Sephadex LH-20 Column Research Articles

Soluble epoxide hydrolase inhibitory activity of components from Leonurus japonicus

One new compound, 10-methoxy-leonurine (1), and four known compounds (2-5) were purified by silica gel, C-18, and Sephadex LH-20 column chromatography from Leonurus japonicus. Their structures were elucidated using one-dimensional (1D)/two-dimensional (2D)-nuclear magnetic resonance (NMR), high-resolution (HR)-electrospray ionization (ESI) mass spectrometry (MS). The compounds were evaluated to determine their inhibition of the catalysis of soluble epoxide hydrolase (sEH). According to the results from in vitro analyses, compounds 1 and 2, which contain guanidine and flavonoid (3), were determined to be potential inhibitors of this enzyme. All compounds were revealed to be non-competitive inhibitors according to Lineweaver-Burk plots. Furthermore, in silico molecular docking indicated that compounds 1-3 are bound to sEH in a similar fashion and have stable binding energies, as calculated by AutoDock 4.2. Molecular dynamics determined the root-mean-square deviation (RMSD), total energy, RMS fluctuation (RMSF), hydrogen bonds, and distance of the complex according to time.

ANTIOXIDANT POTENTIAL, ANTITUMOR ACTIVITY AND PHENOLIC PROFILE OF PHOENIX DACTYLIFERA LINN

Objective: Phoenix dactylifera Linn (Fam.: arecaceae)or date fruits are commercial crops that notarized in holy quran. 70% aqueous MeOH extract of the fruits led to isolation of six compounds; its chemical structures were determined as, β-sitosterol (1), caffeic acid (2), ferulic acid (3), protocatechuic acid (4), p-hydroxybenzoic acid (5) and luteolin (6).Methods: The accurately weighed date fruits were washed, sliced and socked freshly in 70% methanol then exhaustively extracted under reflux for about 2 w and filtered, then fractionated by different solvent; finaly the butanol extract evaporated and fractionated on a polyamide glass column. Using Sephadex LH-20 column to purify the compounds obtained. In our preliminary study, the extracts and compounds were subjected to in vitro cytotoxicity against HepG2 cell line through the MTT assay and the antioxidant potential of the extracts and pure compounds were assayed through in vitro model using (DPPH) and phosphomolybdenum assays.Results: Compounds 2 and 3 exhibited promising antitumor activity with IC50 values of 6 and 10 μg/ml, respectively. Moreover compounds 1, 4, 5 and 6 showed cytotoxic activity with IC50 values of 13, 15, 21 and 35 μg/ml, respectively. The antioxidant potential of the compounds showed the inhibition percentage values (SC50) ranged from 4.36 to 10.25 μg/ml, while the total antioxidant capacity ranged from (583.66 to 702.00 mg AAE/g compound).Conclusion: Our study demonstrated that; dates constituents are powerful as antioxidant and antitumor; hence it is the best potential for pharmaceutical applications.

Chemical constituents from Murraya euchrestifolia

The open silica gel, ODS, and Sephadex LH-20 column chromatography, along with the semi-preparative HPLC was used to isolate and purify the chemical constituents from Murraya euchrestifolia. The structures of the isolates were elucidated by their physiochemical properties, NMR, and MS spectroscopic data, as well as comparison with literature data. Eighteen compounds were isolated from the CH2Cl2 fraction of the 95% aqueous EtOH extract of M. euchrestifolia, and their structures were identified as sakuranetin (1), eriodictyol-7,4'-dimethyl ether (2), isosakuranetin (3), 5-hydroxy-7,4'-dimethoxyflavanone (4), eriodictyol-7-methyl ether (5), lichexanthon (6), 5,6,7-trimethoxycoumarin (7), 5-hydroxy-6,8-dimethoxycoumarin (8), 8-hydroxy-6-methoxy-3-n-pentylisocoumarin (9), ethyl caffeate (10), 4-hydroxy-3,5- dimethoxycinnamic acid ethyl ester (11), methyl 3-(5'-hydroxyprenyl)-coumarate (12), (E)-coniferol (13), β-hydroxypropiovanillone (14), 3-hydroxy-7,8-didehydro-β-ionone (15), 3β-hydroxy-5α, 6α-epoxy-7-megastigmen-9-one (16), grasshopper ketone (17), and 4-hydroxy-3,5-dimethoxybenzaldehyde (18). Compounds 1-15 and 18 were first obtained from the plants of Murraya genus, and compounds 16 and 17 were isolated from M. euchrestifolia for the first time.

Characterization of flavonoids from fern Cheilanthes tenuifolia and evaluation of antioxidant, antimicrobial and anticancer activities

The present study is designed to identify various bioactive flavonoid compounds from the methanolic fern extract (MFE) of Cheilanthes tenuifolia. Flavonoids derived from C. tenuifolia (fern) possess potent anti-cancerous, anti-bacterial, anti-oxidant activities that are responsible for their chemo-preventive potential against selected bacterial panel. A preparative column chromatography with a column Sephadex LH-20 was used to isolate and purify flavonoids from C. tenuifolia. Their structure and chemical bonds were identified by using Nuclear Magnetic Resonance (NMR) spectroscopy and Fourier Transform-Infra Red Spectroscopy (FTIR). Two flavonoids were identified as rutin (2.8mg) and quercetin (3.34mg) from 100g of C. tenuifolia. The minimum inhibitory concentration (MIC) values of (2.25 and 0.45µg/ml) were obtained in purified flavonoids against Staphylococcus aureus and Enterobacter sp., respectively. The two flavonoids (Rutin and quercetin) showed significant in vitro anti-oxidant activity; in this regard, the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging potential of quercetin (86.1%) was higher than that of rutin (73.2%). However, for human hepatoma HepG2 and human carcinoma HeLa cells, quercetin exhibited high anti-cancer activity than rutin. The purified compounds (rutin and quercetin) with having great potential of free radical inactivator in HepG2 cells. The results suggest that MFE of C. tenuifolia could potentially be employed in traditional medicine as they are rich in compounds with anti-oxidant, anti-microbial and anti-cancer properties.

Production and Characterization of a Polyphenol Enriched Date Palm Extract from California Date Palm

Date palm (Phoenix dactylifera) is an important food cultivated in the Coachella Valley in Southern California. Medjool and deglet noor dates are the two most popular varieties and both pack an impressive list of nutrients, including vitamins, minerals, lipids and proteins. Dates are also rich in phenolic compounds including phenolic acid, flavonoids, tannins, anthocyanins and carotenoids. These compounds are associated with a variety of beneficial health effects. However, very few studies have identified the molecular mechanisms underlying these observed effects. Therefore, the aim of the present study was to produce and characterize a date palm extract (DPE) made from California dates that can then be used in subsequent in vitro and in vivo studies to elucidate the mechanisms by which dates confer beneficial health effects. California dates were harvested during the 2014 and 2015 harvest seasons and freeze‐dried date extract was produced from equal part medjool and deglet noor dates by means of liquid extraction and solid phase separation. The extracts were characterized using High Performance Liquid Chromatography (HPLC) and Matrix‐Assisted Laser Desorption Ionization Time‐of‐Flight Mass Spectrometry (MALDI‐TOF MS).HPLC chromatograms show that the predominant polyphenols present within the extracts have spectral characteristics indicative of hydroxycinnamic acids; specifically, the UV‐Visible spectra has a maximum absorbance in the range of 320–330nm. The extracts also have spectral characteristics indicative of proanthocyanidins (PAC). Specifically, the UV‐Visible spectra have an absorbance at 280nm and resolve as a poorly eluting ‘hump’ rather than well‐defined individual peaks. The qualitative and quantitative presence of PAC in the date extract was detected by butanol‐HCl and by separation of the extract on a Sephadex LH20 column prior to HPLC and MALDI‐TOF MS analysis. This separation provides an enriched PAC fraction, which helps with the subsequent characterization of PACs, providing sufficient separation of compounds to allow for the detection of PAC oligomers from 3–7 degrees of polymerization by MALDI‐TOF MS. HPLC chromatograms of the PAC fraction at 280 nm show two broad and unresolved humps, due to the structural heterogeneity of the PAC oligomers. Furthermore, positive reflectron mode MALDI‐TOF MS exhibited a series of compounds which appear to be oligomeric in nature with repeatable extension units. We propose that this mass may correspond to suberin compounds, specifically an ester of ferulic acid with long‐chain w ‐ hydroxyfatty acids (C16, C18, C20). The pattern of MALDI peaks suggests the presence of a series of w ‐ hydroxyfatty acids varying in degree of unsaturation.This study shows that date palm fruits are a good source of phenolic compounds mainly hydroxycinnamic acids, PAC and lipophilic polyphenols, and that this extract can be used for further in vitro and in vivo analysis.Support or Funding InformationFunded by California Date Commission

Characterization and mechanisms of anti-influenza virus metabolites isolated from the Vietnamese medicinal plant Polygonum chinense

BackgroundPolygonum chinense Linn. is a common medicinal plant in Southeast Asia and has been used in traditional medicine in Vietnam. The plant contains phytochemicals with various biological properties; however, its antiviral effect has not yet been demonstrated. This study was aimed to evaluate the anti-influenza virus activity of crude extracts of P. chinense, to characterize antiviral metabolites therefrom and to investigate their mechanisms of antiviral action.MethodsThe methanol (MeOH) extract and organic solvent layers of P. chinense were prepared by extraction and partition with relevant solvents. The ethyl acetate (EtOAc) layer showing antiviral activity was chromatographed repeatedly on SiO2 and Sephadex LH-20 columns to give eight pure metabolites. Their chemical structures were determined by NMR and MS spectral data. Anti-influenza virus activity of the eight metabolites against virus strains A/Puerto Rico/8/34 (H1N1, PR8), A/Hong Kong/8/68 (H3N2, HK) and B/Lee/40 (Lee) was evaluated on the basis of cytopathic effect (CPE) and plaque inhibition assays. Time-of-addition, confocal microscopy and neuraminidase inhibition assay were performed for mode-of-action studies of active ingredients.ResultsThe MeOH extract of P. chinense showed anti-influenza virus activity with EC50 values ranging from 38.4 to 55.5 μg/mL in a CPE inhibition assay. Among the eight pure metabolites isolated from P. chinense, ellagic acid (PC5), methyl gallate (PC7) and caffeic acid (PC8) significantly inhibited viral replication in a dose-dependent manner in both plaque inhibition and CPE inhibition assays with EC50 values ranging from 14.7 to 81.1 μg/mL and CC50 values higher than 300 μg/mL. Mode-of-action studies suggested that PC5 and PC7 suppress virus entry into or replication in cells, while PC8 targets influenza viral neuraminidase, even oseltamivir-resistant one.ConclusionThese results demonstrated that P. chinense and its metabolites possess effective anti-influenza virus activities. The botanical materials of P. chinense could be a promising multitargeted inhibitor of influenza A and B viruses and applied to development of a novel herbal medicine.

Inhibitory effect of proanthocyanidins from Chinese bayberry (Myrica rubra Sieb. et Zucc.) leaves on the lipid oxidation in an emulsion system

Three different Chinese bayberry leaves proanthocyanidins (BLPs) extracts were prepared by a normal extraction method, Sephadex LH-20 column purification (SPBLPs) and ultrasound degradation (UDBLPs). Their peroxyl radical scavenging capacity (PSC) and anti-lipid oxidation activity were evaluated. SPBLPs had a high PSC value at 23000.4 ± 398.5 mg of vitamin C equivalent/100 g. An emulsion model containing cholesterol and linoleic acid was used to evaluate the anti-lipid oxidation ability of BLPs. The inhibition rates of 7-ketocholesterol for the UDBLPs and SPBLPs at 40 μg/mL were 83.8 ± 0.6% and 88.5 ± 0.4% respectively, which were equivalent to that of artificial antioxidant BHT or TBHQ at 20 μg/mL. SPBLPs exhibited great antioxidant activity in prevention of lipid oxidation. The prevention of linoleic acid oxidation was highly correlated to the total phenolic contents (TPC), while the correlation of the inhibition of cholesterol oxidation and the proanthocyanidins contents was higher than that of the TPC. Overall, UDBLPs or SPBLPs could be a natural antioxidant applied for health promotion.

Anti-proliferative effect of Moringa oleifera Lam (Moringaceae) leaf extract on human colon cancer HCT116 cell line

Purpose : To investigate the in vitro anti-proliferative effect and mechanism of action of Moringa oleifera Lam. leaf extract on human colon carcinoma HCT116 cell line. Methods : M. oleifera leaves were extracted with methanol. It was fractionated by Sephadex LH-20 column chromatography. Several fractions were identified by thin layer chromatography (TLC), proton nuclear magnetic resonance (1H NMR) and mass spectrometry (MS). The growth inhibitory activity and mechanism of action of the extracts in HCT116 colon cancer cells were investigated by 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay and Western blotting. Results : Successive fractions from M. oleifera leaf crude extracts by column chromatography were combined into four pooled batches (MOL1 - MOL4) according to their absorbance at 260 nm and TLC pattern. MOL2 and MOL3 contain astragalin and isoquercetin, respectively. The results from MTT assay indicated that cell proliferation was significantly (p < 0.05) inhibited in a concentration-dependent fashion, especially by MOL2, MOL3 and MOL4. MOL2 and MOL3 exhibited a stronger cell growth inhibition than their major ingredients. The anti-proliferative activity of MOL2 - MOL4 in HCT116 colon cancer cells was mediated by downregulation of ERK1/2 phosphorylation. Conclusion : M. oleifera leaf extract has a strong anti-proliferative activity which is exerted by decreasing ERK1/2 phosphorylation. Thus, the extract has a potential for use in cancer chemoprevention. Keywords : Moringa oleifera , Anti-proliferation, Colon cancer, AKT, ERK1/2 phosphorylation, Chemoprevention

Fradiamine A, a new siderophore from the deep-sea actinomycete Streptomyces fradiae MM456M-mF7.

New bioactive substances were identified from several marine actinomycetes strains by LC-HRESI-MS based non-targeted metabolomics. A new siderophore and its derivative, named fradiamines A and B, were isolated from the extract of the deep-sea actinomycetes Streptomyces fradiae MM456M-mF7 by Diaion CHP-20P, Sephadex LH-20 column chromatography and HPLC. Fradiamine A was a new compound, but fradiamine B was previously patented as a sweetness enhancer. Their structures were determined by NMR and LC-HRESI-MS/MS analysis. Fradiamines A and B contained two alkyl amines asymmetrically bonded to citrate, a type of structure derived from actinomycetes and other bacteria and rarely observed in siderophores. Fradiamines A and B showed moderate antibiotic activity against Clostridium difficile with IC50 values of 32 and 8 μg ml-1, respectively.

PRODUCTION OF ANTIMICROBIAL AND ANTICANCER FOM FEATHER-KERATINOLYTIC NOCARDIOPSIS SP. 28ROR AS A NOVEL STRAIN USING FEATHER MEAL MEDIUM

Objective: Production of bioactive secondary metabolites from a feather-degrading actinobacterial species using feather meal medium. Methods: Protease producer actinobacterial isolates (22) recovered from farm soil, poultry farm soil and feather wastes were used to test the antagonistic effect on pathogenic bacteria and fungi, including Staphylococcus aureus, Escherichia coli, Microsporum canis and Trichophyton mentagrophyte using Mueller-Hinton agar and potato dextrose agar (PDA) media. The best isolate was used to produce the active metabolites in feather meal medium composed from (g/l) 10 feather meal, 5 sucrose and 0.3 l cement extract were dissolved in tap water, in addition to the standard medium composting of (g/l) glucose (1%), tryptone (1%), KH2PO4 (0.07%) and K2HPO4 (0.14%) were dissolved in distilled water, both of them at initial pH 9. The secondary metabolites were partially purified by Sephadex LH20 column and the antimicrobial activity and cytotoxic activity were assayed.Results: 31.82% of isolates inhibited the growth of both bacterial and fungal test organisms and the best one was Nocardiopsis sp. 28ROR (GenBank: KC702802.1) at a significant level P˂ 0.05. It produced bioactive metabolites in both feather meal broth and the standard media. The partially purified metabolites inhibit the breast cancer cell line MCF-7 (51%) and normal hepatic cell line WRL-68 (9%), in addition to inhibiting S. aureus andTrichophyton menta agrophyte growth.Conclusion: The actinobacteria has vast abilities to degrade very complex wastes and converted to simple constituents to reprocess in other industries. So the Nocardiopsis sp. 28ROR was a novel strain produced anticancer, antimicrobial substances using feather meal medium as a cheap waste medium.

A new sesquiterpene from Acorus calamus (Ⅱ)

A new quaiane-tgpe sesquiterpene was isolated from the 95% ethanol extract of the rhizomes of Acorus calamus by silica gel and sephadex LH-20 column chromatographic methods. Structure and absolute configuration of the sesquiterpene were elucidated by spectroscopic data and X-ray crystallographic analysis, and named as 1R,5R,7S-guaiane-4R,10R-diol-6-one.

Purification of quercetin-3- O -sophoroside and isoquercitrin from Poacynum hendersonii leaves using macroporous resins followed by Sephadex LH-20 column chromatography

In China, Poacynum hendersonii is frequently used as a substitute for Apoacynum venetum L (Luobuma), which is a famous traditional Chinese medicine. Quercetin-3-O-sophoroside and isoquercitrin are two major flavonoids in Poacynum hendersonii leaves. In this work, a suitable method was established for the large-scale preparation of quercetin-3-O-sophoroside (QOS) and isoquercitrin (ISO) from Poacynum hendersonii leaves using macroporous resin combined with Sephadex LH-20 column chromatography. The adsorption/desorption capacities and desorption ratios of six macroporous resins were evaluated using static experiments. The HPD-300 resin had the best adsorption performance because it had the largest surface area, and was selected for further study. Compared with pseudo-first-order and intraparticle diffusion kinetics models, the pseudo-second-order model could better fit the adsorption kinetics of both QOS and ISO on the HPD-300 resin. In addition, the adsorption isotherms of the two compounds on the HPD-300 resin were fitted well to the Langmuir model. Under optimal conditions, the purities of QOS and ISO in the product were increased from 2.16% and 1.26% to 21.34% and 10.70% with recovery yields of 82.1% and 77.3%, respectively. Subsequently, Sephadex LH-20 column chromatography was employed for improving the purities of the two compounds. After separation by Sephadex LH-20 column chromatography, the purities of QOS and ISO achieved 93.5% and 95.6%, respectively.

云南糯玉米龙须的化学成分研究

本论文研究了云南产的糯玉米的化学成分。采用正相硅胶、凝胶及反相材料进行化合物的分离和纯化，采用IR、NMR、MS等现代波谱分析鉴定化合物的结构。结果：从云南产的糯玉米中分离得到个12化合物，它们分别为：7-羟基-4’-甲氧基异黄酮(1)、柯伊利素-6-C-β-波伊文糖-7-O-β-葡萄糖苷(2)、豆甾-4-烯-3β，6β-二醇(3)、7α-羟基谷甾醇(4)、胡萝卜苷棕榈酸酯(5)、7α-羟基谷甾醇-3-O-β-D-葡萄糖苷(6)、棕榈酸(7)、胡萝卜苷(8)、对羟基桂皮酸(9)、香草酸(10)、大豆脑苷I (11)和 β-谷甾醇(12)，以上化合物为首次从该植物中分离得到。 In order to discovery activity ingredients, the chemical component of style of Zea mays was studied. Chemical constituents of style of Zea mays were isolated by silica gel, sephadex LH-20, and Rp-18 column chromatography. And their structures were elucidated by spectral methods. Twelve compounds were isolated, and their structures were identified as 7-hydroxy-4’-methoxyisoflavone (1), chrysoeriol-6-C-α-boivinopyranosyl-7-O-β-glucopyranoside (2), stigmasta-4-en-3β, 6β-diol (3), 7α-hydroxysitosterol (4), daucosterol plamitate (5), 7α-hydroxysitosterol-3-O-β-D-glucopyranoside (6), palmitic acid (7), daucosterol (8), p-hydroxycinnamic acid (9), vanillic acid (10), soya-cere- broside I (11), β-sitosterol (12). Above all compounds were obtained from this plant for the first time.

Inhibitory Activities of Stauntonia hexaphylla Leaf Constituents on Rat Lens Aldose Reductase and Formation of Advanced Glycation End Products and Antioxidant.

Stauntonia hexaphylla (Thunb.) Decne. (Lardizabalaceae) leaves (SHL) have been used traditionally as analgesics, sedatives, diuretics, and so on, in China. To date, no data have been reported on the inhibitory effect of SHL and its constituents on rat lens aldose reductase (RLAR) and advanced glycation end products (AGEs). Therefore, the inhibitory effect of compounds isolated from SHL extract on RLAR and AGEs was investigated to evaluate potential treatments of diabetic complications. The ethyl acetate (EtOAC) fraction of SHL extract showed strong inhibitory activity on RLAR and AGEs; therefore, EtOAc fraction (3.0 g) was subjected to Sephadex LH-20 column chromatography, for further fractionation, with 100% MeOH solvent system to investigate its effect on RLAR and AGEs. Phytochemical investigation of SHL led to the isolation of seven compounds. Among the isolated compounds, chlorogenic acid, calceolarioside B, luteolin-3′-O-β-D-glucopyranoside, quercetin-3-O-β-D-glucopyranoside, and luteolin-7-O-β-D-glucopyranoside exhibited significant inhibitory activity against RLAR with IC50 in the range of 7.34–23.99 μM. In addition, 3-(3,4-dihydroxyphenyl) propionic acid, neochlorogenic acid, and luteolin-3′-O-β-D-glucopyranoside exhibited the most potent inhibitory activity against formation of AGEs, with an IC50 value of 115.07–184.06 μM, compared to the positive control aminoguanidine (820.44 μM). Based on these findings, SHL dietary supplements could be considered for the prevention and/or treatment of diabetes complication.

Rapid Identification and Isolation of Inhibitors of Rat Lens Aldose Reductase and Antioxidant in Maackia amurensis.

Oxidative stress and aldose reductase activity have been implicated in the development of diabetic complications. In this study, the antioxidant and aldose reductase (AR) inhibitory effects of Maackia amurensis (MA) were investigated. The ethyl acetate fraction of the MA extract showed the highest inhibitory activity in antioxidant and rat lens AR (RLAR). To identify and isolate the active components in the ethyl acetate fraction of the MA extract, high-speed countercurrent chromatography and Sephadex LH-20 column chromatography were performed and guided by an offline HPLC-ABTS assay and HPLC microfractionation AR assay. Four antioxidants, namely, piceatannol (IC50 = 6.73 μM), resveratrol (IC50 = 11.05 μM), trans-ferulic acid (IC50 = 13.51 μM), and chlorogenic acid (IC50 = 27.23 μM), and six AR inhibitors, namely, chlorogenic acid (IC50 = 4.2 μM), tectoridin (IC50 = 50.4 μM), genistein (IC50 = 57.1 μM), formononetin (IC50 = 69.2 μM), resveratrol (IC50 = 117.6 μM), and daidzein (IC50 = 151.9 μM), were isolated and identified. The screening results of the offline HPLC-ABTS assay and HPLC microfractionation AR assay matched the activity of isolated compounds. Thus, MA is potentially valuable for antioxidant and AR inhibitor discovery and efficient drug design for the prevention and treatment of diabetic complications.

2-(2-Phenylethyl)chromones from Endophytic Fungal Strain Botryosphaeria rhodina A13 from Aquilaria sinensis

ObjectiveTo study the characteristic 2-(2-phenylethyl)chromone components of endophytic fungal strain of Aquilaria sinensis by solid culture. MethodsThe compounds were isolated by various chromatographic methods such as silica gel, reverse-phase silica gel, Sephadex-LH20 column chromatography as well as crystallization. ResultsSeven 2-(2-phenylethyl)chromone analogues were isolated from the solid culture of Botryosphaeria rhodina A13. Their structures were established by spectral data as well as physicochemical properties, and identified as 6-hydroxy-7-methoxy-2-(2-phenylethyl)chromone (1), 6,7-dimethoxy-2-(2-phenylethyl) chromone (2), (5S,6R,7S,8R)-2-(2-phenylethyl)-5,6,7,8-tetrahydrchromone (3), 6-hydroxy-2-(2-phenylethyl)chromone (4), 4′-hydroxy-2-(2-phenylethyl)chromone (5), 6-methoxy-2-phenethyl-4H-chromen-4-one (6), and 6-methoxy-2-(4′-methoxy-phenethyl)-4H-chromen-4-one (7). ConclusionAll of the compounds are isolated for the first time from the genus Botryosphaeria. This research opens up a new vista to produce the characteristic components of agarwood by endophytic fungi.

Separation and Identification of Anthocyanins Extracted from Blueberry Wine Lees and Pigment Binding Properties toward β-Glucosidase.

Anthocyanins were isolated from blueberry wine lees using Sephadex LH-20 column chromatography and semipreparative high-performance liquid chromatography (semipreparative HPLC) and then identified by HPLC-DAD-ESI-MS/MS. Our results show that malvidin-3-hexose (Mv-3-hex) and malvidin-3-(6'acetyl)-hexose (Mv-3-ace-hex) are the major components in the anthocyanin extracts of blueberry wine lees (>90%). The binding characteristics of Mv-3-hex and Mv-3-ace-hex with β-glucosidase were investigated by fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking. Spectroscopic analysis revealed that β-glucosidase fluorescence quenched by Mv-3-hex and Mv-3-ace-hex follows a static mode. Binding of Mv-3-hex and Mv-3-ace-hex to β-glucosidase mainly depends on electrostatic force. The result from CD spectra shows that adaptive structure rearrangement and increase of β-sheet structure occur only in the presence of Mv-3-ace-hex. A molecular docking study suggests that Mv-3-ace-hex has stronger binding with β-glucosidase than Mv-3-hex.

Antioxidant, anti-inflammatory and anti-septic potential of phenolic acids and flavonoid fractions isolated from Lolium multiflorum

Context: Interest has recently renewed in using Lolium multiflorum Lam. (Poaceae) (called Italian ryegrass; IRG) silage as an antioxidant and anti-inflammatory diet. Objective: This study investigated the antioxidant, anti-inflammatory and anti-septic potential of IRG silage and identified the primary components in IRG active fractions. Materials and methods: Total 16 fractions were separated from the chloroform-soluble extract of IRG aerial part using Sephadex LH-20 column before HPLC analysis. Antioxidant and anti-inflammatory activities of the fractions at doses of 0–100 μg/mL were investigated using various cell-free and cell-mediated assay systems. To explore anti-septic effect of IRG fractions, female ICR and BALB/c mice orally received 40 mg/kg of phenolic acid and flavonoid-rich active fractions F7 and F8 every other day for 10 days, respectively, followed by LPS challenge. Results: The active fractions showed greater antioxidant and anti-inflammatory potential compared with other fractions. IC50 values of F7 and F8 to reduce LPS-stimulated NO and TNF-α production were around 15 and 30 μg/mL, respectively. Comparison of retention times with authentic compounds through HPLC analysis revealed the presence of caffeic acid, ferulic acid, myricetin and kaempferol in the fractions as primary components. These fractions inhibited LPS-stimulated MAPK and NF-κB activation. Supplementation with F7 or F8 improved the survival rates of mice to 70 and 60%, respectively, in LPS-injected mice and reduced near completely serum TNF-α and IL-6 levels. Discussion and conclusion: This study highlights antioxidant, anti-inflammatory and anti-septic activities of IRG active fractions, eventually suggesting their usefulness in preventing oxidative damage and inflammatory disorders.

Lipopolysaccharide로 유도된 Raw264.7 cell에서 Rhododendron mucronulatum Turcz. Flower으로부터 분리한 myricetin에 의한 염증 억제효과

As a research of inflammation inhibitory activity using natural resource, the inflammation inhibitory activity by purified active compound from Rhododendron mucronulatum flower was experimented. Rhododendron mucronulatum flower components were purified and separated with Sephadex LH-20 and MCI gel CHP-20 column chromatography, Purified compound was confirmed as myricetin by 1HNMR, 13C-NMR and Fast atom bombardment (FAB)-Mass spectrum to have inhibition activity on inflammatory factors secreted by Raw 264.7 cells in response to lipopolysaccharide stimulation. Myricetin inhibited nitric oxide (NO) expression in a concentration dependent manner, approximately 40% inhibition was observed at a concentration of 50 μM. The inhibition effect of myricetin on inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 protein expression was 20% and 80%, respectively, at a concentration of 25 μM. Myricetin also inhibited expression of the inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and prostaglandin E2 (PGE2) in a concentration dependent manner; a concentration of 50 μM, 70%, 80%, 80% and 95% inhibition was observed, respectively. Therefore myricetin isolated from Rhododendron mucronulatum flowers is expected to have an anti-inflammatory effect in Raw 264.7 cell induced by lipopolysaccharides. The results can be expected myricetin from Rhododendron mucronulatum flower to use as functional resource for anti-inflammatory activity.

At-line hyphenation of high-speed countercurrent chromatography with Sephadex LH-20 column chromatography for bioassay-guided separation of antioxidants from vine tea (Ampelopsis grossedentata).

Vine tea (Ampelopsis grossedentata), a widely used healthy tea, beverage and herbal medicine, exhibited strong antioxidant activity. However, systematic purification of antioxidants, especially for those with similar structures or polarities, is a challenging work. Here, we present a novel at-line hyphenation of high-speed countercurrent chromatography with Sephadex LH-20 column chromatography (HSCCC-Sephadex LH-20 CC) for rapid and efficient separation of antioxidants from vine tea target-guided by 1,1-diphenyl-2-picryl-hydrazyl radical-high performance liquid chromatography (DPPH-HPLC) experiment. A makeup pump, a six-port switching valve and a trapping column were served as interface. The configuration had no operational time and mobile phase limitations between two dimensional chromatography and showed great flexibility without tedious sample-handling procedure. Seven targeted antioxidants were firstly separated by stepwise HSCCC using petroleum ether-ethyl acetate-methanol-water (4:9:4:9, v/v/v/v) and (4:9:5:8, v/v/v/v) as solvent systems, and then co-eluted antioxidants were on-line trapped, concentrated and desorbed to Sephadex LH-20 column for further off-line purification by methanol. It is noted that six elucidated antioxidants with purity over 95% exhibited stronger activity than ascorbic acid (VC). More importantly, this at-line hyphenated strategy could sever as a rapid and efficient pathway for systematic purification of bioactive components from complex matrix.