Introduction

Chinese kale (Brassica oleracea var. alboglabra) belongs to the Brassicaceae family, and its edible parts are bolting stems and tender leaves (Sun et al., 2012a, 2019). It is rich in bioactive compounds, including vitamin C, total phenolics, glucosinolates and carotenoids, which can enhance the immunity in consumers and reduce their risk of cancer (Sun et al., 2011, 2012b). Chinese kale belongs to long-day plants and favors a cold, cool and humid climate. It is an important vegetable crop in South China and is cultivated widely (Lei et al., 2017).

Chlorophyll is the major photosynthesis pigment and is widely distributed in green plants (Zhang et al., 2019). Chlorophyll and its various derivatives can be absorbed by the human body in diet rich in chlorophyll pigments, such as green vegetables. In addition, chlorophyll pigments have numerous health benefits (Ferruzzi and Blakeslee 2007), such as their good ability to chelate with several chemical carcinogens and mutagens (Jubert et al., 2009), reduce cancer risk (Cervantes-Paz et al., 2014) and scavenge free radicals (Chen and Roca, 2018).

Carotenoids are the second most abundant natural pigments with more than 750 members (Quinlan et al., 2012). Carotenoids are isoprenoid metabolites that are synthesized by all photosynthetic organisms. They are essential for photoprotection and participate in light harvesting in photosynthetic systems (Rodriguez-Concepcion et al., 2018). The colors of carotenoids range from colorless to yellow, orange and red, with variations reflected in many flowers, fruits and vegetables (Cazzonelli and Pogson, 2010). Carotenoids play an important role in both plants and humans (Amin et al., 2009). In plants, carotenoids and their derivatives participate in many critical biological functions, such as biosynthesis of phytohormones, photosynthesis and photo-protection (Cazzonelli and Pogson, 2010). However, in human body, carotenoids could enhance the antioxidant and anti-inflammatory activity, and prevent several reactive oxygen species (ROS)-mediated chronic diseases (Rodriguez-Concepcion et al., 2018). Previous studies have reported that carotenoids can prevent and treat several cancers and chronic diseases (Amin et al., 2009). Lutein and zeaxanthin can help to prevent eye diseases (Caseli et al., 2013). In addition, several of the carotenoids can be used as an origin of vitamin A. β-carotene is the most important precursor of vitamin A, which can be degraded into vitamin A when the human body requires it. It can cure dry eye disease, night blindness and other diseases caused by a lack of vitamin A. In general, the antioxidant properties of chlorophyll and carotenoids are associated with a lower risk of developing cancer (Britton and Khachik, 2009).

Chinese kale has a number of varieties with a long history of cultivation. Based on maturity, it can be divided into prematurity, medium maturity, late maturity and extremely late maturity types. It can also be classified into two types according to whether the petals are white or yellow (Lei et al., 2017). Numerous studies have reported large differences in phytochemicals among different species, types, varieties and organs of vegetables (Chen et al., 2019; Sun et al., 2011; Takaidza et al., 2018). However, no studies have been reported on pigments of Chinese kale to date. The present experiment measured the differences of chlorophyll and carotenoids content in leaves and bolting stems of 19 Chinese kale varieties with different maturity and color to explore the effects of variety and organ on the pigment content of Chinese kale.

Materials and Methods

Plant materials

The experiment investigated 19 varieties of Chinese kale, including four maturity types (prematurity, medium maturity, late maturity and extremely late maturity) and two flower colors (white and yellow) (Table 1). These varieties of Chinese kale were sown on 10 September 2015, with a row spacing of 30 cm and a plant spacing of 40 cm. A total of 10,000 kg of decomposed fertilizer was applied to each hectare of field planting. After four days of field planting, 75 kg of urea was applied to each hectare, with 150 kg of urea applied to each hectare every 15 days and watered at the same time. Carbendazim was sprayed at seedling stage to prevent sclerotinia and soft rot, and metoclopramide was sprayed to control diamondback moth. All plants were harvested according to different maturities on 15 November 2015, 5 December 2015, 5 January 2016 and 25 January 2016. At each harvesting, strong and healthy plants with identical maturity levels and no pests and diseases were chosen and transported to laboratory immediately. Fifteen plants were collected per variety, five plants were pooled as one repetition, and three replicates were used in total. After wiping the material surface clean, samples were divided according to edible parts into leaves and bolting stems. After that, all samples were lyophilized for 72 h using a vacuum freeze dryer (Songyuan Huaxing, Beijing, China), and grounded to a fine powder and stored at -80°C.

Analysis of chlorophyll and carotenoids

Polypropylene tubes containing 0.2 g of sample powder were filled with 25 mL of acetone for extraction in darkness at 4°C for 12 h. Samples were then centrifuged to 8,000 g at 4°C for 10 min. Cellulose acetate filters (0.22 μm) were used to filter supernatant into a brown chromatographic flask. The high-performance liquid chromatography (HPLC) analysis of chlorophyll and carotenoids was conducted using an Agilent 1260 instrument equipped with a model variable wavelength absorbance detector (VWD). The samples (10 μL) were separated at 30°C on a Waters Spherisorb C18 column (150 × 3.9 mm) using 80% acetonitrile–water and isopropanol at a flow rate of 0.5 mL min^–1. Absorbance was detected at 428 and 448 nm (Shi et al., 2014). The peaks of the samples were qualitatively assessed based on the retention period of individual standards. The standards of chlorophylls (a and b) and carotenoids (neoxanthin, violaxanthin, lutein and β-carotene) were obtained from Solarbio Science & Technology Co. Ltd. (Beijing, China). Chlorophyll and carotenoid concentrations in the samples were calculated according to the standard curve of standards.

Statistical analysis

The raw data were processed by Excel 2018, and the results were shown as mean values of three replicates. The statistical analysis and correlation analysis were performed using SPSS 18.0. The least significant difference test was applied to assess differences (P < 0.05). In order to determine relationships among samples, principal component analysis (PCA) and partial least-square discriminant analysis (PLS-DA) were performed using SIMCA-P 11.5 with unit variance scaling. Variance and correlation analyses were also performed. Hierarchical clustering analyses (HCA) were conducted using the TIGR MeV software (version 4.1) (Sun et al., 2018).

Results

Identification of chlorophyll and carotenoids

The composition and concentrations of chlorophyll and carotenoids in leaves and bolting stems of Chinese kale were analyzed by HPLC. Figure 1 shows that both chlorophyll a and chlorophyll b and four carotenoids (neoxanthin, violaxanthin, lutein and β-carotene) were detected in all samples of 19 varieties and two organs.

Figure 1. HPLC of chlorophyll and carotenoids in the leaves of Chinese kale. 1: neoxanthin, 2: violaxanthin, 3: lutein, 4: chlorophyll b, 5: chlorophyll a and 6: β-carotene.

Chlorophyll concentrations in leaves and bolting stems of Chinese kale

The leaves of distinct varieties of Chinese kale had significant differences in chlorophyll concentrations, and the distribution of chlorophyll a, chlorophyll b and total chlorophyll was identical (Table 2). The lowest concentrations of chlorophyll a (3.11 mg g^–1 DW), chlorophyll b (2.14 mg g^–1 DW) and total chlorophyll (5.24 mg g^–1 DW) were observed in JL-06, while the highest levels of all these were found in JL-03 (12.60, 9.40 and 22.01 mg g^–1 DW, respectively). These were four-fold higher than those in JL-06. However, the ratio of chlorophyll b to chlorophyll a in the leaves of different varieties of Chinese kale remained almost identical, at approximately 0.7.

Significant differences were found in chlorophyll concentrations of bolting stems of different varieties of Chinese kale (Table 3). The distribution of chlorophyll in bolting stems was identical to those in leaves; however, the concentrations were notably lower than in leaves. Among the tested varieties, the lowest concentrations of chlorophyll a (0.53 mg g^–1 DW), chlorophyll b (0.44 mg g^–1 DW) and total chlorophyll (0.97 mg g^–1 DW) were observed in JL-06, while the highest levels were observed in JL-03 (1.84, 1.30 and 3.14 mg g^–1 DW, respectively). These were approximately three-fold higher than those observed in JL-06. Moreover, no significant difference in the ratio of chlorophyll b to chlorophyll a was found in the distinct varieties of Chinese kale, ranging from 0.67 to 0.83.

Carotenoid concentrations in leaves and bolting stems of Chinese kale

Significant differences in carotenoid concentrations were found between the leaves of different varieties of Chinese kale (Table 4). Among the tested varieties, the lowest concentrations of neoxanthin, violaxanthin, lutein, β-carotene and total carotenoids were in JL-06, which were 0.29, 0.06, 0.66, 0.21 and 1.23 mg g^–1 DW, respectively. The highest concentrations of neoxanthin (1.11 mg g^–1 DW), lutein (2.53 mg g^–1 DW), β-carotene (0.74 mg g^–1 DW) and total carotenoids (4.51 mg g^–1 DW) were found in JL-03, and were more than three-fold than found in JL-06. Moreover, the highest concentration of violaxanthin (0.18 mg g^–1 DW) was observed in JL-25, and was 2.8-fold higher than that in JL-06. The differentiation of carotenoid proportion in the leaves of different varieties of Chinese kale was small. Among carotenoids, lutein was predominant, accounting for more than 50% of total carotenoid concentrations, followed by neoxanthin and β-carotene, which accounted for more than 20% and 10%, respectively. The lowest proportion was that of violaxanthin, accounting for 2.7-5.8%, in different varieties.

Significant differences were found in carotenoid concentrations of bolting stems in different varieties of Chinese kale (Table 5). Among varieties, JL-06 has the lowest concentrations of neoxanthin, lutein, β-carotene and total carotenoids, which were 0.11, 0.14, 0.04 and 0.34 mg g^–1 DW, respectively. In addition, concentrations of β-carotene and total carotenoids were also relatively low in JL-24. JL-02 and JL-17 had the lowest concentration of violaxanthin, which was 0.03 mg g^–1 DW for both. JL-03 had the highest concentrations of neoxanthin (0.19 mg g^–1 DW), violaxanthin (0.06 mg g^–1 DW), lutein (0.36 mg g^–1 DW), β-carotene (0.12 mg g^–1 DW) and total carotenoids (0.73 mg g^–1 DW). These were, respectively, 1.8-, 1.8-, 2.6-, 3.1- and 2.2-fold of the corresponding lowest content. No significant difference was found in the proportion of individual carotenoid in different varieties of Chinese kale; lutein accounted for the highest proportion, with approximately 50% of total carotenoid concentration, followed by Neoxanthin and β-carotene accounting for approximately 20% and 10–20%, respectively. Violaxanthin had the lowest proportion, with only 6–16%.

PCA and PLS-DA analysis

Principal component analysis of chlorophyll and carotenoids was performed to investigate directly variation in different organs, varieties and maturities of Chinese kale. As shown in Figure 2A, two principal components were obtained through dimension reduction. Principal component 1 (PC1) played a major role (accounting for 98.4%), while principal component 2 (PC2) played a minor role (accounting for only 1.4%). The sum of both PCs was nearly 100%. Leaves and bolting stems were discriminated from each other by PC1. PC1 and PC2 were located on the positive and negative sides of the abscissa axis, respectively, and distribution of the bolting stem was relatively compact. However, different varieties of Chinese kale could not be separated into different groups as organs or maturities by PC2, which had little effect, because of its low proportion. The results of PLS-DA analysis were consistent with that of PCA. Bolting stems were clearly separated from leaves by PLS-DA1, while different varieties of Chinese kale were not separated into different groups according to organs or maturities by PLS-DA2 (Figure 2B). Therefore, it was concluded that organs formed the main source of differences for chlorophyll and carotenoid concentrations in different varieties of Chinese kale, while maturities and flower colors exerted little effect.

Figure 2. PCA and PLS-DA analysis of leaves and bolting stems of Chinese kale. (A) PCA score plot; (B) PLS-DA score plot; (C) PLS-DA loading plot.

The results of PLS-DA loading plot showed that each index was located at the positive position of abscissa (Figure 2C), and leaves of Chinese kale were located at the positive position of abscissa in PLS-DA score plot (Figure 2B). This suggests that leaves of Chinese kale had relatively high chlorophyll and carotenoid concentrations.

HCA analysis

Hierarchical clustering analysis was performed to visualize chlorophyll and carotenoid concentrations in leaves and bolting stems for different varieties of Chinese kale. The concentration of each pigment was normalized, and the results are shown in Figure 3. The red color in Figure 3 represents relatively high concentrations, and the green color represents relatively low concentrations. The concentrations of chlorophyll and carotenoids in leaves of different varieties of Chinese kale were significantly higher than those in bolting stems. The pigments were divided into two parts. Group A contains violaxanthin, which was highest in the leaves of JL-25 and JL-07, and lowest in the bolting stems of JL-02 and JL-17. Group B contains β-carotene, neoxanthin, lutein, total carotenoids, chlorophyll b, chlorophyll a and total chlorophyll. These were higher in the leaves of JL-03 and JL-19, and lower in the bolting stems of JL-06.

Figure 3. Heatmap and HCA of chlorophyll and carotenoids in Chinese kale. Red and green colors reflect relative concentrations.

Correlation analysis

Correlation analysis was conducted to investigate correlations between chlorophyll and carotenoids in leaves and bolting stems of different varieties of Chinese kale (Table 6). A significantly positive correlation (P < 0.01) was observed between chlorophyll and carotenoids. All correlation coefficients were above 0.9, and the correlation coefficient between chlorophyll a and total chlorophyll was highest (1.000), while the correlation was relatively low between violaxanthin and others. Moreover, the lowest correlation coefficient was found between violaxanthin and total carotenoids (0.939), indicating that the contribution of violaxanthin to total carotenoids was relatively small.

Discussion

Carotenoids have strong antioxidant capacity and play an important role in human health. Chinese kale is a good source of carotenoids and contains high concentrations of carotenoids. The results showed that the highest concentrations of neoxanthin, violaxanthin, lutein, β-carotene and total carotenoids in leaves of Chinese kale were 1.11, 0.18, 2.53, 0.74 and 4.51 mg g^–1 DW, respectively. The highest concentrations of neoxanthin, violaxanthin, lutein, β-carotene and total carotenoids in bolting stems were 0.19, 0.06, 0.36, 0.12 and 0.73 mg g^–1 DW, respectively. Individual and total carotenoid concentrations in Chinese kale were significantly higher than those of mature pepper (Lee et al., 2005). Lutein was identified as the predominant carotenoid, accounting for approximately 50% of total carotenoids, and this result was consistent with the results of Farnham and Kopsell (2009) for broccoli. In addition, JL-03 could be used to breed a functional variety because of its highest concentration of chlorophyll and carotenoids (except violaxanthin in leaves).

Numerous studies showed that significant differences in the composition and concentrations of phytochemicals exist in different varieties or organs of vegetables (Abu-Reidah et al., 2017; Agneta et al., 2014; Amri et al., 2017). This could be due to the genetic diversity of most characters in different varieties and organs (Hanson et al., 2011). In this study, the concentrations of chlorophyll and carotenoids in leaves were significantly higher than those in bolting stems. This result was consistent with previous studies in baby mustard (Brassica juncea var. gemmifera) (Sun et al., 2018) and bitter melon (Momordica charantia) (Tuan et al., 2011). Perhaps, it is because chlorophyll and carotenoids are important photosynthetic pigments in plants, and photosynthesis mainly occurs in leaves (Tuan et al., 2011).

Many studies have indicated that plant species with different colors vary greatly in their carotenoid concentrations (Carvalho et al., 2013). Carotenoid concentrations in Brassica napus with yellow flowers were significantly higher than those with white flowers, and the predominant carotenoid was violaxanthin (Zhang et al., 2015). However, this study did not find significant differences in leaves or bolting stems of Chinese kale with different flower colors. It is possible that carotenoids have different metabolism in flowers with different colors, but the metabolism in other organs was not affected by that in petals (Perrin et al., 2017). This finding implied that flower color exerted a limited effect on chlorophyll and carotenoid concentrations of leaves and bolting stems of Chinese kale; however, it is necessary to analyze whether it affects other phytochemicals as well.

In recent years, many studies have investigated plant maturity, and mainly focused on genetic analysis (Li et al., 2019; Nuñez-Lillo et al., 2019) and cultivation techniques (Lu et al., 2017). The present study, for the first time, has measured chlorophyll and carotenoid concentrations of Chinese kale with different maturities. However, no significant correlations were found between pigment concentrations and maturities, indicating that maturity exerted a limited effect on chlorophyll and carotenoid concentrations of Chinese kale. It could be that the sowing period of Chinese kale in this experiment was not strictly separated, since the maturity of Chinese kale differed in response to the most suitable sowing period. The possible reason requires further study.

Many studies have reported a significant positive correlation between chlorophyll and carotenoids, and the correlation coefficient was highest compared to other indicators (Sun et al., 2018; Yousefi et al., 2019). The correlation analysis in the present experiment showed significant positive correlations between chlorophyll and carotenoids (P < 0.01), which was consistent with results of previous studies (Conesa et al., 2019; Sun et al., 2018).

Conclusion

This study reported the composition and concentrations of chlorophyll and carotenoids in different organs, varieties, maturities and flower colors of Chinese kale. The pigment concentrations were significantly higher in leaves than in bolting stems, indicating that organs exerted a major role in chlorophyll and carotenoid accumulation. This was followed by variety effect, while maturity and flower color exerted little effect. Lutein was the main component of carotenoids and accounted for approximately 50% of total carotenoids. JL-03 had the highest concentrations of chlorophyll and carotenoids, and was thus a promising variety for the future breeding initiatives.