* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
A mixture of 52 g of naringin and 5 w / vpercent of hydrochloric acid was mixed at a solid-to-liquid ratio of 1: 100 g / mL and hydrolyzed at 100 ° C for 1 hour. And then quickly cooled to room temperature, and then put it aside for 12 hours, precipitation of solid, after filtration with purified water to wash the solid to neutral, and then filtered to 25percent moisture content of naringenin wet crude 24. 5 grams; The crude naringenin obtained in the step (1) was added to a 30W / Vpercent acetic acid solution at a solid-to-liquid ratio of 1: 7g / mL, stirred at room temperature for 30 minutes, and then the solid was added to the same concentration Of acetic acid solution for 15 minutes, and then filtered, the solid washed with water to neutral, and then solid-liquid ratio of 1: 10g / mL volume concentration of 95percent ethanol dissolved, and then neutral alumina chromatography column, 100mL volume concentration of 95percent ethanol column chromatography column combined solution after the column, and then added to the solution volume of 10percent activated carbon at room temperature for 30 minutes, after filtration, the filtrate and then add the solution volume 10 The activated carbon was stirred at room temperature for 30 minutes. After filtration, the filtrate was concentrated to 1/3 volume by volume, distilled water was added into the same volume of distilled water, cooled to room temperature and placed in the refrigerator at 5 ° C for 24 hours. And then the white nacre crystals are precipitated and filtered to obtain naringenin wet fine product. The naringenin wet boutique is heated and boiled with 95percent ethanol by solid-liquid ratio of l: 6g / mL, and dissolved in ethanol An equal volume of distilled water was recrystallized once, and the precipitated white needle-like crystals were collected by filtration and then dried to obtain naringenin 14 g. In this case, the content of naringenin was 99.92percent and the yield was 26.9percent.
4.62 g
at 90℃;
Taking 98percent naringin 10g, macroporous resin D101 type 100g (about 200 ml), is placed in the 500 ml triangle flask, add 6percent sulfuric acid (6 ml _AOE 28692X0AO _ 100 ml) 150 ml, is connected to the 30 cm - 40 cm air condensing tube, water bath 90 °C closed hydrolysis 48h (macroporous resin by the white-light yellow, colorless solution), remove and cool, filtered, macroporous resin to a little water to wash, macroporous resin end to burn in the cup, add 300 ml water liquid, adding sodium bicarbonate, to neutral, filtered, 200 ml water washing, drying, using ethanol as solvent, reflux extraction 3 times (macroporous resin recovery white), each 20min, combined ethanol solution, decompression recovering ethanol, to obtain white of the naringenin 4.62g, determine its purity by HPLC is 94.5percent.
Reference：
[1] Patent: CN103467428, 2016, B, . Location in patent: Paragraph 0017-0020
[2] Phytochemistry, 2005, vol. 66, # 14, p. 1698 - 1706
[3] Journal of Agricultural and Food Chemistry, 2013, vol. 61, # 4, p. 931 - 938
[4] Patent: CN103772337, 2017, B, . Location in patent: Paragraph 0030-0033
3
[ 10236-47-2 ]
[ 492-61-5 ]
[ 6014-42-2 ]
[ 480-41-1 ]
Reference：
[1] Journal of Molecular Catalysis B: Enzymatic, 2014, vol. 105, p. 95 - 103
A mixture of 52 g of naringin and 5 w / v% of hydrochloric acid was mixed at a solid-to-liquid ratio of 1: 100 g / mL and hydrolyzed at 100 C for 1 hour. And then quickly cooled to room temperature, and then put it aside for 12 hours, precipitation of solid, after filtration with purified water to wash the solid to neutral, and then filtered to 25% moisture content of naringenin wet crude 24. 5 grams; The crude naringenin obtained in the step (1) was added to a 30W / V% acetic acid solution at a solid-to-liquid ratio of 1: 7g / mL, stirred at room temperature for 30 minutes, and then the solid was added to the same concentration Of acetic acid solution for 15 minutes, and then filtered, the solid washed with water to neutral, and then solid-liquid ratio of 1: 10g / mL volume concentration of 95% ethanol dissolved, and then neutral alumina chromatography column, 100mL volume concentration of 95% ethanol column chromatography column combined solution after the column, and then added to the solution volume of 10% activated carbon at room temperature for 30 minutes, after filtration, the filtrate and then add the solution volume 10 The activated carbon was stirred at room temperature for 30 minutes. After filtration, the filtrate was concentrated to 1/3 volume by volume, distilled water was added into the same volume of distilled water, cooled to room temperature and placed in the refrigerator at 5 C for 24 hours. And then the white nacre crystals are precipitated and filtered to obtain naringenin wet fine product. The naringenin wet boutique is heated and boiled with 95% ethanol by solid-liquid ratio of l: 6g / mL, and dissolved in ethanol An equal volume of distilled water was recrystallized once, and the precipitated white needle-like crystals were collected by filtration and then dried to obtain naringenin 14 g. In this case, the content of naringenin was 99.92% and the yield was 26.9%.
4.62 g
With sulfuric acid; at 90℃;
Taking 98% naringin 10g, macroporous resin D101 type 100g (about 200 ml), is placed in the 500 ml triangle flask, add 6% sulfuric acid (6 ml _AOE 28692X0AO _ 100 ml) 150 ml, is connected to the 30 cm - 40 cm air condensing tube, water bath 90 C closed hydrolysis 48h (macroporous resin by the white-light yellow, colorless solution), remove and cool, filtered, macroporous resin to a little water to wash, macroporous resin end to burn in the cup, add 300 ml water liquid, adding sodium bicarbonate, to neutral, filtered, 200 ml water washing, drying, using ethanol as solvent, reflux extraction 3 times (macroporous resin recovery white), each 20min, combined ethanol solution, decompression recovering ethanol, to obtain white of the naringenin 4.62g, determine its purity by HPLC is 94.5%.
With sulfuric acid; In water; at 90℃; for 2h;
Naringenin (NGE) was obtained from naringin by acid hydrolysis, using a reported method (Robin et al.2007). Briefly, the process consists on hydrolyzing a10% (w/v) aqueous solution of naringin with sulfuric acid at 0.8 M concentration, stirring during 2 h at 90 C. After that, the reaction media was cooled down to 4 C, allowing the naringenin precipitation. It was then washed with cool water and dried at 50 C. This process was done for a second time to ensure a complete conversion.
EXAMPLE 1 616.5 grams naringin were added to a round bottomed flask, followed by the addition of about one liter of 0.5 N hydrochloric acid. Upon stirring and refluxing, the naringin dissolves to form a yellow to red-brown solution. While a brown gummy or oily material initially forms, it eventually disappears. As the reaction proceeds, a yellow solid precipitates. The reaction time is approximately 2 to 4 hours. The solution is cooled, and the yellow solid filtered and washed with copious amounts of water until the washings are no longer acidic. The amount of naringenin produced is about 160 grams.
Particular preference is given to combinations comprising as biological control agent an isoflavone selected from the group consisting of [Group (3)]: (3.3) formononetin, (3.6) hesperetin, (3.7) naringenin,
37
dimethylformamide(DMF)[ No CAS ]
[ 112-82-3 ]
[ 480-41-1 ]
naringenin 7-O-cetyl ether[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With sodium carbonate; In water; ethyl acetate; acetone;
Example 1 Preparation and Analysis of <strong>[480-41-1]naringenin</strong> 7-O-cetyl ether(Compound 4, NG5006) 10 g(36.73 mmol) of <strong>[480-41-1]naringenin</strong> was dissolved in a mixture of 100 ml of acetone and 100 ml of dimethylformamide(DMF). 13.5 ml of hexadecanyl bromide and 4.70 g of sodium carbonate were added to the mixture and stirred in a water bath at 80C. for 12 hours. The resulting solution was cooled, and then, 100 ml of water and 800 ml of EtOAc were added thereto and the mixture was extracted with EtOAc. The extract thus obtained was washed with water and concentrated under a reduced pressure. The solid formed was filtered using a glass filter and dried under a reduced pressure to give 10.1 g of <strong>[480-41-1]naringenin</strong> 7-O-cetyl ether.
With sodium carbonate; In methanol; water; ethyl acetate; N,N-dimethyl-formamide; acetone;
Example 2 Preparation and analysis of <strong>[480-41-1]naringenin</strong> 7-O-stearyl ether(compound 7) 10 g(36.73 mmol) of <strong>[480-41-1]naringenin</strong> was dissolved in a mixture of 20 ml of acetone and 20 ml of DMF. 1.5 ml of octadecanyl bromide and 470 mg of sodium carbonate were added to the mixture and stirred in a water bath at 80C. for 19 hours. The resulting solution was cooled, and then, 20 ml of water and 200 ml of EtOAc were added thereto and the mixture was extracted with EtOAc. The extract thus obtained was washed with water and concentrated under a reduced pressure. A small amount of MeOH was added to the solid thus formed and the mixture was stirred with glass stick. The solid thus obtained was filtered and washed with EtOAc to give 1.25g of <strong>[480-41-1]naringenin</strong> 7-O-stearyl ether as a very pale yellow solid. m.p.: 117-119C 1H NMR (CDCl3)delta 12.0 (s, 1H), 7.32 (d, J=8.4 Hz, 2H), 6.87 (d, J=8.4 Hz, 2H), 6.04 (d, J=2.0 Hz, 1H), 6.02 (d, J=2.0 Hz, 1H), 5.34 (dd, J=12.8, 2.8 Hz, 1H), 3.94 (t, J=6.8 Hz, 2H), 3.07 (dd, J=17.2, 12.8 Hz, 1H), 2.77 (dd, J=17.2, 2.8 Hz, 1H), 1.75 (quin, J=6.8 Hz, 2H), 1.44-1.36 (m, 2H), 1.34-1.22 (m, 28H), 0.89 (t, J=6.8 Hz, 3H) ppm. 13C-NMR(CDCl3) delta 195.8, 167.5, 164.0, 162.7, 156.0, 130.6, 127.9, 115.6, 103.0, 95.5, 94.6, 78.9, 68.6, 43.2, 32.0, 29.74-29.65 (8 carbons), 29.62, 29.57, 29.4, 29.3, 28.9, 25.9, 22.7, 14.2 ppm. 1D NOESY: NOE contacts were observed between H (6.04 and 6.02 ppms) and H (3.94 ppm).
With carbon dioxide; In methanol; at 20℃; under 90009.0 Torr;Resolution of racemate;
The analysis was performed using a 4.6 mm 100 mm ChiralpakAD-H column (Chiral Technologies, West Chester, PA, USA)with 3 mm particle and 1000 A pore sizes. The flow rate was 4.0 mL/min with the outlet back pressure set to 120 bars. The sample wasdissolved in methanol at a concentration of 1 mg/1 mL, and theinjection volume was 2 mL. The composition of the mobile phaseconsisted of carbon dioxide and methanol with a gradient profile of10e60% methanol in 3.7 min. The column temperature was ambient room temperature. The MSD positive scan range was100e800 amu with source temperature at 450 C vaporization and325 C drying gas at a flow rate of 7 L/min. The capillary voltagewas2500 V with a corona current of 5 mA. Purification was performedusing a 21.1 mm 250 mm Chiralpak AD-H column (Chiral Technologies,West Chester, PA, USA) with 5 mm particle and 1000 Apore sizes. The flow rate was 70 mL/min with the outlet backpressure set to 150 bars. The sample was dissolved in methanol at aconcentration of 65 mg/mL, and the injection volume was 1.5 mL,stacked in 3.25-min intervals. The mobile phase consisted of carbondioxide and methanol at 50% (v/v), and the column temperaturewas set to 40 C.
With alpha-L-rhamnosidase from Alternaria alternata SK37.001; In aq. acetate buffer; at 40℃; for 0.166667h;pH 5.5;Enzymatic reaction;
Two millilitres of 0.1% naringin in 0.5 M sodium acetate buffer (pH 5.5) was incubated with the crude alpha-L-rhamnosidase at 40 C for 10 min. The crude enzyme dosage was 15 U g-1 of naringin. After the reaction was stopped by placement in a boiling water bath for 10 min, the reaction solution was mixed with 1.9 ml of methanol and centrifuged at 8000g for 5 min. The supernatant was analyzed using high performance liquid chromatography (HPLC) equipped with Sepax C18 column at 35 C. The mobile phase was methanol:water 50:50 (v:v) at a flow rate of 1 ml min-1. The products were detected using an ultraviolet detector at 280 nm.
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In tetrahydrofuran; at 60℃; for 2.5h;Inert atmosphere;
<strong>[480-41-1]Naringenin</strong> (174 mg, 0.62 mmol), WSCHCl (79 mg, 0.42 mmol), and a small amount of N,N-dimethylaminopyridine (DMAP) were added to a solution of PJ-1 succinate ester (180 mg, 0.32 mmol) in tetrahydrofuran (2.0 mL), and stirred at 60 C for 2.5 h under a nitrogen atmosphere. A small amount of distilled water was added to the reaction mixture, which was extracted with ethyl acetate. The organic layer was washed with brine, dried over MgSO4, and condensed in vacuo to give an amorphous powder, which was purified by silica gel column chromatography (n-hexane : AcOEt = 5/1-4/1), followed by HPLC (n-hexane : AcOEt = 2/1) to afford compound 1 (133.6 mg, 51%) as a colorless powder. Mp 145-147 C (MeOH-CHCl3); inlMMLBox -57.4 (c 0.05, CHCl3). 1H NMR delta 2.75-2.85 (6H, m, H-3beta, <strong>[480-41-1]naringenin</strong> H-3alpha, and -O-CO-CH2-CH2-CO-O-), 3.03 (1H, dd, J = 13.0, 16.5 Hz, <strong>[480-41-1]naringenin</strong> H-3beta), 3.31 (3H, s, OMe), 4.73 (1H, br t, J = 2.6, H-21alpha), 5.31 (1H, m, H-15), 5.39 (1H, dt, J = 13.0, 3.2 Hz, <strong>[480-41-1]naringenin</strong> H-2), 5.98 (1H, br s, <strong>[480-41-1]naringenin</strong> H-8), 6.00 (1H, br s, <strong>[480-41-1]naringenin</strong> H-6), 6.85 (1H, br s, <strong>[480-41-1]naringenin</strong> OH), 7.15 (2H, d, J = 8.7 Hz, <strong>[480-41-1]naringenin</strong> H-3' and H-5'), 7.44 (2H, d, J = 8.7 Hz, <strong>[480-41-1]naringenin</strong> H-2' and H-6'), 12.02 (1H, s, <strong>[480-41-1]naringenin</strong> OH); 13C NMR delta 29.4 and 29.5 (-O-CO-CH2-CH2-CO-O-), 43.3 (<strong>[480-41-1]naringenin</strong> C-3), 57.1 (OMe), 78.6 (<strong>[480-41-1]naringenin</strong> C-2), 79.3 (C-21), 86.0 (C-3), 95.5 (<strong>[480-41-1]naringenin</strong> C-8), 96.9 (<strong>[480-41-1]naringenin</strong> C-6), 121.7 (C-15), 121.9 (<strong>[480-41-1]naringenin</strong> C-3' and C-5'), 127.3 (<strong>[480-41-1]naringenin</strong> C-2' and C-6'), 138.7 (C-14), 171.0 and 171.8 (-O-CO-CH2-CH2-CO-O-), 195.4 (<strong>[480-41-1]naringenin</strong> C-4); MS (FAB) m/z: 779 [M - OMe]+; HRFABMS (C49H63O8, 779.4515; calculated for 779.4523) [M - OMe]+.
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In tetrahydrofuran; at 60℃; for 2.5h;Inert atmosphere;
General procedure: <strong>[480-41-1]Naringenin</strong> (174 mg, 0.62 mmol), WSCHCl (79 mg, 0.42 mmol), and a small amount of N,N-dimethylaminopyridine (DMAP) were added to a solution of PJ-1 succinate ester (180 mg, 0.32 mmol) in tetrahydrofuran (2.0 mL), and stirred at 60 C for 2.5 h under a nitrogen atmosphere. A small amount of distilled water was added to the reaction mixture, which was extracted with ethyl acetate. The organic layer was washed with brine, dried over MgSO4, and condensed in vacuo to give an amorphous powder, which was purified by silica gel column chromatography (n-hexane : AcOEt = 5/1-4/1), followed by HPLC (n-hexane : AcOEt = 2/1) to afford compound 1 (133.6 mg, 51%) as a colorless powder.
With hydroxylamine hydrochloride; sodium acetate; In methanol; at 40℃; for 48h;Reflux;
<strong>[480-41-1]Naringenin</strong> oxime was obtained by adding <strong>[480-41-1]naringenin</strong> (N) (200.9mg, 0.73mmol) to a solution of hydroxylamine hydrochloride (76.9mg, 1.10mmol) and anhydrous sodium acetate (90.6mg, 1.10mmol) in 5mL of methanol. The reaction mixture was stirred at 40C for 24h, then refluxed for 24h to accomplish the reaction. Next, the mixture was poured into cold water with ice to obtain cream-coloured solid. The product was filtered, washed with cold water and air dried. The crude product was purified using column chromatography to give 186.4mg of cream-coloured solid. Yield: 87.9%. Mp: 222-223C. HR-MS: m/z=286.0681 [M-H+]-, clcd. m/z=286.0721 [M-H+]-. The cream-coloured NOX is stable at room temperature, soluble in methanol, ethanol, acetone and diethyl ether. It is insoluble in water, hexane, chloroform, dichloromethane and ethyl acetate.
76%
With hydroxylamine hydrochloride; sodium acetate trihydrate; In ethanol; water; for 4h;Reflux;
<strong>[480-41-1]Naringenin</strong> oxime was prepared by treating 0.01 mol (2.72 g) <strong>[480-41-1]naringenin</strong> with hydroxylamine hydrochloride (0.01 mol, 0.69 g) in ethanol and sodium acetate trihydrate (0.01 mol, 1.36 g) in water. The solution was heated on a water bath for 4 h with constant stirring. On cooling and water addition, the cream compound precipitated out. It was filtered, washed with water, and dried under vacuum over P2O5. The product was recrystallized from ethanol-water (1:4 v/v); yield 2.2 g (76%). Melting point: 233-235 C. Elemental analyses results were; found (%): C 62.34; H 4.78; N 4.58; calculated for [C15H13NO5] [atomic mass 287.27]: C 62.72; H 4.56; N 4.88%.
General procedure: <strong>[480-41-1]Naringenin</strong> (0.03mM, 8.2mg) and CD (0.01mM) were completely dissolved in a mixed solution of ethanol and water (ca. 10mL, v:v=1:4, given the poor water solubility of <strong>[480-41-1]naringenin</strong>, ethanol was used), and the mixture was stirred for 5 days at room temperature. After evaporating the ethanol from the reaction mixture, the uncomplexed <strong>[480-41-1]naringenin</strong> was removed by filtration. The filtrate was evaporated under reduced pressure to remove the solvent and dried in vacuum to produce the <strong>[480-41-1]naringenin</strong>/CD complexes.
General procedure: <strong>[480-41-1]Naringenin</strong> (0.03mM, 8.2mg) and CD (0.01mM) were completely dissolved in a mixed solution of ethanol and water (ca. 10mL, v:v=1:4, given the poor water solubility of <strong>[480-41-1]naringenin</strong>, ethanol was used), and the mixture was stirred for 5 days at room temperature. After evaporating the ethanol from the reaction mixture, the uncomplexed <strong>[480-41-1]naringenin</strong> was removed by filtration. The filtrate was evaporated under reduced pressure to remove the solvent and dried in vacuum to produce the <strong>[480-41-1]naringenin</strong>/CD complexes.
General procedure: <strong>[480-41-1]Naringenin</strong> (0.03mM, 8.2mg) and CD (0.01mM) were completely dissolved in a mixed solution of ethanol and water (ca. 10mL, v:v=1:4, given the poor water solubility of <strong>[480-41-1]naringenin</strong>, ethanol was used), and the mixture was stirred for 5 days at room temperature. After evaporating the ethanol from the reaction mixture, the uncomplexed <strong>[480-41-1]naringenin</strong> was removed by filtration. The filtrate was evaporated under reduced pressure to remove the solvent and dried in vacuum to produce the <strong>[480-41-1]naringenin</strong>/CD complexes.
With potassium carbonate; In N,N-dimethyl-formamide; at 50℃; for 72h;
A mixture of NG (2.72 g, 10 mmol), potassium carbonate(0.69 g, 5 mmol) and 1,2-dibromoethane (21.6 mL, 250mmol) in 100 mL DMF was stirred at 50 C for 3 days. Theresultant mixture was cooled to room temperature and filtrated.The filtrate was mixed with 1 L H2O, and thenextracted with ethyl acetate (150 mL × 3). The organic layerwas dried over Na2SO4 and concentrated. The residue waspurified with a silica gel column and was eluted with Petroleumether/EtOAc (2:1) to afford 1 (0.95 g, 25%): mp180.0-181.0 C. 1H nuclear magnetic resonance (NMR)(400 MHz, dimethylsulfoxide (DMSO)-d6): delta 2.73 (dd, J =2.4, 17.2 Hz, 1H, H-3cis), 3.33 (m, 1H, H-3trans), 3.78 (t, J= 5.2 Hz, 2H, H-2??), 4.38 (t, J = 5.2 Hz, 2H, H-1??), 5.49(dd, J = 2.4, 14.4 Hz, 1H, H-2), 6.10 (d, J = 1.6 Hz, 1H, H-8), 6.12 (d, J = 1.6 Hz, 1H, H-6), 6.79 (d, J = 8.4 Hz, 2H,H-3?,5?), 7.33 (d, J = 8.4 Hz, 2H, H-2?,6?), 9.61 (s, 1H, 4?-OH), 12.10 (s, 1H, 5-OH).
With potassium carbonate; In N,N-dimethyl-formamide; at 70℃;
General procedure: A flavonoid 1 (5 mmol) was dissolved in 30 mL of DMF, followed by addition of 1,2-dibromoethane (4.3 mL, 50 mmol) and K2CO3 (20 mmol). The resulted mixture was stirred at 70 C for 10-15 h. Three times volume of ice-cooled water was added with vigorously stirring, which was acidified with HCl and extracted with EtOAc for three times. The combined organic layer was washed with saturated brine and then dried over MgSO4. Removal of solvent under reduced pressure yield a light yellow powder. Flash chromatography (petroleum ether/EtOAc, from 3/1 to 1/1) furnished 2 in good yield.
With Novosphingobium sp. PP1Y protein extract; In aq. phosphate buffer; at 35℃; for 3h;pH 8.5;Enzymatic reaction;
General procedure: In experiments of pH monitoring, 2 mM solutions of naringinwere prepared in HCl/KCl, Na-acetate, Na-phosphate, Tris/HCl, Na-carbonate buffers, in order to cover the pH range between 2.2 and10.9 (Table 2). 100 L of PP1Y crude extract were added to 0.5 mLof each solution and incubated at 35C under magnetic stirring.Reactions were checked every 20 min by TLC analyses (solvent sys-tem A) and naringin consumption was evaluated by using solutionsof pure naringin, narigenin, rhamnose and glucose as chromato-graphic standards. Blank reactions at different pHs were performedto evaluate the chemical degradation of the flavanone glycoside,occurring preferentially in strongly basic conditions.Similarly, solutions of 2 mM naringin in 50 mM Tris/HCl bufferpH 8.5 containing 1, 10 and 50% v/v of DMSO or CH3CN (Table 2)were placed under constant agitation at 35C in the presence of200 L of PP1Y crude enzymatic extract (0.74 mg total protein)/mLof reaction. As previously reported, reactions were monitored byTLC analysis (solvent system A).
With Trametes hirsuta laccase; In methanol; ethyl acetate; acetonitrile; at 35℃; for 1h;pH 6.4;Enzymatic reaction;
General procedure: Prior to monitoring oxygen consumption the effect of pH on the maximum absorbance value was studied as described below. Buffer solutions were prepared using 20 mM ammonium acetate buffer for pH 4.5-5 and 20 mM phosphate buffer for pH 6.9. The effect of pH on absorbance maxima was studied by dissolving solubilized <strong>[480-41-1]naringenin</strong> in 98% acetone in the various pH values as above and recording the wavelength scans (900-200 nm) after 30 min incubations using a Hitachi U-2001UV-vis spectrophotometer. The reaction mixture contained <strong>[480-41-1]naringenin</strong> (0.5 mM)and one of the polyhydroxylated monoaromatics compound (2 mM) in the molar ratio of 1:4 (in order to increase the probability of coupling), 40% (v/v) of organic solvent (methanol, acetonitrile and ethyl acetate), and 0.67 nkat ml-1 final laccase activity in 20 mM ammonium acetate buffer for pH 6.4. Reactions were carriedout at 35 C while shaking at 600 rpm using a rotary shaker for 1 h. To the incubation mixtures an equal volume of methanol was added and the mixture was centrifuged for 15 min at 14,000 x g and 600 mul aliquots were transferred into cleanvials.
With Trametes hirsuta laccase; In methanol; ethyl acetate; acetonitrile; at 35℃; for 1h;pH 6.4;Enzymatic reaction;
General procedure: Prior to monitoring oxygen consumption the effect of pH on the maximum absorbance value was studied as described below. Buffer solutions were prepared using 20 mM ammonium acetate buffer for pH 4.5-5 and 20 mM phosphate buffer for pH 6.9. The effect of pH on absorbance maxima was studied by dissolving solubilized <strong>[480-41-1]naringenin</strong> in 98% acetone in the various pH values as above and recording the wavelength scans (900-200 nm) after 30 min incubations using a Hitachi U-2001UV-vis spectrophotometer. The reaction mixture contained <strong>[480-41-1]naringenin</strong> (0.5 mM)and one of the polyhydroxylated monoaromatics compound (2 mM) in the molar ratio of 1:4 (in order to increase the probability of coupling), 40% (v/v) of organic solvent (methanol, acetonitrile and ethyl acetate), and 0.67 nkat ml-1 final laccase activity in 20 mM ammonium acetate buffer for pH 6.4. Reactions were carriedout at 35 C while shaking at 600 rpm using a rotary shaker for 1 h. To the incubation mixtures an equal volume of methanol was added and the mixture was centrifuged for 15 min at 14,000 x g and 600 mul aliquots were transferred into cleanvials.
With Trametes hirsuta laccase; In methanol; ethyl acetate; acetonitrile; at 35℃; for 1h;pH 6.4;Enzymatic reaction;
General procedure: Prior to monitoring oxygen consumption the effect of pH on the maximum absorbance value was studied as described below. Buffer solutions were prepared using 20 mM ammonium acetate buffer for pH 4.5-5 and 20 mM phosphate buffer for pH 6.9. The effect of pH on absorbance maxima was studied by dissolving solubilized <strong>[480-41-1]naringenin</strong> in 98% acetone in the various pH values as above and recording the wavelength scans (900-200 nm) after 30 min incubations using a Hitachi U-2001UV-vis spectrophotometer. The reaction mixture contained <strong>[480-41-1]naringenin</strong> (0.5 mM)and one of the polyhydroxylated monoaromatics compound (2 mM) in the molar ratio of 1:4 (in order to increase the probability of coupling), 40% (v/v) of organic solvent (methanol, acetonitrile and ethyl acetate), and 0.67 nkat ml-1 final laccase activity in 20 mM ammonium acetate buffer for pH 6.4. Reactions were carriedout at 35 C while shaking at 600 rpm using a rotary shaker for 1 h. To the incubation mixtures an equal volume of methanol was added and the mixture was centrifuged for 15 min at 14,000 x g and 600 mul aliquots were transferred into cleanvials.
With Trametes hirsuta laccase; In methanol; ethyl acetate; acetonitrile; at 35℃; for 1h;pH 6.4;Enzymatic reaction;
General procedure: Prior to monitoring oxygen consumption the effect of pH on the maximum absorbance value was studied as described below. Buffer solutions were prepared using 20 mM ammonium acetate buffer for pH 4.5-5 and 20 mM phosphate buffer for pH 6.9. The effect of pH on absorbance maxima was studied by dissolving solubilized <strong>[480-41-1]naringenin</strong> in 98% acetone in the various pH values as above and recording the wavelength scans (900-200 nm) after 30 min incubations using a Hitachi U-2001UV-vis spectrophotometer. The reaction mixture contained <strong>[480-41-1]naringenin</strong> (0.5 mM)and one of the polyhydroxylated monoaromatics compound (2 mM) in the molar ratio of 1:4 (in order to increase the probability of coupling), 40% (v/v) of organic solvent (methanol, acetonitrile and ethyl acetate), and 0.67 nkat ml-1 final laccase activity in 20 mM ammonium acetate buffer for pH 6.4. Reactions were carriedout at 35 C while shaking at 600 rpm using a rotary shaker for 1 h. To the incubation mixtures an equal volume of methanol was added and the mixture was centrifuged for 15 min at 14,000 x g and 600 mul aliquots were transferred into cleanvials.
General procedure: Sulfuric acid (2.0mL, 0.037mmol) was added dropwise to a beaker (100 mL) containing scutellarin (50 mg, 0.11 mmol). It was shaken or agitated by ultrasound agitated to dissolve the substrate in the acid at room temperature. Water (2.0 mL) was then added carefully dropwise. When the evolution of heat ceased (in 10 minutes), the mixture was added to water (15 mL) in one portion with stirring with a glass rod. The yellow crystals that were deposited were collected by suction filtration and washed by water (5 mL). In most cases, such products were pure enough for direct use. Moreover, it could be further purified by recrystallisation from aqueous methanol (70%, v/v) or column chromatography on silica gel (eluent:ethyl acetate/formic acid/water=100/4/3, v/v/v, Rfs of SCU and SCUE were 0.1 and 0.8 on silica gel GF254 respectively). Light yellow crystals were obtained after recrystallisation (28.5mg, 93% yield); m.p. 285-287C (>300C)27. IR (KBr, cm-1): numax 3442, 3331, 3098, 1671, 1619, 1587, 1509. 1H NMR (500MHz, DMSO-d6): delta 12.80 (s, 1H, 5-OH), 10.48 (s, 1H, 7-OH), 10.33 (s, 1H, 4?-OH), 8.75 (s, 1H, 6-OH), 7.92 (d, 2H, J=8.6Hz, C2?, C6?-H), 6.92 (d, 2H, J=8.6Hz, C3?, C5?-H), 6.75 (s, 1H, C3-H), 6.58 (s, 1H, C8-H). HR-ESI-MS (m/z): 309.0363 for [M+Na]+, calcd 309.0370.
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