The ginger extract (50 g) was chromatographed on a Sephadex LH-20 column with 95% ethanol as eluant to remove the nonphenolic compounds (Fraction 1, 21.8 g) and to generate the gingerols and shogaols enriched fraction (Fraction 2, 28 g). Fraction 2 was then loaded into a Diaion HP-20 column eluted first with water to remove the water soluble compounds and then with 40% aqueous ethanol to obtain fraction A (9 g) followed by 95% aqueous ethanol to obtain fraction B (11g). Fraction A (5g) was subjected to a normal phase silica gel column with a stepwise gradient of hexane/ethyl acetate [9:1; 8:2, and 7:3] to give pure [6]- (2g), [8]- (0.5 g), and [10]-gingerol (0.4 g). Fraction B (5 g) was also subjected to a normal phase silica gel column with a stepwise gradient of hexane/ethyl acetate [9:1 and 8:2] to generate 13 fractions. Fraction B5 (1g) was subjected to a C-18 reverse phase column eluted with a stepwise gradient of methanol/water [3:2, 7:3, and 4:1] to give [6]-paradol (40 mg), [1]-dehydrogigerdione (60 mg), and [10]-shogaol (120 mg). Following a similar procedure, fraction B7 (1.5 g) gave 200 mg [8]-shogaol and 1 g of [6]-shogaol. The purification procedure was guided by TLC and HPLC analysis. The structures of these eight compounds were confirmed based on their 1H and 13C NMR analysis (Figure 1 ).
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Sephadex LH 20
Sephadex LH 20
Sephadex LH-20 is a cross-linked dextran-based gel filtration resin used for the purification and separation of molecules based on size and molecular weight.
It is commonly utilized in biochemistry and analytical chemistry to isolate and purify proteins, peptides, nucleic acids, and other biomolecules.
Sephadex LH-20 offers high resolution and reproducible results, making it a valuable tool for research involving complex biological samples.
Its versatile applications include purification of enzymes, removal of salts and small molecules, and fractionation of macromolecules.
With the advent of AI-driven platforms like PubCompare.ai, researchers can now easily access optimized protocols and compare best practices to enhance the reproducibility and effeciency of their Sephadex LH-20 experiments.
It is commonly utilized in biochemistry and analytical chemistry to isolate and purify proteins, peptides, nucleic acids, and other biomolecules.
Sephadex LH-20 offers high resolution and reproducible results, making it a valuable tool for research involving complex biological samples.
Its versatile applications include purification of enzymes, removal of salts and small molecules, and fractionation of macromolecules.
With the advent of AI-driven platforms like PubCompare.ai, researchers can now easily access optimized protocols and compare best practices to enhance the reproducibility and effeciency of their Sephadex LH-20 experiments.
Most cited protocols related to «Sephadex LH 20»
Carbon-13 Magnetic Resonance Spectroscopy
Diaion HP 20
Ethanol
ethyl acetate
ginger extract
gingerol
Hexanes
High-Performance Liquid Chromatographies
Methanol
sephadex LH 20
shogaol
Silica Gel
GBS pigment was purified as previously described (Rosa-Fraile et al., 2006 (link)) with some modifications. In brief, WT GBS were grown at 37°C in New Granada Media (de la Rosa et al., 1992 (link)) until the broth turned red (48–72 h). Bacterial cells were pelleted, washed three times with distilled water and twice with DMSO. The cell pellet was then resuspended in DMSO:0.1% TFA overnight to extract the pigment, cell debris was pelleted, and the supernatant containing the pigment was saved. The above process was repeated until the supernatant obtained from GBS cells was clear. Pigment was then precipitated by addition of 25% NH4OH to a final concentration of 0.25% as previously described (Vanberg et al., 2007 (link)). Precipitated pigment was washed three times with HPLC grade water and twice in DMSO, redissolved in DMSO:0.1%TFA, and purified using a Sephadex LH-20 (GE Healthcare) column as previously described (Rosa-Fraile et al., 2006 (link); Vanberg et al., 2007 (link)). Fractions containing purified pigment were pooled and precipitated with NH4OH (Scientific Products) as described above, washed three times with HPLC grade water, twice with DMSO, and lyophilized. As a control, GBSΔcylE was also grown in New Granada Media and pigment extraction protocol was followed as described above. For NMR analysis, purified pigment or control ΔcylE samples were resuspended in DMSO-d6:0.1% d-TFA (Sigma-Aldrich). 1H,13C, 1H-COSY NMR experiments were performed at 298K on a Bruker AV-500 NMR Spectrometer. Residual DMSO-d5 was used to calibrate chemical shifts. For MS experiments, lyophilized pigment or control ΔcylE samples were dissolved in DMSO:0.1%TFA and analyzed by Fourier Transform Ion Cyclotron Resonance mass spectrometry on a Bruker AutoFlex APEX Qe 47e instrument. For hemolytic and cytotoxic assays, lyophilized pigment or control ΔcylE extract was dissolved in DTS to a final concentration of 1mM. The samples were incubated overnight at room temperature in the dark before use.
Hemolytic titer assays was performed using methods described with some modifications (Nizet et al., 1996 (link)). In brief, twofold serial dilutions of purified pigment or control ΔcylE extract in DTS was performed in PBS + 0.2% glucose in a final volume of 100 µl. These samples were then incubated with 100 µl of heparin-treated hRBCs (1%) in 96-well plates at 37°C for 1 h, after which the plates were spun for 4 min at 3,000 g to pellet unlysed hRBC. The supernatants were transferred to a replica 96-well plate and hemoglobin release was measured by recording the absorbance at 420 nm. Positive and negative controls included wells that contained hRBC with 0.1% SDS or PBS, respectively. Solvent control for each pigment concentration was included in the analysis. The effective concentration 50 is the concentration of pigment that produces 50% hemoglobin release compared with the SDS control and was determined using non linear regression. The experiment was performed in triplicate using three independent preparations of purified pigment.
For proteinase K treatment of the pigment before hemolytic assays, pigment and control ΔcylE samples in DTS was lyophilized and dissolved in proteinase K buffer (20 mM Tris, pH 8.0, and 1 mM CaCl2). Each sample was divided into two and proteinase K was added at a final concentration of 0.25 mg/ml, as previously described (Vanberg et al., 2007 (link)), to one of the aliquots and all samples were incubated at 37°C for 1 h. Hemolytic titer assays were then performed on pigment and control samples that were treated with and without proteinase K. Buffer controls were also included. The activity of proteinase K used in these experiments was confirmed by digesting 100 µg BSA with 0.25 mg/ml proteinase K at 37°C for 1 h followed by 12% SDS-PAGE and SYPRO Ruby staining.
Hemolytic titer assays was performed using methods described with some modifications (Nizet et al., 1996 (link)). In brief, twofold serial dilutions of purified pigment or control ΔcylE extract in DTS was performed in PBS + 0.2% glucose in a final volume of 100 µl. These samples were then incubated with 100 µl of heparin-treated hRBCs (1%) in 96-well plates at 37°C for 1 h, after which the plates were spun for 4 min at 3,000 g to pellet unlysed hRBC. The supernatants were transferred to a replica 96-well plate and hemoglobin release was measured by recording the absorbance at 420 nm. Positive and negative controls included wells that contained hRBC with 0.1% SDS or PBS, respectively. Solvent control for each pigment concentration was included in the analysis. The effective concentration 50 is the concentration of pigment that produces 50% hemoglobin release compared with the SDS control and was determined using non linear regression. The experiment was performed in triplicate using three independent preparations of purified pigment.
For proteinase K treatment of the pigment before hemolytic assays, pigment and control ΔcylE samples in DTS was lyophilized and dissolved in proteinase K buffer (20 mM Tris, pH 8.0, and 1 mM CaCl2). Each sample was divided into two and proteinase K was added at a final concentration of 0.25 mg/ml, as previously described (Vanberg et al., 2007 (link)), to one of the aliquots and all samples were incubated at 37°C for 1 h. Hemolytic titer assays were then performed on pigment and control samples that were treated with and without proteinase K. Buffer controls were also included. The activity of proteinase K used in these experiments was confirmed by digesting 100 µg BSA with 0.25 mg/ml proteinase K at 37°C for 1 h followed by 12% SDS-PAGE and SYPRO Ruby staining.
Even if cranberry is a supplement food, the study has been approved by the different research ethics committees (1/Comité d'éthique Sud Méditerranée III, Nîmes, France; 2/Comité Etico de Investigaciòn Clinica (CEIC) de Fundació Puigvert, Barcelona, Spain; 3/Ethical committee of Jahn Ferenc South-Pest Teaching Hospital, Hungary; 4/Ethics Committee of Medicine and Medical Care, University of Occupational and Environmental Health, Japan), in each country and has been conducted according to the principles expressed in the Declaration of Helsinki. Thirty-two female, sexually-active volunteers over the age of 18, with normal renal function were recruited through four urology departments in Japan (Kyushu University Hospital), Hungary (South-Pest Hospital, Budapest), Spain (Fundació Puigvert, Barcelona), and France (Hôpital Foch, Suresnes) (eight volunteers/department) to participate in this randomized, double-blind, placebo-controlled cross-over study. Volunteers were cleared through ethics committees and provided informed consent. Exclusion criteria included antibiotic use within six months prior to the study, pregnancy, known allergy or intolerance of cranberry products, or routine consumption of any food supplements consisting of vitamins, minerals or trace elements. Throughout the study, volunteers were instructed not to alter their dietary or lifestyle habits in any way. However, during the capsule consumption, volunteers were told to avoid all Vaccinium-containing foods, drinks and supplements including other forms of V. macrocarpon (cranberry), V. myrtillus (bilberry or European blueberry), V. angustifolium (wild or lowbush blueberry), V. corymbosum (highbush blueberry), and V. vitis-ideae (lingonberry). In addition, volunteers were instructed to limit consumption of chocolate, tea, grape and wine. All medications taken during the study were reported to the doctor responsible for following the study at each center.
The study was carried out using commercially available capsules of cranberry powder (Urell®/Ellura™ Pharmatoka, Rueil Malmaison, France) and capsules of placebo composed of colloidal silica, magnesium stearate, cellulose and gelatin. Capsules were opaque to conceal the color of the contents. The PACs in the cranberry powder were quantified by Brunswick Laboratories (Norton, MA) using a colorimetric assay, an updated dimethylaminocinnamaldehyde method (DMAC method), taking advantage of the selective colorimetric reaction between PACs and DMAC after open column gel chromatography on Sephadex® LH-20 (Amersham). The capsule dosages were standardized to deliver 18 or 36 mg of PAC equivalents in the cranberry powder.
The volunteers in Japan and Hungary received 0, 36 or 72 mg PAC equivalents per day and those in France and Spain received 0, 18 or 36 mg PAC equivalents per day. Each volunteer received successively three regimens (always 2 capsules) distributed in random order, consisting of: (1) cranberry (2 PAC dosage levels), or (2) placebo, or (3) 1 capsule of cranberry at each PAC dosage and 1 capsule of placebo, with a washout period of at least one week between each regimen. Volunteers consumed the capsules in the morning at 8:00 AM. The first urines were collected from 9:00 AM-2:00 PM following capsule consumption, and pooled. The second collections were made the following morning (8:00 AM). Pre-cranberry consumption urines collections (0 h) were obtained at the beginning of the study in each volunteer. Various biological and physicochemical parameters of the urine samples (collected at each regimen) were measured using the Multistix® (Bayer) system. Urines with an abundance of leukocytes and nitrites were excluded. Remaining samples were centrifuged at 4000 g for 15 minutes, sterilized by filtration (0.45 μm), separated in 3 aliquots and stored at -20°C.
A uropathogenic E. coli strain previously isolated from a patient with UTI (NECS978323) [12 (link)], with P-fimbriae papG and type-1 pili was utilized. To allow the direct observation of adherent bacteria by fluorescence microscopy, NECS978323 was genetically modified to express green fluorescent protein (GFP) using a pBBR-derived non-mobilizable plasmid carrying a GFP expression cassette [24 (link)]. Bacteria were grown in trypticase soy broth (bioMerieux, Marnes La Coquette, France) or colonization factor antigen agar for 16 h at 37°C to enhance expression of P-fimbriae.
To insure product potency, the PAC-standardized cranberry powder was tested for in vitro uropathogenic bacterial anti-adhesion activity prior to consumption by the volunteers, utilizing a MRHA, to detect anti-adhesion activity in uropathogenic P-fimbriated E. coli [11 (link)]. Briefly, the anti-adhesion bioactivity of the powder was tested by measuring the ability of the fractions to suppress agglutination of human red blood cells (HRBC) (A1, Rh+) following incubation with uropathogenic P-fimbriated E. coli. Bacteria were suspended in phosphate-buffered saline (PBS) solution at pH 7.0 at a concentration of 5 × 108 bacteria/mL. The powder was dissolved in PBS at a starting concentration of 60 mg/mL, and the pH adjusted to neutrality with NaOH. A serial 2-fold dilution was prepared, and each dilution (30 mL) was incubated with 10 mL of bacterial suspension on a 24-well polystyrene plate for 10 min at room temperature on a rotary shaker. A 3% v/v suspension of HRBCs in PBS was prepared, and 10 mL of the diluted blood was added to the test suspension. The suspension was incubated for 20 min on a rotary shaker at 21C and evaluated microscopically for the ability to prevent agglutination. The final dilution concentration was recorded at which agglutination suppression by the cranberry fraction occurred. Wells containing bacteria plus PBS, HRBC plus PBS, bacteria plus test fraction, and HRBC plus test fraction served as negative controls for agglutination, and wells containing bacteria plus HRBC served as a positive control for agglutination. Assays were performed in triplicates.
Urines were tested ex vivo for anti-adhesion activity before and after the treatment regimes, utilizing two separate assays. The MRHA assay described above was modified by substituting urine for a cranberry solution. Briefly, a 30-μL drop of each urine was incubated with 10 μL of the bacterial suspension on a 24-well polystyrene plate for 10 min at 21C on a rotary shaker. The HRBCs were added to the urines, incubated for 20 min on a rotary shaker at 21C and evaluated microscopically for the ability to prevent agglutination. If no agglutination was observed, the urine was considered to contain anti-adhesion metabolites and was recorded as possessing Anti-Adhesion Activity (AAA). The results were expressed as a percentage of anti-adhesion activity (0% MRHA = 100% AAA, 50% MRHA = 50% AAA and 100% MRHA = 0% AAA). Assays were repeated 4 times on triplicate urine samples and the standard error calculated.
In the second ex vivo urine assay, bacterial adhesion was evaluated utilizing the human T24 epithelial cell-line (ATCC HTB-4). A new technology was developed using fluorescent NECS978323 to enhance detection of strain adhesion. Monolayers of epithelial cells were grown at 37°C in McCoy's 5a medium containing 10% (v/v) fetal calf serum, 1.5 mM glutamine, and antibiotics (50 U/mL penicillin and 50 mg/mL of streptomycin), on coverslips in 24-well Falcon tissue culture plates. Bacteria were grown overnight in the urine samples containing 5% (v/v) Luria broth. Bacterial were harvested by centrifugation, resuspended at DO600 0.1 in McCoy's medium, added to the tissue culture and incubated for 2.5 h at 37°C. After washes with PBS, the cells were fixed with 4% paraformaldehyde, incubated 20 min at room temperature, and examined under a fluorescent microscope. An adhesion index (AI) corresponding to the mean number of adherent bacteria per cell for 100 cells was calculated. This index was expressed as the mean of at least three independent assays.
C. elegans has been used to develop an easy model system of host-pathogen interactions to identify basic evolutionarily conserved pathways associated with microbial pathogenesis. This test is based upon the capacity of E. coli to be ingested by C. elegans nematodes leading to infection and ultimately involving the killing death of the worms [25 (link)]. Percentage of killed nematodes in presence of the E. coli strain is an indirect marker of virulence potential of this strain. The C. elegans infection assay was carried out as described by Lavigne et al. [25 (link)] using Fer-15 mutant line, which has a temperature sensitive fertility defect. Fer-15 was provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). Briefly to synchronize the growth of worms, eggs were collected using the hypochlorite method. E. coli strain was grown for overnight in human urine containing 5% (v/v) nematode growth medium (NGM). Bacteria cells were harvested by centrifugation, washed once and suspended in phosphate buffered saline solution (PBS) at pH 7.0 at a concentration of 105 CFU/ml. NGM agar plates were inoculated with 10 μl of strain and incubated at 37°C for 8-10 h. Plates were allowed to cool to room temperature and seeded with L4 stage worms (20-30 per plate). Plates were then incubated at 25°C and scored each day for live worms under a stereomicroscope (Leica MS5). At least three replicates repeated 5 times were performed for each selected clone. A worm was considered dead when it no longer responded to touch. Worms that died as a result of becoming stuck to the wall of the plate were excluded from the analysis. Lethal Time 50% (LT50) and death (LT100) corresponded to time (in hours) required to kill 50% and 100% of the nematode population, respectively. OP50 is an avirulent E. coli strain, an international standard food for nematodes used as control. The number of bacteria within the C. elegans digestive tract was carried out as described by Garsin et al. [26 (link)]. Five C. elegans were picked at 72 h, and the surface bacteria were removed by washing the worms twice in 4 μl drops of M9 medium on a NGM agar plate containing 25 μg/ml gentamicin. The nematodes were placed in a 1.5 ml Eppendorf tube containing 20 μl of M9 medium with 1% Triton X-100 and were mechanically disrupted by using a pestle. The volume was adjusted to 50 μl with M9 medium containing 1% Triton X-100 which was diluted and plated on Luria-Bertani agar containing 50 μg/ml ampicillin. At least three replicates were performed for each assay.
The quantitative variables were described by the median values, the range and the mean, and standard deviation. The qualitative variables were described by figures and percentages. The 95% confidence intervals were assessed by the exact method of Clopper-Pearson. Frequencies between AAA = 0% and AAA > 0% were compared according to the criteria using a Fisher exact test and the index values were compared using a Kruskal-Wallis test. The index value was modelled by the hours, the country and the dose using a Generalized Estimating Equation model. Survival curves of the worms were explored in a univariate analysis (Kaplan-Meier curves). The median values of survival times were given. The survival curves were compared using log-rank test. A multivariate survival analysis was then performed (Cox model). No procedure of variables selection was performed. The assumptions of proportional hazards were checked. A value of p ≤ 0.05 given by the SAS®/ETS software (version 8.1) (SAS Institute Inc, Cary, NC, USA) was considered statistically significant.
The study was carried out using commercially available capsules of cranberry powder (Urell®/Ellura™ Pharmatoka, Rueil Malmaison, France) and capsules of placebo composed of colloidal silica, magnesium stearate, cellulose and gelatin. Capsules were opaque to conceal the color of the contents. The PACs in the cranberry powder were quantified by Brunswick Laboratories (Norton, MA) using a colorimetric assay, an updated dimethylaminocinnamaldehyde method (DMAC method), taking advantage of the selective colorimetric reaction between PACs and DMAC after open column gel chromatography on Sephadex® LH-20 (Amersham). The capsule dosages were standardized to deliver 18 or 36 mg of PAC equivalents in the cranberry powder.
The volunteers in Japan and Hungary received 0, 36 or 72 mg PAC equivalents per day and those in France and Spain received 0, 18 or 36 mg PAC equivalents per day. Each volunteer received successively three regimens (always 2 capsules) distributed in random order, consisting of: (1) cranberry (2 PAC dosage levels), or (2) placebo, or (3) 1 capsule of cranberry at each PAC dosage and 1 capsule of placebo, with a washout period of at least one week between each regimen. Volunteers consumed the capsules in the morning at 8:00 AM. The first urines were collected from 9:00 AM-2:00 PM following capsule consumption, and pooled. The second collections were made the following morning (8:00 AM). Pre-cranberry consumption urines collections (0 h) were obtained at the beginning of the study in each volunteer. Various biological and physicochemical parameters of the urine samples (collected at each regimen) were measured using the Multistix® (Bayer) system. Urines with an abundance of leukocytes and nitrites were excluded. Remaining samples were centrifuged at 4000 g for 15 minutes, sterilized by filtration (0.45 μm), separated in 3 aliquots and stored at -20°C.
A uropathogenic E. coli strain previously isolated from a patient with UTI (NECS978323) [12 (link)], with P-fimbriae papG and type-1 pili was utilized. To allow the direct observation of adherent bacteria by fluorescence microscopy, NECS978323 was genetically modified to express green fluorescent protein (GFP) using a pBBR-derived non-mobilizable plasmid carrying a GFP expression cassette [24 (link)]. Bacteria were grown in trypticase soy broth (bioMerieux, Marnes La Coquette, France) or colonization factor antigen agar for 16 h at 37°C to enhance expression of P-fimbriae.
To insure product potency, the PAC-standardized cranberry powder was tested for in vitro uropathogenic bacterial anti-adhesion activity prior to consumption by the volunteers, utilizing a MRHA, to detect anti-adhesion activity in uropathogenic P-fimbriated E. coli [11 (link)]. Briefly, the anti-adhesion bioactivity of the powder was tested by measuring the ability of the fractions to suppress agglutination of human red blood cells (HRBC) (A1, Rh+) following incubation with uropathogenic P-fimbriated E. coli. Bacteria were suspended in phosphate-buffered saline (PBS) solution at pH 7.0 at a concentration of 5 × 108 bacteria/mL. The powder was dissolved in PBS at a starting concentration of 60 mg/mL, and the pH adjusted to neutrality with NaOH. A serial 2-fold dilution was prepared, and each dilution (30 mL) was incubated with 10 mL of bacterial suspension on a 24-well polystyrene plate for 10 min at room temperature on a rotary shaker. A 3% v/v suspension of HRBCs in PBS was prepared, and 10 mL of the diluted blood was added to the test suspension. The suspension was incubated for 20 min on a rotary shaker at 21C and evaluated microscopically for the ability to prevent agglutination. The final dilution concentration was recorded at which agglutination suppression by the cranberry fraction occurred. Wells containing bacteria plus PBS, HRBC plus PBS, bacteria plus test fraction, and HRBC plus test fraction served as negative controls for agglutination, and wells containing bacteria plus HRBC served as a positive control for agglutination. Assays were performed in triplicates.
Urines were tested ex vivo for anti-adhesion activity before and after the treatment regimes, utilizing two separate assays. The MRHA assay described above was modified by substituting urine for a cranberry solution. Briefly, a 30-μL drop of each urine was incubated with 10 μL of the bacterial suspension on a 24-well polystyrene plate for 10 min at 21C on a rotary shaker. The HRBCs were added to the urines, incubated for 20 min on a rotary shaker at 21C and evaluated microscopically for the ability to prevent agglutination. If no agglutination was observed, the urine was considered to contain anti-adhesion metabolites and was recorded as possessing Anti-Adhesion Activity (AAA). The results were expressed as a percentage of anti-adhesion activity (0% MRHA = 100% AAA, 50% MRHA = 50% AAA and 100% MRHA = 0% AAA). Assays were repeated 4 times on triplicate urine samples and the standard error calculated.
In the second ex vivo urine assay, bacterial adhesion was evaluated utilizing the human T24 epithelial cell-line (ATCC HTB-4). A new technology was developed using fluorescent NECS978323 to enhance detection of strain adhesion. Monolayers of epithelial cells were grown at 37°C in McCoy's 5a medium containing 10% (v/v) fetal calf serum, 1.5 mM glutamine, and antibiotics (50 U/mL penicillin and 50 mg/mL of streptomycin), on coverslips in 24-well Falcon tissue culture plates. Bacteria were grown overnight in the urine samples containing 5% (v/v) Luria broth. Bacterial were harvested by centrifugation, resuspended at DO600 0.1 in McCoy's medium, added to the tissue culture and incubated for 2.5 h at 37°C. After washes with PBS, the cells were fixed with 4% paraformaldehyde, incubated 20 min at room temperature, and examined under a fluorescent microscope. An adhesion index (AI) corresponding to the mean number of adherent bacteria per cell for 100 cells was calculated. This index was expressed as the mean of at least three independent assays.
C. elegans has been used to develop an easy model system of host-pathogen interactions to identify basic evolutionarily conserved pathways associated with microbial pathogenesis. This test is based upon the capacity of E. coli to be ingested by C. elegans nematodes leading to infection and ultimately involving the killing death of the worms [25 (link)]. Percentage of killed nematodes in presence of the E. coli strain is an indirect marker of virulence potential of this strain. The C. elegans infection assay was carried out as described by Lavigne et al. [25 (link)] using Fer-15 mutant line, which has a temperature sensitive fertility defect. Fer-15 was provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). Briefly to synchronize the growth of worms, eggs were collected using the hypochlorite method. E. coli strain was grown for overnight in human urine containing 5% (v/v) nematode growth medium (NGM). Bacteria cells were harvested by centrifugation, washed once and suspended in phosphate buffered saline solution (PBS) at pH 7.0 at a concentration of 105 CFU/ml. NGM agar plates were inoculated with 10 μl of strain and incubated at 37°C for 8-10 h. Plates were allowed to cool to room temperature and seeded with L4 stage worms (20-30 per plate). Plates were then incubated at 25°C and scored each day for live worms under a stereomicroscope (Leica MS5). At least three replicates repeated 5 times were performed for each selected clone. A worm was considered dead when it no longer responded to touch. Worms that died as a result of becoming stuck to the wall of the plate were excluded from the analysis. Lethal Time 50% (LT50) and death (LT100) corresponded to time (in hours) required to kill 50% and 100% of the nematode population, respectively. OP50 is an avirulent E. coli strain, an international standard food for nematodes used as control. The number of bacteria within the C. elegans digestive tract was carried out as described by Garsin et al. [26 (link)]. Five C. elegans were picked at 72 h, and the surface bacteria were removed by washing the worms twice in 4 μl drops of M9 medium on a NGM agar plate containing 25 μg/ml gentamicin. The nematodes were placed in a 1.5 ml Eppendorf tube containing 20 μl of M9 medium with 1% Triton X-100 and were mechanically disrupted by using a pestle. The volume was adjusted to 50 μl with M9 medium containing 1% Triton X-100 which was diluted and plated on Luria-Bertani agar containing 50 μg/ml ampicillin. At least three replicates were performed for each assay.
The quantitative variables were described by the median values, the range and the mean, and standard deviation. The qualitative variables were described by figures and percentages. The 95% confidence intervals were assessed by the exact method of Clopper-Pearson. Frequencies between AAA = 0% and AAA > 0% were compared according to the criteria using a Fisher exact test and the index values were compared using a Kruskal-Wallis test. The index value was modelled by the hours, the country and the dose using a Generalized Estimating Equation model. Survival curves of the worms were explored in a univariate analysis (Kaplan-Meier curves). The median values of survival times were given. The survival curves were compared using log-rank test. A multivariate survival analysis was then performed (Cox model). No procedure of variables selection was performed. The assumptions of proportional hazards were checked. A value of p ≤ 0.05 given by the SAS®/ETS software (version 8.1) (SAS Institute Inc, Cary, NC, USA) was considered statistically significant.
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Bromides
Cells
Colon Adenocarcinomas
diphenyl
ginger extract
gingerol
HCT116 Cells
High-Performance Liquid Chromatographies
Histocompatibility Testing
Homo sapiens
Lung Cancer
Macrophage
Mus
sephadex LH 20
Silica Gel
Solvents
Tetrazolium Salts
Chemicals Porcine pancreas α-amylase type VI (EC 3.2.1.1), α-glucosidase type I from Baker’s Yeast (EC 3.2.1.20), 3,5-dinitrosalicylic acid (DNS), p-nitrophenyl-α-D-glucopyranose (PNPG), maltose and acarbose were obtained from Sigma-Aldrich (Paris, France). Soluble starch, sodium dihydrogenphosphate (NaH2PO4), sodium potassium tartrate and sodium chloride were purchased from Merck. Analytical grade solvents for extraction and HPLC grade solvents for chromatography were from Scharlau (Barcelona, Spain). HPLC grade water was obtained by an EASY-pure II (Barnstead, Dubuque IA, USA) water purification system. Deuterated solvents were purchased from Armar Chemicals (Döttingen, Switzerland).
General Analytical HPLC separations were carried out on a system consisting of a 1100 series binary high-pressure mixing pump with degasser module, column oven and a 1100 series PDA detector (all Agilent, Waldbronn, Germany). A Gilson 215 liquid handler with a Gilson 819 injection module and 50 μL loop was used as autosampler. The HPLC was coupled to an Esquire 3000 Plus ion trap mass spectrometer equipped with an electrospray (ESI) interface (Bruker Daltonics, Bremen, Germany). Data acquisition and processing was performed using HyStar 3.0 software (Bruker Daltonics). Semi-preparative HPLC separations were carried out on an Agilent 1100 series HPLC system consisting of a 1100 series quaternary low-pressure mixing pump with degasser module, column oven, a 1100 series PDA detector, and an autosampler with a 1000 μL loop. The preparative HPLC system consisted of a Shimadzu SCL-10VP controller and binary pump (LC-8A), a UV–vis SPD-M10A VP detector and Class-VP 6.12 as software. NMR spectra were recorded on an Avance III spectrometer operating at 500 MHz and 125 MHz for 1H and 13C, respectively (Bruker Biospin, Fällanden, Switzerland). A 1 mm TXI probe was used, and data processing was performed with Topspin 2.1 (Bruker). Absorbance of enzyme-assay reaction mixture was measured by BioTek microplate reader (XS2).
Plant materialThe aerial parts of S. chloroleuca Rech. f. & Aell. were collected from Shahrestanak, Tehran province of Iran, in June 2008 at an altitude of 2300 m. The plant was botanically identified by Dr. Ali Sonboli of Biology Department of Medicinal Plants and Drug Research Institute, Shahid Beheshti University, Tehran, Iran. Voucher specimen (MPH 845) has been deposited at the herbarium of Medicinal Plants and Drugs, Research Institute, Shahid Beheshti University, Tehran, Iran.
Extraction and isolation Dried leaf material (100 g) was ground with a ZM 1 ultra-centrifugal mill (Retsch, Haan, Germany) equipped with a 0.75 mm Conidur ring sieve, and extracted by successive percolation with n-hexane, ethylacetate and methanol (2 L each). After evaporation to dryness under reduced pressure, 20 g of methanol extract was obtained. The extract was suspended in distilled water and loaded onto a Diaion HP-20 column (5 × 40 cm) i.d. After washing with water, the column was eluted with methanol (3 L), to provide a fraction enriched in phenolic compounds (8.1 g). This fraction was subjected to column chromatography over sephadex LH-20 (2×50 cm) i.d, eluted with methanol. After screening by TLC the obtained fractions with similar compositions were pooled, to yield 5 combined fractions (F1-F5). These main fractions were assayed for their α-amylase and α-glucosidase inhibition activities. The most active fractions were separated by preparative HPLC (SunFire C18, 5 μm, 150 × 30 mm i.d., Waters) with 10-100 % of methanol in water (both containing 0.1 % formic acid), over 40 min at a flow rate of 20 mL/min, and injection volume of 200 µL. Collected peaks from preparative HPLC were evaporated and subjected to semi-preparative HPLC (SunFire C18, 5 μm, 150 × 10 mm i.d., Waters) with 10-100 % methanol in water (both containing 0.1 % formic acid) over 40 min, at a flow rate of 4 mL/min. Several injections yielded compounds 1 (8 mg), 2 (5 mg) from F3 and 3 (6 mg) from F4. The n-hexane extract was separated on silica gel using n-hexane-ethylacetate mixtures as eluent. Fractions obtained with 40% ethylacetate (250 mg) were purified by semi-preparative HPLC, and yielded the known compound salvigenin (4 (link)) (20 mg). The detailed purification process of active components (1 (link)-4 (link)) was performed by the flowchart scheme described in
Luteolin 7-O-glucoside (1)1H NMR (500 MHz, DMSO-d6) δ 3.16-3.46 (m, sugar-H), 3.69(d, J = 11.0 Hz, H-5″), 5.02 (d, J = 7.4 Hz, H-1″), 6.41 (d, J = 2.0 Hz, H-6), 6.67 (s, H-3), 6.74 (d, J = 2.0 Hz, H-8), 6.87 (d, J = 8.3 Hz, H-5′), 7.37-7.40 (m, H-2′,6′). UV λmax 254 nm, 350 nm. MS (m/z) 447.1 [M-H]-.
Luteolin 7-O-glucuronide (2)1H NMR (500 MHz, DMSO-d6) δ 3.28-3.51 (m, sugar-H), 3.98 (d, J = 9.3 Hz, H-5″), 5.23 (d, J = 7.2 Hz, H-1″), 6.45 (d, J = 2.0 Hz, H-6), 6.70 (s, H-3), 6.79 (d, J = 2.0 Hz, H-8), 6.91 (d, J = 8.5 Hz, H-5′), 7.40-7.45 (br s, H-2′, 6′). UV λmax 254 nm, 350 nm. MS (m/z) 461.1 [M-H]-.
Diosmetin 7-O-glucuronide (3)1H NMR (500 MHz, DMSO-d6) δ 3.33-3.45 (m, sugar-H), 3.90 (s, OMe-4′), 4.02 (d, J = 9.6 Hz, H-5″), 5.25 (d, J = 7.3 Hz, H-1″), 6.47 (d, J = 2.0 Hz, H-6), 6.86 (d, J = 2.0 Hz, H-8), 6.93 (s, H-3), 6.95 (d, J = 8.3 Hz, H-5′), 7.55-7.40 (m, H-2′, 6′). UV λmax 268 nm, 345 nm. MS (m/z) 475.1 [M-H]-.
Salvigenin (4)1H NMR (500 MHz, CDCl3) δ 3.89 (s, OMe-4′), 3.92 (s, OMe-7), 3.96 (s, OMe-6), 6.54 (s, H-8), 6.58 (s, H-3), 7.02 (d, J = 9.0 Hz, H-3′, 5′), 7.84 (d, J = 9.0 Hz, H-2′, 6′). UV λmax 274 nm, 330 nm. MS (m/z) 329.1 [M+H]+.
α-Amylase inhibition assayα-Amylase inhibition activity was assessed by a previously reported procedure with some modifications (17 ). The assay system, which was carried out in 96-well plates, comprised the following components in a total volume of 250 µL: 100 mM sodium phosphate (pH 6.8), 17 mM NaCl, 1.5 mg soluble starch, 50 µL of inhibitor solution in DMSO at various concentrations (for pure compounds 12.5, 25, 50, 100 and 150 µM), and 10 µL of enzyme solution (25 unit/mL). After incubation at 37 °C for 30 min, the reaction was stopped by addition of 20 µL NaOH (2N) and 20 µL color reagent (4.4 µM of 3,5-dinitrosalisylic acid, 106 µM of potassium sodium tartarate tetrahydrate and 40 µM of NaOH) followed by a 20 min incubation at 100 °C water bath. α-Amylase activity was determined by measuring the absorbance of the mixture, due to the maltose generated at 540 nm. Individual blanks were prepared to correct for the blank ground absorbance, where the enzyme was replaced with buffer as follows:
Corrected absorbance of test sample = Absorbance of sample –absorbance of blank
From the net absorbance obtained, the % (w/v) of maltose generated was calculated from the equation obtained from the maltose standard calibration curve (0–0.1%, w/v, maltose).
Control incubations, representing 100% enzyme activity, were conducted in the same manner replacing the plant extract with DMSO. The percentage of α-amylase inhibition was calculated by the following equations:
% α-amylase inhibition activity= 100 - % reaction
α-Glucosidase inhibition assayThe α-glucosidase inhibition was measured according to an earlier reported bioassay method (18 ). The mixture contained 20 µL α-glucosidase (0.5 unit/mL), 120 µL of 0.1 M phosphate buffer (pH 6.9) and 10 µL of test sample at varying concentrations (for pure compounds 5, 10, 15, 30 and 50 µM). The mixed solution was incubated in 96-well plates at 37 °C for 15 min. After preincubation, the enzymatic reaction was initiated by adding 20 µL of 5 mM p-nitrophenyl-α-D-glucopyranoside solution in 0.1 M phosphate buffer (pH 6.9), and the reaction mixture was incubated for another 15 min at 37 °C. The reaction was stopped by adding 80 µL of 0.2 M sodium carbonate solution and then the absorbance was measured by microplate reader at 405 nm. The reaction system without plant extracts was used as control and the system without α-glucosidase was used as blank for correcting the background absorbance. The inhibitory rate of sample on α-glucosidase was calculated by the following formula:
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Most recents protocols related to «Sephadex LH 20»
After mixing with silica 60 RP-C18, the MSA fraction (23.9 g) was placed on an RP-C18 column (50 g) and eluted with H2O/MeOH (100:0 → 0:100, v/v) to obtain 12 fractions (Fr.1–Fr.12). Fr.9 (1.617 g) was separated with a Sephadex LH-20 column (chloroform–methanol, 1:1) to obtain nine fractions (Fr.9.1-9). Fr.9.3 (1.254 g) was separated using a silica gel column (200–300 mesh) and eluted with petroleum ether/acetone (100:1 → 6:4, v/v) to obtain five fractions (Fr.9.3.1-5). Fr.9.3.2 (135 mg) was loaded on a silica gel column eluting with chloroform/acetone (100:1 → 6:4, v/v) and then purified by Sephadex LH-20 (acetone) to obtain compound 8 (3 mg). Fr.9.3.4 (180 mg) was subjected to Sephadex LH-20 chromatography (chloroform–methanol, 1:1) to obtain four fractions (Fr.9.3.4.1-4). Fr.9.3.4.3 (132 mg) underwent separation on a silica gel column eluted with chloroform/acetone (100:1 → 8:2, v/v) and was subsequently purified by a Sephadex LH-20 (acetone) to obtain compound 9 (2 mg). Fr.9.3.5 (113 mg) was loaded on a Sephadex LH-20 (acetone) and purified on a silica gel column via elution with chloroform/methanol (100:1 → 8:2, v/v) to obtain compound 10 (8 mg). Fr.5 (259 mg) was subjected to Sephadex LH-20 (chloroform–methanol, 1:1) chromatography to obtain five fractions (Fr.5.1-5). Fr.5.4 (54 mg) was further separated using a silica gel column via elution with chloroform/acetone (100:1 → 6:4, v/v) and purified by Sephadex LH-20 chromatography (acetone) to obtain compound 2 (7 mg). Fr.5.5 (80 mg) was subjected to silica gel column chromatography and eluted with chloroform/acetone (100:0 → 8:2, v/v) and purified by Sephadex LH-20 chromatography (methanol) to obtain compound 7 (5 mg). Fr.4 (1.6 g) was loaded on a Sephadex LH-20 column (chloroform–methanol, 1:1) to obtain seven fractions (Fr.4.1-7). Fr.4.5 (637 mg) was further separated using Sephadex LH-20 (methanol) to obtain four fractions (Fr.4.5.1-4). Fr.4.5.2 (439 mg) was subjected to semi-preparative HPLC with gradient elution of MeOH–H2O (30:70 → 35:65 and 40:60 → 100:0) for 50 min to obtain five fractions (Fr.4.5.2.1-5). Fr.4.5.2.3 (120 mg) was separated using a Sephadex LH-20 column (methanol) to obtain three fractions (Fr.4.5.2.3.1-3). Fr.4.5.2.3.3 (108 mg) was further separated using a silica gel column via elution with chloroform/acetone (100:1 → 0:100, v/v) to obtain eight fractions (Fr.4.5.2.3.3.1-8). Fr.4.5.2.3.3.6 (51 mg) was separated using a Sephadex LH-20 column (methanol) to obtain compound 6 (28 mg). Fr.4.5.2.5 (64 mg) was separated using a Sephadex LH-20 column (methanol) to obtain two fractions (Fr.4.5.2.5.1-2). Fr.4.5.2.5.2 (59 mg) was further separated using a column of silica gel via elution with chloroform/acetone (100:1 → 0:100, v/v) and purified by Sephadex LH-20 (methanol) to obtain compound 3a/b (5 mg). Fr.3 (2.135 g) was separated using a Sephadex LH-20 column (chloroform–methanol, 1:1) to obtain five fractions (Fr.3.1-5). Fr.3-5 (1.232 g) was separated using a Sephadex LH-20 column (chloroform–methanol, 1:1) to obtain four fractions (Fr.3.5.1-4). Fr.3.5.4 (1.081 g) was subjected to semi-preparative RP-C18 HPLC with a gradient elution of MeOH:H2O (10:90 → 45:55 and 60:40 → 100:0) for 40 min to obtain three fractions (Fr.3.5.4.1-3). Fr.3.5.4.3 (34 mg) was further separated using a silica gel column via elution with chloroform/acetone (50:1 → 0:100, v/v) to obtain four fractions (Fr.3.5.4.3.1-4). Fr.3.5.4.3.2 (8 mg) was separated using a Sephadex LH-20 column (methanol) to obtain compound 4 (5 mg).
The WB fraction (30.0 g) was placed on an RP-C18 column (60 g) and eluted with H2O/MeOH mixtures (100:0 → 0:100, v/v) to obtain 15 fractions (Fr.1-15). Fr.15 (878 mg) was separated using a silica gel column via elution with petroleum ether/ethyl acetate (300:1 → 6:4, v/v) to obtain five fractions (Fr.15.1-5). Fr.15.2 (153 mg) was separated using a silica gel column via elution with petroleum ether/ethyl acetate (100:1 → 8:2, v/v) and then purified with a silica gel column via elution with petroleum ether/ethyl acetate (200:1 → 8:2, v/v) to obtain compound11 (1 mg). Fr.15.4 (123 mg) was separated using a silica gel column via elution with chloroform/acetone (10:1 → 6:4, v/v) and then purified by Sephadex LH-20 (acetone) to obtain compound 8 (2 mg). Fr.6 (385 mg) was loaded on a Sephadex LH-20 column (methanol) to obtain six fractions (Fr.6.1-6). Fr.6.3 (108 mg) was subjected to a silica gel column (100:1 → 7:3, v/v) and purified by Sephadex LH-20 (methanol) to obtain compound 1 (6 mg). Fr.4 (1.15 g) was subjected to Sephadex LH-20 column (chloroform–methanol, 1:1) to obtain five fractions (Fr.4.1-5). Fr.4.1 (715 mg) was loaded on a silica gel column via elution with chloroform/acetone (100:1 → 8:2, v/v) to obtain four fractions (Fr.4.1-4). Fr.4.1.3 (83 mg) was subjected to a Sephadex LH-20 column (methanol) and purified by a silica gel column via elution with chloroform/acetone (100:1 → 8:2, v/v) to obtain compound 5 (15 mg).
The WB fraction (30.0 g) was placed on an RP-C18 column (60 g) and eluted with H2O/MeOH mixtures (100:0 → 0:100, v/v) to obtain 15 fractions (Fr.1-15). Fr.15 (878 mg) was separated using a silica gel column via elution with petroleum ether/ethyl acetate (300:1 → 6:4, v/v) to obtain five fractions (Fr.15.1-5). Fr.15.2 (153 mg) was separated using a silica gel column via elution with petroleum ether/ethyl acetate (100:1 → 8:2, v/v) and then purified with a silica gel column via elution with petroleum ether/ethyl acetate (200:1 → 8:2, v/v) to obtain compound
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The dried flow branch (170 kg) of G.elata were re-extracted 3 times with 95% ethanol, each time for 2 h, the extraction solution was obtained, and then extracted 3 times with ethyl acetate and other volumes, and the ethyl acetate phase and aqueous phase were obtained after decompression and concentration. The ethyl acetate phase is detected by silica gel column chromatography (petroleum ether-acetone (elution of 100:1–0:1) gradient), TLC detection and merger of the same polarity part to obtain 8 components (Fr.1–Fr.8). Through TLC detection and color development results of color developers, select Fr.5. Fr.7 and Fr.8 LC–MS analysis was performed, and according to the analysis results, Fr.7 and Fr.8, were separated and purified. Drag Fr.7 (124 g) normal-phase silica gel column chromatographic section with petroleum ether/acetone as eluent and yielded 14 parts: Fr.7.1–Fr.7.14. Drag Fr. Part 7.10 was obtained by Sephadex LH-20 gel column chromatography (chloroform: methanol 1:1), and then by repeated atmospheric pressure normal-phase column chromatography (200–300 mesh), Sephadex LH-20 gel column chromatography (methanol) to obtain Compound 7 (yellow powder, 10 mg). Drag Fr.7.11 Part is obtained by Sephadex LH-20 gel column chromatography (chloroform: methanol 1:1), and then by repeated atmospheric pressure normal-phase column chromatography (200–300 mesh), Sephadex LH-20 gel column chromatography (methanol) to obtain compound 5 (yellow powder, 12 mg). Drag Fr.7.13 Part is obtained by Sephadex LH-20 gel column chromatography (chloroform: methanol 1:1), and then subjected to repeated atmospheric pressure normal-phase column chromatography (200–300 mesh) and recrystallization to obtain compound 11 (yellow amorphous powder, 50 mg). Drag Fr.8 (743 g) Atmospheric pressure normal-phase silicone column chromatographic section with chloroform/acetone as eluent yielded 13 parts: Fr.8.1–Fr.8.13. Drag Fr.8.6 (26 g) Atmospheric pressure normal-phase silicone column chromatographic section with chloroform/acetone as eluent yielded 6 parts: Fr.8.6.1–Fr.8.6.6. Fr.8.6.6 (5.3 g) partially purified by Sephadex LH-20 gel column chromatography (chloroform: methanol 1:1), repeated atmospheric pressure normal-phase column chromatography (200 to 300 mesh) and Sephadex LH-20 gel column chromatography, semi-prepared HPLC to obtain compounds 1 (yellow powder, 5 mg), 2 (yellow powder, 23 mg), 3 (yellow powder, 4 mg), 4 (yellow powder, 0.8 mg), 8 (yellow powder, 13 mg) and 12 (yellow powder, 22 mg). Drag Fr.8.8 Removed by MCI gives seven parts, Fr.8.8.1 to Fr.8.8.7. Fr.8.8.1 Compound 9 (black powder, 29 mg) was obtained by repeated atmospheric pressure normal-phase column chromatography (200–300 mesh), Sephadex LH-20 gel column chromatography (chloroform: methanol 1:1) and Sephadex LH-20 gel column chromatography (methanol), and further semi-prepared HPLC purification to give compound 6 (yellow powder, 3 mg). Drag Fr.8.8.2 Compound 10 (light yellow powder, 7 mg) and Compound 13 (black amorphous powder, 53 mg) was purified by repeated atmospheric pressure normal-phase column chromatography (200–300 mesh), Sephadex LH-20 gel column chromatography (200 to 300 mesh), Sephadex LH-20 gel column chromatography (methanol), and semi-prepared HPLC (Fig. 4 ).![]()
Flow chart of extraction and separation of the flow branch of Gastrodiaelata
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The air-dried aerial parts (3.2 kg) of A. aureum were powdered and extracted three times with MeOH (3 × 10 L) at 45 o C for 1 h under sonication. After removal of solvent under reduced pressure, the crude extract was suspended in H2O and successively partitioned with n-hexane (3 × 1 L) and EtOAc (3 × 1 L). The EtOAc soluble fraction (61.0 g) was fractionated on a silica gel column eluted with a CH2Cl2/MeOH (100:1 to 1:1, v/v) gradient to afford six fractions A-1 to A-6. Fraction A-2 (2.79 g) was purified by a Sephadex LH-20 column with MeOH followed by chromatography with silica gel eluted with CH2Cl2/MeOH (15:1, v/v) to yield compounds 12 (2.7 mg) and 4 (75.4 mg). Fraction A-3 (1.16 g) was subjected to Sephadex LH-20 CC eluted with MeOH to give two subfractions A-3.1 and A-3.2. Subfraction A-3.1 was further separated by RP-C18 silica gel CC eluted with MeOH/H2O (4:1, v/v) to obtain compounds 1 (50.9 mg) and 2 (37.7 mg). Compound 6 (2.4 mg) was obtained from the subfraction A-3.2 by RP-C18 silica gel CC eluted with MeOH/H2O (2:1, v/v). Fraction A-4 (11.35 g) was chromatographed repeatedly on a silica gel column eluted with CH2Cl2/MeOH (15:1, 10:1, 5:1, 1:1, v/v) and five subfractions A-4.1 -A-4.5 were obtained. Subfraction A-4.1 was purified by Sephadex LH-20 CC with MeOH, followed by recrystallization in acetone to yield compound 3 (140.2 mg). Purification of the A-4.3 by Sephadex LH-20 CC eluted with MeOH to give compound 10 (60.7 mg). Subfraction A-4.4 was purified by a Sephadex LH-20 column with MeOH followed by chromatographed with RP-C18 silica gel eluted with MeOH-H2O (1:2, v/v) to afford compounds 11 (13 mg) and 13 (9 mg). Compound 5 (5.6 mg) was obtained from the subfraction A-4.5 by RP-C18 silica gel CC eluted with MeOH/H2O (2:3, v/v). Fraction A-5 (7 g) was further separated by silica gel CC eluted with CH2Cl2/MeOH (10:1, v/v) to give two subfractions A-5.1 and A-5.2. These subfractions were purified by Sephadex LH-20 CC with MeOH elution and then recrystallized in acetone to yield compounds 7 (35.0 mg) and 8 (80 mg). Fraction A-6 (2.5 g) was subjected to Sephadex LH-20 CC eluted with MeOH to give two subfractions A-6.1 and A-6.2. Subfraction A-6.1 was further separated by silica gel CC eluted with CH2Cl2/MeOH (2:1, v/v) to afford compound 9 (9.1 mg). 13 C NMR (125 MHz, acetone-d6) data are given in Table 1.
The frozen animals (260.5 g, dry weight after extraction) were cut into pieces and extracted exhaustively with acetone at room temperature (2.0 L × 4). The organic extract was evaporated to give a brown residue, which was then partitioned between Et2O and H2O. The Et2O solution was evaporated to give a dark brown residue (4.0 g). The obtained residue was subjected to gradient silica gel (200–300 mesh) column chromatography (CC) [Et2O/petroleum ether (PE) 0→100%] and yielded nine fractions (Fr. 1–9). Fr. 2 was divided into three subfractions (Fr. 2A–2C) by Sephadex LH-20 CC (PE/CH2Cl2/MeOH, 2:1:1). Following two-stage purification including Sephadex LH-20 CC (CH2Cl2, 100%) and silica gel CC (300–400 mesh, PE/Et2O 100:1→10:1), the subfraction Fr. 2C13 was purified by semi-preparative HPLC (MeCN, 100%, 3.0 mL/min) and analytical HPLC (MeOH/H2O, 80:20, 1.0 mL/min) to yield compounds 1 (2.0 mg, tR = 16.4 min), 2 (0.3 mg, tR = 18.4 min) and 4 (1.0 mg, tR = 15.3 min), respectively. Fr. 6 was split by Sephadex LH-20 CC (PE/CH2Cl2/MeOH, 2:1:1) to give two subfractions (Fr. 6A and Fr. 6B). Next, Fr. 6B was purified by Sephadex LH-20 CC (CH2Cl2, 100%), yielding subfraction Fr. 6BA. Final purification of Fra. 6BA was achieved by semi-preparative HPLC (MeOH/H2O, 80:20, 2.8 mL/min) to afford compounds 7 (0.5 mg, tR = 24.5 min) and 6 (1.0 mg, tR = 22.0 min) and the mixture of compounds 3 and 5 . The mixture was further purified by analytical HPLC (MeCN/H2O, 60:40, 1.0 mL/min) to yield pure 3 (1.1 mg, tR = 13.0 min) and 5 (1.0 mg, tR = 14.6 min), respectively. Fr. 9 was subjected to a column of Sephadex LH-20 eluted with CH2Cl2/MeOH, 1:1, to yield two subfractions (Fr. 9A and 9B). Fr. 9A was first split by Sephadex LH-20 column chromatography (PE/CH2Cl2/MeOH, 2:1:1) to give five subfractions (Fr. 9AA–Fr. 9AE). Fr. 9AE was purified by RP-HPLC (MeCN/H2O, 60:40, 3.0 mL/min), yielding a subfraction (Fr. 9AEC, tR = 8.0 min). Since there are two points observed on the thin-layer chromatography (TLC), Fr. 9AEC was purified by silica gel CC (300–400 mesh, CH2Cl2/MeOH, 96:4) to give compounds 12 (1.0 mg) and 14 (0.5 mg). Fr. 9AD was purified with silica gel CC (300–400 mesh, Et2O/PE, 1:1), followed by semi-preparative HPLC (MeCN/H2O, 60:40, 3.0 mL/min) to afford 13 (5.4mg, tR = 2.1 min), 11 (3.2mg, tR = 8.8 min) and fraction 9ADFG (tR = 13.7min). In a similar manner, Fr. 9ADFG was purified by silica gel CC (300–400 mesh, CH2Cl2/MeOH, 98:2) to give compound 10 (2.6 mg). Moreover, compound 9 (2.7 mg, tR = 7.3 min) was obtained from Fr. 9B through Sephadex LH-20 CC (PE/CH2Cl2/MeOH, 2:1:1) followed by RP-HPLC (MeCN/H2O, 60:40, 3.0 mL/min), while compound 15 (2.7 mg, tR = 7.3 min) was obtained from the Fr. 9B through Sephadex LH-20 CC (PE/CH2Cl2/MeOH, 2:1:1), silica gel CC (200–300 mesh, Et2O/PE 50%→100%), followed by RP-HPLC (MeCN/H2O, 60:40, 3.0 mL/min).
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The fresh mycelium of Talaromyces sp. CS-258 was cultured on potato dextrose broth medium for 3 days and then incubated in autoclaved 1 L Erlenmeyer flasks with rice culture medium (70 g rice, 0.2 g corn steep liquor, 0.5 g yeast extract, 0.3 g peptone, 0.6 g gourmet powder, and 100 mL seawater collected from the Huiquan Gulf of the Yellow Sea near the campus of IOCAS) for 30 days at room temperature. After incubation, the EtOAc crude extract was obtained by exhaustive concentration with MeOH and successive extraction with EtOAc/H2O four times.
The crude extract was fractionated by silica gel column vacuum liquid chromatography (VLC) elution with increasing polarity gradient of petroleum ether (PE)/EtOAc and CH2Cl2/MeOH to yield nine fractions (Fr.A–Fr.I). Fr.E was further split by column chromatography (CC) using Sephadex LH-20 to produce three subfractions from E-1 to E-3. Fr.E-1 was rechromatographed over silica gel elution with a slow CH2Cl2/MeOH gradient from 200:1 to 20:1 to yield three mixtures of E.1.1–E.1.3. Then, mixture E.1.1 was followed by semipreparative HPLC separation (Elite ODS-BP column, 5 μm, 10 × 250 mm, MeOH-H2O, 75:25, 3 mL/min) to afford compounds12 (6.0 mg, tR 8 min) and 10 (10.2 mg, tR 10 min). Mixtures of E.1.2 and E.1.3 were also subjected to semipreparative HPLC purification to yield 7 (3.3 mg, tR 20 min, 70% MeOH-H2O), 30 (2.0 mg, tR 18 min, 75% MeOH-H2O), and 31 (5.1 mg, tR 28 min, 65% MeOH-H2O). Compound 4 (21.1 mg) was obtained via recrystallization from Fr.E-2. Another semipreparative HPLC collection with 75% MeOH-H2O was applied for Fr.E-3 to give compounds 18 (4.2 mg, tR 22 min), 19 (3.2 mg, tR 16 min), and 20 (4.0 mg, tR 18 min).
Fr.F was subjected to reversed-phase CC using Lobar LiChroprep RP-18 from 10% to 90% MeOH-H2O to produce three subfractions, Fr.F-1 to F-3. Fr.F-1 was sequentially recrystallized to obtain compound2 (100.0 mg). Fr.F-2 was subjected to Sephadex LH-20 CC to afford compound 24 (65.9 mg) and two mixed components. Then, the former component was purified by HPLC separation with MeOH-H2O (52:48) and identified as 8 (13.7 mg, tR 25 min). The latter was subjected to preparative TLC (developing solvents: CH2Cl2−MeOH, 20:1) to obtain 27 (3.1 mg). Treated with successive CC on Sephadex LH-20 and HPLC with 58% MeOH-H2O, Fr.F-3 was found to produce compounds 9 (16.5 mg, recrystallization), 21 (15.3 mg, tR 16 min), 22 (9.5 mg, tR 18 min), and 23 (4.9 mg, recrystallization) totally.
Fr.G was subjected to further CC over Lobar LiChroprep RP-18 in the MeOH−H2O solvent system and thus gave 6 subfractions, Fr.G-1 to Fr.G-6. Subsequently, compounds25 (4.0 mg, tR 14 min) and 26 (2.5 mg, tR 12 min) were provided from Fr.G-1 by Sephadex LH-20 (MeOH) and semipreparative HPLC elution with 42% MeOH−H2O; meanwhile, compound 1 (11.0 mg, tR 16 min) was obtained from another mixture of Fr.G-1 via HPLC in 30% MeOH−H2O. Fr.G-2 was subjected to a series of CC on Sephadex LH-20 and the HPLC separation system of 42% MeOH/H2O and 45% MeOH/H2O to afford 11 (6.7 mg, tR 18 min) and 5 (24.6 mg, tR 15 min). Fr.G-3 was further purified by Sephadex LH-20 and then by preparative TLC (developing solvents: CH2Cl2−MeOH, 15:1) and by HPLC with 80% MeOH/H2O to obtain compounds 13 (90.0 mg, tR 27 min), 14 (14.6 mg, tR 35 min), and 15 (4.8 mg, tR 25 min). The remaining subfractions, Fr.G-4 to Fr.G-6, were partitioned using nearly the same method through CC over Sephadex LH-20, silica gel, and semipreparative HPLC to produce compounds 16 (5.5 mg, from Fr.G-4, tR 10 min, 52% MeOH/H2O), 17 (5.0 mg, from Fr.G-6, tR 28 min, 51% MeOH/H2O), and 6 (6.0 mg, from Fr.G-5).
After CC on reversed-phase RP-18 and Sephadex LH-20, Fr.I was fractionated and purified to give compound29 (3.2 mg, by silica gel column and following Sephadex LH-20), 28 (2.0 mg, by preparative TLC), 3 (13.0 mg, by semipreparative HPLC collection of 46% MeOH-H2O at tR 35 min), and 32 (2.4 mg, by preparative TLC and Sephadex LH-20).
The crude extract was fractionated by silica gel column vacuum liquid chromatography (VLC) elution with increasing polarity gradient of petroleum ether (PE)/EtOAc and CH2Cl2/MeOH to yield nine fractions (Fr.A–Fr.I). Fr.E was further split by column chromatography (CC) using Sephadex LH-20 to produce three subfractions from E-1 to E-3. Fr.E-1 was rechromatographed over silica gel elution with a slow CH2Cl2/MeOH gradient from 200:1 to 20:1 to yield three mixtures of E.1.1–E.1.3. Then, mixture E.1.1 was followed by semipreparative HPLC separation (Elite ODS-BP column, 5 μm, 10 × 250 mm, MeOH-H2O, 75:25, 3 mL/min) to afford compounds
Fr.F was subjected to reversed-phase CC using Lobar LiChroprep RP-18 from 10% to 90% MeOH-H2O to produce three subfractions, Fr.F-1 to F-3. Fr.F-1 was sequentially recrystallized to obtain compound
Fr.G was subjected to further CC over Lobar LiChroprep RP-18 in the MeOH−H2O solvent system and thus gave 6 subfractions, Fr.G-1 to Fr.G-6. Subsequently, compounds
After CC on reversed-phase RP-18 and Sephadex LH-20, Fr.I was fractionated and purified to give compound
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