Seasonal Variation in Nutritional Substances in Varieties of Leafy Chinese Kale (Brassica oleracea var. alboglabra): A Pilot Trial

Abstract

Chinese kale (Brassica oleracea var. alboglabra), a native Chinese vegetable, is usually grown for its bolting stems as the common edible part. However, the tender leaves of the vegetable have higher nutritional value. To investigate the effects of cultivation seasons on the nutritional substances in leafy Chinese kale, we conducted a pilot trial to analyze the differences in the content of nutritional substances, including glucosinolates, in five varieties of leafy Chinese kale (JLYC-01, JLYC-02, JLYC-03, JLYC-04, JLYC-05) cultured in fall, winter, and spring. The plant weight was 27.2 g–40.4 g in spring, 20.0 g–38.6 g in winter, and 20.3 g–34.0 g in fall, and the JLYC-05 variety showed superiority among the varieties, with weights of 34.0 g in fall, 38.6 g in winter, and 39.7 g in winter. Overall, the nutritional substance content in leafy Chinese kale cultivated in spring and fall was better than that of those cultivated in winter, providing a key reference for leafy Chinese kale planting. Among the five varieties, JLYC-04 and JLYC-05 are excellent candidates for future breeding programs, since JLYC-04 has a higher content of total phenols (10.1 mg GAE g⁻¹ DW–10.7 mg GAE g⁻¹ DW) and glucosinolates (5.8 μmol g⁻¹ DW–7.1 μmol g⁻¹ DW), exhibiting strong antioxidant capacity, while JLYC-05 contains more chlorophyll (157 mg 100 g⁻¹ FW–214 mg 100 g⁻¹ FW) and carotenoids (31.8 mg 100 g⁻¹ FW–39.1 mg 100 g⁻¹ FW).

1. Introduction

Chinese kale (Brassica oleracea var. alboglabra), a biennial or annual herbaceous plant of the genus Brassica in the family Brassicaceae, is widely cultivated in southern China [1]. Chinese kale is rich in various nutritional substances, such as vitamin C and bioactive compounds [2,3]. Among these, glucosinolates, a class of nitrogen- and sulfur-containing secondary metabolites, contribute to its distinctive flavor, which is highly favored by consumers [4].

Glucosinolates can be classified into aliphatic, indole, and aromatic glucosinolates based on their side chain structures. Certain types of these compounds and their degradation products may confer benefits to human health at appropriate concentrations. For example, isothiocyanates are known for their diverse biological functions, including potent anticancer effects in various types of cancer. Therefore, glucosinolates are a key indicator in evaluating the nutritional value of Chinese kale [5].

Changes in environmental factors (e.g., light, temperature, water), caused by the changing seasons, can significantly affect the nutritional substance and flavor of vegetables [6,7,8,9]. Extended exposure to light (20 h/day) has been shown to increase levels of aliphatic glucosinolates and the biomass of pak choi (Brassica rapa subsp. chinensis) [10]. Studies on Kimchi cabbages have shown that summer-grown cabbages are more bitter and less sweet compared to winter-grown ones, which is largely due to differences in glucosinolate levels. This difference is primarily attributed to variations in glucosinolate levels, which are promoted by high temperatures and drought stress [11]. The total glucosinolates concentration in Brassica oleracea leaves grown at 32 °C was found to be 114% higher than that at 22 °C [12]. High temperature also causes sucrose to decompose into reducing sugars, thereby reducing the sweetness of radish [13]. The contents of carotenoids and chlorophyll in kale have been found to decrease under drought stress [14]. On the contrary, the content of glucosinolates in pak choi (Brassica rapa subsp. chinensis) was found to increase when the crop was subjected to drought stress [10]. Phenolic substances in tea plants exhibit significant seasonal variations, with the content of epigallocatechin and gallocatechin being higher in autumn than in spring and summer [15]. Chlorophyll and carotenoid levels in Amaranthus decrease in summer, while ascorbic acid and total phenolic contents increase in winter and summer, respectively, and the accumulation of total phenolic contents plays an important role in drought stress tolerance [16]. Additionally, the antioxidant capacity of Hedera helix leaves show a high increase in winter [17].

In addition to the cultivation season, the nutritional substance of horticultural products is distinctive across different varieties. Zhang et al. [18] found that the chlorophyll and carotenoid contents in different varieties of Chinese kale varied significantly, by up to 3.2 and 2.1 times, respectively. Significant differences were also observed in phenolic content and antioxidant capacity among different varieties of pomegranate [19]. Sun et al. conducted a nutritional quality assay on 27 varieties of Chinese kale and found that both the type and content of glucosinolates varied across the varieties [20].

Chinese kale is usually grown for its bolting stems as the common edible part; however, studies have revealed that the tender leaves have higher nutritional value than the stems [18]. Therefore, five different varieties of leafy Chinese kale were selected to detect the plant weight, reducing sugars, soluble proteins, soluble solids, chlorophyll, carotenoids, vitamin C, total phenols, antioxidant capacity, and glucosinolates in different seasons, aiming to facilitate understanding of the effects of cultivation season on the content of nutritional substances among different varieties. The results will provide a useful reference for leafy Chinese kale production.

2. Materials and Methods

2.1. Plant Materials

Five Chinese kale varieties—JLYC-01, JLYC-02, JLYC-03, JLYC-04, and JLYC-05 (Figure 1)—were selected as test materials. The seeds of these varieties can be obtained by contacting the corresponding authors. Seeds were sown in the greenhouse at the Vegetable Research Institute of Zhejiang University (120°5′29″ E, 30°17′47″ N). After 3 weeks, the seedlings were transplanted into plastic greenhouses where the row spacing was 40 × 30 cm, and harvested after 45 days in fall (1 September–15 October), 80 days in winter (1 December–18 February), and 50 days in spring (1 March–19 April), and the climate data during the cultivation periods were recorded (Supplementary Table S1). The fertilization regime was designed based on the crop growth stages: urea (46% N) was band-applied at 75 kg ha⁻¹ at 3 days after transplanting to promote early root development, while compound fertilizer (15-15-15) was side-dressed at 225 kg ha⁻¹ during the 6–7 leaf stage. In fall, irrigation was applied every 2–4 days, while during winter and spring, the interval was extended to every 5–7 days. Plants were harvested when they had developed 8–10 true leaves, prior to shooting. For each variety, healthy, uniformly mature plants, free from pests, diseases, or mechanical damage, were selected. Ten plants per variety were used as one replication, with a total of three replications. The harvested plants were quickly transported back to the laboratory in foam boxes lined with ice, cleaned, and sampled. Samples were freeze-dried using a freeze-dryer (Alpha 1–2 LD plus, CHIRST, Osterode am Harz, Germany).

2.2. Plant Height, Plant Spreading, and Plant Weight

One replicate of every 10 plants was selected for each cultivar per cultivation season, with three replications prior to harvest. The distance between the lowest part of the plant and the highest point was measured with a tape measure as the height of the plant; the maximum distance of the natural development of the leaves was used as the plant spreading value; and the weight was recorded with a balance.

2.3. Reducing Sugars

The lyophilized powder (0.1 g) was mixed with distilled water (25 mL), incubated in a water bath at 50 °C for 20 min, then centrifuged. Next, 2 mL of the test solution was mixed with 1.5 mL of 3,5-dinitrosalicylic acid reagent, and the mixture was heated in a boiling water bath for 5 min before being cooled to room temperature. The absorbance was measured at 540 nm. A standard curve was prepared using glucose, and the content of reducing sugar in the samples was calculated according to the standard curve [21].

2.4. Soluble Proteins

The lyophilized powder (0.1 g) was mixed with distilled water (25 mL) and ground. After standing for 30 min, the mixture was centrifuged. A total of 5 mL of Coomassie Brilliant Blue G-250 (0.01%) was then added to 1mL of the supernatant, and after standing for an additional 5 min, the absorbance was measured at 595 nm. The content of soluble proteins in the samples was calculated according to the standard curve prepared using bovine serum albumin [22].

2.5. Soluble Solids

The content of soluble solids was determined using an Abbe refractometer (WYA-2WAJ, Shanghai LICHEN-BX Instrument Technology Co., Ltd., Shanghai, China). Ten grams of fresh leaves were juiced into a centrifuge tube, and 1 mL of supernatant was used for data detection.

2.6. Chlorophyll and Carotenoids

Based on the method of Dere et al. [23] with modifications, fresh samples (0.2 g) were ground and extracted with 10 mL of 96.0% ethanol. After centrifugation at 4000× g for 5 min, the supernatant was collected. The total chlorophyll and carotenoids contents were measured using a spectrophotometer by reading the absorbance at 665 nm (A₆₆₅), 649 nm (A₆₄₉), and 470 nm (A₄₇₀). Then, the contents of chlorophyll and carotenoids in the samples were calculated according to the formula:

C_a = 13.95 × A₆₆₅ − 6.88 × A₆₄₉

C_b = 24.96 × A₆₄₉ − 7.32 × A₆₆₅

C_t = C_a + C_b

Cx = (1000 × A₄₇₀ − 2.05 × C_a − 114.8 × C_b)/245

where C_a is the chlorophyll a concentration, in mg L⁻¹; C_b is the chlorophyll b concentration, in mg L⁻¹; C_t is the total chlorophyll concentration, in mg L⁻¹; and Cx is the carotenoid concentration, mg L⁻¹.

2.7. Vitamin C

Based on the method of Nisperos-Carriedo et al. [24] with modifications, 3 g of fresh sample was accurately weighed and ground in a mortar with 1% oxalic acid solution. The residue was washed with 1% oxalic acid solution, and the filtrates were combined and adjusted to a final volume of 25 mL. The residue was washed with oxalic acid solution, and the filtrates were combined and adjusted to 25 mL. The mixture was centrifuged, and the supernatant was filtered for HPLC (Shimadzu, Kawasaki, Japan) analysis. The HPLC conditions were as follows: Spherisorb C18 column (5 μm, 250 mm × 4.6 mm; Elite Analytical Instruments Co., Ltd., Dalian, China), column temperature 30 °C, mobile phase 0.1% oxalic acid solution, flow rate 1 mL min⁻¹, and detection wavelength 243 nm. L-ascorbic acid standards at different concentrations were analyzed to construct a standard curve, and the vitamin C content in the sample was calculated using the external standard method.

2.8. Total Phenols

Referring to the method of Volden et al. [25] with modification, 0.2 g of lyophilized powder was added to 5 mL ethanol and placed in a water bath for 1 h. After centrifugation, the supernatant (0.3 mL) was aspirated and mixed with 1.5 mL Folin–Ciocalteu reagent (0.2 mol L⁻¹) and 1.2 mL sodium carbonate solution (7.5%), then left to stand for 2 h. Absorbance values were measured at 760 nm, and the total phenol content in the samples was calculated according to the external standard method, using gallic acid to create the standard curve.

2.9. Ferric Reducing Antioxidant Power (FRAP)

The lyophilized powder (0.2 g) was mixed with 5 mL ethanol (30%), and the mixture was thoroughly ground before being diluted to a final volume of 25 mL. After incubation in a water bath at 80 °C for 1 h and centrifugation, the supernatant (0.3 mL) was aspirated and mixed with the FRAP working solution (prepared by combining 10 mL of 10 mmol L⁻¹ TPTZ solution, 10 mL of 20 mmol L⁻¹ ferric chloride solution, and 250 mL of acetic acid buffer solution at pH 3.6). The absorbance value was measured at 593 nm after 10 min in a water bath at 37 °C. The antioxidant capacity of the samples was calculated using ferrous sulfate solution as the standard curve [26].

2.10. Glucosinolates

Glucosinolates were extracted from 100 mg frozen powder by boiling in 5 mL of water for 10 min. The supernatant was collected and applied to a DEAE-Sephadex A-25 (Sigma -Aldrich, St. Louis, MO, USA) column and treated with aryl sulfatase to convert glucosinolates to desulphoglucosinolates, which were eluted with water. The same device used to detect vitamin C levels was used. The elution procedure included isocratic elution with 1.5% acetonitrile for 5 min, a linear gradient to 20% acetonitrile over 15 min, and then isocratic elution with 20% acetonitrile for 13 min. Absorbance was monitored at 226 nm using Ortho-nitrophenyl-β-D-galactopyranoside as the internal standard [20,27].

2.11. Statistical Analyses

All assays were performed in triplicate. Statistical analysis was performed using the SPSS package, version 18 (SPSS Inc., Chicago, IL, USA). Data were analyzed using two-way analysis of variance to assess the main effects of season and variety, as well as their interaction effect. If a statistically significant interaction between season and variety was detected (α = 0.05), simple effects analysis was subsequently performed to examine the influence of one independent variable at specific levels of the other variable. For pairwise comparisons within significant simple effects, Tukey’s Honest Significant Difference (Tukey’s HSD) post-hoc test was applied to control the family-wise error rate. All statistical analyses were conducted at a significance level of p < 0.05. Principal component analysis (PCA) was performed in SIMCA-P 11.5 Demo (Umetrics, Umeå, Sweden) with unit-variance (UV) scaling to decipher the relationships among samples. The correlation analysis was performed using Pearson correlation coefficient, and the results were visualized using Cytoscape v. 3.5.1 (The Cytoscape Consorti-um, New York, NY, USA). Data were filtered based on |R²| ≥ 0.65.

3. Results

3.1. Plant Height, Plant Spreading, and Plant Weight

The plant height of JLYC-03 and JLYC-04 in fall was significantly lower than in winter and spring, whereas JLYC-01 showed the opposite trend (Figure 2A). JLYC-01 had the highest plant height in fall (27.6 cm) but the lowest in winter (22.3 cm) and spring (23.4 cm) (Figure 2A).

The plant spreading of JLYC-02 was greater in winter than in fall and spring (Figure 2B). Regardless of the season, JLYC-05 exhibited the greatest plant spreading (40.6 cm in fall, 37.3 cm in winter, and 31.6 cm in spring), while JLYC-01 had the smallest (22.8 cm in fall, 27.4 cm in winter, and 26.7 cm in spring) (Figure 2B).

For plant weight, the values were highest in spring (27.2 g–40.4 g), followed by winter (20.0 g–38.6 g), and lowest in fall (20.3 g–34.0 g) (Figure 2C). Among the different varieties, JLYC-05 had the highest plant weight, while JLYC-01 had the lowest (Figure 2C).

3.2. Reducing Sugars, Soluble Proteins, Soluble Solids, and Chlorophyll

The contents of reducing sugars (Figure 3A), soluble proteins (Figure 3B), soluble solids (Figure 3C), and chlorophyll (Figure 3D) varied significantly across the three cultivation seasons. The levels of reducing sugars and soluble proteins were highest in spring, soluble solids were highest in fall, and chlorophyll content was higher in winter and spring.

For reducing sugars, the highest content was observed in spring (96.1 mg g⁻¹ DW to 191 mg g⁻¹ DW) and the lowest in winter (71.2 mg g⁻¹ DW to 96.7 mg g⁻¹ DW) (Figure 3A). Among the varieties, JLYC-01 had the highest content across all seasons (108.8 mg g⁻¹ DW in fall, 95.7 mg g⁻¹ DW in winter, and 191 mg g⁻¹ DW in spring), while JLYC-02 had the lowest (79.1 mg g⁻¹ DW in fall, 75.6 mg g⁻¹ DW in winter, and 96.1 mg g⁻¹ DW in spring) (Figure 3A).

The soluble protein content followed a similar seasonal trend, with the highest values in spring, followed by winter, and the lowest in fall (except for JLYC-01 and JLYC-02) (Figure 3B). There were no significant differences in soluble protein content among the varieties.

For soluble solids, the highest values were recorded in fall, followed by spring and winter, with no significant differences among the varieties in the same season (Figure 3C).

Chlorophyll content ranged from 123 mg 100 g⁻¹ FW to 232 mg 100 g⁻¹ FW (Figure 3D). For the same variety, chlorophyll content showed no significant difference between winter and spring, but was significantly higher in both seasons than in fall. JLYC-05 had the highest chlorophyll content (157 mg 100 g⁻¹ FW in fall, 227 mg 100 g⁻¹ FW in winter, and 232 mg 100 g⁻¹ FW in spring), while JLYC-01 had the lowest (127 mg 100 g⁻¹ FW in fall, 171 mg 100 g⁻¹ FW in winter, and 170 mg 100 g⁻¹ FW in spring) (Figure 3D).

3.3. Carotenoids, Vitamin C, Total Phenols, and Antioxidant Capacity

Although there were no significant differences in carotenoids content among the different seasons (Figure 4A), JLYC-05 had the highest carotenoid content among the varieties, with 31.8 mg 100 g⁻¹ DW in fall, 37.8 mg 100 g⁻¹ DW in winter, and 39.1 mg 100 g⁻¹ DW in spring (Figure 4A). The vitamin C content ranged from 64.0 mg 100 g⁻¹ FW to 154 mg 100 g⁻¹ FW (Figure 4B), with higher levels in fall compared to the other two seasons. Among the varieties, JLYC-04 had the highest vitamin C content (155 mg 100 g⁻¹ FW in fall, 110 mg 100 g⁻¹ FW in winter, and 121 mg 100 g⁻¹ FW in spring), while JLYC-01 had the lowest (91.0 mg 100 g⁻¹ FW in fall, 64.0 mg 100 g⁻¹ FW in winter, and 87.4 mg 100 g⁻¹ FW in spring) (Figure 4B).

The total phenols content in JLYC-02 and JLYC-03 was higher in spring than in fall and winter, while in JLYC-01, it was lower in spring than in the other two seasons (Figure 4C). Among the varieties, JLYC-04 had the highest total phenol content, while JLYC-01 had the lowest (Figure 4C).

The antioxidant capacity of JLYC-01 and JLYC-02 were significantly higher in fall compared to winter and spring; however, the opposite trend was observed for JLYC-05 (Figure 4D). JLYC-04 had the highest antioxidant capacity (160 μmol g⁻¹ DW in fall, 150 μmol g⁻¹ DW in winter, and 151 μmol g⁻¹ DW in spring), while JLYC-01 had the lowest (134 μmol g⁻¹ DW in fall, 121 μmol g⁻¹ DW in winter, and 108 μmol g⁻¹ DW in spring) (Figure 4D).

3.4. Glucosinolates

We detected 13 types of glucosinolates in leafy Chinese kale, including 8 aliphatic glucosinolates, 4 indole glucosinolates, and 1 aromatic glucosinolate. Aliphatic glucosinolate was the predominant type.

Gluconapin was the most abundant aliphatic glucosinolate, ranging from 1.15 μmol g⁻¹ DW to 3.59 μmol g⁻¹ DW (

Table 1. Glucosinolate content in different varieties of leafy Chinese kale cultured in different seasons.

Season	Variety	GNA (μmol g−1 DW)	SIN (μmol g−1 DW)	PRO (nmol g−1 DW)	GRA (nmol g−1 DW)	GIB (nmol g−1 DW)	GNL (nmol g−1 DW)	GAL (nmol g−1 DW)	GER (nmol g−1 DW)	AGS (μmol g−1 DW)	GBS (μmol g−1 DW)	4-OMGBS (nmol g−1 DW)	NGBS (nmol g−1 DW)	4-OHGBS (nmol g−1 DW)	IGS (μmol g−1 DW)	GST/RGS (nmol g−1 DW)	GS (μmol g−1 DW)
Fall	JLYC-01	2.63 ± 0.87 Aab	0.89 ± 0.22 Bab	97.5 ± 11.0 Aa	33.9 ± 12.3 Aa	5.9 ± 1.5 Ab	24.8 ± 10.8 Aa	11.9 ± 3.3 Ba	17.7 ± 16.5 Aa	3.71 ± 0.98 Aab	0.86 ± 0.05 Ac	67.9 ± 8.2 Aa	92.0 ± 18.4 Bbc	10.5 ± 5.6 ABa	1.03 ± 0.06 Ac	22.3 ± 20.9 Aa	4.76 ± 1.05 Aab
	JLYC-02	1.96 ± 0.24 Ab	0.59 ± 0.16 Bab	34.6 ± 1.2 Bb	71.4 ± 42.1 Aa	10.8 ± 2.5 Bb	13.0 ± 7.4 Aab	8.9 ± 3.9 Ba	3.4 ± 3.5 Ba	2.68 ± 0.45 Ab	2.52 ± 0.40 Aa	89.5 ± 15.0 Aa	135.3 ± 9.3 Aa	7.7 ± 5.0 ABa	2.75 ± 0.42 Aa	19.4 ± 8.4 Aa	5.45 ± 0.35 Aab
	JLYC-03	1.46 ± 0.28 Ab	0.50 ± 0.10 Bb	20.6 ± 2.5 Bb	41.2 ± 1.9 Aa	7.0 ± 1.3 Ab	7.4 ± 1.3 Ab	6.4 ± 3.2 Ba	10.2 ± 4.6 ABa	2.05 ± 0.39 Bb	1.83 ± 0.28 Aab	62.5 ± 18.0 Ba	35.7 ± 19.9 Bd	7.9 ± 2.9 ABa	1.93 ± 0.30 Aab	35.2 ± 7.6 Ba	4.02 ± 0.69 Abb
	JLYC-04	3.59 ± 0.71 Aa	0.96 ± 0.14 Ba	47.7 ± 28.9 Bb	39.9 ± 7.1 Ba	18.9 ± 4.3 Bb	10.5 ± 5.8 ABab	9.2 ± 5.0 Ba	3.4 ± 1.2 Aa	4.68 ± 0.80 Aa	1.89 ± 0.43 Aab	74.9 ± 8.7 Ba	65.5 ± 11.8 Bcd	8.8 ± 4.2 Ba	2.04 ± 0.41 Aab	18.1 ± 3.9 Ba	6.74 ± 1.18 Aa
	JLYC-05	2.35 ± 0.22 Aab	0.87 ± 0.14 Bab	104.0 ± 19.6 Ba	43.6 ± 13.4 Aa	43.8 ± 19.8 Aa	14.1 ± 0.7 Aab	13.0 ± 3.2 Aa	10.8 ± 6.9 Ba	3.44 ± 0.40 ABab	1.54 ± 0.11 Abc	68.6 ± 4.4 Ba	108.0 ± 10.5 Bab	11.9 ± 1.1 Ba	1.73 ± 0.13 Abc	35.2 ± 11.6 Aa	5.21 ± 0.27 Aab
Winter	JLYC-01	1.47 ± 0.51 Ab	0.33 ± 0.13 Cb	20.7 ± 7.8 Bb	32.6 ± 8.6 Ab	3.8 ± 1.5 Ac	9.6 ± 2.6 ABa	8.5 ± 5.2 Bab	3.7 ± 2.1 Aa	1.88 ± 0.65 Bb	0.62 ± 0.19 Aba	72.2 ± 48.1 Ab	62.5 ± 0.8 Bab	2.0 ± 0.4 Ba	0.76 ± 0.24 Aa	25.5 ± 4.6 Aa	2.67 ± 0.45 Bb
	JLYC-02	1.84 ± 0.45 Ab	0.69 ± 0.07 Bb	62.6 ± 7.5 Ba	60.9 ± 17.6 Aab	23.9 ± 3.4 Abc	13.5 ± 2.1 Aa	21.0 ± 10.0 Ba	5.5 ± 1.8 Ba	2.72 ± 0.51 Ab	0.85 ± 0.25 Ba	109.6 ± 50.7 Ab	57.2 ± 14.0 Bb	5.7 ± 3.4 Ba	1.02 ± 0.30 Ba	38.8 ± 35.5 Aa	3.77 ± 0.83 Bb
	JLYC-03	1.20 ± 0.28 Ab	0.43 ± 0.13 Bb	26.9 ± 5.3 Bb	45.2 ± 18.7 Ab	9.0 ± 0.7 Abc	7.8 ± 2.8 Aa	12.9 ± 2.2 Bab	5.9 ± 1.8 Ba	1.74 ± 0.39 Bb	0.80 ± 0.27 Ba	97.0 ± 9.5 Aa	38.8 ± 15.5 Bb	1.8 ± 0.8 Ba	0.93 ± 0.29 Ba	44.4 ± 15.2 ABa	2.71 ± 0.57 Bb
	JLYC-04	2.94 ± 0.40 Aa	1.18 ± 0.15 Aa	77.9 ± 8.0 Ba	118.4 ± 37.9 Aa	73.5 ± 24.3 Aa	16.5 ± 5.3 Aa	13.3 ± 1.8 Bab	4.9 ± 0.7 Aa	4.43 ± 0.52 Aa	0.94 ± 0.24 Ba	267.8 ± 78.9 Aa	75.9 ± 16.9 Bab	4.8 ± 0.9 Ba	1.28 ± 0.34 Aa	76.3 ± 37.4 Aa	5.79 ± 0.83 Aa
	JLYC-05	1.98 ± 0.34 Aab	0.41 ± 0.27 Bb	27.7 ± 3.6 Cb	39.5 ± 15.2 Ab	40.2 ± 15.4 Aab	7.5 ± 3.3 Ba	3.8 ± 1.0 Bb	4.0 ± 1.1 Ba	2.51 ± 0.04 Bb	1.05 ± 0.31 Ba	106.1 ± 41.5 Ab	106.4 ± 29.9 Ba	3.1 ± 1.4 Ba	1.26 ± 0.38 Aa	38.2 ± 17.1 Aa	3.81 ± 0.36 Bb
Spring	JLYC-01	2.70 ± 0.20 Aa	1.29 ± 0.09 Aa	71.7 ± 21.8 Ab	37.3 ± 10.7 Aa	3.7 ± 1.8 Aa	ND	39.9 ± 8.6 Aab	11.6 ± 2.9 Acd	4.16 ± 0.27 Aab	0.51 ± 0.07 Bb	40.7 ± 6.8 Ac	130.5 ± 14.7 Ac	13.7 ± 3.3 Aa	0.69 ± 0.09 Ab	24.0 ± 5.4 Ac	4.87 ± 0.35 Abc
	JLYC-02	1.15 ± 0.06 Bc	1.11 ± 0.10 Aa	154.2 ± 53.8 Ab	36.9 ± 7.7 Aa	ND	ND	48.1 ± 10.2 Ba	24.3 ± 3.9 Aab	2.53 ± 0.17 Ab	1.23 ± 0.13 Ba	72.3 ± 7.4 Ab	78.9 ± 4.9 Bd	18.3 ± 4.6 Aa	1.40 ± 0.15 Ba	49.8 ± 9.0 Ab	3.97 ± 0.26 Bc
	JLYC-03	1.58 ± 0.19 Abc	1.72 ± 0.46 Aa	122.7 ± 14.8 Ab	41.6 ± 12.6 Aa	ND	ND	33.8 ± 6.8 Aab	15.5 ± 1.5 Abc	3.51 ± 0.64 Ab	1.44 ± 0.18 Aa	51.8 ± 8.2 Bbc	86.1 ± 13.6 Ad	13.3 ± 5.2 Aa	1.59 ± 0.20 ABa	67.0 ± 2.4 Aa	5.17 ± 0.44 Ab
	JLYC-04	2.75 ± 0.30 Aa	1.63 ± 0.30 Aa	1012.0 ± 171.6 Aa	39.2 ± 16.2 Ba	ND	ND	43.5 ± 15.2 Aab	5.1 ± 1.8 Ad	6.00 ± 0.77 Aa	1.29 ± 0.08 ABa	71.0 ± 10.5 Bb	181.4 ± 13.1 Ab	21.4 ± 1.5 Aa	1.56 ± 0.09 Aa	32.4 ± 1.3 Abc	7.07 ± 0.19 Aa
	JLYC-05	2.08 ± 0.13 Ab	1.51 ± 0.26 Aa	175.9 ± 8.9 Ab	32.6 ± 6.8 Aa	ND	ND	18.3 ± 4.9 Ab	21.6 ± 1.5 Aab	4.43 ± 1.22 Aab	1.20 ± 0.05 ABa	154.5 ± 8.7 Aa	281.2 ± 25.7 Aa	16.3 ± 4.7 Aa	1.65 ± 0.06 Aa	57.3 ± 8.4 Aab	5.55 ± 0.41 Ab

GNA: gluconapin; SIN: sinigrin; PRO: progoitrin; GRA: glucoraphanin; GIB: glucoiberin; GNL: gluconapoleiferin; GAL: glucoalyssin; GER: glucoerucin AGS: total aliphatic glucosinolates; GBS: glucobrassicin; 4-OMGBS: 4-methoxy glucobrassicin; NGBS: neoglucobrassicin; 4-OHGBS: 4-hydroxy glucobrassicin; IGS: total indole glucosinolates; GST: gluconasturtiin; RGS: total aromatic glucosinolates; GS: total glucosinolates; ND: not detected. Uppercase letters show seasonal differences within varieties, while lowercase letters indicate varietal differences within seasons. Values not sharing a common letter are significantly different at p < 0.05. Values are means ± SD.

). JLYC-04 had the highest content of gluconapin, while JLYC-03 had the lowest. The content of sinigrin was highest in spring (Table 1), with no significant differences among the varieties. The content of progoitrin was highest in spring (except for JLYC-01) (Table 1). JLYC-04 had the highest content in winter (77.9 nmol g⁻¹ DW) and spring (1012.0 nmol g⁻¹ DW). The content of glucoraphanin ranged from 32.6 nmol g⁻¹ DW to 118.4 nmol g⁻¹ DW (Table 1). Leafy Chinese kale cultivated in spring did not contain glucoiberin (Table 1) or gluconapoleiferin (Table 1). In contrast, the contents of glucoalyssin (Table 1) and glucoerucin (Table 1) were highest in spring. The aliphatic glucosinolate content ranged from 1.74 to 6.00 μmol g⁻¹ DW (Table 1), with the highest levels in spring (2.53 μmol g⁻¹ DW to 6.00 μmol g⁻¹ DW) and the lowest levels in winter (1.74 μmol g⁻¹ DW to 4.43 μmol g⁻¹ DW). Among different varieties, JLYC-04 had the highest aliphatic glucosinolate content.

Glucobrassicin was the major indole glucosinolate, ranging from 0.51 μmol g⁻¹ DW to 2.52 μmol g⁻¹ DW (Table 1). Among the different seasons, the highest content of glucobrassicin was observed in fall (2.52 μmol g⁻¹ DW in JLYC-02). The contents of 4-methoxyglucobrassicin (Table 1), neoglucobrassicin (Table 1), and 4-hydroxyglucobrassicin (Table 1) ranged from 40.7 to 267.8 nmol g⁻¹ DW, 35.7 to 281.2 nmol g⁻¹ DW, and 1.8 to 21.4 nmol g⁻¹ DW, respectively. The content of indole glucosinolates in different varieties of leafy Chinese kale exhibited the highest content in fall (1.03 μmol g⁻¹ DW–2.75 μmol g⁻¹ DW) and the lowest in winter, with the exception of JLYC-01 (Table 1).

Gluconasturtiin was the only aromatic glucosinolate detected, which presented at a very low level, ranging from 18.1 nmol g⁻¹ DW to 76.3 nmol g⁻¹ DW (Table 1).

The total glucosinolate (Table 1) content ranged from 2.7 μmol g⁻¹ DW to 7.1 μmol g⁻¹ DW. The content in leafy Chinese kale cultured in winter was significantly lower than those cultured in the other seasons. Among the five varieties, JLYC-04 was the one with the highest content.

3.5. Principal Component Analysis

A comprehensive evaluation of the nutritional indicators was conducted using principal component analysis (PCA). The PCA score plot demonstrates clear separation of samples based on their cultivation season. The first principal component (PC1), accounting for 29.4% of the total variance, effectively distinguishes between spring, fall, and winter samples. PC2 contributes an additional 19.3% variance (Figure 5A). Spring and fall samples showed overlapping yet distinct groupings, while winter samples formed a separate cluster, indicating substantial differences in nutritional substances influenced by seasonal variation. According to the loading plot (Figure 5C), the major contributor to the nutritional substances of leafy Chinese kale in fall was glucobrassicin, those in winter were 4-methoxyglucobrassicin, glucoraphanin, and glucoiberin, and those in spring were sinigrin, soluble proteins, and glucoalyssin.

3.6. Correlation Analysis

A total of 81 correlations were obtained from the correlation analysis, with all correlations being positive except for the one between gluconapoleiferin and soluble proteins (Figure 6, Supplementary Table S2). The strongest correlation with antioxidant capacity was observed for total phenols (R² = 0.681). The correlation coefficients for reducing sugars with sinigrin and 4-hydroxyglucobrassicin were 0.723 and 0.666, respectively. Carotenoids showed correlation coefficients of 0.797 with chlorophyll and 0.663 with neoglucobrassicin. Additionally, aliphatic glucosinolates were most strongly correlated with total glucosinolates, with gluconapin showing the strongest correlation with aliphatic glucosinolates (R² = 0.777), and glucobrassicin demonstrating the strongest correlation with indole glucosinolates (R² = 0.986).

3.7. Variance Analysis

As shown in

Table 2. Estimated proportions of variance components for nutritional substances and antioxidant capacity in five varieties of leafy Chinese kale.

Parameter	Vs/Vp	Vv/Vp	Vsv/Vp
Plant height	0.103 **	0.110 **	0.459 **
Plant spreading	0.0441 **	0.577 **	0.156 **
Plant weight	0.265 **	0.390 **	0.0790 **
Reducing sugars	0.557 **	0.273 **	0.123 **
Soluble proteins	0.667 **	0.234 **	0.0340
Soluble solids	0.776 **	0.0762 **	0.0609 *
Chlorophyll	0.667 **	0.234 **	0.034
Carotenoids	0.288 **	0.477 **	0.0930 *
Vitamin C	0.331 **	0.380 **	0.130 *
Total phenols	0.0273	0.714 **	0.0402
Antioxidant capacity	0.0546 **	0.669 **	0.197 **
Gluconapin	0.0794 **	0.592 **	0.139 *
Sinigrin	0.602 **	0.123 **	0.147 **
Progoitrin	0.249 **	0.255 **	0.470 **
Glucoraphanin	0.116 *	0.199 **	0.354 **
Glucoiberin	0.312 **	0.00912	0.563 **
Gluconapoleiferin	0.552 **	0.0690 *	0.172 **
Glucoalyssin	0.650 **	0.097 **	0.117 **
Glucoerucin	0.321 **	0.107 *	0.286 **
Glucobrassicin	0.433 **	0.291 **	0.158 **
4-Methoxyglucobrassicin	0.195 **	0.222 **	0.405 **
Neoglucobrassicin	0.330 **	0.340 **	0.285 **
4-Hydroxyglucobrassicin	0.684 **	0.0526	0.0526
Aliphatic glucosinolates	0.270 **	0.0320	0.541 *
Indole glucosinolates	0.374 **	0.313 **	0.174 **
Aromatic glucosinolates/ Gluconasturtiin	0.182 **	0.158 *	0.273 *
Total glucosinolates	0.233 **	0.0423 **	0.584 **

V_S/V_P: ratio of cultivation season variance to phenotypic variance; V_V/V_P: ratio of variety variance to phenotypic variance; V_SV/V_P: ratio of cultivation × variety interaction variance to phenotypic variance. * and ** indicate the significance at 0.05 and 0.01 probability levels in the same column, respectively.

, the variance ratios for cultivation season were significant at the 0.01 level, with the exception of total phenols and glucoraphanin. The variance ratios for variety were significant at the 0.01 or 0.05 level, except for glucoiberin, glucoerucin, gluconapoleiferin, 4-hydroxyglucobrassicin, and aliphatic glucosinolates. The interaction between cultivation season and variety was also significant at the 0.01 or 0.05 level, except for soluble proteins, chlorophyll, total phenols, and 4-hydroxyglucobrassicin.

Seasonal effects were most pronounced on the content of chlorophyll, reducing sugars, soluble proteins, soluble solids, sinigrin, glucoalyssin, glucoerucin, gluconapoleiferin, 4-hydroxyglucobrassicin, glucobrassicin, and indole glucosinolates. On the other hand, plant spreading, antioxidant capacity, carotenoids, vitamin C, total phenols, gluconapin, neoglucobrassicin, aliphatic glucosinolates, and total glucosinolates were more influenced by variety. The remaining traits were primarily affected by the interaction between season and variety.

4. Discussion

Chinese kale is a native Chinese vegetable, widely distributed in South China and Southeast Asia. However, little research has focused on the leaves of Chinese kale. In this study, we selected leafy Chinese kale as the research material and systematically analyzed the nutritional substances of different varieties under various cultivation seasons. The results showed that the leaves of Chinese kale contain high levels of vitamin C, chlorophyll, carotenoids, total phenols, and glucosinolates. The detected nutritional substance levels were generally consistent with those reported by previous studies in other Brassica vegetables, and some, such as the vitamin C content, were even significantly higher than those of other Brassica species [25,28,29,30,31,32,33].

Previous studies have shown that cultivation season, meaning the variable growing environment, affects the yield and nutritional quality of Brassica vegetables [34,35,36,37,38]. Chinese kale cultivated in spring has been reported to have a higher glucosinolate content than that cultivated in fall [34], and similar observations have been made in Brassica oleracea var. acephala [36]. Charron et al. [35] found that the glucosinolate content of cabbage varied between spring and fall, with the highest total glucosinolate content being observed when the crop was harvested under high temperatures and long sunlight exposure. Consistently, in the present study, total glucosinolates, total aliphatic glucosinolates, total indole glucosinolates, gluconapin, and glucobrassicin were found to be higher in the warmer cultivation seasons (spring and fall) than in the cooler season (winter). This may be due to the higher temperature inducing the accumulation of glucosinolates [39]. Moreover, the higher glucosinolate content of leafy Chinese kale cultivated in fall and spring may be related to the longer photoperiod as reported by the finding of Liu et al. [40] that glucosinolate content in cabbage increases under long-day conditions. In addition to glucosinolates, other nutritional indexes were also affected by the cultivation season. Free sugars such as sucrose, glucose, and fructose were higher in spring- and summer-cultivated broccoli compared to fall- and winter-cultivated broccoli, whereas the opposite trend was observed in kale and Portuguese cabbage [41]. Our study found that spring-cultivated leafy Chinese kale had higher levels of reducing sugars. Additionally, a higher content of carotenoids was found in the leafy Chinese kale cultivated in spring, which is consistent with the finding that longer light duration increases carotenoids content in broccoli [42]. Fall-cultivated leafy Chinese kale had higher levels of vitamin C and antioxidant capacity. This enhancement in antioxidants and antioxidant capacity may be related to the temperature changes associated with seasonal adaptations [17]. In summary, to maximize glucosinolate content in leafy Chinese kale, it is advisable to cultivate in spring or fall due to higher levels observed in these warmer seasons compared to winter. If the focus is on other nutrients like reducing sugars, soluble proteins, and carotenoids, spring cultivation is recommended, while for higher soluble solids, vitamin C, and antioxidant capacity, fall cultivation is preferable, as these nutrients tend to be more abundant in their respective seasons. Our present study implicated the significant effect of cultivation season on the contents of nutrient substances. However, only meteorological data were collected in this pilot study. Environmental data collection in the greenhouse is necessary to clarify further mechanisms behind why cultivation season affects nutritional substance content. This needs to be paid attention in a future full-scale study.

Numerous studies have shown that the nutritional substances of Brassica vegetables vary significantly depending on both species and variety. The levels of vitamin C, total phenols, and glucosinolates in vegetables such as broccoli, cauliflower, and Chinese kale have been found to differ considerably [28,29,30]. According to Verkerk et al. [43], the composition and content of glucosinolates vary greatly among different crop varieties. Vallejo et al. [32] found significant differences in the vitamin C and glucosinolate content among different varieties of Brassica oleracea. Similarly, significant variations in vitamin C, protein, and mineral content have been found across different varieties of Chinese kale shoots. Consistent with previous studies, the results of the present experiment also revealed significant differences in the nutritional substances of various leafy Chinese kale varieties. JLYC-04 exhibited high levels of vitamin C, total phenols, antioxidant capacity, total glucosinolates, total aliphatic glucosinolates, and 3-butenyl glucosinolates, while JLYC-05 was rich in soluble solids, chlorophyll, and carotenoids. In contrast, JLYC-01 showed significantly lower levels of chlorophyll, carotenoids, vitamin C, total phenols, antioxidant capacity, glucobrassicin, and total indole glucosinolates compared to other varieties. Based on this pilot study, JLYC-04 and JLYC-05 exhibited superior performance in terms of these detected nutrients, making them more suitable candidates for breeding aimed at developing nutrient-functional vegetables.

5. Conclusions

Cultivation season and variety significantly influenced the contents of nutrient substances in leafy Chinese kale. Generally, plants cultivated in spring and fall performed better than those cultivated in winter, providing a reference for leafy Chinese kale planting. Among the five varieties, JLYC-04 and JLYC-05 are excellent candidates for future breeding programs, since JLYC-04 has a higher content of vitamin C, total phenols, and glucosinolates, exhibiting strong antioxidant capacity, while JLYC-05 contains more chlorophyll and carotenoids.