Color, Antioxidant Capacity and Flavonoid Composition in Hibiscus rosa-sinensis Cultivars

Hibiscus rosa-sinensis plants are mainly cultivated as ornamental plants, but they also have food and medicinal uses. In this work, 16 H. rosa-sinensis cultivars were studied to measure their colorimetric parameters and the chemical composition of hydroethanolic extracts obtained from their petals. These extracts were characterized using UHPLC-ESI+-Obitrap-MS, and their antioxidant activity was evaluated using the ORAC assay. The identified flavonoids included anthocyanins derived from cyanidin, glycosylated flavonols derived from quercetin and kaempferol, and flavan-3-ols such as catechin and epicatechin. Cyanidin-sophoroside was the anthocyanin present in extracts of lilac, pink, orange, and red flowers, but was not detected in extracts of white or yellow flowers. The total flavonol concentration in the flower extracts was inversely proportional to the total anthocyanin content. The flavonol concentration varied according to the cultivar in the following order: red < pink < orange < yellow ≈ white, with the extract from the red flower presenting the lowest flavonol concentration and the highest anthocyanin concentration. The antioxidant activity increased in proportion to the anthocyanin concentration, from 1580 µmol Trolox®/g sample (white cultivar) to 3840 µmol Trolox®/g sample (red cultivar).


Introduction
Hibiscus is a genus that belongs to the Malvaceae family and comprises more than 200 species, among which are herbaceous, annual or perennial plants. Plants of this genus are native to Southeast Asia; they grow in warm environments and tropical and subtropical regions, produce large and magnificent flowers of various colors, and some are used for ornamental purposes [1]. The Hibiscus rosa-sinensis flower is Malaysia's national flower [2]. In South Asia, these flowers are used in traditional medicine to relieve bronchial catarrh and diarrhea [3]. In Colombia, these plants are known as "cayenos", and they are cultivated for ornamental purposes due to their brightly colored flowers. The mechanism that regulates the biosynthesis of pigments responsible for H. rosa-sinensis petal colors is not known in detail [4]. In nature, colors are important signals that show warnings (predator deterrence) or rewards (pollinator attraction). Betalains, carotenoids, flavonoids, and anthocyanins confer color to flowers and fruits [5,6]. The yellow and red colors are associated with the biosynthetic pathways of betalains and carotenoids, whereas the anthocyanin biosynthesis pathway generates a wide variety of colors ranging from yellow (λ = 480 nm) to orange, blue and red (λ = 730 nm) [6]. Several flavonoid derivatives participate in this biosynthetic pathway, from colorless compounds such as flavonols to colored pigments such as anthocyanins, tannins and proanthocyanidins [7].
Until now, no studies have been found that relate flavonoid content with color and antioxidant capacity in H. rosa-sinensis cultivars (Figure 1). The objective of this study was to determine a potential relationship between the type and quantity of flavonoids and the color and antioxidant capacity of extracts from H. rosa-sinensis flowers. In total, 20 flavonoids present in the petal extracts of 16 H. rosa-sinensis cultivars of different colors were identified and quantified by UHPLC-ESI+-Orbitrap-MS. The antioxidant capacity of all extracts was measured using the ORAC method, which permitted the selection of specific cultivars as potential sources of natural ingredients.

Identification by UHPLC-ESI+-Orbitrap-MS of Flavonoids Present in the Extracts of H. rosa-sinensis Flowers
UHPLC-ESI + -Orbitrap-MS analyses allowed the detection of 20 flavonoids in the flower extracts of 16 H. rosa-sinensis cultivars (Table 1). Three anthocyanins, eleven flavonols, two flavan-3-ols, two flavanonols, one flavone, and one flavanone were identified. Catechin and epicatechin were the only aglycones, and the remaining detected compounds were glycosylated derivatives of the corresponding flavonoids. Figure 2 shows some chromatographic profiles obtained by LC/MS (Orbitrap) of the hydroalcoholic extracts of four H. rosa-sinensis cultivars. Quercetin-hexuronide-hexoside, cathechin, quercetindihexoside, kaempferol-deoxyhexoside-hexoside, kaempferol-deoxyhexoside, quercetinhexuronide, quercetin-3-glucoside, and apigenin-hexuronide were the compounds common to all H. rosa-sinensis hydroalcoholic extracts examined. The identification of the chromatographic peaks appearing in Figure 1 is presented in Table 1.   common to all H. rosa-sinensis hydroalcoholic extracts examined. The identification of the chromatographic peaks appearing in Figure 1 is presented in Table 1. Chromatographic peak identification appears in Table 1. Figure 2. Chromatographic profiles obtained by LC/MS of the hydroalcoholic extracts of four H. rosa-sinensis cultivars. Chromatographic peak identification appears in Table 1.
The analysis of the mass spectra obtained with different energies (10, 20, 30 or 40 eV) in the HCD (Higher Energy Collision Dissociation Cell) was based on the study of the characteristic fragmentation patterns for flavonoids (Figure 3), which include the typical loss of molecules such as hexoses (C 6 H 10 O 5 ), hexuronides (C 6 H 8 O 6 ), H 2 O and CO, in addition to the rupture suffered by the C ring.
Apigenin-hexuronide ( Figure 2, peak N • 20) was the only flavone detected in the H. rosa-sinensis extracts. In its mass spectrum (HCD, 20 eV), the loss of hexuronide (C 6 H 8 O 6 ) was observed, accompanied by the formation of the product ion [(M+H)-C 6 Table 2 presents the total flavonoid contents (mg/g) according to each subfamily, the colorimetric parameters (L*, a* and b*) of the CIELAB system, and the chromatic representations of the RGB system for the 16 flower cultivars, together with their extract ORAC antioxidant capacity. The antioxidant activity of H. rosa-sinensis flower extracts has been determined using several in vitro methods [36]. In this research, the ORAC method was used because in the hydrogen-atom transfer mechanism the peroxyl radicals are used, which predominate in food and biological systems [37]. The codes of the different flowers are shown in Figure 1. Table 3 reports the content of phenolic compounds (mg/g) in H. rosa-sinensis flower hydroalcoholic extracts of the different cultivars studied in this work.  Table 2 presents the total flavonoid contents (mg/g) according to each subfamily, the colorimetric parameters (L*, a* and b*) of the CIELAB system, and the chromatic representations of the RGB system for the 16 flower cultivars, together with their extract ORAC antioxidant capacity. The antioxidant activity of H. rosa-sinensis flower extracts has been determined using several in vitro methods [36]. In this research, the ORAC method was used because in the hydrogen-atom transfer mechanism the peroxyl radicals are used, which predominate in food and biological systems [37]. The codes of the different flowers are shown in Figure 1. Table 3 reports the content of phenolic compounds (mg/g) in H. rosa-sinensis flower hydroalcoholic extracts of the different cultivars studied in this work.

Relationship of Chemical Composition with Color and Antioxidant Capacity in the H. rosa-sinensis Cultivars under Study
271.05997 (45%). As products of ring C cleavage, the fragments [(M+H)-C6H8O6-C8H6O] + were generated at m/z 153.01824 (C7H5O4 + , 17%) and [(M+H)-C6H8O6-C8H6O3] + at m/z 121.02857 (1%), previously reported by several authors [33][34][35]. Table 2 presents the total flavonoid contents (mg/g) according to each subfamily, the colorimetric parameters (L*, a* and b*) of the CIELAB system, and the chromatic representations of the RGB system for the 16 flower cultivars, together with their extract ORAC antioxidant capacity. The antioxidant activity of H. rosa-sinensis flower extracts has been determined using several in vitro methods [36]. In this research, the ORAC method was used because in the hydrogen-atom transfer mechanism the peroxyl radicals are used, which predominate in food and biological systems [37]. The codes of the different flowers are shown in Figure 1. Table 3 reports the content of phenolic compounds (mg/g) in H. rosa-sinensis flower hydroalcoholic extracts of the different cultivars studied in this work.

Relationship of Chemical Composition with Color and Antioxidant Capacity in the H. rosasinensis Cultivars under Study
were generated at m/z 153.01824 (C7H5O4 + , 17%) and [(M+H)-C6H8O6-C8H6O3] + at m/z 121.02857 (1%), previously reported by several authors [33][34][35]. Table 2 presents the total flavonoid contents (mg/g) according to each subfamily, the colorimetric parameters (L*, a* and b*) of the CIELAB system, and the chromatic representations of the RGB system for the 16 flower cultivars, together with their extract ORAC antioxidant capacity. The antioxidant activity of H. rosa-sinensis flower extracts has been determined using several in vitro methods [36]. In this research, the ORAC method was used because in the hydrogen-atom transfer mechanism the peroxyl radicals are used, which predominate in food and biological systems [37]. The codes of the different flowers are shown in Figure 1. Table 3 reports the content of phenolic compounds (mg/g) in H. rosa-sinensis flower hydroalcoholic extracts of the different cultivars studied in this work.

Relationship of Chemical Composition with Color and Antioxidant Capacity in the H. rosasinensis Cultivars under Study
were generated at m/z 153.01824 (C7H5O4 , 17%) and [(M+H)-C6H8O6-C8H6O3] at m/z 121.02857 (1%), previously reported by several authors [33][34][35]. Table 2 presents the total flavonoid contents (mg/g) according to each subfamily, the colorimetric parameters (L*, a* and b*) of the CIELAB system, and the chromatic representations of the RGB system for the 16 flower cultivars, together with their extract ORAC antioxidant capacity. The antioxidant activity of H. rosa-sinensis flower extracts has been determined using several in vitro methods [36]. In this research, the ORAC method was used because in the hydrogen-atom transfer mechanism the peroxyl radicals are used, which predominate in food and biological systems [37]. The codes of the different flowers are shown in Figure 1. Table 3 reports the content of phenolic compounds (mg/g) in H. rosa-sinensis flower hydroalcoholic extracts of the different cultivars studied in this work.  Table 2 presents the total flavonoid contents (mg/g) according to each subfamily, the colorimetric parameters (L*, a* and b*) of the CIELAB system, and the chromatic representations of the RGB system for the 16 flower cultivars, together with their extract ORAC antioxidant capacity. The antioxidant activity of H. rosa-sinensis flower extracts has been determined using several in vitro methods [36]. In this research, the ORAC method was used because in the hydrogen-atom transfer mechanism the peroxyl radicals are used, which predominate in food and biological systems [37]. The codes of the different flowers are shown in Figure 1. Table 3 reports the content of phenolic compounds (mg/g) in H. rosa-sinensis flower hydroalcoholic extracts of the different cultivars studied in this work.  Table 2 presents the total flavonoid contents (mg/g) according to each subfamily, the colorimetric parameters (L*, a* and b*) of the CIELAB system, and the chromatic representations of the RGB system for the 16 flower cultivars, together with their extract ORAC antioxidant capacity. The antioxidant activity of H. rosa-sinensis flower extracts has been determined using several in vitro methods [36]. In this research, the ORAC method was used because in the hydrogen-atom transfer mechanism the peroxyl radicals are used, which predominate in food and biological systems [37]. The codes of the different flowers are shown in Figure 1. Table 3 reports the content of phenolic compounds (mg/g) in H. rosa-sinensis flower hydroalcoholic extracts of the different cultivars studied in this work.
Shen et al. [19] isolated quercetin-sophoroside from the hydroethanolic extract of H. rosa-sinensis flowers and determined its therapeutic potential against Alzheimer's disease. The authors demonstrated that this glycosylated flavonol decreased memory impairment and improved learning in mice with scopolamine-induced cognitive dysfunction, and then assumed that quercetin-sophoroside could be used as a neuroprotectant. In the same investigation [19], due to the lack of a certified reference substance, quercetin-dihexoside was only tentatively identified.
This study detected cyanidin-3-glucoside, described by Li et al. [20], cyanidin-sophoroside (found at high concentrations, 21 mg/g extract, in the red flower extract, Table 3), which had been isolated for the first time by Nakamura et al. [22] from the H. rosa-sinensis flower extract and characterized by NMR, and cyanidin-sambubioside, which had been reported in H. sabdariffa extracts [28]. The phenolic content of H. rosa-sinensis red flower extracts was 60% cyanidin-sophoroside. Although there is a large variability in the number of flowers per plant, an average of 400 harvested flowers per year per shrub can be used to estimate a potential production of this anthocyanin-rich extract. A density of 1600 plants/ha and an average 10 g flower weight would lead to an annual production of 6.4 ton of flowers per ha. The solvent extraction yield was calculated by dividing the hydroethanolic extract weight by the plant material weight. The yield obtained in this study was 23 ± 2% (w/w). Thus, a hectare of red H. rosa-sinensis plants would permit the production of 1.4 ton per year, of an extract containing anthocyanins, flavan-3-ols and flavanonols, which may be useful as ingredients for pharmaceutical and cosmetic products. Anthocyanins, flavonols, flavan-3-ols, flavononols and flavones contribute to the antioxidant activity of the extracts of many plant species (Table 4). Zaki et al. [24] isolated luteolin-7-glucoside from the hydromethanolic extract of H. rosa-sinensis leaves and evaluated its antioxidant and hypoglycemic effects; the authors reported that in diabetic rats treated with the hydromethanolic extract of H. rosa-sinensis, the hypoglycemic and hypocholesterolemic effects decreased, which led to hepatic improvement. Among the components found in the cultivars under study, luteolin-7-glucoside was not detected, but its isomer, kaempferol-3-glucoside, was confirmed.  [35,[38][39][40][41][42][43]. SOD: superoxide radical scavenging activity. ABTS +• : radical scavenging assay. Figure 4 shows the 3D scatter bubble plots with color scale, where the results of the CIELAB colorimetric coordinates are shown. The size of the bubble represents the antioxidant activity: the larger the bubble size, the higher the ORAC value. The antioxidant activity of the floral extracts varied from 1580 to 3840 µmol Trolox ® /g of extract; the bubble of the white cultivar ( Figure 1P, 1580 ± 41 µmol Trolox ® /g of extract) was the smallest, and the bubble of the red cultivar ( Figure 1A, 3840 ± 74 µmol Trolox ® /g of extract) was the largest, with the highest antioxidant activity value. The color scale changed, depending on the extract of the cultivar analyzed, for a total of six colors, namely, white, lilac, yellow, orange, pink and red. Figure 4A,B show that the bubble corresponding to the extract of the dark red flower is located at a greater distance from the other red bubbles because it presents lower L* values (28) compared to the other red bubbles from the red group (L* values 54-61). The lighter the color of the flower (white, lilac and yellow cultivars), the larger the L* values were, compared to the rest of the flowers studied. These results agree with those reported by Wan et al. [44], who showed that at higher concentrations of pelargonidin-3,5-diglucoside in rose petals, the L* value was lower, thus concluding that the red color of rose petals was due to a high anthocyanin content.
Anthocyanins confer colors to flowers and fruits that can vary from orange to red, pink and purple to blue [45]. Their color is related to the structure, e.g., the number of hydroxyl groups, their degree of methylation, the nature or number of sugar molecules attached to the aglycone, the pH, and the copigmentation that can result from various intermolecular associations, between two identical anthocyanins (self-association) or between an anthocyanin and other phenolic compounds (intermolecular copigmentation) [46], and the formation of π-π-type complexes with a metal ion. Gordillo et al. [47] suggested that flavonols and flavan-3-ols, despite being colorless compounds, may play an important role in color generation through copigmentation with anthocyanins, which would increase the stability of the latter. Bakowska et al. [48] reported the formation of complexes of cyandin-3-glucoside and flavonoids isolated from Scutellaria baicalensis.
Molecules 2023, 28, x FOR PEER REVIEW 13 of 20 suggested that flavonols and flavan-3-ols, despite being colorless compounds, may play an important role in color generation through copigmentation with anthocyanins, which would increase the stability of the latter. Bakowska et al. [48] reported the formation of complexes of cyandin-3-glucoside and flavonoids isolated from Scutellaria baicalensis. The mechanism that describes the pigmentation of Hibiscus flower cultivars is poorly studied [4]. To explore the relationships of the color of the flowers, their antioxidant capacity and the content of flavonoids present in the petals, the Pearson correlation coefficients of the color parameters, the ORAC value and the content of anthocyanins, flavonols, flavan-3-ols, flavanonols, flavones, and flavanones were determined. These relationships are shown in a heatmap ( Figure 5). The color was associated in the following way: the parameters a*, h and C were positively correlated with the ORAC value, the L * values were negatively correlated with the antioxidant capacity, and the parameter b* presented a small negative correlation. The clearer the shade of the flowers (white, yellow, or lilac), the higher the L* values and the lower the antioxidant capacity values of their extracts. The opposite effect was observed for flowers with darker shades (orange < pink < red), with lower L* values, higher antioxidant capacity, and high content of anthocyanins, flavan-3-ols and flavanonols in their extracts.
Studies have explored the relationship between the color and chemical composition of different flowers, e.g., roses [44], water lilies [49], daffodils [50], crabapples [51] and canola [52]. The results obtained in this comparative study of H. rosa-sinensis cultivars were consistent with those reported by Wan et al. [44], who studied the relationship between color and chemical composition of various rose cultivars and reported that the red cultivar had a higher concentration of anthocyanins compared to that of the yellow, orange, and pink cultivars. However, in the latter, the concentrations of carotenoids and flavonols were higher. The authors [44] reported that kaempferol and quercetin aglycones were the predominant flavonols in roses. Sheyth and De [53] reported that the red cultivar had the highest total phenolic content and antioxidant activity compared with the pink, yellow and white cultivars. According to Tai et al. [51], the highest concentration of anthocyanins in wild apple flowers was related to the amount of the enzyme chalcone synthase, which regulates anthocyanin biosynthesis and, therefore, was responsible for the red coloration and color variation in the petals of apple cultivars.
According to Yin et al. [52], the main components detected in the extracts of red and pink petals of Brassica napus were derivatives of epicatechin, quercetin and isorhamnetin, The mechanism that describes the pigmentation of Hibiscus flower cultivars is poorly studied [4]. To explore the relationships of the color of the flowers, their antioxidant capacity and the content of flavonoids present in the petals, the Pearson correlation coefficients of the color parameters, the ORAC value and the content of anthocyanins, flavonols, flavan-3-ols, flavanonols, flavones, and flavanones were determined. These relationships are shown in a heatmap ( Figure 5). The color was associated in the following way: the parameters a*, h and C were positively correlated with the ORAC value, the L * values were negatively correlated with the antioxidant capacity, and the parameter b* presented a small negative correlation. The clearer the shade of the flowers (white, yellow, or lilac), the higher the L* values and the lower the antioxidant capacity values of their extracts. The opposite effect was observed for flowers with darker shades (orange < pink < red), with lower L* values, higher antioxidant capacity, and high content of anthocyanins, flavan-3-ols and flavanonols in their extracts.
Studies have explored the relationship between the color and chemical composition of different flowers, e.g., roses [44], water lilies [49], daffodils [50], crabapples [51] and canola [52]. The results obtained in this comparative study of H. rosa-sinensis cultivars were consistent with those reported by Wan et al. [44], who studied the relationship between color and chemical composition of various rose cultivars and reported that the red cultivar had a higher concentration of anthocyanins compared to that of the yellow, orange, and pink cultivars. However, in the latter, the concentrations of carotenoids and flavonols were higher. The authors [44] reported that kaempferol and quercetin aglycones were the predominant flavonols in roses. Sheyth and De [53] reported that the red cultivar had the highest total phenolic content and antioxidant activity compared with the pink, yellow and white cultivars. According to Tai et al. [51], the highest concentration of anthocyanins in wild apple flowers was related to the amount of the enzyme chalcone synthase, which regulates anthocyanin biosynthesis and, therefore, was responsible for the red coloration and color variation in the petals of apple cultivars.
According to Yin et al. [52], the main components detected in the extracts of red and pink petals of Brassica napus were derivatives of epicatechin, quercetin and isorhamnetin, whereas those from yellow and white petals were found to be kaempferol derivatives. In the Hibiscus flower extracts studied here, glycosylated derivatives of quercetin were the major components in pink, orange, and yellow flowers. Yin et al. [52] reported that derivatives of hydroxycinnamic acid, sinapoyl malate, naringenin- 7-O-glucoside, cyanidin-3glucoside, cyanidin-3,5-di-O-glucoside, petunidin-3-O-β-glucopyranoside, isorhamnetin -3 whereas those from yellow and white petals were found to be kaempferol derivatives. In the Hibiscus flower extracts studied here, glycosylated derivatives of quercetin were the major components in pink, orange, and yellow flowers. Yin et al. [52] reported that derivatives of hydroxycinnamic acid, sinapoyl malate, naringenin- 7-O-glucoside, cyanidin-3glucoside, cyanidin-3,5-di-O-glucoside, petunidin-3-O-β-glucopyranoside,   Flavan-3-ols (catechin and epicatechin) were mainly detected in the extracts of red cultivars. This can be explained through the biosynthetic pathway ( Figure 6): the precursor of catechin is leucoanthocyanidin, from which the anthocyanidins, precursors of epicatechin, and anthocyanins are biosynthesized. The increased bioavailability of Flavan-3-ols (catechin and epicatechin) were mainly detected in the extracts of red cultivars. This can be explained through the biosynthetic pathway ( Figure 6): the precursor of catechin is leucoanthocyanidin, from which the anthocyanidins, precursors of epicatechin, and anthocyanins are biosynthesized. The increased bioavailability of anthocyanidins can lead to the biosynthesis of flavan-3-ols. In the extracts from white flowers (Table 2), neither anthocyanins nor anthocyanidins were detected, and the concentration of flavan-3-ol was 120 times lower than that found in the extracts of red cultivars (Table 2). flowers (Table 2), neither anthocyanins nor anthocyanidins were detected, and the concentration of flavan-3-ol was 120 times lower than that found in the extracts of red cultivars (Table 2).
Patel et al. [13] carried out a comparative study of the extracts of red, pink, orange, yellow and white cultivars of leaves, stems and roots of Hibiscus rosa-sinensis and found the highest and lowest total phenol content in the red and white cultivars, respectively. To the best of our knowledge, our research is the first comparative study of flavonoids present in extracts from different cultivars of Hibiscus rosa-sinensis flowers. Patel et al. [13] carried out a comparative study of the extracts of red, pink, orange, yellow and white cultivars of leaves, stems and roots of Hibiscus rosa-sinensis and found the highest and lowest total phenol content in the red and white cultivars, respectively. To the best of our knowledge, our research is the first comparative study of flavonoids present in extracts from different cultivars of Hibiscus rosa-sinensis flowers.
Anthocyanins, flavan-3-ol and flavanonols, among the flavonoids present in H. rosa-sinensis flowers, showed a high positive correlation with the ORAC value, and the higher content of these compounds, the higher the antioxidant capacity value measured by the ORAC method. Some glycosylated derivatives of quercetin and total quercetins also showed positive correlations with the ORAC value, but lower correlations compared with other substances. A negative correlation was observed for flavonols, anthocyanins, flavan-3-ols, and flavanonols: while the concentration of flavonols increased in the extracts obtained from pink, orange, yellow and white Hibiscus cultivars, these compounds decreased in the red cultivars.
The flavonol content, which was inversely proportional to the content of anthocyanins, flavan-3-ols and total flavanonols, was associated with lower antioxidant capacity. The extract of the red cultivar, with the highest content of anthocyanins, had a higher antioxidant capacity than the extract of the white flower, characterized by a high content of flavonols; however, its antioxidant capacity was the lowest compared with that of the other floral extracts studied.

Color Measurement
The color of the flowers of the Hibiscus rosa-sinensis cultivars (Figure 1) was measured on the adaxial surface of fresh petals with an STS-VIS microspectrophotometer (Ocean Optics, Dunedin, FL, USA.), size 40 × 42 × 24 mm, with wavelength ranges of λ = 190-650 nm (UV) and λ = 350-800 nm (Vis). The CIELAB (International Commission on Illumination) system was used. The colorimetric coordinates (L*, a*, b*, Cab* and hab) were obtained with Ocean View 1.5.0 software (Ocean Optics, Dunedin, FL, USA) After color measurement, the petals were extracted with solvent.

Antioxidant Assay: Oxygen Radical Absorption Capacity
The oxygen radical absorbance capacity (ORAC) was determined for each extract following the methodologies described by Ou et al. [58] and Huang et al. [59], with some modifications. The antioxidant capacity was measured from the difference between the area under the curve (AUC) of each sample and the AUC of the blank. Fluorescein (FL) (150 µL, 8.16 × 10 −5 mM) was added to the sample dilutions in phosphate buffer (pH 7.4), and the mixtures were incubated (37 • C, 18 min) followed by AAPH [2,2 -azinobis-(2amidino-propane) dihydrochloride] addition (25 µL, 153 mM). The decrease in fluorescein fluorescence (FL) was recorded for 80 min using wavelengths of λ = 490 nm for excitation and λ = 510 nm for emission. Trolox ® (97%, Sigma-Aldrich, St. Louis, MO, USA) was used as a control standard. The net area obtained was the basis for calculating the µmol Trolox ® /g of extract using a calibration curve. Determinations were made in triplicate on a Turner Biosystems Inc., ModulusTM II Microplate Multimode Reader (Sunnyvale, CA, USA with 96-well poly(styrene) microplates and a fluorescence module. They were expressed as the mean value ± the standard deviation (n = 3).

Conclusions
A positive correlation was found between the antioxidant capacity of the H. rosa-sinensis flower extracts and the content of anthocyanins, flavan-3-ols and total flavanonols, whereas the correlation was negative with flavonols. The antioxidant capacity and the highest amounts of anthocyanins, flavan-3-ols and flavononols were found in the extracts of the darker red flowers. H. rosa-sinensis red flowers can be a source of natural ingredients and bioactive compounds such as anthocyanins, flavan-3-ols and flavanonols for pharmaceutical and cosmetic products. Yellow and white H. rosa-sinensis cultivars can serve as natural sources for the extraction of flavonols. Knowing the composition of flavonoids present in flowers based on their color can serve as a basis for understanding in more detail the mechanisms that regulate color and its origin in flowers, particularly in cultivars of Hibiscus spp.