The same extraction procedure was carried out to analyse the content of anthocyanin, polyphenol, and antioxidant properties according to Nowacka et al. [69 (link)] with slight modifications. The dried material was ground in an analytical mill (IKA A11 basic; IKA-Werke GmbH, Staufen, Germany). For a falcon with a capacity of 15 mL, about 0.3 g of the material and 10 mL of the extraction reagent (80% ethyl alcohol + 0.1 M hydrochloric acid in the ratio 85:15) was added. Anthocyanin is more stable under acid conditions [70 (link)]. Extraction was performed on a shaker (Multi Reax, Heidolph Instruments, Schwabach, Germany) for 12 h at room temperature and with limited access to light. The solution was centrifuged for 2 min at 3000 rpm in a laboratory centrifuge (MegaStar 600, VWR, Leuven, Belgium). The supernatant was placed in 0.2 mL PCR tubes. Two extractions were made for each sample.
Megastar 600
Manufactured by Avantor
Sourced in Belgium
The MegaStar 600 is a high-performance centrifuge designed for laboratory applications. It features a large capacity and high-speed operation, making it suitable for a variety of sample processing tasks.
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Lab products found in correlation
4 protocols using megastar 600
1
Anthocyanin, Polyphenol, and Antioxidant Extraction
2
Extraction and Analysis of Snack Compounds
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The baked and cooled snacks were ground in an analytical mill A 11 basic (IKA Poland Sp. z o.o., Warsaw, Poland). Approx. 0.3 g of ground material was weighed on an analytical balance to the nearest 0.0001 g into a 15 mL plastic falcon and mixed with 10 mL of extraction reagent (80% ethyl alcohol). Extraction was carried out on a shaker Multi Reax (Heidolph Instruments GmbH & Co., Schwabach, Germany) for 24 h at 6000 rpm at room temperature. Samples were centrifuged for 2 min at 5000 rpm using a laboratory centrifuge MegaStar 600 (VWR International Sp. z o.o., Gdańsk, Poland). The supernatant was transferred to resealable 0.2 mL tubes. Three extracts were made for each sample.
3
Polyphenol Extraction from Plant Samples
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After the extraction times were complete, all the extracts were left to cool at room temperature followed by centrifugation at 8400 rpm for 10 min. (MegaStar 600, VWR, Leuven, Belgium). The supernatants were pooled and syringe filtered through 0.45 μm PTFE filters for free phenolics, and PVDF filters for bound phenolic extracts. Aliquots (20 mL) of the liquor supernatants were acidified by adding hydrochloric acid solution (37%) until the pH reached 6.5 and subsequently subjected to liquid-liquid partitioning in EtOAc:water (1:1 v/v, 3 times) to obtain polyphenol-enriched fractions. The EtOAc fractions were evaporated to dryness under nitrogen and reconstituted in 20 mL 50% methanol. All the extractions were carried out in triplicate and stored at −25 °C until further use.
4
Spectrophotometric Determination of Chlorogenic Acid
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The determination was carried out using the spectroscopic method based on a color reaction of the analyte with the Folin Ciocalteau (F-C) reagent [55 (link)]. The material was ground using an analytical mill (IKA A11 basic, IKA-Werke GmbH & Co., Staufen, Germany).Then, 0.3 g of dried material was weighed into the test tube with an accuracy of ±0.0001 g and diluted with 10 mL of 80% ethyl alcohol. The solution was shaken on an orbital shaker (Multi Reax, Heidolph Instruments, Schwabach, Germany) at 20 °C and after 12 h it was centrifuged (MegaStar 600, VWR, Leuven, Belgium) at 4350 rpm for 2 min. Such extract was used for the further analysis.
In a well of a 96-well plate, 10 µL of the supernatant was placed, which was diluted twice with distilled water. Then, 40 µL of F-C reagent (5 times diluted with distilled water) was added to the solution, and after 3 min, 250 µL of 7% sodium carbonate solution was added. The samples were incubated for 60 min at 20 °C and protected from light. Absorbance was measured at a wavelength of 750 nm on a plate reader (against reagent blank). The result was expressed in mg/100 g of dry matter of chlorogenic acid using a calibration curve for chlorogenic acid standard at a concentration of 0–100 µL. The assays were performed in duplicate for each extract.
In a well of a 96-well plate, 10 µL of the supernatant was placed, which was diluted twice with distilled water. Then, 40 µL of F-C reagent (5 times diluted with distilled water) was added to the solution, and after 3 min, 250 µL of 7% sodium carbonate solution was added. The samples were incubated for 60 min at 20 °C and protected from light. Absorbance was measured at a wavelength of 750 nm on a plate reader (against reagent blank). The result was expressed in mg/100 g of dry matter of chlorogenic acid using a calibration curve for chlorogenic acid standard at a concentration of 0–100 µL. The assays were performed in duplicate for each extract.
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