ± 0.01 nM Trolox equivalent (TE)/mg extract for DPPH, 0.87 ± 0.02 and 1.58 ± 0.02 nM TE/mg extract for FRAP, 14.42 ± 0.41 and 19.25 ± 0.23 nM TE/mg extract for ABTS, and enzyme inhibitory effects with IC50 values of 95.21 ± 2.21 and 74.55 ± 3.85 for α-glucosidase, and 142.53 ± 11.45 and 128.76 ± 13.85 µg/mL for pancreatic lipase. Multivariate analysis indicated that the TPC and TFC were positively related to the antioxidant activities. Further phytochemical purification led to the isolation of ten compounds (1–10). 6-O-Caffeoylarbutin (7) showed significant inhibitory effects on α-glucosidase and pancreatic lipase enzymes with values of 38.38 ± 1.84 and 97.56 ± 7.53 µg/mL, and had the highest antioxidant capacity compared to the other compounds.

Purification and characterization of hydrolysable tannins extracted from Coriaria nepalensis bark using macroporous resin and their application in gallic acid production

As an indispensable chemical raw material, tannins are widely used in industrial production. The crude tannins extracted from plants are difficult to be used directly because of their low purity. Therefore, tannins purification are particularly important. In this study, an economical and efficient method was developed for the purification of novel vegetable tannin resources from Coriaria nepalensis bark by macroporous resin. The purity of the obtained tannin was increased significantly from 32.5%–70.6% by optimal adsorption and desorption process conditions with D101 macroporous resin. The isothermal adsorption results illustrated by Langmuir and Freundlich equations showed that the adsorption of tannin with macroporous resin was in accordance with the exothermic process. Furthermore, the structure of the purified tannins was investigated by MALDI-TOF MS. The results indicate that the main structural compositions of the obtained tannins were Tri-O-galloylglucose, Tetra-O-galloylglucose, Penta-O-galloylglucose and Hexa-O-galloylglucose. The hydroxyl and DPPH radical scavenging rates of the purified tannins at a concentration of 256 μg mL−1 increased to 86.33% and 89.08%, respectively. When the concentration was 512 μg mL−1, the reducing ability of ferricyanide to the ferrous form reached to 0.88. Moreover, the minimal inhibitory concentrations for Staphylococcus aureus and Escherichia coli were 32 μg mL−1, which is superior to commercial tannic acid. In addition, further application experiment results show that Coriaria nepalensis bark tannins can be effectively hydrolyzed under the action of tannase to obtain a high yield of gallic acid (53.2%).

Preparation and characterization of antioxidant flavonoid-enriched extract from saffron by-product: a combination of ultrasound-assisted extraction and macroporous resin purification

The stigma of saffron, a precious spice widely used in the world, takes only 7.4% (w/w) of the total weight of the flower, resulting in wasting the rest 92.6% of the flower. The utilization of the waste saffron floral bio-residues (SFB) has hence attracted people’s attention. In this paper, ultrasound-assisted extraction (UAE) associated with macroporous resin (MPR) purification step was successfully applied to enrich antioxidant flavonoid in SFB extract for the first time, resulting in significant increase in flavonoid content compared with other extraction method reported in literatures. The yield of the extract was up to 10%, and the content of kaempferol-3-O-sophoroside (KS) in the obtained extract was more than 50%, which was much higher than the total flavonoid content (2.05–19.2%) reported in other literatures. UAE procedure was optimized by studying the influence of parameters, e.g., extracting solvent, time, temperature and sample–solvent ratio, based on contents of KS and delphinidin-3,5-di-O-glucoside (DG) and the antioxidant activity of the extract. Further enrichment of flavonoid was achieved by D-101 MPR with optimum purification conditions. Furthermore, 52 flavonoids, 8 alkaloids and 10 polyols were identified or putatively characterized from the SFB extract using UPLC-MS, and the contents of KS and DG were determined to be 501.90 and 22.01 mg/g, respectively. Besides, the SFB flavonoid extract showed good antioxidant activities with IC50 33.79 μg/mL for DPPH scavenging effect and 39.65 μmol/mg trolox for ORAC values

Gracillin Isolated from Reineckia carnea Induces Apoptosis of A549 Cells via the Mitochondrial Pathway.

BackgroundReineckia carnea is commonly used to treat cough, pneumonia and other diseases in China. In our previous study, it was found that the ethanol extracts of Reineckia carnea have a strong inhibitory effect on the proliferation of human lung cancer A549 cells. Here, we isolated gracillin from ethanol extracts for the first time.PurposeClarify the antiproliferation effect of gracillin on A549 cells and further explore its mechanisms via the mitochondrial pathway.MethodsGracillin was isolated and purified by silica gel, D-101 macroporous resin and preparative RP-HPLC, then identified by NMR and HR-MS. The inhibitory effects of gracillin on the proliferation of A549 cells were detected by the MTS method. Its mechanisms were further explored by flow cytometry and Western blot.ResultsA steroid saponin, gracillin, was isolated and identified from Reineckia carnea for the first time. In a concentration-dependent and time-dependent manner, gracillin significantly inhibited the proliferation of A549 cells with an IC50 value at 2.54 μmol/L and induced morphological changes. The results of flow cytometry analysis showed that the apoptosis rate of A549 cells was significantly increased (p < 0.05), and the cells proportion was obviously arrested in S phase. The concentration of intracellular calcium was raised (p < 0.01), and the mitochondrial membrane potential was greatly decreased (p < 0.01). In addition, the expression levels of Bax, caspase-3, cleaved caspase-3, and cytochrome C were dramatically up-regulated while Bcl-2 was down-regulated (p < 0.05) in A549 cells.ConclusionThis study confirmed that gracillin has a significant antiproliferative effect on A549 cells. Gracillin could induce the apoptosis of A549 cells through the mitochondrial pathway, which might be associated with regulation of the concentration of intracellular calcium, the mitochondrial membrane potential and the expression levels of Bax, Bcl-2, caspase-3, cleaved caspase-3, and cytochrome C.

Extract from Rostellularia procumbens (L.) Nees Inhibits Thrombosis and Platelet Aggregation by Regulating Integrin β3 and MAPK Pathways.

Aim of study: The main objective of this study was to investigate the antithrombotic and antiplatelet effect of the extract from Rostellularia procumbenss (L.) Nees and understand the mechanisms by which it exerts its antithrombotic and antiplatelet mechanisms. Materials and methods: The antithrombotic effective parts (RPE) were isolated using D101 macroporous adsorption resin and potential active ingredients (JAC) were isolated using the preparative liquid-phase method. The lactate dehydrogenase kit was used to determine the toxicity of RPE and JAC to platelets. The antiadhesion effect of RPE and JAC on platelets was observed by fluorescence microscopy with rhodamine phalloidin. Antithrombotic efficacy of RPE and JAC in vivo was evaluated by establishing a rat tail thrombosis model. Contents of p-selectin, TXB2, and 6-keto-PGF1α in rat serum were measured using an enzyme-linked immunosorbent (ELISA) assay, and the rat black tail rate was measured to prove the protective effect of RPE and JAC on the tail thrombus rat model. Western blot was used for detection of serum-related proteins in the tail thrombus rat model. Results: The results showed that RPE had antithrombotic and antiplatelet effects. RPE and JAC have no toxicity to platelets. In vitro experiments showed that RPE and JAC had antiadhesion effects on platelets. In vivo experiments showed that RPE significantly inhibited the increase of p-selectin and TXB2 and significantly increased the content of 6-keto-PGF1α in the serum of rats. Western blot results demonstrated that RPE and JDB significantly inhibited the phosphorylation of the MAPK protein family in the platelets of rats, and RPE also significantly inhibited the phosphorylation of β3 protein. Conclusions: RPE has antithrombotic and antiplatelet activity in vivo and vitro. Its mechanism may be via preventing integrin αIIbβ3 activation, which in turn leads to the inhibition of the phosphorylation of the MAPK family and further suppresses TXA2, which leads to the antithrombotic and antiplatelet effects.

A sustainable and efficient preparation process of anthocyanins from blue honeysuckle fruit and comprehensive bioactivity assessment

In the present study, an integrated process for preparation of anthocyanins components from blue honeysuckle fruits using deep eutectic solvent-based negative pressure cavitation extraction (DES-NPCE) was explored rationally. Single factor optimization combined with Box-Behnken Design (BBD) were applied to confirm the influential parameters. The maximum extraction yields of total anthocyanins and main constituent cyanidin-3-glucoside (C3G) reached 6.601 mg/g and 4.839 mg/g, which were 1.197–1.565 folds of other conventional methods. Moreover, the anthocyanins products exhibited higher stability from DES-NPCE extraction than that of conventional extraction solvents, which was conducive to the preservation and utilization of anthocyanins. After enrichment with macroporous resin D101, the contents of total anthocyanins and C3G reached 25.72% and 19.05%, and the recovery yields were 81.35% and 82.24%, respectively. Furthermore, the anthocyanins products exhibited more powerful chemical and cellular antioxidant activities, which were similar or stronger than that of positive control Vc or BHT. These results revealed that the proposed process was definitely an efficient and sustainable strategy for the stabilization preparation of thermo sensitive biologically active natural products. The developed method has also proved to have sufficient potential for application in food, nutrition and pharmaceutical industries.

Abstract 1856: Effects of different ethanol elutefractions of Qigesan, Tongyoutang, Shashenmaidongtang and Buqiyunpitang on expression of secretory proteins in esophageal carcinoma EC9706cell

Abstract Purpose: To investigate the effect of different concentration of ethanol elution of Chinese medicine prescriptions(Qigesan, Tongyoutang, Shashenmaidongtang and Buqiyunpitang) on the proliferation, cell cycle and MMP1, MMP11, PCSK9, TREM2, STX1A secretory proteins expression in esophageal cancer EC9706 cells line, so as to explore the connotation of TCM syndrome pathogenesis and molecular biology. Methods: Water extracts of Qigesan, Tongyoutang, Shashenmaidong tang and Buqiyunpitang were made gradient elution and got different concentration of ethanol elution fractions by macroporous resin, ultrapure water, 30% ethanol, 50% ethanol, 70% ethanol, and anhydrous ethanol. MTT methods was used to screen the the above four prescriptions for inhibiting the proliferation of EC9706 cell. The effects of most obvious elution fractions of esophageal four prescriptions on the proliferation, cell cycle and secretory proteins expression were investigated by MTT assay, flow cytometry and Western blot analysis, respectively. Results: Different concentration of ethanol elution fractions of Qigesan, Tongyoutang, Shashenmaidong tang and Buqiyunpitang were got through macroporous resin D101 gradient elution with different concentrations of alcohol. 30% ethanol elution fraction of Qigesan, 50% ethanol elution fraction of Tongyoutang and Sashenmaidongtang, anhydrous ethanol elution fractions of Buqiyunpitang had obviously inhibitory effect on the proliferation of EC9706 cell in dose-dependent and time-dependent manner, changed morphology of EC9706 cell, which appeared as round with rough edges, karyopyknosis, and karyorrhexis. Ic50 values of Qigesan 30% ethanol elution fraction, Tongyoutang 50% ethanol elution fraction, Sashenmaidongtang 50% ethanol elution fraction, Buqiyunpitang anhydrous ethanol elution fractions for EC9706 were 825.09ug·ml-1,860.66 ug·ml-1, 270.43 ug·ml-1, 634.60 ug·ml-1, which all induced G0/G1 phase arrest in EC9706 cells. Qigesan 30% ethanol elution fractions could inhibit the expression of MMP1, MMP11, TREM2, IGFPB3; Tongyoutang 50% alcohol fractions could inhibit STX1A expression; Sashenmaidongtang 50% ethanol elution fractions could inhibit MMP1, PCSK9, TREM2, IGFPB3, STX1A expression; Buqiyunpitang anhydrous ethanol fractions could inhibit MMP1, MMP11, PCSK9, TREM2, IGFPB3 and STX1A expression. Conclusion: Qigesan 30% ethanol elution fractions, Tongyoutang and Sashenmaidongtang 50% ethanol elution fractions, Buqiyunpitang anhydrous ethanol elution fractions can inhibit the proliferation of EC9706 cell, which have close relationship with MMP1, MMP11, PCSK9, TREM2, IGFPB3, STX1A proteins expression. This study provides new basis for the development of anti-esophageal carcinoma drugs in traditional Chinese medicine. Key words: esophageal carcinoma; Qigesan; Tongyoutang; Shashenmaidongtang; Buqiyunpitang; secretory protein Citation Format: Fuchun Si. Effects of different ethanol elutefractions of Qigesan, Tongyoutang, Shashenmaidongtang and Buqiyunpitang on expression of secretory proteins in esophageal carcinoma EC9706cell [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1856.

Preparation and characterization of a low-phenylalanine whey hydrolysate using two-step enzymatic hydrolysis and macroporous resin adsorption

A low-phenylalanine (Phe) diet is indispensable for phenylketonuria patients. The objectives of this study were to develop a two-step enzymatic method to effectively release Phe from whey protein, and then to prepare a low-Phe hydrolysate (LPH) by using adsorbent. Thermolysin (T) or Pancreatin (P) combined with Protease A2SD (A2) showed the degree of hydrolysis ranging from 42.62% to 45.43% and released more than 90% Phe from whey protein. The D101 macroporous resin column removed 98.38% Phe from TA2 hydrolysate (TA2H), compared to 64.50% of Phe removal by activated carbon. The protein content of LPH prepared by a magnified D101 resin column was 60.41 g/100 g hydrolysate and its Phe content was 1.38 ± 0.11 mg/g protein equivalent. After adsorption, the percentage of peptides below 600 Da significantly increased from 50.79% to 93.60%, while the intensity of bitterness and astringency measured by electronic-tongue also increased. However, aroma-active compounds such as Millard reaction and lipid oxidation products were reduced after absorption. The LPH prepared in our study could be a suitable protein substitute to make phenylketonuria food.

Optimization of the fermentation conditions of Bacillus nematodes and screening of macroporous adsorption resin

Bacillus sp. SMrs28, metabolites from which had significant nematicidal activity, was isolated from the rhizosphere soil of Stellera chamaejasme. To determine the optimal fermentation conditions of the strain and the resin type of preliminary purified active ingredient, fermentation conditions were optimized by single factor experiment, while the macroporous resin types were screened in a static adsorption experiment. The results showed that the optimal fermentation conditions of SMrs28 strain were as follows: glucose and yeast powder were the best carbon source and nitrogen source, fermentation for 48 h, inoculum volume of 10%, temperature at 28 ℃,a rotation speed of 180 r·min-1, liquid volume of 30 mL in 150 mL triangular flask, and with an initial pH of 7.2. The static adsorption experiments showed that the adsorption and desorption of active ingre-dients in the fermentation broth by the macroporous adsorption resin D101 was significantly better than that of XAD-4, HP20 and AB-8, with the nematicidal activity of the desorption liquid being significantly improved. The nematicidal activity of fermentation broth was significantly improved by the optimization of fermentation conditions and the screening of optimal macroporous adsorption resins. These results laid a foundation for the further isolation and purification of active ingredients from SMrs28 strain, and provided theoretical basis for the development and utilization of microbial nematicides.

In vitro and in vivo anti-inflammatory effects of different extracts from Epigynum auritum through down-regulation of NF-κB and MAPK signaling pathways

Ethnopharmacological relevanceEpigynum auritum has been historically used as a “dai” or traditional medicine for the treatment of inflammation, swelling and severe pain during injury; these may reduce risk of disease and lead to healthier aging. Apart from this, Epigynum auritum extract was also used in arhritis treatment which is also a type of inflammation. Previous phytochemical studies of E. auritum revealed that steroids are main characteristic components with a number of biological activities (especially immunosuppressive and anti-inflammatory activity) Nevertheless, the underlying mechanism of the E. auritum on inflammatory diseases is still unresolved. Aim of the studyThis study aimed to comparatively investigate the anti-inflammatory potential of different fractions from the extract of E. auritum (EAE), with their possible active ingredients to reveal the underlying mechanism. Materials and methodsThe EAE was fractionated by column chromatography with macroporous resin D101 which yielded six fractions. The potential anti-inflammatory properties of different fractions of EAE were evaluated in in vitro and in vivo model. The lipopolysaccharide (LPS)-induced RAW264.7 macrophages cells were used for in vitro studies however two typical acute inflammation murine models (xylene-induced ear edema and carrageenan-induced paw edema) were used for anti-inflammatory studies. The important molecular mechanisms related to inflammation were also analyzed by ELISA, western blotting and immunofluorescence. UHPLC-MS/MS was used to analyze the chemical composition of 100% EAE fraction. ResultsDifferent EAE fractions (especially the Fr. 100% of MeOH:H2O) significantly reduced the productions of NO, ROS, TNF-α, and IL-6 by LPS-induced RAW264.7 macrophages and increased the expression of IL-10. The expression levels of iNOS and COX-2 enzymes were significantly down-regulated by 100% EAE fraction. Furthermore, 100% EAE fraction inhibited the phosphorylation of the ERK1/2, JNK, and p38 MAPK, and reduced the nuclear translocation of NF-κB which prevents its activation by blocking the phosphorylation and degradation of inhibitor protein of IκBα. In addition two inflammatory animal models; xylene-induced ear edema and carrageenan-stimulated paw edema were also developed with significantly ameliorated inflammatory cytokines. The treatment of these inflammatory models with 100% EAE fraction (Fr. 100%) suppressed the expressions of elevated inflammatory cytokines. Besides the UHPLC-HRMS/MS analysis was also carried out in which the androstane analogues were found to be as a main chemical components. ConclusionDifferent fractions (especially Fr. 100%) exert inhibitory effect on inflammation by regulating the release of inflammatory mediators through the NF-κB and MAPK signaling pathways. The androstane and its derivatives might be performing an important role in the observed anti-inflammatory activity. Therefore, Fr. 100% of EAE could be applied as a potential drug candidate for the prevention and treatment of inflammatory diseases.

Enhancing effect of macroporous adsorption resin on gamma-aminobutyric acid production by Enterococcus faecium in whole-cell biotransformation system.

Gamma-aminobutyric acid (GABA) biosynthesis depended to a great extent on the biotransformation characterization of glutamate decarboxylase (GAD) and process conditions. In this paper, the enhancing effect of D101 macroporous adsorption resin (MAR) on the GABA production was investigated based on the whole-cell biotransformation characterization of Enterococcus faecium and adsorption characteristics of D101 MAR. The results indicated that the optimal pH for reaction activity of whole-cell GAD and pure GAD was 4.4 and 5.0, respectively, and the pH range retained at least 50% of GAD activity was from 4.8 to 5.6 and 4.0-4.8, respectively. No substrate inhibition effect was observed on both pure GAD and whole-cell GAD, and the maximum activity could be obtained when the initial L-glutamic acid (L-Glu) concentration exceeded 57.6mmol/L and 96.0mmol/L, respectively. Besides, GABA could significantly inhibit the activity of whole-cell GAD rather than pure GAD. When the initial GABA concentration of the reaction solution remained 100mmol/L, 33.51 ± 9.11% of the whole-cell GAD activity was inhibited. D101 MAR exhibited excellent properties in stabilizing the pH of the conversion reaction system, supplementing free L-Glu and removing excess GABA. Comparison of the biotransformation only in acetate buffer, the GABA production, with 50g/100mL of D101 MAR, was significantly increased by 138.71 ± 5.73%. D101 MAR with pre-adsorbed L-Glu could significantly enhance the production of GABA by gradual replenishment of free L-Glu, removing GABA and maintaining the pH of the reaction system, which would eventually make the GABA production more economical and eco-friendly.

Optimization of extraction for Anemarrhena asphodeloides Bge. using silica gel-based vortex-homogenized matrix solid-phase dispersion and rapid identification of antioxidant substances.

A novel and simple method was established for the extraction and determination of seven compounds in Anemarrhena asphodeloides Bge. using silica gel-based vortex-homogenized matrix solid-phase dispersion and ultra-high performance liquid chromatography quadrupole-time of-flight mass spectrometer. The conditions for the extraction were optimized. Silica gel was used as the dispersant, 50% methanol-water was selected as an elution solvent and the grinding time was 3min. Compared with the traditional ultrasonic-assisted extraction, the developed method was rapid and efficient. In order to screen potential antioxidants, extract dealing with the optimized method was applied to a polyamide chromatography column and a D-101 macroporous resin column. Fr.2.2 showed the highest antioxidant activities with the most content of flavonoid. A total of 25 peaks were identified from the active fraction. A 2,2'-diphenyl-1-picrylhydrazyl ultra-high performance liquid chromatography coupled with mass spectrometry approach was adopted for the rapid and exact screening and identification of antioxidant compounds. It indicated that flavonoids exhibited potential antioxidant activities. The antioxidant activities of nine monomeric compounds in vivo were tested. Structure-activity relationships were discussed. Five flavonoids with the concentration of 500 µg/mL would reduce the oxidative stress of PC12 cells that were induced with 2,2'-azobis[2-methylpropionamidine] dihydrochloride.

Study on non-flavonoids chemical constituents from Spatholobi Caulis

To study the non-flavonoids chemical constituents in water extract of Spatholobi Caulis. Some purification and analysis techniques like silica gel, D101-macroporous adsorptive resins, and Sephadex LH-20 column chromatographies as well as reversed phase high-performance liquid chromatography were used to isolate and analyze the phenolic acid esters and other type compounds from Spatholobi Caulis integrally. The structures of these compounds were identified by spectroscopic techniques such as nuclear magnetic resonance and high resolution mass spectrometries. Twenty-seven compounds, including phenolic acid, coumarin, lignan, terpene, alkaloid, and steroid compounds, were isolated from ethyl acetate and n-butanol fractions in water extract of Spatholobi Caulis, and they were identified as β-sitosterol(1), feruli acid methyl ester(2), syringaresinol(3),(+)-medioresinol(4),(+)-epipinoresinol(5), p-acetylphenol(6), bolusanthin Ⅳ(7), evofolin B(8), salicylic acid(9), trans-p-hydroxy-cinnamic acid(10), abscisic acid(11), m-hydroxyphenol(12), C-veratroylglycol(13), p-hydroquinone(14), 8,9-dihydroxymegastigma-4,6-dien-3-one(15), p-hydroxybenzoic acid(16), 6,9-dihydroxymegastigma-4,7-dien-3-one(17), protocatechuic acid(18), protocatechuic acid methyl ester(19), 5,7-dihydroxycoumarin(20), isolariciresinol(21), nicotinic acid(22), daucosterol(23),(+)-pinoresinol(24), stigmasterol(25), allantoin(26) and koaburaside(27), respectively. Furthermore, compounds 2-15, 19-22, 24 and 26 were isolated from genus Spatholobus for the first time.

Investigation of Chemical Composition, Antioxidant Activity, and the Effects of Alfalfa Flavonoids on Growth Performance.

The flavonoids were extracted from alfalfa using ethanol assisted with ultrasonic extraction and purified by D101 macroporous resin column chromatography. The chemical composition and content of ethanol elution fractions (EEFs) were assessed by ultrahigh-performance liquid chromatography and hybrid quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS) and aluminum nitrate-sodium nitrite-sodium hydroxide colorimetric method. The in vitro antioxidant activity of two EEFs was conducted by scavenging DPPH free radical, and the main antioxidants of 75% EEFs were screened using DPPH-UHPLC. Moreover, the in vivo antioxidant activity of 75% EEFs and the growth performance of broilers were studied. The results showed that the content of 30% and 75% EEFs was 26.20% and 62.57%. Fifteen compounds were identified from 75% EEFs, and five of them were reported in alfalfa for the first time. The scavenging activity of 75% and 30% EEFs (200 μg/mL) against DPPH was 95.51% and 78.85%. The peak area of 5,3′,4′-trihydroxyflavone and hyperoside was decreased by 82.69% and 76.04%, which exhibited strong scavenging capacities. The total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) level of three treated groups against the normal control group (NC) fed with basal diet significantly increased by 3.89-24.49%, 0.53-7.39%, and 0.79-11.79%, respectively. While the malondialdehyde (MDA) decreased by 0.47-18.27%. Compared with the NC, the feed to gain ratio (F : G) of three treated groups was lowered by 2.98-16.53% and survival rate of broilers significantly increased. Consequently, 75% EEFs extracted from alfalfa exhibited powerful antioxidant activities and might be a potential feed additive to poultry and livestock.

Study on Antiviral in Vitro And Long-Term Toxicity to Rats of the Fruit from Prunus Cerasifera Ehrhar F

Background: In recent years, many literatures have pointed out that the fruit of Prunus cerasifera Ehrhar f. has anti-oxidation effect. Based on the anti-oxidation principle of traditional Chinese medicine in antiviral aspect the antiviral effect of the fruit was studied herein and the problem of the toxicity of the fruit has not been systematically investigated yet. To clarify the toxic effects of the fruit and its material basis, the toxicity test of the fruit was carried out. Methods:The fruit was percolated with water and the percolated liquid was concentrated to collect the concentrated liquid. Viral infection model in vitro was used to evaluate the antiviral effects of different viruses in vitro. CPE was observed under a microscope to determine the inhibitory effects of different viruses. The best type of macro-porous resin was determined by the method of macro-porous resin adsorption. The types of active ingredients were determined by physical and chemical identification, and their contents were determined by UV. By intragastric administration the long-term toxicity of percolating juice of the fruit to rats was observed including the nature severity, dose-toxicity relationship, time-toxicity relationship, main target organs, toxicity reversible degree, delayed toxicity, etc Results: showed that percolation solution of the fruit had obvious inhibitory effect on HSV-1 virus, and the TI value was 109.22. The inhibition effect of 60% ethanol precipitation on HSV-1 was the best, and the TI value was 148.84. D101 macro-porous resin presented an excellent separation effect at the volume of the first column eluted by 25% ethanol and TI value was 200.85. Chemical polyphenols were determined by physical and chemical identification and the content was 18.15 mg/g. Comparison of each dose group of percolation of the fruit and blank group there were not abnormalities in appearance signs, behavioral activities, body weight, food intake, hematological examination, serum biochemistry, organ quality and organ coefficient; Morphological examination of organs such as heart, liver, spleen, lung, kidney, ovary, testis, etc. didn’t show pathological changes related to medication; Reversible observation showed that the fruit did not cause delayed toxicity to rats. There was not statistically significant difference in the above indicators.