Variability in lutein and zeaxanthin content, fatty acid and phytosterols profiles, and genetic parameters of some Tagetes spp. cultivars

Abstract

Lutein and zeaxanthin are oxygenated carotenoids with strong antioxidant and photoprotective properties, widely used in pharmaceuticals, cosmetics, and food. To identify Tagetes cultivars with higher pigment content, ten cultivars from T. erecta and T. patula were cultivated using a randomized complete block design with five replications. Agronomic and phytochemical traits, including carotenoids, lutein, zeaxanthin, fatty acids, and phytosterols, were evaluated. Significant variability was observed, particularly for flower diameter, total carotenoids, lutein, fresh flower weight (FFW), and antioxidant activity. FFW ranged from 10.28 to 23.28 g per plant, and dry flower weight from 2.70 to 5.67 g. Carotenoids, lutein, and zeaxanthin contents varied from 6.20 to 15.92, 2.44–6.42, and 1.05–2.48 mg/g DW, with cultivar TP5 showing the highest levels. Palmitic acid (34.12–49.15%) and linoleic acid (18.4–29.12%) were the dominant fatty acids, while TE2 had the highest β-sitosterol (207.34 µg/g FW). Genetic analysis indicated high heritability and genetic advance for carotenoids, lutein, zeaxanthin, antioxidant activity, and flower diameter, suggesting additive gene effects. This study provides the integrated morphological, phytochemical, and genetic assessment across diverse Tagetes cultivars, identifying promising candidates for breeding programs and industrial exploitation.

Introduction

Carotenoids represent a diverse class of naturally occurring tetraterpenoid pigments that are widely distributed across plants, algae, fungi, and numerous bacterial species [1]. Within the human body, these compounds contribute to a broad spectrum of physiological processes essential for health maintenance [2]. Based on their structural characteristics, carotenoids are subdivided into two major groups: carotenes and xanthophylls. Carotenes are hydrocarbons devoid of oxygen atoms, typically terminating in cyclic hydrocarbon moieties, whereas xanthophylls are oxygenated derivatives of carotenoids. Representative members of the carotene group include α-carotene, β-carotene, β-cryptoxanthin, and lycopene, while xanthophylls encompass lutein, zeaxanthin, meso-zeaxanthin, canthaxanthin, and astaxanthin [3, 4]. Among the xanthophylls, lutein and zeaxanthin have attracted significant attention due to their wide-ranging biological activities. These compounds are reported to exhibit neuroprotective, cardioprotective, ocular, antioxidant, anti-inflammatory, anti-neoplastic, anti-diabetic, anti-obesity, and antimicrobial properties, in addition to their potential roles in osteoporosis prevention [5, 6]. Furthermore, both molecules exert photoprotective effects by mitigating ultraviolet (UV)-induced skin damage, thereby reducing the risk of dermatological disorders [7]. Their health-promoting functions are primarily attributed to their strong radical-scavenging capacity, inhibition of oxidative damage, and attenuation of oxidative stress [8]. Structurally, lutein is a polyene hydrocarbon composed of eight isoprene units, containing 40 carbon atoms, nine conjugated double bonds, and two hydroxyl substituents positioned on β-ionone rings. Zeaxanthin is a structural isomer of lutein, differing in the location of a double bond within the cyclic moiety [4]. Unlike other carotenoids, lutein and zeaxanthin possess hydroxyl groups at both molecular termini, conferring hydrophilic characteristics. This polarity enhances their interaction with singlet oxygen, enabling more efficient quenching of reactive oxygen species compared to nonpolar carotenoids [9]. The primary dietary sources of carotenoids are colorful fruits and vegetables [10]. Nevertheless, alternative natural reservoirs such as microalgae, macroalgae, and agro-industrial byproducts have recently been explored for carotenoid extraction [11, 12]. Among terrestrial sources, the petals of marigold flowers (genus Tagetes), particularly T. erecta (African marigold) and T. patula (French marigold), are considered among the richest sources of lutein and zeaxanthin [13]. These species, belonging to the Asteraceae family, are cultivated extensively due to their commercial relevance. Approximately 40–50% of the flower biomass comprises brightly pigmented petals ranging from yellow to deep orange, which are enriched in bioactive metabolites such as flavonoids, phenolics, and especially carotenoids [14]. Owing to their elevated lutein concentration, marigolds are exploited industrially for the production of natural lutein used in functional foods, animal feed, cosmetics, and pharmaceutical formulations [12]. Additionally, marigold-derived pigments are incorporated into poultry feed to enhance yolk coloration, which is associated with superior nutritional quality and consumer preference in eggs [15, 16].

Given the potential and high consumption of lutein and zeaxanthin in various pharmaceutical and cosmetic industries and the importance of cultivating their sources on a large scale, no comprehensive study has been conducted on different Tagetes spp. cultivars in Iran so far. Investigations on other medicinal plants such as Ocimum basilicum [17] and Tribulus terrestris [18] have successfully identified superior populations suitable for cultivation, yet such studies are lacking for Tagetes spp. cultivar in Iran. However, studies including Saini et al. [13], who measured carotenoids, fatty acids, tocopherols, and phytosterols in several cultivars of Tagetes.

The novelty of the present study lies in providing the first comprehensive, comparative assessment of ten Tagetes (T. erecta L. and T. patula L.) cultivars in Iran, combining advanced chromatographic profiling (HPLC, GC–FID, GC–MS) with genetic parameters (heritability, genetic advance, PCA, clustering, and path analysis). This integrated approach enables the identification of superior pigment-rich cultivars and offers a robust framework for targeted breeding and large-scale industrial applications.

Materials and methods

Plant materials

Seeds of various Tagetes spp. cultivars were purchased from Pakan Seed Company in Isfahan, Iran. The cultivars were from two species, T. erecta and T. patula. The cultivars of the species T. erecta included Hawaii (TE1), Aranysarga (TE2), Garuda Deep (TE3), Antigua Orange (TE4), and Incall Orange (TE5), and the cultivars of the species T. patula included Durango Bee (TP1), Superboy Orange (TP2), Bolero (TP3), Konstance (TP4), and Queen Sophia (TP5) (Table 1). Cultivars were cultivated under a randomized complete block design (with five repetitions) with a spacing of 30 × 50 cm at the Medicinal Plant Research Farm of Shahid Beheshti University (N 35°48′, E 51°23′, altitude 1773 m) two years (2024–2025). Each plot contained 50 plants arranged in five rows. The plants were irrigated every three weeks throughout the growing season using a drip irrigation system. The study site is situated within a semi-arid climatic zone, exhibiting a long-term average temperature of 14.7 °C. Throughout the crop growth period, the mean daily maximum and minimum temperatures were recorded at 21.5 °C and 10.1 °C, respectively. The location receives an annual precipitation of approximately 386.5 mm. Plants were grown without application of fertilizers or pesticides. Soil analysis indicated a silty loam texture with an electrical conductivity of 2.87 dS/m, pH 7.8, organic matter content of 1.27%, nitrogen concentration of 0.084%, available phosphorus of 68.2 mg/kg, and potassium of 351.1 mg/kg (Table 2).

Table 1.

Characteristics and codes of the various Tagetes spp. Cultivars studied

Botanical Name	Cultivar	Flower Color	Code
Tagetes erecta L.	Hawaii	Orange	TE1
Tagetes erecta L.	Aranysarga	Yellow	TE2
Tagetes erecta L.	Garuda Deep	Yellow	TE3
Tagetes erecta L.	Antigua Orange	Orange	TE4
Tagetes erecta L.	Incall Orange	Orange	TE5
Tagetes patula L.	Durango Bee	Yellow	TP1
Tagetes patula L.	Superboy Orange	Orange	TP2
Tagetes patula L.	Bolero	Red	TP3
Tagetes patula L.	Konstance	Orange	TP4
Tagetes patula L.	Queen Sophia	Orange	TP5

Table 2.

Geographical positions, climatic data, and soil properties of the cultivated region

Geographic positions	Climatic data	Soil properties
Altitude (m): 1773	Average maximum annual temperature (ºC): 21.5	Texture: silty loam
Longitude (E): 51° 23ʹ	Average minimum annual temperature (ºC): 10.1	EC (ds m-1): 2.87
Latitude (N): 35° 48ʹ	Average annual temperature (ºC): 14.7	pH : 7.8
	Rainfall (mm): 386.5	Total N (%): 0.084Available P (mg kg-1): 68.2Available K (mg kg-1): 351.1

Phenotypic and functional assessments

At the full flowering stage, morphological characteristics, including plant height and flower diameter, were recorded using a ruler and digital caliper. Then, after harvesting the plants, the shoot fresh weight (SFW), flower fresh weight (FFW), and root fresh weight (RFW) were measured using a digital scale. Following, plants shade-dried, and subsequently evaluated for functional traits, specifically shoot dry weight (SDW), flower dry weight (FDW) and root dry weight (FDW) per plant, determined with a digital balance.

Total carotenoids

For the quantification of carotenoids, 0.1 g of dried petal tissue was homogenized in a mortar using 10 mL of 80% acetone until a uniform extract was obtained. The resulting suspension was subjected to centrifugation at 5000 rpm for 5 min to separate the supernatant. Absorbance values of the clarified extract were subsequently determined with a UV–visible spectrophotometer (LABOMED UVS-2800) at 480 and 510 nm. The carotenoid concentration was expressed in mg/g DW according to the formula proposed by Arnon [19]:

where V represents the extract volume (mL) and W denotes the dry mass of the petal sample (g).

Extraction and quantification of lutein and Zeaxanthin by HPLC

To isolate carotenoids, approximately 500 mg of finely ground petal material was subjected to ultrasonic-assisted extraction using an Elma S120H system (Elma, Germany). The plant powder was suspended in 100 mL of an ethanol, water mixture (90:10, v/v) and treated for 20 min under sonication. The resulting suspension was then clarified by centrifugation at 1,400 × g for 10 min at 4 °C using a Rotanta 460r centrifuge (Hettich, Germany). The supernatant was subsequently concentrated under reduced pressure at 35 °C with a rotary evaporator. The dried residue was re-dissolved in 1 mL of ethanol and passed through a 0.22 μm syringe filter before analysis.

Chromatographic determination of lutein and zeaxanthin was carried out using high-performance liquid chromatography (HPLC). Separation was achieved on a YMC C30 column (150 mm × 2.0 mm, 3 μm particle size; YMC Co., Japan) equipped with a guard cartridge containing the same stationary phase. Analyses were performed with a Knauer Wellchrom-K1001 system (Knauer, Germany) coupled to a photodiode array detector (model K2800). The mobile phase consisted of two solvents: methanol with ammonium acetate (98:2, v/v) as solvent A, and pure ethyl acetate as solvent B. Gradient elution was programmed as follows: 0–10 min, 0–80% B; 10–13 min, 80–100% B; 13–14 min, hold at 100% B; 14–24 min, return to 0% B. The flow rate was maintained at 0.37 mL/min. For quantification, absorbance was monitored at 453 nm for zeaxanthin and 546 nm for lutein, following the procedure described by Li and Engelberth [20]. Calibration curves were constructed by injecting standard solutions at the seven concentrations of 2, 10, 50, 100, 250, 500, and 1000 ppm. The results were presented in mg/g DW.

Fatty acid analysis

The total crude oil fraction of the petals was obtained using a maceration-based extraction approach [21]. In brief, 1 g of finely powdered, dried floral tissue was suspended in 10 mL of n-hexane and exposed to ultrasonic treatment for 20 min, with the sonication step repeated three consecutive times. Following extraction, the mixture was left undisturbed at room temperature (23 ± 2 °C) for 72 h. The solvent phase was then separated by filtration through Whatman No. 1 filter paper, and the n-hexane was subsequently removed by evaporation under ambient conditions. The recovered oil was preserved in dark, airtight containers at − 18 °C until subsequent analysis.

For fatty acid profiling, 50 mg of the crude oil was subjected to derivatization into fatty acid methyl esters (FAMEs). Initially, the oil was treated with methanolic potassium hydroxide prepared by dissolving 50 mg of KOH in 4 mL of methanol (corresponding to a 1:80 w/v ratio relative to the oil). The mixture was homogenized by vortexing, then centrifuged at 4,000 rpm for 10 min. Transesterification of glyceride-bound fatty acids was first achieved with KOH, followed by conversion of residual free fatty acids using boron trifluoride in methanol. This sequential procedure ensured complete transformation of the lipid fraction into methyl esters.

Fatty acid methyl esters were analyzed using a gas chromatograph (Agilent 7890 A Plus) equipped with an automated injector and an FID detector. Separation was performed on a CP-Sil 88 capillary column (100 m × 0.25 mm, 0.20 μm film). Data were processed with ChemStation software, and the relative content of each fatty acid was obtained from peak area ratios. Identification was confirmed by comparison with a commercial standard mixture containing 28 fatty acids (GLC-462, Nu-Chek Prep, USA).

Phytosterol analysis

Phytosterol quantification was performed through solid-phase extraction on aminopropyl cartridges, using 5α-cholestane as the internal reference [22]. After sequential solvent washes to remove esterified lipids and neutral interferences, free sterols were selectively eluted, dried, and silylated. The derivatives were analyzed by GC–MS on a DB-5MS column under programmed temperature conditions, with nitrogen as the carrier gas. Compound identities were verified through comparison with authentic standards and by evaluating characteristic ionization and fragmentation patterns.

Quantification of total phenol content (TPC) and flavonoid content (TFC)

Phenolic compounds were extracted from 200 mg of powdered petal using ultrasound-assisted extraction with methanol/water (80:20, v/v) for 30 min, followed by centrifugation (1,400 g, 10 min, 4 °C). The supernatant was concentrated under reduced pressure at 35 °C, re-dissolved in methanol, and filtered through a 0.22 μm membrane. The TPC of petal extracts was measured using the Folin–Ciocalteu method in microplate format, with results expressed as mg gallic acid equivalents (GAE) per gram of plant. TFC was assessed using the aluminum chloride colorimetric assay, also in microplate format, with results expressed as mg rutin equivalents (RE) per gram of plant. Absorbance readings were taken at 760 nm for TPC and 420 nm for TFC after specified incubation periods (30 and 15 min, respectively) [23, 24].

Measurement of antioxidant power via the FRAP technique

The evaluation of antioxidant potential was carried out through the FRAP method, originally developed by Benzie and Strain [25], with slight procedural adjustments applied in the present study. Briefly, samples were reacted with freshly prepared FRAP reagent (acetate buffer, TPTZ in HCl, and FeCl₃, 10:1:1 v/v) and incubated at 37 °C for 30 min. Absorbance of the Fe²⁺–TPTZ complex was measured at 593 nm, and antioxidant activity was calculated from a ferrous sulfate calibration curve (0.25–8 mM).

Statistical analysis

Each experimental treatment was conducted in five independent replicates, and data are presented as mean (two-year data) values with their corresponding standard deviations (SD). Since the effect of year×cultivar was not statistically significant, the two-year data were averaged for analysis. Assumptions of ANOVA were verified prior to analysis using the Shapiro–Wilk test for normality and Levene’s test for homogeneity of variances. Statistical significance was assessed through one-way analysis of variance (ANOVA), followed by post-hoc comparisons using Duncan’s multiple range test at a significance threshold of p < 0.05. Variance analysis was performed in SAS software (version 9.4) (Table 3). Genetic parameters for each agro-morphological and phytochemical characteristic were estimated according to the following equation:

Table 3.

Expected mean squares for evaluation Tagetes spp. Cultivars

S.O.V	df	SS	MS	Expected mean squares
Block	r-1	SS block	MSr
Treatment	t-1	SS treatment	MSt
Error	(r-1) (t-1)	SS error	MSe

S.O.V Sources of variance, df Degree of freedom, r Number of block, t Number of treatments, SS Sum of squares, MS Mean squares

1

2

3

4

5

6

7

8

In this equation, r represents the number of replications, MSe denotes the mean square of error, MSg refers to the mean square of genotype, µ indicates the overall sample mean, and k corresponds to the standardized selection differential [26]. Statistical computations were carried out in R (v.4.3.2) environment utilizing the “variability” package for data analysis. Path coefficient analysis was performed to explore the relationship between essential oil yield and linalool content. Pearson correlation coefficients were calculated to assess trait associations. Both the PCA biplot and the cluster analysis were performed using Origin software (version 2022), with the cluster analysis based on the Euclidean distance coefficient and Ward’s method. Graphs and Heatmaps were generated using Origin Lab 2022.

Result and discussion

Quantitative traits variability

A considerable level of variation was detected among the Tagetes spp. cultivars with respect to both agro-morphological and phytochemical parameters, suggesting the potential for selective breeding based on these characteristics (Table 4). The traits exhibiting the highest coefficients of variation (CV) included flower diameter (34.76%), carotenoids content (31.22%), lutein (29.63%), FFW (28.93%), and antioxidant activity (28.18%), whereas the lowest CV was recorded for SFW (18.97%) (Table 4). Elevated CV values reflect broader quantitative dispersion within a trait, thereby offering breeders greater opportunities for selection [27]. The CV has been widely applied in assessing agro-morphological diversity in medicinal plant species such as Alcea spp [26]., Satureja rechingeri [27], and Tribulus terrestris [18].

Table 4.

Statistical descriptive parameters for agro-morphological and phytochemical traits of different Tagetes spp. Cultivars

Variable	Unit	Mean	Max	Min	SD	CV (%)
Plant height	cm	39.41	57.45	25.30	8.09	20.53
Shoot fresh Weight	g/plant	394.81	552.00	301.00	74.92	18.97
Shoot dry Weight	g/plant	54.68	77.74	37.16	11.62	21.25
Root fresh Weight	g/plant	24.46	36.00	15.00	5.84	23.88
Root dry Weight	g/plant	6.04	9.00	3.57	1.57	26.06
Flower diameter	cm	4.62	7.40	2.30	1.60	34.76
Flower fresh Weight	g/plant	15.22	25.20	9.30	4.40	28.93
Flower dry Weight	g/plant	3.82	6.14	2.44	1.05	27.53
Carotenoids content	mg/g DW	9.98	16.55	6.05	3.11	31.22
Total phenol content	mg GAE/g DW	10.64	15.92	7.27	2.28	21.47
Total flavonoid content	mg RE/g DW	8.06	10.82	5.07	1.58	19.65
Antioxidant activity	µmol Fe+ 2/g DW	18.65	29.35	11.05	5.25	28.18
Zeaxanthin	mg/g DW	1.68	2.57	1.03	0.46	27.73
Lutein	mg/g DW	4.57	6.70	2.31	1.35	29.63

Min Minimum, Max Maximum, SD Standard deviation, CV Coefficient of variation

Agronomic traits

The results of the variance analysis indicated that the examined Tagetes spp. cultivar exhibited statistically significant variation across the evaluated traits (p < 0.05). In our study, plant height was ranged from 28.86 to 55.39 cm. The highest plant height belonged to the TP2 (55.39 cm), followed by TP5 (49.25 cm), and TP4 (45.27 cm) (Fig. 1a). TP3 (544.7 g/plant) and TE2 (307.2 g/plant) had the highest and lowest SFW, respectively (Fig. 1b). The SDW, RFW, RDW, and flower diameter varied between 37.92 and 76.71 g/plant, 15.80 to 34.04 g/plant, 3.76 to 8.51 g/plant, and 2.52 to 7.06 cm, respectively (Fig. 1c, d,e, f). The average FFW exhibited cultivar-dependent variation, ranging between 10.28 g per plant in TE5 and 23.28 g per plant in TP3 (Fig. 1g). The highest FDW was related to the TP3 cultivar (5.67 g/plant), followed by TP5 (5.08 g/plant), and TE2 (4.89 g/plant), and the lowest to the TE5 population (2.70 g/plant) (Fig. 1h). The present investigation revealed considerable differences in agronomic characteristics among the Tagetes spp. cultivars. For efforts aimed at the large-scale cultivation, and genetic improvement of medicinal species, it is essential to examine diversity both within and between cultivar. Analysis of morphological variability provides breeders with critical information that can guide the development of improved cultivars with enhanced agronomic performance [26].

Fig. 1.

Histogram of plant height (a), shoot fresh weight (b), shoot dry weight (c), root fresh weight (d), root dry weight (e), flower diameter (f), flower fresh weight (g) and flower dry weight (h) among Tagetes spp. cultivar. The mean comparisons were performed using the Duncan test at p ≤ 0.05 significant level. Means followed by the same letter(s) are not significantly different

Total carotenoids, lutein and Zeaxanthin

Significant variation in total carotenoids, lutein and zeaxanthin were observed among the studied cultivar (p < 0.05). Total carotenoids ranged between 6.20 and 15.92 mg/g DW (Fig. 2a). The maximum level was observed in TP5 (15.92 mg/g DW) followed by TP1 (13.72 mg/g DW), while the minimum amount was displayed by TE4 (6.20 mg/g DW). The lutein was ranging from 2.44 to 6.42 mg/g DW (Fig. 2b). The TP5 exhibited the highest lutein (6.42 mg/g DW), while the TP1 (6.14 mg/g DW) and TP2 (5.75 mg/g DW) revealed lower levels, with the least amount in TE4 (2.44 mg/g DW). The amount of zeaxanthin varied between 1.05 and 2.48 mg/g DW, the lowest and highest of which was observed in ecotypes TE4 and TP5, respectively (Figs. 2c and 3). In addition to genetic variation as one of the factors influencing the wide diversity of carotenoids, it has been reported that environmental factors also affect the synthesis of lutein, zeaxanthin, and other pigments in plants [28]. Tatarowska et al. [29] reported the total carotenoid content, lutein, and zeaxanthin in cultivars of potato as 5.57 to 20.20, 2.92 to 6.66, and 1.44 to 3.05 mg/kg FW, respectively. In addition, the lutein, and zeaxanthin content of different accessions of corn has been reported as 0.76 to 1.43 µg/g FW, 1.32 to 2.24 µg/g FW, respectively [30].

Fig. 2.

Carotenoids content (a), lutein (b), and zeaxanthin (c) content among the cultivars of Tagetes spp. The mean comparisons were performed using the Duncan test at p ≤ 0.05 significant level. Means followed by the same letter(s) are not significantly different

Fig. 3.

The HPLC-PDA chromatogram of Tagetes spp. extract (in TP5 cultivar)

Fatty acid profile

GC analysis was employed to characterize the fatty acid composition of petal oils from various Tagetes spp. cultivars, with the outcomes presented in Table 5. Across the examined samples, seven distinct fatty acids were detected, namely lauric, myristic, palmitic, stearic, oleic, linoleic, and α-linolenic acids, accounting for approximately 98.57–99.69% of the total oil fraction. Saturated fatty acids (SFA) represented the largest portion, varying between 60.49% in cultivar TP5 and 74.5% in cultivar TP2. In contrast, monounsaturated fatty acids (MUFA) contributed a relatively minor fraction (1.45–3.31%), while polyunsaturated fatty acids (PUFA) were present at levels ranging from 22.37% to 35.77%. Among the SFAs, palmitic and stearic acids were most abundant. Palmitic acid concentrations extended from 34.12% in TP3 to 49.15% in TP2, whereas stearic acid ranged from 15.1% in TE3 to 24.34% in TE5. Linoleic acid, the predominant PUFA, exhibited considerable variability, with values spanning 18.4–29.12% and peaking in cultivar TP5. Earlier research corroborates these findings. Gong et al. [31] reported that linoleic acid (26.41%), palmitic acid (24.22%), and oleic acid (20.12%) were the principal fatty acids in T. erecta floral lipids. Similarly, Saini et al. [13] documented that palmitic acid (33.36–47.43%) and linoleic acid (11.30–30.25%) predominated in the petals of Tagetes spp. In alignment with these studies, the present investigation identified palmitic acid and linoleic acid as the dominant fatty acids across the analyzed Tagetes spp. cultivars, with notable inter-cultivar variability.

Table 5.

Fatty acid profiles of the studied Tagetes spp. Cultivars

Fatty acid (%)	TE1	TE2	TE3	TE4	TE5	TP1	TP2	TP3	TP4	TP5
Lauric acid (C12:0)	0.76 ± 0.03e	0.91 ± 0.02d	1.3 ± 0.28c	1.5 ± 0.16a	0.83 ± 0.07e	0.64 ± 0.09f	0.43 ± 0.10g	0.34 ± 0.06h	0.24 ± 0.01i	1.42 ± 0.11b
Myristic acid (C14:0)	14.23 ± 0.45b	15.4 ± 1.08ab	16.2 ± 1.33a	11.4 ± 0.82c	7.5 ± 0.17e	8.4 ± 0.12d	3.5 ± 0.19h	6.5 ± 0.29g	8.34 ± 0.41d	7.12 ± 0.53f
Palmitic acid (C16:0)	42.8 ± 2.65b	39.1 ± 2.18c	35.75 ± 2.85d	40.45 ± 3.12c	34.27 ± 2.41d	35.78 ± 2.08d	49.15 ± 3.08a	34.12 ± 2.73d	39.28 ± 2.79c	34.61 ± 2.79d
Stearic acid (C18:0)	16.34 ± 1.24e	18.4 ± 1.25d	15.1 ± 1.01f	18.34 ± 1.29d	21.45 ± 1.58c	24.34 ± 1.21a	21.42 ± 1.15c	23.16 ± 1.03b	20.13 ± 1.24c	17.34 ± 1.23de
Oleic acid (C18:1n9c)	2.13 ± 0.20e	1.73 ± 0.12g	1.83 ± 0.24f	2.64 ± 0.41d	1.45 ± 0.13h	1.82 ± 0.23f	2.64 ± 0.12d	3.82 ± 0.14a	2.82 ± 0.18c	3.31 ± 0.27b
Linoleic acid (C18:2n6c)	19.53 ± 1.78de	18.4 ± 1.63e	23.34 ± 1.67b	20.34 ± 1.69d	28.34 ± 1.85a	24.51 ± 1.62b	19.23 ± 1.26de	23.53 ± 1.58b	22.41 ± 1.71c	29.12 ± 1.83a
α-Linolenic acid (C18:3n3)	3.9 ± 0.37e	5.34 ± 0.51c	6.1 ± 0.31b	4.62 ± 0.21d	5.34 ± 0.38c	4.2 ± 0.51de	3.14 ± 0.13f	7.1 ± 0.61a	5.73 ± 0.45c	6.65 ± 0.44ab
SFAs	74.13 ± 3.91a	73.81 ± 3.62ab	68.35 ± 3.15cd	71.69 ± 3.01b	64.05 ± 2.63e	69.16 ± 2.71bc	74.5 ± 2.85a	64.12 ± 2.14e	67.99 ± 2.19d	60.49 ± 1.82f
MUFA	2.13 ± 0.20e	1.73 ± 0.12g	1.83 ± 0.24f	2.64 ± 0.41d	1.45 ± 0.13h	1.82 ± 0.23f	2.64 ± 0.12d	3.82 ± 0.14a	2.82 ± 0.18c	3.31 ± 0.27b
PUFAs	23.43 ± 1.05e	23.74 ± 1.16e	29.44 ± 1.09cd	24.96 ± 1.25e	33.68 ± 1.42b	28.71 ± 1.62d	22.37 ± 1.70f	30.63 ± 1.84c	28.14 ± 1.36d	35.77 ± 1.92a

Data are mean ± standard deviation (n = 5)

Values followed by the same letter within each row are significantly different (p < 0.05)

SFA saturated fatty acids, MUFA monounsaturated fatty acids, PUFA polyunsaturated fatty acids

Phytosterols

Based on GC-MS, β-sitosterol, stigmasterol, and campesterol compounds were identified in the studied cultivars (Table 6). The lowest (91.64 µg/g FW) and highest (207.34 µg/g FW) β-sitosterol were recorded for the TP4 and TE2 cultivars, respectively. The TE3 cultivar exhibited the maximum stigmasterol (196.45 µg/g FW), while the GP7 cultivar showed the minimum stigmasterol (75.34 µg/g FW). The minimum and maximum of campesterol were 11.43 µg/g FW and 22.53 µg/g FW related to TE3 and TP1 cultivars, respectively. The amount of total sterol varied between 182.32 and 377.31 µg/g FW, the lowest and highest of which was observed in cultivars TP4 and TE3, respectively (Table 6). Previous studies also reported β-sitosterol as the dominant sterol composition in Tagetes spp. (127.08–191.99 µg/g FW) [13], Perilla frutescens (27.7–37.9 µg/g FW) [31], and Moringa oleifera (175.9 µg/g FW) [32].

Table 6.

Phytosterol composition of the studied Tagetes spp. Cultivars

Cultivar	Campesterol	Stigmasterol	β-Sitosterol	Total Sterol
	(µg/g FW)
TE1	15.32 ± 0.93de	172.56 ± 2.81b	180.13 ± 2.61b	368.01 ± 5.19c
TE2	14.34 ± 0.63e	153.23 ± 2.19c	207.34 ± 3.41a	374.91 ± 4.81b
TE3	11.43 ± 0.84f	196.45 ± 2.83a	169.43 ± 2.59c	377.31 ± 5.39a
TE4	16.34 ±.71d	172.23 ± 2.59b	183.23 ± 2.13b	371.8 ± 5.42bc
TE5	25.0 ± 1.19a	112.41 ± 2.10e	143.42 ± 2.26f	280.83 ± 4.19f
TP1	22.53 ± 1.04b	139.23 ± 2.17d	150.31 ± 2.37e	312.07 ± 4.78e
TP2	18.34 ± 0.83c	142.13 ± 1.93d	164.87 ± 1.98d	325.34 ± 4.41d
TP3	19.0 ± 1.06c	89.34 ± 1.53g	97.28 ± 1.57h	205.62 ± 4.28h
TP4	15.34 ± 0.70de	75.34 ± 1.23h	91.64 ± 1.94i	182.32 ± 3.96i
TP5	21.24 ± 1.14b	109.34 ± 1.58f	118.83 ± 1.69g	249.41 ± 4.09g

Values followed by the same letter within each column are significantly different (p < 0.05)

TPC, TFC and antioxidant activity

Phenolic compounds have long been recognized for their therapeutic potential, particularly as natural antioxidants, owing to their biologically active properties that contribute positively to human health [33]. Substantial differences were identified across cultivars with respect to TPC, TFC, and antioxidant activity (Fig. 4). A comparable trend of variation was also evident for both TPC and TFC when cultivars were evaluated comparatively. TPC and TFC displayed considerable variability, ranging from 7.41 to 15.18 mg GAE/g DW and 5.14 to 10.48 mg RE/g DW, respectively. Notably, TE3 exhibited maximum TPC and TFC content, contrasting with TP1, which showed the lowest values for these parameters (Fig. 4a and b). In another study, the TPC and TFC of different marigold cultivars were reported to be 428.58–592.71.58.71 mg GAE/100 g and 135.06–233.39.06.39 mg QE/100 g, respectively [34]. Also, Kushwaha et al. [35] reported the TPC and TFC in different T. patula cultivars as 72.12–86.33 mg/100 g and 31.46–38.0.46.0 mg QE/100 g, respectively. The same as similar pattern of TPC and TFC, there was a relationship between the mentioned traits and the antioxidant activity of Tagetes spp. cultivars (Fig. 4c) as the maximum and minimum amount of antioxidant activity was the same with those shown in total phenols and flavonoids i.e. TE3 had the maximum (28.09 µmol Fe²⁺/g DW) antioxidant activity while TP2 showed the minimum (11.61 µmol Fe²⁺/g DW) activity (Fig. 5c). The findings indicated that petals extracts with a higher diversity of bioactive constituents exhibited greater antioxidant potential. The redox-protective potential of polyphenolic molecules is primarily attributed to their structural configuration, which enables them to scavenge reactive free radicals and bind transition metal ions, ultimately mitigating cellular oxidative stress. Consequently, these metabolites demonstrated considerable antioxidant efficiency. Previous investigations have likewise reported a significant positive association between phenolic concentration and antioxidant activity [36]. The biosynthesis of polyphenols in plants can occur through multiple metabolic pathways, and their accumulation varies across tissues depending on several regulatory mechanisms. Factors such as genetic background, environmental and climatic conditions, as well as the choice of solvent during extraction, play a decisive role in determining the quantitative levels of phenolic and flavonoid constituents obtained from plant materials [37].

Fig. 4.

Total phenol content (a), total flavonoids content (b), and antioxidant activity (c) among the cultivars of Tagetes spp. The mean comparisons were performed using the Duncan test at p ≤ 0.05 significant level. Means followed by the same letter(s) are not significantly different

Fig. 5.

Path coefficient analysis of lutein (LUT) with all the agro-morphological and phytochemical traits of the studied Tagetes spp. cultivars. PH: Plant height; SDW, Shoot dry weight; SDW: Shoot dry weight; RFE: Root fresh weight; RDW: Root dry weight; FD: Flower diameter; FFW: Flower fresh weight; FDW: Flower dry weight, TPC: Total phenol content; TFC: Total flavonoid content; FRAP: antioxidant activity; CC: Carotenoids content, ZEA: Zeaxanthin

Genetic parameters

The estimated genetic parameters are summarized in Table 7. Among the evaluated traits, the flower diameter (36.08%), carotenoids content (32.39%), lutein (30.82%), and antioxidant activity (29.11%) exhibited the highest GCV, whereas SFW displayed the lowest GCV (19.76%). PCV values ranged from 19.80% to 36.25%, with flower diameter and SFW showing the lowest and highest PCV, respectively. The close proximity of PCV and GCV values indicates that these traits are primarily governed by genetic factors, with minimal environmental influence [36]. These findings align with previous studies [37, 38], suggesting that phenotypic selection can be effective for improving traits largely independent of environmental effects. Traits with high GCV are particularly valuable for breeding programs aimed at developing high-yielding varieties through hybridization and selection, as a greater GCV generally results in increased variability among segregating progeny [39]. In Ocimum basilicum ecotypes, Bakhtiar et al. [17] reported maximum phenotypic and genetic variability for herb weight, number of lateral branches, plant height, and petal color. Estimates of GCV and heritability provide critical insight into the expected genetic gains achievable via phenotypic selection [40]. Broad-sense heritability (h²b) was high (≥ 0.90) for most traits, highlighting its importance in predicting gains in plant improvement programs [41]. The highest heritability was observed for carotenoids (0.99), followed by lutein (0.99), Zeaxanthin (0.99), flower diameter (0.99), TFC (0.97), SFW (0.99) and SDW (0.99). GA was greatest for SWF (160.42%), SDW (24.16%) and plant height (16.71%), while zeaxanthin showed the lowest GA (1.0%). GAM was highest for flower diameter (73.99%), carotenoids content (66.41%), lutein (66.33%), antioxidant activity (59.41%), and zeaxanthin (59.39%), with SFW again showing the lowest GAM (40.63%). High heritability combined with elevated GAM for traits such as flower diameter, carotenoids content, lutein, antioxidant activity, and zeaxanthin suggests the predominance of additive gene action, indicating that these traits can be effectively enhanced through pedigree-based selection [42, 43]. Conversely, traits like days to SFW and plant height, which displayed high heritability but low GAM, are likely influenced by non-additive gene action. Such traits are more suited to improvement via hybridization or exploitation of heterosis, and direct selection is generally not recommended [44].

Table 7.

Genetic parameters for studied traits of Tagetes spp. Cultivars

Character	𝜎𝑝2	𝜎𝑔2	GCV (%)	PCV (%)	hb2	GA	GAM
Plant height	70.81	68.27	20.96	21.35	0.96	16.71	42.40
Shoot fresh weight	6111.69	6088.32	19.76	19.80	0.99	160.42	40.63
Shoot dry weight	147.17	146.73	22.15	22.18	0.99	24.91	45.56
Root fresh weight	37.01	34.63	24.05	24.86	0.93	11.72	47.94
Root dry weight	2.69	2.54	26.39	27.13	0.94	3.19	52.88
Flower diameter	2.81	2.78	36.08	36.25	0.99	3.42	73.99
Flower fresh Weight	20.93	19.59	29.08	30.05	0.93	8.82	57.96
Flower dry Weight	1.19	1.11	27.52	28.57	0.92	2.09	54.62
Carotenoids content	10.55	10.45	32.39	32.54	0.99	6.62	66.41
Lutein	1.99	1.98	30.82	30.89	0.99	2.89	63.33
Zeaxanthin	0.23	0.23	28.88	28.94	0.99	1.00	59.39
Total phenol content	5.69	5.59	22.22	22.41	0.98	4.83	45.40
Total flavonoid content	2.73	2.72	20.46	20.50	0.99	3.39	42.07
Antioxidant activity	30.06	29.50	29.11	29.39	0.98	11.08	59.41

𝜎_𝑝² Genotypic variance, 𝜎_𝑔² Phenotypic variance, GCV Genotypic coefficients of variation, PCV Phenotypic coefficients of variation, GA Genetic advance, GAM Genetic advance as percentage of the mean, h²_b Broad-sense heritability

Path analysis

Path coefficient analysis was conducted to elucidate the relationships amid agro-morphological and phytochemical attributes of Tagetes spp. cultivars and their contribution to lutein (Fig. 5). The traits exerting the most substantial direct effects on lutein were FFW (3.36), RFW (1.21), and zeaxanthin concentration (0.77). Conversely, FDW (–3.08), RDW (–1.38), flower diameter (–0.82), and SDW (–0.47) negatively influenced lutein. Indirect contributions to lutein were most pronounced through FFW, particularly from plant height (1.03) and SFW (0.70). Collectively, these results highlighted FFW, RFW and zeaxanthin as primary determinants of lutein, with plant height and plant height exerting secondary, yet significant, effects. The subsequent path analysis exploring the determinants of zeaxanthin concentration (Fig. 6) indicated that FDW (1.64), SFW (0.63), carotenoids (0.55), and lutein content (0.34) had strong positive direct effects, whereas FFW (–1.75), SDW (–0.37), and SDW (–0.20) negatively influenced zeaxanthin content. The pronounced direct effects of FDW, SFW, carotenoids, and lutein content suggest that these parameters represent key selection criteria for breeding initiatives aimed at enhancing zeaxanthin levels in Tagetes spp. cultivars. Recent investigations into genotypic path analysis with essential oil yield as the dependent variable in parental lines and hybrids of Ocimum basilicum demonstrated that fresh herb yield per plant and chavibetol concentration were the primary contributors to oil yield, with additional influences arising from linalool proportion, essential oil percentage, plant stature, methyl cinnamate level, flowering time (50%), and citral content [45]. Similarly, Ibrahim et al. [46] reported that, among fifteen sweet basil genotypes, dry herb yield, stem dry biomass, and essential oil content exerted the most pronounced direct effects on oil productivity. As emphasized by Baretta et al. [47], the identification and validation of traits exhibiting both strong correlations with the target character and substantial direct effects in the desired direction represent critical considerations in the selection strategies of breeding programs.

Fig. 6.

Path coefficient analysis of zeaxanthin (ZEA) with all the agro-morphological and phytochemical traits of the studied Tagetes spp. cultivars. PH: Plant height; SDW, Shoot dry weight; SDW: Shoot dry weight; RFE: Root fresh weight; RDW: Root dry weight; FD: Flower diameter; FFW: Flower fresh weight; FDW: Flower dry weight, TPC: Total phenol content; TFC: Total flavonoid content; FRAP: antioxidant activity; CC: Carotenoids content, LUT: Lutein

Correlation, PCA and cluster analysis

The correlation analysis revealed distinct clustering patterns between growth attributes, carotenoid accumulation, fatty acid composition, and antioxidant traits in Tagetes spp. cultivars (Fig. 7). Biomass-related parameters (fresh and dry weight of flowers, shoots, and roots, together with plant height and flower diameter) were strongly and positively associated with each other, indicating that enhanced vegetative vigor generally coincides with improved floral development. Carotenoid pigments, particularly lutein and zeaxanthin, displayed tight positive intercorrelations, highlighting their coordinated biosynthesis and suggesting that genotypes with elevated levels of one xanthophyll are likely to accumulate the others. These pigments were also positively linked to flower biomass, implying that physiological allocation to growth is accompanied by enrichment in nutritionally relevant metabolites. In contrast, saturated fatty acids, especially palmitic acid, were negatively correlated with both carotenoid concentration and growth parameters, suggesting a metabolic trade-off between structural lipid accumulation and secondary metabolite pathways. Polyunsaturated fatty acids, including linoleic and α-linolenic acids, exhibited more favorable associations with biomass traits, whereas their relationship with sterols was inverse, pointing to divergent allocation routes within lipid metabolism. Moreover, antioxidant indices (TPC, TFC, FRAP) formed a coherent cluster with sterols (stigmasterol, β-sitosterol, and total sterol), underscoring a shared biochemical framework underlying antioxidant potential. Collectively, these findings indicate that high-performing Tagetes spp. cultivars are characterized by a concurrent increase in carotenoids and vegetative vigor, while elevated SFA levels mark a decline in both quality and yield-related traits.

Fig. 7.

Linear correlation between the agro-morphological and phytochemical traits of Tagetes spp. cultivars

In the present study, principal component analysis (PCA) was applied to a dataset integrating agronomic attributes, phytochemical profiles, and the concentrations of lutein and zeaxanthin in multiple Tagetes spp. cultivars. The outcome of the analysis, summarized in Table 8, illustrates the distribution of traits and their contribution to cultivar differentiation. The first six components collectively explained 94.79% of the observed variation, with PC1, PC2, and PC3 accounting for 51.86%, 15.28%, and 9.93% of the total variance, respectively. PC1 was largely determined by biomass-related traits (SFW, SDW, RFW, RDW, flower diameter, FFW, and FDW) together with carotenoid content, lutein, zeaxanthin, and oleic acid. PC2 was strongly shaped by antioxidant-related variables, including TPC, TFC, FRAP, and α-linolenic acid, while PC3 was mainly driven by lauric and myristic acids. The separation of cultivars across the components highlights the principal factors underlying variation within the studied populations. Although all cultivars were grown under identical conditions, the PCA results suggest that the observed similarities and differences in phytochemical composition are more strongly attributable to inherent genetic makeup than to geographical provenance. These findings provide a valuable framework for identifying promising genotypes and for harnessing carotenoids, fatty acids, and bioactive phytochemicals in targeted breeding programs and diverse industrial applications, particularly in the food, pharmaceutical, cosmetic, and health sectors [48].

Table 8.

Eigenvalues of the principal component axes from the multiple regression analysis of the studied parameters in Tagetes spp. Cultivars

Traits	Component
	1	2	3	4	5	6
Plant height	0.931	0.201	0.105	−0.142	−0.062	0.070
Shoot fresh weight	0.940	0.243	0.114	−0.138	−0.043	0.081
Shoot dry weight	0.949	0.134	0.070	−0.088	−0.034	0.145
Root fresh weight	0.909	0.223	−0.014	−0.045	−0.035	0.199
Root dry weight	0.933	0.147	0.032	−0.110	−0.010	0.242
Flower diameter	−0.937	0.212	0.040	0.139	−0.031	−0.181
Flower fresh weight	0.694	0.158	0.377	−0.131	0.416	−0.394
Flower dry weight	0.665	0.105	0.365	−0.108	0.455	−0.437
Carotenoids content	0.780	−0.366	0.032	0.236	0.332	0.198
Total phenol content	−0.511	0.676	−0.373	−0.150	0.247	0.152
Total flavonoid content	−0.505	0.664	−0.267	−0.354	0.314	0.056
Antioxidant activity	−0.472	0.657	−0.406	−0.232	0.303	0.081
Zeaxanthin	0.838	−0.351	0.015	0.157	0.275	0.118
Lutein	0.816	−0.444	−0.014	0.055	0.213	0.061
Lauric acid	−0.424	0.270	0.505	0.428	0.211	0.401
Myristic acid	−0.732	0.047	0.588	0.042	−0.170	−0.155
Palmitic acid	−0.291	−0.688	−0.303	−0.467	0.171	0.256
Stearic acid	0.568	−0.117	−0.561	0.080	0.016	−0.448
Oleic acid	0.744	0.039	0.285	−0.449	0.013	0.233
Linoleic acid	0.468	0.593	−0.105	0.600	−0.004	0.199
α-Linolenic acid	0.415	0.742	0.483	−0.063	−0.097	−0.122
Campesterol	0.480	0.133	−0.530	0.584	0.151	−0.115
Stigmasterol	−0.820	−0.167	0.241	0.070	0.240	0.240
β-Sitosterol	−0.857	−0.295	0.116	0.098	0.352	−0.110
Total Sterol	−0.863	−0.237	0.160	0.122	0.322	0.065
Eigenvalues	12.44	3.66	2.38	1.72	1.28	1.24
% of variance	51.86	15.28	9.93	7.16	5.36	5.17
Cumulative variance %	51.86	67.14	77.08	84.25	89.61	94.79

The outcomes of the cluster analysis, performed on different populations using agronomic and phytochemical parameters, are illustrated in Fig. 8. The analysis demonstrated that the populations could be organized into three distinct major clusters. The TP3, TP5, and TP4 cultivars were allocated to the first group (I), which were superior to other cultivars in terms of SFW, SDW, RFW, RDW, flower diameter, FFW, FDW, carotenoids, lutein and zeaxanthin. The second group (II) included populations TP1, TP2 and TE5. Group III consisted of the TE2, TE3, TE4, and TE1 populations, which exhibited the highest levels of fatty acids and sterol constituents compared with the other groups.

Fig. 8.

Cluster analysis of Tagetes spp. cultivars based on agro-morphological and phytochemical using Euclidean distances

The biplot constructed from the first two principal components (PC1 = 53.1% and PC2 = 15.1% of total variance) illustrates clear differentiation among the evaluated Tagetes spp. cultivars based on agronomic, and phytochemical attributes (Fig. 9). The biplot confirmed that the patterns observed for agronomic traits, phytochemical profiles, and the distribution of fatty acids and sterols were in strong agreement with the outcomes obtained through cluster analysis. Cultivars TP3, TP4, and TP5 clustered in the positive quadrant of PC1, closely associated with plant height, SFW, FDW, RFW, FDW, and flowers, as well as carotenoid pigments including lutein and zeaxanthin. This indicates that these genotypes combine superior vegetative growth with elevated carotenoid accumulation, which are desirable traits for both yield improvement and nutraceutical potential. Conversely, TE3 and TE5 were positioned in the upper-left quadrant along PC2, aligning with higher TPC, TFC, and FRAP, suggesting their value as sources of bioactive antioxidants. In contrast, TE1 and TP2 were separated toward the negative side of PC1, primarily associated with sterols (stigmasterol, β-sitosterol, and total sterol) and palmitic acid, while TP1 showed proximity to lauric and myristic acids. These separations reflect distinct metabolic specializations, where some cultivars prioritize fatty acid and sterol accumulation over carotenoid and biomass production. These findings suggest that genotype selection strategies in Tagetes spp. cultivars should consider both productivity-related traits and phytochemical richness, depending on the breeding or utilization objectives. Shakuri et al. [33] applied principal component analysis (PCA) to assess various ecotypes of Iranian oak, where the evaluated morphological and phytochemical attributes were primarily grouped within the first two principal components, explaining 68.53% of the overall variance. Similarly, Bakhtiar et al. [48] conducted a PCA on Medicago species, in which both morphological and phytochemical parameters were mainly represented in the first two components, accounting for 78.06% of the total variation.

Fig. 9.

Bi-plot graph for the first and second principal components based on the agro-morphological and phytochemical traits for cultivars of Tagetes spp

Conclusion

This investigation revealed substantial variability among Tagetes spp. cultivars in agronomic, phytochemical, and genetic attributes, highlighting their strong potential for targeted breeding and large-scale utilization. The positive associations observed between biomass traits and carotenoid pigments, particularly lutein and zeaxanthin, suggest a coordinated regulation of growth and nutritionally valuable metabolites. In contrast, the predominance of saturated fatty acids, especially palmitic acid, was inversely related to plant vigor and carotenoid accumulation, reflecting a metabolic trade-off between structural lipid deposition and secondary metabolite pathways. Genetic parameter estimates underscored the predominance of additive gene action for traits such as flower diameter, carotenoid content, and antioxidant capacity, underscoring their suitability for direct selection within breeding programs. Integrating correlation analyses, PCA, and clustering further demonstrated that cultivars TP3, TP4, and TP5 represent elite genotypes combining superior vegetative performance with enhanced carotenoid accumulation, making them promising candidates for crop improvement and nutraceutical exploitation. Conversely, genotypes such as TE3 and TE5, which were distinguished by elevated phenolic compounds and antioxidant activity, constitute valuable resources for developing health-promoting formulations. Collectively, these findings establish a solid framework for the selective improvement of Tagetes cultivars and point toward diverse applications of their bioactive metabolites across food, pharmaceutical, cosmetic, and health-related industries. These findings provide valuable baseline information for breeding programs focused on developing cultivars with enhanced pigment content, improved bioactive profiles, and superior nutritional value.

Authors’ contributions

GE: Sample collection, methodology, carried out field and lab work, data curation, data analysis, conceptualization, supervision, data curation and analysis, writing-original draft. HSH: Methodology, review, and editing.

Funding

Not applicable.

Data availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Declarations

Ethics approval and consent to participate

This manuscript is an original research and has not been published or submitted in other journals.

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Competing interests

The authors declare no competing interests.

Conflict of interest

The authors declare no conflict of interest.

Ethical review

This study does not involve any human or animal testing.

Clinical trial number

Not applicable.