From the Field to the Pot: Phytochemical and Functional Analyses of Calendula officinalis L. Flower for Incorporation in an Organic Yogurt

Edible flowers have been used as ingredients because of their biological activities, taste, and overall appearance. This research was aimed to characterize the chemical composition and in vitro antioxidant activity of the marigold flower (Calendula officinalis L.) extracted with different proportions of water and ethyl alcohol, and the lyophilized extract with higher content of antioxidant compounds was incorporated into an organic yogurt. Results showed that the hydroalcoholic extract (50:50 v/v) presented the highest total phenolic content (TPC), flavonoids, and antioxidant activity (ferric reducing antioxidant power (FRAP), total reducing capacity (TRC), and Cu2+/Fe2+ chelating ability). Phenolic acids and flavonoids were quantified in the extract by LC-DAD, while 19 compounds were tentatively identified by ESI-MS/MS. The lyophilized marigold extract (LME) also inhibited 12% of Wistar rat’s brain lipid oxidation in vitro, inhibited α-amylase, and α-glucosidase activities, but showed no cytotoxicity towards cancerous cells (HCT8 and A549). However, marigold flower extract protected human erythrocytes against mechanical stress. When added into an organic yogurt model (0 to 1.5%), LME increased TPC and antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH) and TRC), and the sensory analysis showed that the organic yogurt had an acceptance of 80.4%. Our results show that the use of LME may be a technological strategy to increase the content of bioactive compounds in yogurts.


Chemical Composition, Instrumental Color, and Antioxidant Activity of the Extracts
The total carotenoids (TC) of C. officinalis flower extracts were quantified according to the colorimetric method described by Rodriguez-Amaya [18], and the results were expressed in µg of β-carotene per 100 g of flowers. The total phenolic content (TPC) was determined by the Prussian Blue method [19], and the results were expressed in mg of gallic acid equivalents per 100 g of flowers. Total flavonoids (TF) were quantified by the UV-Vis spectrophotometric method (Shimadzu UV-1800, Kyoto, Japan) according to the methodology described by Lees et al. [20]. The TF content was expressed as mg quercetin equivalents per 100 g of flowers. The ortho-diphenolics were quantified according to Durán et al. [21], and the results were expressed in mg of chlorogenic acid equivalents per 100 g of flowers.
Color intensity = Abs 420 nm + Abs 520 nm + Abs 620 nm The antioxidant activity (AA) by capturing the DPPH radical was analyzed according to Brand-Williams et al. [23], and ferric reducing antioxidant power (FRAP) was verified according to Benzie et al. [24]. The results of DPPH and FRAP were expressed in mg of ascorbic acid equivalents per 100 g of flowers. The reducing capacity of the Folin-Ciocalteu reagent (RCFC) was determined according to Singleton et al. [25], and the results were expressed in mg of gallic acid equivalents per 100 g of flowers. The chelating activity in relation to Cu 2+ (CCA) and Fe 2+ (FCA) ions was determined according to the experimental conditions described by Santos et al. [26], and the results were expressed as the percentage of inhibition of the formation of the Cu 2+ -pyrocatechol violet complex and Fe 2+ -ferrozine complex, respectively. All analyses were performed in triplicate.

Extract Selection by Principal Component Analysis
The extract with higher chemical content, in vitro antioxidant activity, and color parameters was selected using principal component analysis (PCA). Firstly, the triplicate values were transformed into z-scores to standardize the results in unit variance. Subsequently, the correlation matrix of Calendula officinalis flower extracts (n = 15) in rows was elaborated, and the response variables (n = 14) in columns, totaling 210 data points [27]. The projection in the two-dimensional plane (PC1xPC2) was performed with the variables with higher load factors or equal to 0.60. Approximately 4 L of the selected extract were divided into 50 mL plastic containers, which were subjected to lyophilization under vacuum of 830 µmL Hg for 120 h (Terroni, model LD 1500A, São Carlos, Brazil).

Individual Phenolic Composition of the Selected Extract
The individual phenolic compounds from the hydroalcoholic extract (50:50 v/v) of C. officinalis flowers were quantified using high-performance liquid chromatography, HPLC (Shimadzu, model LC-20T Prominence, Kyoto, Japan) with photodiode array detector (DAD; model SPD-M20A, Shimadzu, Kyoto, Japan). Table 1 contains the chromatographic conditions for quantification of phenolic acids (p-coumaric, caffeic, ferulic, and ellagic) and flavonoids (procyanidin A2 and quercetin-3-rutinoside) quantified in the study. Chromatographic separation was performed using reversedphase and a C 18 column (150 mm × 4.6 mm, particle size 3.5 µm; Agilent, Santa Clara, CA, USA). The mobile phase consisted of water acidified with 0.2% (v/v) formic acid (solvent A) and acetonitrile (solvent B). The elution gradient applied was 0 to 10 min 97% A, 10 to 15 min 90% A, 15 to 17 min 88% A, 17 to 23 min 45% A, 23 to 30 min 35% A, 30 to 35 min 100% A, and 35 to 47 min 100% A. Throughout the chromatographic separation the temperature of the column was maintained at 40 • C, and the sample injection volume was 10 µL with the flow rate of 0.5 mL/min. The quantification of phenolic compounds was performed by employing analytical curves, and the results were expressed in mg per 100 g of dried flowers, according to Fidelis et al. [28].
The qualitative identification of phenolic compounds from hydroalcoholic extract of C. officinalis flowers was performed using a mass spectrometry [ESI-MS/MS] Xevo ® (Waters, MA, USA). The negative ionization mode [M-H]was used, and the capillary and cone voltages were set at 30 V and  2.6. In Vitro Functional Properties of the Lyophilized Marigold Extract (LME) The antimicrobial activity of LME was evaluated by the diffusion method in the agar-well plate as described by Cleeland et al. [31]. The standardized cell suspension (10 6 CFU/mL) of Pseudomonas aeruginosa (IAL 1853), Salmonella Enteritidis (S 2887), Salmonella Typhimurium (IAL 2431), and Escherichia coli (IAL 2064) were grown in nutrient broth at 37 • C for 24 h; Bacillus cereus (ATCC 14579) and Staphylococcus aureus (ATCC 13565) were grown in nutrient broth at 30 • C for 24 h; Listeria monocytogenes (ATCC 7644) was grown in soy tryptone broth added with 0.6% yeast extract at 37 • C for 24 h; and Saccharomyces cerevisiae (NCYC 1006) was grown in malt extract broth at 30 • C for 24 h. The extract was dissolved with dimethyl sulfoxide (DMSO), and 2 mg added in each well (0.7 cm). The ampicillin (10 µg/mL) was used as a positive control, except for S. cerevisiae cicloheximide (1 µg/mL) was used, and sterile water as a negative control. The results were expressed as inhibition halo (cm).
The antihemolytic effect of LME was evaluated under hypotonic and isotonic conditions according to Zhang et al. [32]. The blood with O + blood typing was obtained from the UEPG University The inhibition of lipid oxidation of the brain tissue of Wistar rats was evaluated according to the experimental conditions described by Migliorini et al. [33]. The UEPG Animal Research Ethics Committee approved process n • 047/2017 for the use of brain tissue from Wistar rats. The extract was solubilized in ultrapure water with a concentration of 1000 mg/L, and the quercetin standard (50 mg/L) was used as a positive control. The results were expressed as percentage inhibition of lipid oxidation.
The in vitro antiproliferative activity was evaluated for A549, HCT8, and IMR90 cells. The cell lines were maintained as described by Escher et al. [35]. Cells were plated into 96-well plates at a density of 1 × 10 4 cells/well (A549 and HCT8), and 5 × 10 3 (IMR90), 100 µL/well and they were treated with different concentrations (100-1000 µL/mL) of LME. The cell viability test was performed as proposed by Santos et al. [36], by the MTT assay. All experiments were carried out in quadruplicate, and the dose-response analysis was determined by non-linear regression. The IC 50 , GI 50 , and LC 50 parameters were calculated per the method described by Carmo et al. [37].
The generation of intracellular ROS was measured by a ROS assay with DCFH-DA, as described by Escher et al. [35]. Briefly, cancerous A549 cells (6 × 10 4 per well) and healthy IMR90 cells (2 × 10 4 per well) were treated with different concentrations of lyophilized marigold extract (10, 50, and 100 µg/mL), 15 µmol/L hydrogen peroxide (positive control), or culture medium (negative control) diluted in DCFH-DA solution (25 mmol/L). Then, the cells were incubated at 37 • C for 1 h with the extract concentrations, and subsequently, they were washed with PBS and a H 2 O 2 solution (15 µmol/L) was added. The ROS level was determined by fluorescence (excitation of 485 nm and emission of 538 nm), and results were reported as the percentage of fluorescence intensity.

Yogurt Manufacture and Analysis
Yogurts (~2 L per formulation) were prepared according to Perina et al. [38]. First, the organic whole bovine milk (Escher Organics, Campo Magro, Brazil) was added with 110 g/L organic sucrose (União, Barra Bonita, Brazil; IBD Certified), and the mixture was pasteurized at 65 • C/30 min. After cooling the mixture to 42 • C, 0.05 g/L of lyophilized bacterial culture (Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) was added. The mixture was incubated at 42 • C until the clot point (pH 4.6). The curd was cooled to 7 • C, homogenized, and added at different concentrations of lyophilized marigold extract: 0.25, 0.50, 1.00, and 1.50 g/100 g yogurt. The negative control was elaborated with 0 g extract/100 g yogurt. The yogurts were stored at 7 • C until the time of analysis.
To determine the total phenolic content and antioxidant activity of yogurts, 5 g samples were extracted with 5 mL of methyl alcohol (1:1 v/v) and vortexed for 5 min. Subsequently, the mixture was centrifuged at 900× g for 15 min, and the upper phase layer collected and analyzed immediately. The TPC content (mg gallic acid equivalents (GAE)/100 g) and antioxidant activity by DPPH (mg AAE/100 g) were determined according to Margraf et al. [19] and Brand-Williams et al. [23], respectively. The total reducing capacity was measured using the Folin-Ciocalteu method modified by Berker et al. [39], and the results were expressed in mg quercetin equivalents per 100 g of yogurt.
A preliminary sensory analysis of the five yogurt formulations (data not shown) performed by untrained assessors indicated that lyophilized marigold extract (LME) concentrations above 0.25 g/100 g enhanced the bitter and astringent taste in yogurt, which is an undesirable feature of polyphenols. Therefore, the formulation containing 0.25 g LME/100 g yogurt was selected for the analysis of proximal composition, physicochemical parameters, instrumental texture, and sensory analysis.
The proximal composition of organic yogurt added at 0 g and 0.25 g LME/100 g was determined according to AOAC [40]: proteins (Nx6.38), total lipids (Bligh-Dyer method), moisture (105 • C/24 h), ashes (550 • C), and total carbohydrates was estimated by difference. The results were expressed in g/100 g of yogurt. The total caloric value was determined using Atwater factors (4 kcal/g for carbohydrates and proteins, and 9 kcal/g for lipids), and expressed in kcal/100 g of yogurt. The pH was measured at 25 • C in pH meter (Model HSP-3B, Labmeter, Brazil) previously calibrated. The total titratable acidity was verified using NaOH solution (0.1 mol/L), and the results were expressed as g lactic acid/100 g yogurt.
The instrumental color of yogurts (0 g and 0.25 g LME/100 g) using the MiniScan ® EZ 4500L colorimeter (Hunter Lab, Reston, USA) was expressed in scale parameters developed by the Commission Internationale de Eclairage (CIE): lightness (L*), green-red component (a* axis), and blue-yellow component (b* axis). The instrumental texture was determined using the Universal TA-XT2 Texture Analyzer (Stable Micro Systems, Surrey, UK). The double compression test with an aluminum cylinder (25 mm diameter) with a depth of 20 mm and a speed of 1 mm/s was used. The firmness (g) and consistency (g.s) parameters were measured by the Texture Expert v. 1.20 program using the algorithm Fracture TPA.
Sensory analysis with 76 untrained assessors (17 men and 59 women between 18 and 35 years) was carried out after signing the consent form approved by the Ethics Committee of the UEPG (approval code-CAAE: 65493717.9.0000.0105). The yogurt containing 0.25 g LME/100 g (20 g) was served in a glass identified with three random digits. A structured 9-point hedonic scale (1-extremely disliked to 9-extremely liked) was used to evaluate the degree of liking of odor, taste, texture, color, and overall impression. The assessors were asked if they consider yogurt a healthy product (yes/no). Moreover, the assessors opined on the overpayment (R$ 0 to R$ 3.00) for a 100 g unit of "yogurt rich in natural antioxidant compounds" and "organic yogurt" compared to "yogurt without antioxidant" and "conventional yogurt" (R$ 1.00), respectively.

Statistical Analyses
The results were expressed as mean ± standard deviation (n = 3). The Brown-Forsythe test was applied to evaluate the homoscedasticity of the data. The one-way analysis of variance (ANOVA) was applied to evaluate the differences between the mean values, followed by Fisher's least significant difference test (p ≤ 0.05). The linear correlations between the results (n = 15) were evaluated by Pearson's correlation coefficient (p ≤ 0.05). The results of antimicrobial activity, lipid oxidation inhibitory activity, proximal composition, physicochemical parameters, and instrumental texture were evaluated by unpaired Student t test (p ≤ 0.05). The TIBCO Statistica software v. 13.3 (TIBCO Statistica™ Ltd., Palo Alto, CA, USA) was used in the analyses. Table 3 shows the chemical composition and instrumental color of C. officinalis flower extracts. The β-carotene content ranged from 97 to 637 µg/100 g (p ≤ 0.05). The highest content of β-carotene was extracted with 100% EtOH, and significant differences were not observed between extracts with 50 and 75% EtOH. Carotenoid contents ranging from 200 to 3510 mg/100 g were quantified in different C. officinalis flower varieties [17,41]. The marigold flowers as well as β-carotene containing α-carotene, γ-carotene, lutein, luteoxanthin, flavoxanthin, rubixanthin, and other carotenoids [42,43].
Ferreira et al. [15] compared chloroform, ethyl alcohol, methyl alcohol (MeOH), and hydromethanolic (MeOH:H 2 O, 70:30 v/v) solvents in the extraction of phenolic compounds from marigold flowers. The authors found that hydromethane was more effective in extracting phenolic compounds because it was highly polar. Pires et al. [44] quantified in the extracts of marigold petals containing MeOH:H 2 O (80:20 v/v) the contents of 1131 mg/100 g of phenolic compounds and 1115 mg/100 g of flavonoids, and in the aqueous extract the contents of 747 mg/100 g and 737 mg/100 g, respectively. The literature shows that methyl alcohol and hydromethanolic solutions extract higher levels of phenolic compounds. However, in this research, it was chosen to use ethyl alcohol because of its low toxicity. In addition, the contents of bioactive compounds (carotenoids and phenolic compounds) may vary due to different C. officinalis flower varieties and cultivation sites [41,45].
The color indices of the extracts were influenced by the combination of the solvents H 2 O and EtOH in which the color intensity varied from 1.1 to 2.1 u. a. and the hue ranged from 4.9 to 12.6 u. a. (p ≤ 0.05). The highest intensity of color and hue was verified in the extract obtained with 100% EtOH which presented the highest content of β-carotene (637 µg/100 g). According to Khalid et al. [45], carotenoids are mainly responsible for the yellow-orange coloration of C. officinalis inflorescences. In the correlation analysis, color intensity was significantly correlated (p ≤ 0.05) with β-carotene (r = 0.8959), ortho-diphenolics (r = 0.6150), and TPC (r = −0.5393). The hue showed a significant correlation (p ≤ 0.05) with β-carotene (r = 0.7874) and TPC (r = −0.8702).
The chemical antioxidant activity of the extracts was influenced by the different proportions of the solvents H 2 O and EtOH (p ≤ 0.05) ( Table 4). The extract obtained with 75% H 2 O:25% EtOH showed higher antioxidant activity through electron transfer evaluated by DPPH, FRAP, and RCFC. In the analysis by FRAP and RCFC, no significant differences were observed between the extracts with 75% H 2 O:25% EtOH and 50% H 2 O:50% EtOH. Additionally, the metal chelating activity of the extracts was evaluated by inhibiting the formation of the Cu 2+ -pyrocatechol violet complex and the Fe 2+ -ferrozine complex. The extract with 50% H 2 O:50% EtOH showed the highest chelating activity with inhibition of 60% and 53% of the formation of the Cu 2+ -pyrocatechol violet complex and Fe 2+ -ferrozine complex, respectively.

Principal Component Analysis (PCA)
In the principal component analysis, the relationship of extracts with chemical compounds, instrumental color, and antioxidant activity was explored. The two-dimensional projection explained 90% of the data variability, and the main components 1 and 2 explained 65.94% and 23.95% of the data variability, respectively (Figure 1). The main component 1 showed that the extracts with 75% H 2 O:25% EtOH (assay 2) and 50% H 2 O:50% EtOH (assay 3) showed higher content of TPC, RP, YP, and antioxidant activity by DPPH, FRAP, RCFC, and CCA. The extract with 100% EtOH (assay 5) showed higher hue, color intensity, yellow pigment, and β-carotene content, but low antioxidant activity. In the principal component 2, the 25% H 2 O:75% EtOH extract (assay 4) correlated with flavonoids, ortho-diphenolics, and Fe 2+ chelating activity, while the 100% H 2 O extract (assay 1) showed no correlation with chemical compounds, color parameters, and antioxidant activity. The PCA indicated that the extract with 50% H 2 O:50% EtOH showed higher values of antioxidant activity and bioactive compounds concerning the other extracts. Therefore, the extract obtained with 50% H 2 O:50% EtOH was selected for the analysis of the individual phenolic composition, in vitro functional properties, and incorporation in organic yogurt.

Principal Component Analysis (PCA)
In the principal component analysis, the relationship of extracts with chemical compounds, instrumental color, and antioxidant activity was explored. The two-dimensional projection explained 90% of the data variability, and the main components 1 and 2 explained 65.94% and 23.95% of the data variability, respectively (Figure 1). The main component 1 showed that the extracts with 75% H2O:25% EtOH (assay 2) and 50% H2O:50% EtOH (assay 3) showed higher content of TPC, RP, YP, and antioxidant activity by DPPH, FRAP, RCFC, and CCA. The extract with 100% EtOH (assay 5) showed higher hue, color intensity, yellow pigment, and β-carotene content, but low antioxidant activity. In the principal component 2, the 25% H2O:75% EtOH extract (assay 4) correlated with flavonoids, ortho-diphenolics, and Fe 2+ chelating activity, while the 100% H2O extract (assay 1) showed no correlation with chemical compounds, color parameters, and antioxidant activity. The PCA indicated that the extract with 50% H2O:50% EtOH showed higher values of antioxidant activity and bioactive compounds concerning the other extracts. Therefore, the extract obtained with 50% H2O:50% EtOH was selected for the analysis of the individual phenolic composition, in vitro functional properties, and incorporation in organic yogurt.

Selected Extract: Individual Phenolic Composition and in Vitro Functional Properties
The individual phenolic composition of the hydroalcoholic extract (50:50 v/v) of C. officinalis flowers were determined by HPLC-DAD ( Figure S1). The phenolic acids quantified were p-coumaric (5.8 mg/100 g), caffeic (9.2 mg/100 g), ferulic (18.3 mg/100 g), and ellagic acid, 3.7 mg/100 g. The quantified flavonoids were procyanidin A2 (42.5 mg/100 g) and quercetin-3-rutinoside (46.1 mg/100 g). Olennikov et al. [17] quantified caffeic acid (92 mg/100 g) and quercetin-3-rutinoside (226 mg/100 g) in the hydroalcoholic extract, 60% EtOH, of C. officinalis flowers var. Greenheart Orange. Pires et al. [44] quantified caffeic acid (1 mg/100 g) and quercetin-3-rutinoside (30 mg/100 g) in the hydromethanolic extract (MeOH:H Figure S2. The compounds tentatively identified in this study corroborate data obtained by Miguel et al. [29] and Faustino et al. [30]. The antimicrobial activity of the lyophilized marigold extract is presented in Table 5. The extract showed the inhibitory effect of the growth of L. monocytogenes, P. aeruginosa, S. Typhimurium, S. Enteritidis, B. cereus, E. coli, and S. aureus (0.27 cm). These results corroborate Efstratiou et al. [46], who found that the ethanolic extract of C. officinalis petals inhibited the growth of P. aeruginosa (0.7 cm), B. cereus (1 cm), E. coli (0.3 cm), and S. aureus (2.2 cm). The hydromethanolic extract (MeOH:H 2 O, 80:20 v/v) and the infusion of petals C. officinalis demonstrated inhibitory effect against Gram-positive and Gram-negative bacteria [44]. According to Faustino et al. [30] methanolic extract of some C. officinalis varieties showed low antibacterial activity due to the lower content of antibacterial phenolic compounds such as caffeic acid. The antihemolytic effect of LME analyzed under hypotonic and isotonic conditions is shown in Figure 2. Figure 2A shows that using the 50 mg/L of the LME, it was possible to calculate the H 50 , being the NaCl concentration where 50% of the erythrocytes is present in the sample are lysed. The lower this value, the more efficient the extract will be in inhibiting hemolysis caused by low extracellular osmotic pressure. The control, in the absence of the extract, had an H 50 value of 0.413%, while the extract had a lower H 50 value (0.356%). These results suggest that the bioactive compounds present in LME may interact with erythrocyte membrane phospholipids in order to reduce the flow parameter of the lipid bilayer [47], which increases the resistance of cells to hypotonic hemolysis [48].  Figure 2B shows that the hemolysis rate is reduced, 0.4% NaCl, with increasing LME concentration (50-110 mg/L). The C. officinalis flowers are rich in saponins, compounds that have hemolytic activity by solubilizing membranes [48]. The results showed that the extract showed antihemolytic activity, indicating the absence or low content of saponins in C. officinalis flowers. The antihemolytic activity of C. officinalis extracts had already been tested in the literature under oxidative and thermal stress conditions and, in both cases, the hydroalcoholic extract (EtOH:H2O, 80:20 v/v) was efficient in inhibiting hemolysis [49]. LME showed 12% inhibition of lipid oxidation of Wistar rat brains at a concentration of 1000 mg/L, statistically differing (p < 0.001) from quercetin inhibition capacity (50 mg/L) (Figure 3). The superiority of quercetin was already expected since it is a standard of high purity and with a recognized ability to inhibit lipid oxidation. However, the inhibitory capacity presented by the LME is an essential feature because it shows that it is active in biological media inhibiting the oxidation of brain tissue lipids. This test shows the ability of the compounds present in the extract to transfer H + atom to free radicals generated by lipid-induced oxidation [50]. The ability of C. officinalis extracts to inhibit lipid oxidation was also observed in an in vivo study by Tanideh et al. [51]. The same authors found that the hydroalcoholic extract (EtOH:H2O, 80:20 v/v) of C. officinalis flowers, administered orally to male Sprague-Dawley rats, showed decreased lipid oxidation.  Figure 2B shows that the hemolysis rate is reduced, 0.4% NaCl, with increasing LME concentration (50-110 mg/L). The C. officinalis flowers are rich in saponins, compounds that have hemolytic activity by solubilizing membranes [48]. The results showed that the extract showed antihemolytic activity, indicating the absence or low content of saponins in C. officinalis flowers. The antihemolytic activity of C. officinalis extracts had already been tested in the literature under oxidative and thermal stress conditions and, in both cases, the hydroalcoholic extract (EtOH:H 2 O, 80:20 v/v) was efficient in inhibiting hemolysis [49]. LME showed 12% inhibition of lipid oxidation of Wistar rat brains at a concentration of 1000 mg/L, statistically differing (p < 0.001) from quercetin inhibition capacity (50 mg/L) (Figure 3). The superiority of quercetin was already expected since it is a standard of high purity and with a recognized ability to inhibit lipid oxidation. However, the inhibitory capacity presented by the LME is an essential feature because it shows that it is active in biological media inhibiting the oxidation of brain tissue lipids. This test shows the ability of the compounds present in the extract to transfer H + atom to free radicals generated by lipid-induced oxidation [50]. The ability of C. officinalis extracts to inhibit lipid oxidation was also observed in an in vivo study by Tanideh et al. [51]. The same authors found that the hydroalcoholic extract (EtOH:H 2 O, 80:20 v/v) of C. officinalis flowers, administered orally to male Sprague-Dawley rats, showed decreased lipid oxidation. LME, 20 mg/mL, inhibited 27% of α-amylase enzymatic activity, indicating a dose-dependent effect with increasing extract concentration (1-15 mg/mL) ( Figure 4A). The LME, 500 µg/mL, inhibited 43% of α-glucosidase enzymatic activity. The concentrations of 50 to 100 µg/mL and 200 to 300 µg/mL of LME showed no significant differences in the inhibitory activity of α-glucosidase (Figure 4). The extract from the leaves of C. officinalis was evaluated in the inhibitory activity of the α-amylase enzyme as described by Olennikov et al. [52]. The authors found that 38.02 µg/mL of hydroalcoholic extract (EtOH:H 2 O, 60:40 v/v) of leaves inhibited 50% of α-amylase activity. In another study, hydroalcoholic extract (50:50 v/v) from C. officinalis leaves reduced serum glucose levels in diabetic rats [53]. recognized ability to inhibit lipid oxidation. However, the inhibitory capacity presented by the LME is an essential feature because it shows that it is active in biological media inhibiting the oxidation of brain tissue lipids. This test shows the ability of the compounds present in the extract to transfer H + atom to free radicals generated by lipid-induced oxidation [50]. The ability of C. officinalis extracts to inhibit lipid oxidation was also observed in an in vivo study by Tanideh et al. [51]. The same authors found that the hydroalcoholic extract (EtOH:H2O, 80:20 v/v) of C. officinalis flowers, administered orally to male Sprague-Dawley rats, showed decreased lipid oxidation. LME, 20 mg/mL, inhibited 27% of α-amylase enzymatic activity, indicating a dose-dependent effect with increasing extract concentration (1-15 mg/mL) ( Figure 4A). The LME, 500 µg/mL, inhibited 43% of α-glucosidase enzymatic activity. The concentrations of 50 to 100 µg/mL and 200 to 300 µg/mL of LME showed no significant differences in the inhibitory activity of α-glucosidase ( Figure 4). The extract from the leaves of C. officinalis was evaluated in the inhibitory activity of the α-amylase enzyme as described by Olennikov et al. [52]. The authors found that 38.02 µg/mL of hydroalcoholic extract (EtOH:H2O, 60:40 v/v) of leaves inhibited 50% of α-amylase activity. In another study, hydroalcoholic extract (50:50 v/v) from C. officinalis leaves reduced serum glucose levels in diabetic rats [53]. According to the cytotoxicity results ( Figure 5), the lyophilized marigold extract enhanced the cell viability parameter (IC50), stimulated the growth index (GI50), and it was not able to cause cell death (LC50) in both cancerous (HCT8, A549) and noncancerous (IMR90) cell lines, suggesting no apparent cytotoxicity. Similarly, Pires et al. [44] also observed that not all the C. officinalis samples inhibited the growth of MCF-7, NCI-H460, HeLa, and HepG2 tumor cells. Herein, considering the cell proliferation behavior, exogenous antioxidants (i.e., flavonoids and carotenoids) may exert cytotoxic effects in several cancerous cell lines, but whether an antioxidant supplement would be helpful, harmful, or neutral depends on the specific antioxidant, its dose, the chemotherapy drugs being used, and the type and stage of cancer [54]. Regarding the growth of A549, IMR90, and HCT8 cells, we hypothesize that the bioactive compounds in C. officinalis may favor mitogenic mechanisms, which involves complex pathways that can be related to overexpression of essential cell cycle regulatory proteins, such as cyclin-dependent kinases, D-type cyclins, polo-like kinases, and aurora kinases, which stimulate the cell cycle progression in the M/G1/S phases [55]. Bearing these concerns, more research is needed to explain the possible mechanisms involved in C. officinalis-induced cell growth.
Regarding the cell-based antioxidant assay, LME did not induce ROS generation ( Figure 6) at the higher tested concentrations in both cancerous (A549) and noncancerous (IMR90) cells. Palozza et al. [56] observed that carotenoids in the C. officinalis extract have already been associated with antiproliferative activity and cytotoxicity through their prooxidant ability. Thus, although the C. officinalis extract presented an interesting chemical profile, antioxidant, antimicrobial, and anti-hemolytic capacities, it did not exhibit cytotoxic effects against the cancerous cell lines. According to the cytotoxicity results ( Figure 5), the lyophilized marigold extract enhanced the cell viability parameter (IC 50 ), stimulated the growth index (GI 50 ), and it was not able to cause cell death (LC 50 ) in both cancerous (HCT8, A549) and noncancerous (IMR90) cell lines, suggesting no apparent cytotoxicity. Similarly, Pires et al. [44] also observed that not all the C. officinalis samples inhibited the growth of MCF-7, NCI-H460, HeLa, and HepG2 tumor cells. Herein, considering the cell proliferation behavior, exogenous antioxidants (i.e., flavonoids and carotenoids) may exert cytotoxic effects in several cancerous cell lines, but whether an antioxidant supplement would be helpful, harmful, or neutral depends on the specific antioxidant, its dose, the chemotherapy drugs being used, and the type and stage of cancer [54]. Regarding the growth of A549, IMR90, and HCT8 cells, we hypothesize that the bioactive compounds in C. officinalis may favor mitogenic mechanisms, which involves complex pathways that can be related to overexpression of essential cell cycle regulatory proteins, such as cyclin-dependent kinases, d-type cyclins, polo-like kinases, and aurora kinases, which stimulate the cell cycle progression in the M/G1/S phases [55]. Bearing these concerns, more research is needed to explain the possible mechanisms involved in C. officinalis-induced cell growth.
Regarding the cell-based antioxidant assay, LME did not induce ROS generation ( Figure 6) at the higher tested concentrations in both cancerous (A549) and noncancerous (IMR90) cells. Palozza et al. [56] observed that carotenoids in the C. officinalis extract have already been associated with antiproliferative activity and cytotoxicity through their prooxidant ability. Thus, although the C. officinalis extract presented an interesting chemical profile, antioxidant, antimicrobial, and anti-hemolytic capacities, it did not exhibit cytotoxic effects against the cancerous cell lines.

Characterization of the Organic Yogurt with LME
The TPC and antioxidant activity (DPPH and total reducing capacity (TRC)) of organic yogurts manufactured with different concentrations of LME are shown in Table 6. Increasing LME concentration, 0.25 to 1.5 g/100 g, increased the TPC and in vitro antioxidant activity of organic

Characterization of the Organic Yogurt with LME
The TPC and antioxidant activity (DPPH and total reducing capacity (TRC)) of organic yogurts manufactured with different concentrations of LME are shown in Table 6. Increasing LME concentration, 0.25 to 1.5 g/100 g, increased the TPC and in vitro antioxidant activity of organic yogurts, and thus indicating a dose-dependence effect. These results corroborate with Demirkol et

Characterization of the Organic Yogurt with LME
The TPC and antioxidant activity (DPPH and total reducing capacity (TRC)) of organic yogurts manufactured with different concentrations of LME are shown in Table 6. Increasing LME concentration, 0.25 to 1.5 g/100 g, increased the TPC and in vitro antioxidant activity of organic yogurts, and thus indicating a dose-dependence effect. These results corroborate with Demirkol et al. [57] who determined an increase in TPC (20 to 52 mg/100 g yogurt) and DPPH value (263 to 1100 mg/mL for 50% inhibition of the radical) in yogurt added with grape pomace (1 to 5 g/100 g milk). Abdel-Hamid et al. [58] measured an increase in antioxidant activity using the DPPH (43 to 78% radical inhibition) and ABTS (54 to 91% radical inhibition) assays in probiotic yogurt supplemented with 0.5 to 2% of Siraitia grosvenorii fruit extract. Table 6. Total phenolic content and chemical antioxidant activity of organic yogurt with different concentrations of lyophilized marigold extract (C. officinalis L.).

Parameters
Formulations ( The proximal composition, physicochemical characterization, color, and instrumental texture of organic yogurt with LME (0.25 g LME/100 g) and without extract (0 g LME/100 g, control) is presented in Table 7. The protein, lipid, ash content, titratable acidity, hardness, and consistency of organic yogurt manufactured with LME did not show statistically significant differences with the control. The organic yogurts with and without LME show statistically significant differences (p ≤ 0.05) in carbohydrate content, moisture, energy, and pH. These results are similar to those found by Karnopp et al. [2] in organic yogurt manufactured with Bordeaux grape skin flour. The same authors found the following properties in the yogurt: carbohydrate (9.35 g/100 g), protein (3.80 g/100 g), lipid (6.23 g/100 g), ash (0.81 g/100 g), moisture (76.49 g/100 g) contents, pH (4.19), titratable acidity (0.61 g lactic acid/100 g), hardness (20.88 g), and consistency (493.13 g/s). The incorporation of the LME in the organic yogurt caused a color change (L*, a*, and b*) compared to the control (p < 0.001) ( Table 7). Demirkol et al. [57] also found a reduction in the brightness (L*) and increased redness (a*) in yogurt manufactured with grape pomace flour. According to Pires et al. [6], edible flower petal extracts have great potential for use as natural colorants and are an excellent source of bioactive compounds to develop innovative products with novel sensory characteristics and antioxidant activity.
The organic yogurt with 0.25 g LME/100 g presented a general sensory acceptance rate of 80.4%. The average scores given for odor (7.6 ± 1.5), taste (6.4 ± 2.2), consistency (7.2 ± 2.0), color (7.7 ± 1.5), and overall impression (7.2 ± 1.7) were ranked between "slightly liked" and "moderately liked ". The bitter residual taste reported by some assessors may have influenced the taste attribute (average grade <7). All evaluators (100%, n = 76) consider yogurt a healthy product. Regarding the commercial value of the yogurt, 75% of assessors would pay between R$ 2.00 and R$ 3.00 for yogurt rich in natural antioxidant compounds (100 g) compared to yogurt without natural antioxidants, and 75% would pay between R$ 2.00 and R$ 3.00 for organic yogurt (100 g) compared to conventional yogurt. Table 7. Proximate composition, physicochemical characteristics, instrumental color, and texture of organic yogurts with and without the lyophilized marigold extract (LME).

Parameters
Organic Yogurt with LME 1

Conclusions
The hydroalcoholic extract (50:50 v/v) of Calendula officinalis flowers presented the highest total phenolic content (TPC), flavonoids, and in vitro antioxidant activity (FRAP, TRC, and Cu 2+ /Fe 2+ chelating ability). Quercetin-3-rutinoside, procyanidin A2, ferulic, caffeic, p-coumaric, and ellagic acids were quantified in the extract by LC-DAD, while 19 compounds were tentatively identified by ESI-MS/MS. The lyophilized marigold extract presented antimicrobial activity, antihemolytic activity, inhibition of lipid oxidation of Wistar rat's brain, and inhibited α-amylase/α-glucosidase activities. The LME showed no cytotoxicity towards cancerous (A549 and HCT8) and noncancerous (IMR90) cells, and it did not induce intracellular ROS generation. The incorporation of different concentrations of LME in organic yogurt increased the total phenolic content and antioxidant activity. The organic yogurt with 0.25 g LME/100 g had a high acceptability index, demonstrating that the use of LME may be a technological and functional-led strategy to increase the content of bioactive compounds in yogurts.