乙醇提取法

乙醇提取法
一、参考文献：2011Antioxidant Activity of Uruguayan Propolis. In Vitro Ethanolic Extracts Preparation. Propolis samples from the
southern region of Uruguay were provided as raw material by the Uruguayan Beekeepers Association (SAU), collected in late spring/early summer, and stored at 20 C in the dark until use. Propolis ethanolic extracts (EEP, 40 g/L) were prepared by adding 20 mL of 75% ethanol
to 2 g of raw propolis previously milled. The suspension was heated to 50-60 C for 30 min under agitation and then filtered. This procedure was repeated twice over each sample, and the collected extracts were combined to a final volume of 50.0 mL. EEP were gently bubbled
with nitrogen and stored at room temperature in the dark. The UV absorption spectra were performed in a Cary 50 spectrophotometer (Varian, USA)
Total Polyphenols and Flavonoids Determination.The
relative content in polyphenols was determined according to the
Folin Ciocalteu (FC) method.Briefly, dilutions of EEP or gallic acid
(standard) were mixed with FC reagent, and 10% Na
2CO
3
was added.Absorbance
at 760 nm was measured in a Varioskan Flash microplate reader (Thermo Electron Corp.) after 2 h of incubation at room temperature.
Flavonoid content was determined by mixing dilutions of EEP or quercetin (standard) with 5% Al2Cl3;21 the mixture was left in the dark for 30 min, and the absorbance was measured at 425 nm in the microplate reader.
二、参考文献：2004Antioxidant activity of propolis of various geographic origins
样品的制备：Crude propolis materials were extracted with ethanol at room
temperature for 24 h. The ethanol suspension was separated by centrifugation, and the supernatant was concentrated under reduced pressure to give EEP.
Total polyphenol contents in EEP were determined by the
Folin-Ciocalteau colorimetric method (Kumazawa,Taniguchi et al., 2002; Singleton, Orthofer, & Lamuela-Raventos, 1999). EEP solution (0.5 ml) was mixed with 0.5 ml of the Folin-Ciocalteau reagent (Kanto Chemicals, Tokyo, Japan) and 0.5 ml of 10% Na2CO3, and the absorbance was measured at 760 nm after 1 h incubation at room temperature. EEP samples were evaluated at the final concentration of 20 mg/ml. Total polyphenol
contents were expressed as mg/g (gallic acid equivalents).
Total flavonoid contents in EEP were determined by the method of
Woisky and Salatino (1998), with minor modifications. To 0.5 ml of EEP solution, 0.5ml of 2% AlCl3 ethanol solution was added. After 1 h at room temperature, the absorbance was measured at 420 nm. EEP samples were evaluated at the final concentration of 20 mg/ml. Total flavonoid contents were calculated as quercetin (mg/g) from a calibration curve.
三、参考文献：Chemical and Functional Characterization of Italian Propolis Obtained by Different Harvesting Methods
Propolis Extraction. As previously mentioned, three different
solvents were used for the extraction: ethanol, acetone, and chloroform. For each extract, 1 g of minced propolis was extracted with 10 mL of solvent under continuous stirring at room temperature for 30 min. The extraction was performed a second time after 24 h of continuous stirring. The ethanolic extract was filtered in a 25 mL volumetric flask and filled to volume with ethanol. The acetone and chloroform extracts were filtered and evaporated under vacuum at approximately 55 °C. Then, each residue was dissolved in ethanol, and the volume was filled to 25 mL.原因，好在哪里
Total Phenolics Determination. T he total phenolics content (TP)
was estimated by a properly modified Folin−Ciocalteu method.A volume of 50 μL of extract diluted to 1:50 (v/v) in ethanol was mixed with 2.5 mL of the Folin−Ciocalteu reagent 1:10 (v/v) and 2.0 mL of a hot saturated
solution of Na
2CO
3
. The absorbance was measured at 760 nm after 5 min of
incubation at 50 °C in the dark. Gallic acid was used for the calibration curve (20−800 μg/mL). TP was expressed as milligrams of gallic acid equivalents per gram of propolis (GAEs/g).
Total Flavones and Flavonols Determination. T he total flavones
and flavonols (TFF) were estimated according to an aluminum
chloride method based on the procedure described by Woisky and Salatino。

For the calibration curve, four standard solutions of quercetin in 80% ethanol (25, 50, 100, and 200 μg/mL) were prepared.
A 0.5 mL portion of standard solutions was separately mixed with
1.5 mL of 95% ethanol, 0.1 mL of 10% AlCl3 in water (w/v), 0.1 mL of
1 M potassium acetate, and 2.8 mL of 80% ethanol. After incubation at 20 °C for 30 min, the absorbance was measured at 425 nm. The 10% AlCl
3 was substituted by the same quantity of distilled water in the blank sample. Similarly, 0.5 mL of each extract diluted to 1:50 (v/v) in 80% ethanol was analyzed as described above. The results are expressed as TFF % w/w.
水提取法
一、参考文献：Seasonal effect on chemical composition and biological
activities
Total phenolic content
Total phenolic content of propolis extracts was determined as described by Popova et al. (2004, 2005) with slight modifications(Popova et al., 2004; Singleton & Rossi, 1965; Woisky & Salatino,1998). Forty microlitres of propolis extract were diluted with 300 ul of distilled water and 80 ul of Folin–Ciocalteu reagent and 120ul of a 20% sodium carbonate solution were added. The reaction mixture was brought to 1 ml with distilled water and incubated in the dark (at room temperature) during 2 h. The absorbance was measured at 760 nm in a spectrophotometer (Aquamate Plus UV–Vis, Thermo Scientific). The total phenolic content was estimated using a mixture of pinocembrin and galangin(2:1 w/w) standard.
Flavone and flavonol content
The total flavone and flavonol content of propolis samples was estimated by the colorimetric method based on aluminium chloride complex formation as described by Popova et al. (Bonvehi &Coll, 1994; Popova et al., 2004). Forty microlitres of propolis extract were diluted with 400 ul of methanol
(w/v) were added. The volume was brought to 1 ml with and 20ul of 5% AlCl
3
methanol.The mixture was incubated for 30 min (at room temperature). The absorbance was read at 425 nm in a spectrophotometer (Aquamate Plus UV –Vis, Thermo Scientific). The results were expressed as milligrams per gram of propolis of rutin equivalents.
二、参考文献：Preparation of Water Extract of Propolis.Propolis samples were frozen at -18 o C and ground into powder by a mill. Four grams of powder was dissolved in 20 mL of distilled water at 60 o C for 7 h. The crude extract was filtered, and the residue was re-extracted under the same conditions. Both extracts were centrifuged at 28000g for 30 min, and the supernatants were concentrated under reduced pressure to produce the WEP. The average WEP yield of 26 propolis samples was 6.0 ( 1.6%. The WEP solution (10 mg/mL H2O) was used as the sample solution for further experiments.
Total Polyphenol Contents. Total polyphenol contents in WEP were determined by the Folin Ciocalteu colorimetric method according to the method of Tawaha et al.Briefly, 20 μL of WEP (10 mg/mL) was mixed with 3.0 mL of the Folin Ciocalteu reagent and 2.0 mL of 20% Na2CO3 and then agitated and diluted to 25 mL using distilled water. The absorbance was measured at 765 nm after 1.5 h of incubation at 30 o C with intermittent shaking. Total polyphenol contents were expressed as gallic acid
equivalent (mg/g).
Flavone Flavonol and Flavanone Contents. The flavone-flavonol and flavanone contents were measured using the method of Ivan et al.31 with minor modifications. To determine the flavone-flavonol content, 1.0 mL of WEP (10 mg/mL) was mixed with 3.0 mL of 95% alcohol, then 2.5 mL of 10% AlCl3 and 2.5 mL of 1 mol/L KAc were added, and the mixture was agitated and then diluted to 25 mL using distilled water. After 15 min at room temperature, the absorbance was measured at 415 nm. Flavone-flavonol contents were calculated as quercetin equivalent (mg/g).
此文献还测了可溶性碳酸盐溶液的含量这个指标。