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.

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