Page 51 - Phytochemistry -1 (PG404) / Clinical Pharmacy 2nd level students ( 2019 )

Clinical pharmacy PharmD program Third level Phytochemistry-1 (PG-504)

Flavonoid Glycosides

UV Spectrophotometric Assay of Flavonoids

Principle:

The method depends on the UV absorption of free flavonoids. Most
flavonoids show maximum absorbance at wave length 370 nm. At this
wavelength, most other extractives do not interfere in the assay.

The total flavonoid concentration is calculated by reference to a standard
curve of the most abundant flavonoid, this procedure should be proceeded
by prior chromatographic separation.

Procedure
1. A 5 ml portion of the preparation (tincture or extract), accurately

measured is transferred to a small flask, and is then hydrolyzed by
heating on .a water bath with 10 ml of 10% H 2SO 4 for 30 minutes.
2. The original volume is then reduced to one-half and the mixture is

cooled on ice for 15 minutes where the flavonoid aglycones are
precipitated.
3. Filter the cooled solution. The solid on the filter (flavonoids

aglycones) is dissolved by pouring 50 ml of warm 95% ethanol
(warmed to about 50°C), receiving the filtrate in a 100 ml volumetric
flask. Complete to volume with 95% EtOH.
4. Pipette 5 ml aliquot into a 25 ml volumetric flask and dilute to volume

with 50% EtOH.
5. The absorbance of the resulting solution is measured at 370 nm using
a suitable spectrophotometer, against a blank of 50% EtOH.

6. Calculate the flavonoid concentration by reference to the standard
curve of pure quercetin.
Preparation of The standard Curve for Quercetin:

1. Prepare 0.01% solution of quercetin in 95% EtOH (1ml =0.1 mg = 100 g).
2. Transfer different volumes (0.25, 0.5, 0.75, 1, 1.5, 2, 2.5 and 3 ml equivalent to
25, 50, 75, 100, 150, 250, 300 g of quercetin respectively), each to a clean and
dry 25 ml volumetric flasks. Complete each to 25 ml with 50% EtOH and mix.
3. Measure the absorbance of the resulting solution in each flask at 370 nm against
a blank in which quercetin solution is replaced by 50% EtOH.
4. The Concentration (C) is then plotted against the absorbance (A).
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