Flavonoid Content During the Growth and Floral Development of Calendula officinalis L.

Our study uses an accessible and reliable colorimetric method to quantify total flavonoid, facilitating data analysis in phytochemistry laboratories and supporting the research of compounds with therapeutic properties.

The flavonoid content in calendula is studied during floral development to identify the optimal harvest time and maximize its medicinal and commercial potential.

Our protocol is a sensitive and efficient micro-technique that enables the quantification of flavanoids using small sample amounts, optimizing solvent use, and analysis with plant material is limited.

[Instructor] To begin, dry the plant material in a forced-air oven at 40-degrees Celsius for 48 hours. Once dry, remove it from the oven, transfer the dried material into paper envelopes, and store them in the dark at room temperature. Freeze the plant material using liquid nitrogen to prepare for grinding. Then, grind the frozen material in a porcelain mortar until it reaches a uniform texture. Prepare the samples by weighing 25 milligrams of the pulverized material from each sample. Add 500 microliters of 80% methanol to the weighed material. To extract flavonoids, incubate the mixture at 70-degrees Celsius for one hour. After incubation, centrifuge the mixture at 731 g for 13 minutes. Now, prepare aliquots by taking 150 microliters of the obtained extract, and adding 37 microliters of 80% methanol to it. Take 50 microliters of the extract and add 100 microliters of one molar potassium acetate, and 100 microliters of 10% aluminum chloride. Complete the solution by bringing the total volume of the mixture up to five milliliters using distilled water. Set the absorbance wavelength of a spectrophotometer to 415 nanometers, and measure the absorbance of the solution. Generate the calibration curve by preparing quercetin solutions ranging from 50, 100, 175, and 350 milligrams per milliliter. To prepare calibration samples, pipette 500 microliters of the quercetin solution from the last point of the calibration curve, and add 100 microliters of 10% aluminum chloride, and 100 microliters of one molar potassium acetate to the solution. Then, add 1.5 milliliters of 80% methanol and 2.8 milliliters of distilled water. Let the calibration solutions rest for 40 minutes at room temperature. Set the absorbance wavelength of the spectrophotometer to 415 nanometers, and measure the absorbance of the calibration samples to construct the calibration curve. Finally, calculate the flavonoid concentration and flavonoid content per gram of dry material. This table summarizes how the flavonoid concentration and flavonoid production per plant vary from the initial bud formation to full bloom and fruit development in calendula flower heads. Higher levels of total flavonoid concentration were observed between stages eight and 11, corresponding to buds with separated sepals to fully opened flower heads. In earlier stages, such as buds with united sepals, and in later stages such as senescent flower heads, flavonoid concentrations were 22% to 27% lower compared to fully opened flower heads. This figure shows the measurement of total flavonoid concentration in dry matter of calendula flower heads, ligulate flowers, and tubular flowers across floral development stages. The results suggest that understanding the variation of flavonoid concentration among different floral structures and stages is useful for determining the optimal harvest time to maximize flavonoid content in calendula flower heads.