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

Summary

Here, we present the aluminum chloride colorimetric method, a direct analytical technique to determine flavonoids in calendula quantitatively. This approach utilizes a straightforward chemical reaction involving treating the calendula extract with an aluminum chloride reagent, forming a colored complex. The color intensity, assessed using spectrophotometry, correlates with the flavonoid concentration.

Abstract

Pot marigold (Calendula officinalis L.) with multiple uses, notably its medicinal applications due to its richness and diversity in secondary metabolites, such as flavonoids, which possess anti-inflammatory and antioxidant properties.This study analyzed plant growth and biomass distribution, as well as total flavonoid concentration in tubular, ligulate flowers, and flower heads throughout floral development. Floral initiation and differentiation occurred five weeks after transplantation, following a sigmoid pattern of biomass accumulation in its organs as well as in plant height. Aerial organs reached a maximum biomass of 76 g/ g/plant on day 69. The maximum absolute growth rate was 2.38 g/day at 62 days, while the highest relative growth rate was recorded in reproductive organs on day 48 (0.137 g/g/day). Flavonoids, expressed as quercetin equivalents, showed higher concentrations during stages ranging from flower buds to fully opened flower heads (145-177 mg/g of dry matter). These findings emphasize the importance of harvesting at optimal stages to maximize flavonoid content and leverage their potential for therapeutic and commercial applications.

Introduction

The growing interest in secondary metabolites from medicinal plants is driven by their diverse applications in pharmaceutical, therapeutic, cosmetic, and other industries. Among them is Calendula officinalis L., an annual herbaceous plant of the Asteraceae family native to the Mediterranean region¹ but widely cultivated worldwide, has gained recognition due to the various uses of its flowers, including ornamental, medicinal, industrial, and culinary applications². Currently, England is the world's largest producer of C. officinalis³.  

The flower heads are the most utilized organ of this plant as they contain bioactive compounds such as flavonoids, carotenoids, terpenoids, essential oils, tannins, coumarins, carbohydrates, and fatty acids^3,4,5,6. These natural compounds contribute to its pharmacological properties, including anti-inflammatory, antioxidant, antimicrobial, and wound-healing effects⁷. Historically, C. officinalis, commonly known as pot marigold, has been used in traditional medicine systems such as Ayurveda and homeopathy for alleviating a wide range of ailments, from skin wounds and gastrointestinal disorders to menstrual irregularities and inflammatory conditions⁸. Modern applications extend to the pharmaceutical, food, and cosmetic industries, where calendula extracts are incorporated into creams, serums, tinctures, and drug delivery systems⁹. Despite extensive research, challenges remain in fully exploiting the therapeutic potential of C. officinalis. Variability in the concentration of bioactive compounds due to environmental and cultivation factors highlights the need for standardized extraction and formulation processes. The biological activity of flavonoids and their identification in plant tissues issues are essential aspects of quality control^10,11.

In Mexico, pot marigold cultivation often occurs without a detailed understanding of its growth processes and floral development, thus limiting its physiological performance, yield, and the correlation between bioactive compounds and agronomic factors. The concentration and distribution of flavonoids in plant tissues are influenced by growth and cultivation conditions, emphasizing the importance of studying the physiological and developmental processes of this species.

This study aimed to analyze biomass distribution and sink-source relationships in vegetative and reproductive organs of C. officinalis, for identifying key floral development events, and to quantify the total flavonoid concentration in flower heads by means of a microspectrophotometric method proposed here. These findings aim to optimize agricultural practices and enhance the quality of products derived from this plant.

Protocol

1. General materials and methods

1. Field experiment for detecting floral initiation
   1. Set up the experiment under field conditions. Transplant calendula seedlings over an area of 153 m² with a planting density of plants per square meter, spaced 60 cm apart.
   2.  Establish the localized drip irrigation system with 16/8000-gauge NOTE: The seed lot used in this experiment was harvested  from a single genotype,then selected by the  mass selection breeding method across  three previous cultivation cycles. For a temperate climate, sowing time is best at the Spring season, followed by  transplanting approximately 2 months later, when the seedlings are ready. Irrigation is recommended once a week. Pests and diseases should be avoided, and agronomic management should be carried out based on the crop's needs.
2. Collect samples from selected plants every other day, starting 14 days after transplanting. Watch the apical meristem under a stereoscopic microscope (about 40X) in order to determine the date of inflorescence primordium appearance.
   NOTE: When the floral meristem (FM) appears in at least half of the samples, then this date can be recorded as the proper age for FM. The experimental unit to observe floral initiation was five meristems in a single plant, with eight replications (plants). Growth and floral development analysis
   1. Collect the apical capitulum when the floral bud opens.
      NOTE: Make sure the ligulate flowers are visible.
   2. Carry out floral organ collections during the last seven floral stages¹² (Figure 1).
      NOTE: In this experiment, five capitulums from eight different plants were used at each developmental stage. In all cases, the flowers were collected from the apical part of the plant.
   3. Sample whole plants at six times through the biological cycle.
   4. Dry the collected samples for 48 h. Measure the dry weight of vegetative and reproductive organs.
      NOTE: Drying is recommended at 40 °C in an air-circulating oven.
   5. Calculate the Sink Strength (SS) and Sink Activity (SA) for both vegetative and reproductive organs¹³.
      NOTE: For SS and SA, use the formulas equivalent to the Absolute Growth Rate (AGR) and Relative Growth Rate (RGR), respectively.
   6. Plot growth curves using a spreadsheet.

2. Quantification of total flavonoids¹⁴

1. Dry the plant material in a forced-air oven at 40 °C for 48 h.
2. Store the dried material in paper bags. Keep them in the dark at room temperature (RT).
3. Freeze the plant material using liquid nitrogen to prepare it for grinding.
4. Grind the frozen material in a porcelain mortar until it reaches a uniform texture.
   NOTE: Be cautious when handling liquid nitrogen, as it may splash upon contact with the plant material.
5. Sample preparation: Weigh 25 mg of pulverized dry matter from each sample. Add 500 µL of 80% methanol.
6. Extraction of flavonoids: Incubate the mixture in an ultrasound device at 70 °C for one h. Then centrifuge it at 731 g for 13 min¹⁵.
7. Prepare aliquots: Take 150 µL of the obtained extract and add 37 µL of 80% methanol.
8. Mix 50 µL of the extract, 100 µL 10% aluminum chloride, and 100 µL 1 M potassium acetate. Add distilled water to bring the total volume to 5 mL.
9. Allow the solution to rest for 30 minutes at room temperature.
10. A spectrophotometer was used to measure the absorbance at 415 nm.
11. Generate the calibration curve: Prepare a stock solution by dissolving 2.7 mg of quercetin in 10 mL of 80% methanol. From this stock, prepare a series of 5 quercetin solutions ranging from 50, 100,175, 250, and  350 µL., and dilute to 10 mL.
12. Prepare calibration samples: For each quercetin solution (500µL), add 100 µL 10%  aluminum chloride  and 100 µL 1M potassium acetate,1.5 mL of 80% methanol, and 2.8 mL of distilled water.
13. Let the solution rest for 40 minutes at room temperature.
14. Measure the absorbance of the calibration samples at 415 nm with the spectrophotometer. Record the absorbance values to construct the calibration curve.
    NOTE: Compare the total flavonoid concentration in ray flowers, tubular flowers, and whole flower heads. This experiment used a completely randomized experimental design with five repetitions per treatment. The response variable was the flavonoids concentration; the treatment means were compared with the Tukey test (SAS Institute, 2003).

Results

Growth of C. officinalis exhibited sigmoid biomass accumulation kinetics throughout the plant and its organs. This observation aligns with reports from other authors, which consistently display a sigmoid growth curve. Among the aerial organs, stems accumulated the most biomass (Figure 3). Initial growth in all aerial organs was slow, with flowering and bud development starting on day 41. The plant reached its maximum dry biomass on day 69, averaging 76 g per plant.

Discussion

Two types of flowers, ligulate and tubular, were identified (Figure 5). We found that ligulate flowers contain a lower concentration of flavonoids than tubular flowers, with differences ranging from 11% to 53%. This indicates that tubular flowers accumulate more flavonoids than ligulate flowers, because the tubular flowers have more biomass and a higher concentration of these compounds.

When the stages of floral development were compared, it was found that the fla...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Air-circulating oven	Precision 17 GCA Corp.	N/A	Equipment
Bug clean	Alternagro	N/A	Control of agricultural pests with plant oils and extracts
Methanol analytical grade	merck	822283	Reagent
Nitrofoska special 12+12+17+2 MgO+8 S	EUROCHEM	7/548/CEE,1999/45/CE	NPK - granular fertilizer contains: ammonium nitrate, ammonium salts, phosphates, calcium salts, potassium, in some cases magnesium and trace elements. 1H-Pyrazole, 3,4-dimethyl-,phosphate (1:1)
PHC Healthy Start	PHC Mexico	N/A	PHC Healthy Start 12-16-12 is a fertilizer to improve plant nutrition, color and vigor.
Quercetin	Sigma-Aldrich	204-187-1	Standard substance