Comparative analysis for antioxidant capacities, important plant pigments and total phenolic contents in different Brassica vegetables  

Chander Parkash , S.S. Dey , Raj Kumar , M.R. Dhiman , Munish Dhiman , Satish Kumar , Vinod Kumar
Indian Agricultural Research Institute, Regional Station, Katrain (Kullu Valley), H.P.-175129 India
Author    Correspondence author
Molecular Plant Breeding, 2015, Vol. 6, No. 10   doi: 10.5376/mpb.2015.06.0010
Received: 27 Feb., 2015    Accepted: 11 May, 2015    Published: 09 Jun., 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Parkash et al., Comparative analysis for antioxidant capacities, important plant pigments and total phenolic contents in different Brassica vegetables, Molecular Plant Breeding, 2015, Vol.6, No. 10 1-7 (doi: 10.5376/mpb.2015.06.0010)

Abstract

A study to analyze the quantitative comparative variation of antioxidant capacity within the Brassica family was undertaken with ten cultivars representing 5 brassica vegetables. It was observed that sprouting broccoli cultivar Pusa Broccoli KTS-1 exhibited significantly highest contents of antioxidants viz., CUPRAC (17.91 µ mol tolox/g) and FRAP (14.34 µ mol trolox/g) whereas, significantly higher contents of total phenolics (6120.0 µg gallic acid/gfw), anthocyanin (3.28 mg/100 g), ascorbic acid (80.832 mg/100g of sample), lycopene (8.02 mg in 100 g sample), total carotenoids (52.82 mg/100g) and β–carotene (11.32 µg/100 g) were recorded in kale cultivar KTK-64 in comparison to other vegetables. High genetic advance coupled with high heritability was observed for all the traits which is attributed to considerable additive genetic effects and selection for these traits would be most effective. CUPRAC has shown significant and positive correlation at both genotypic and phenotypic levels with all the other 7 traits whereas FRAP was significantly correlated with total phenolics and anthocyanin only. Based on D2 analysis the 10 genotypes were classified into 4 clusters. The cluster IV had maximum 4 genotypes and cluster II had only one genotype.

Keywords
Brassica vegetables; Anti-oxidant capacity; Phytochemicals; Variability; Correlation; Diversity
In the past years, most studies were concentrated on the role of diet in human health. It has been studied that plant products are much more beneficial to reduce a high risk of many diseases such as cancer, cardiovascular diseases, diabetes and atherosclerosis etc. (Goldman, 2004). There are a number of phytochemicals such as phenols, ascorbic acid, anthocyanin, chlorophyll, carotenoids, CUPRAC, FRAP etc. which provide basic nutrition to furnish the defensive mechanism to reduce the risk of many chronic diseases in humans (Deng et al., 2013). Objective of this study was to analyze the quantitative comparative variation of antioxidant capacity, phenols and other compounds within the Brassica family.
Brassica vegetables such as cabbage, cauliflower, broccoli, kohlrabi and kale possess both antioxidant and anti-carcinogenic properties (Cohen et al., 2000; Chu et al., 2002; Verhoeven et al., 1997). All plants of the genus Brassica like broccoli, Brussels sprouts, cabbage, cauliflower, collard greens and kohlrabi contain glucosinolates which are known to have fungicidal, bactericidal and cancer protective characteristics (Fahey, 2003). These vegetables are rich sources of glucosinolates, sulfur-containing compounds that impart a pungent aroma and bitter taste. The hydrolysis of glucosinolates by a class of plant enzymes called myrosinase results in the formation of biologically active compounds, such as indoles and isothiocyanates. Myrosinase is physically separated from glucosinolates in intact plant cells. However, when brassica vegetables are chopped or chewed, myrosinase comes in contact with glucosinolates and catalyzes their hydrolysis. Therefore, the potential for high intakes of brassica vegetables as well as several glucosinolate hydrolysis products to prevent cancer is of interest. Natural antioxidant defence mechanism is present in human beings (Ali et al., 2001), however, this natural antioxidant defence mechanism can be insufficient hence dietary intake of antioxidant compounds become important (Halliwell, 1994).
Several studies showed that high consumption of vegetables and fruits had an inverse relationship with the incidence of degenerative diseases including heart diseases (Hertog et al., 1993; Lee et al., 2004), cancer (Block et al., 1992; Byers and Perry, 1992; Sugimura, 2002), inflammation (Giugliano et al., 2006; Rahman et al., 2006) and brain dysfunction (Youdim and Joseph, 2001; Trewavas and Stewart, 2003). Oxidative stress results from either a decrease of natural cell antioxidant capacity or an increased amount of reactive oxygen species (ROS) in organisms. When the balance between oxidants and antioxidants in the body is shifted by the overproduction of free radicals, it will lead to oxidative stress and DNA damage. (Ames et al., 1993; Liu, 2002). Brassica vegetables contain many bioactive compounds, especially organosulphur phytochemicals possessing anticarcinogenic activity and other phytochemicals, which are known to possess antioxidant activity. Dietary antioxidants present in these vegetables, such as water-soluble vitamin C and phenolic compounds, as well as lipid-soluble vitamin E and carotenoids, contribute to both the first and the second defence lines against oxidative stress (Lampi et al., 2002; Krinsky, 2001; Davey et al., 2000).
The role of antioxidants is to protect all bio-molecules from the oxidative damage of free radicals, therefore antioxidants can delay the progress of degenerative diseases. Antioxidants can act by scavenging reactive oxygen species (SOD removing O2-), by inhibiting their formation (by blocking activation of phagocytes), by binding transition metal ions and preventing formation of OH and/or decomposition of lipid hydroperoxides by repairing damage (alpha-tocopherols repairing peroxyl radicals so terminating the chain lipid peroxidation) or in combination of any of the above (Niwa et al., 2001). Human body can produce few antioxidants especially many enzymatic systems (superoxide dismutase, catalase and glutathione peroxidase) and can obtain some low molecular weight antioxidants from foods, in particular vegetables and fruits (vitamin C, vitamin E, carotenoids and phenolic compounds), which can scavenge some reactive species (superoxide anion and hydrogen peroxide) and inhibit a chain reaction. Consumption of foods naturally bearing antioxidant power is the most efficient way of combating such undesired transformations and health risks. Consequently, the opportunity for improving health by improving diet is great (Ames et al., 1993).
1 Materials and Methods
1.1 Basic experimental materials
Experimental materials for the study comprised of ten varieties of five brassica vegetables out of which four varieties were of cabbage namely Golden Acre, Pusa Cabbage-1, KTCBH-84 and Quisto, two each of cauliflower (Early Snowball and Pusa Himjyoti) and kohlrabi (Pusa Virat and KKS-2) and one each of kale (KTK-64) and sprouting broccoli (Pusa Broccoli KTS-1).
1.2 Field experiment
One month old seedlings were transplanted during 2013-14 at Naggar research farm of IARI Regional Station, Katrain, Kullu, HP, India. The farm is located at 32.120N latitude and 77.130E longitude with an altitude of 1688 m above the mean sea level, and receives an average annual rainfall and snowfall of 100–110 cm and 110–130 cm, respectively. The plot size was 3 m x 3 m with inter-and intra-row spacing of 45 cm. Each variety was replicated three times in a randomized block design (CRBD). The crop was uniformly fertilized with recommended doses of chemical fertilizers @120 kg N, 60 kg P2O5 and 50 kg K2O per ha supplied as urea, single super phosphate and murate of potash, respectively. Half dose of N and full doses of P2O5 and K2O were applied as basal dressings at the time of transplanting while remaining half dose of N was top-dressed at 30 days after transplanting.
1.3 Sampling and laboratory analysis
True-to-type representatives (5 heads/curds/knobs/ leaves) of each variety of the five brassica vegetables in each replication were harvested at marketable stage. These were chopped, homogenized and a fresh sample of 5 g of each was stored immediately under refrigerated conditions (-20℃) until assay. Ethanol extract was prepared from the homogenized 5 g sample in 15 ml absolute ethanol. Then it was centrifuged at 10,000 rpm for 15 min at 4℃. The supernatant was stored at -20℃.
1.4 Anti-oxidant capacities and total phenolics assay
The method described by Apak et al. (2006) was followed with minor modifications. For cupric ion reducing antioxidant capacity (CUPRAC) analysis, 100µl sample was mixed with 4 ml of CUPRAC reagent (1 ml neucuproine, 1 ml ammonium acetate, 1ml CuCl2 and 1 ml of distilled water; pH 7.4). Absorbance was recorded 450 nm in spectrophotometer. The results were expressed as µ mol trolox/g. The ferric reducing ability of plasma (FRAP) assay was performed based on the procedure described by Benzie and Strain (1996) with slight modifications. In this assay, 100 µl of the diluted sample were added to 3 ml of the FRAP reagent and the reaction was monitored after 4 min at 593 nm. The results were expressed as µmol Fe(II)/g fresh weight (FW) of vegetable. Total phenolic contents were determined with Folin–Ciocalteu method (Singleton and Rossi 1965). Briefly, 0.50 ml extract was mixed with 2.5 ml of 1:10 diluted Folin–Ciocalteu reagent. After 4 min, 2 ml of saturated sodium carbonate solution was added. The mixture was incubated in dark for 2 h at room temperature and its absorbance was detected at 760 nm. Gallic acid was used for calibration, and the results were expressed as mg of gallic acid equivalents (mg GAE) per 100 g fresh weight (FW) of vegetable.
2 Plant pigments and ascorbic acid content
2.1 Anthocyanin
10 g of sample was blend with 10 ml of ethanolic HCl with the help of pestle and mortar and transferred in 50 ml conical flask by using 10 ml ethanolic HCl for washing. Solution is stored overnight at 40C. Then the solution was filtered with Whatman No. 1 filter paper. Final volume was made upto 100 ml and stored in dark for 2 hours. O.D. was taken at 535 nm (S. Ranganna, 2008).
Total Anthocyanin (mg/100 g) =

Where e = 98.2 (absorbance of a solution containing 0.1 mg/ml anthocyanin)
2.2 Carotenoids
5 g of sample was taken and crushed with acetone until the residue became colorless. Filtrate was transferred in the separating funnel containing 20 ml of petroleum ether. 2-3 drops of 5% sodium sulphate were added in separating funnel. Then 20 ml of petroleum ether was added to make 2 separate phases. Lower phase was re-extracted with additional petroleum ether till it become colourless. Final volume was made up to 50 ml and absorbance was taken at 503nm and 452nm using petroleum ether as a blank (S. Ranganna, 2008).
2.3 Lycopene
Absorbance (1 unit) = 3.1206 µg Lycopene/ml
Lycopene (mg in 100 g sample) =

2.4 Total Carotenoids
Total carotenoids (mg/100g) =



2.5 β-Carotene
µg/100 g =
2.6 Ascorbic acid
10 g of sample was crushed with 4% oxalic acid and volume made up to 100 ml. Then sample was filtered wit Whatman filter paper No. 1 and filtrate was used for further analysis. 10 ml of the prepared sample was titrated against the 2,4-dichlorophenolindophenol dye till the faint pink color was appeared (S. Ranganna, 2008).
Ascorbic acid (mg/100g of sample) =


2.7 Statistical analysis
The analysis of variance of the data was carried out following Panse and Sukhatme (1967). The variability estimates (genotypic, phenotypic and environmental variance; and genotypic and phenotypic coefficient of variation) were worked out through analysis of variance (ANOVA), while correlation coefficients were determined by covariance and variance between the traits. D2 analysis was performed following Mahalanobis (1936).
3 Results and Discussion
3.1 Comparative analysis
Analysis of variance revealed highly significant differences among the genotypes for all the 8 phytochemicals under study (data not presented). The comparative analysis of ten varieties representing 5 different brassica vegetables for antioxidant capacities, plant pigments and total phenolics as presented in Table 1 revealed that sprouting broccoli cultivar Pusa Broccoli KTS-1 exhibited highest contents of antioxidants viz., CUPRAC (17.91 µ mol trolox/g) and FRAP (14.34 µ mol trolox/g) whereas highest contents of total phenolics (6120.0 µg gallic acid/gfw), anthocyanin (3.28 mg/100 g), ascorbic acid (mg/100g of sample), lycopene (8.02 mg in 100 g sample), total carotenoids (52.82 mg/100g) and β–carotene (11.32 µg/100 g) recorded in kale cultivar KTK-64. Superiority of kale in terms of total carotenoids and polyphenols has also been reported by Kaulmann et al. (2014).


Table 1 Mean performance of ten varieties of 5 Brassica vegetables for 8 phytochemicals

3.2 Genetic variance
The variance and coefficient of variation provide the extent of variability present among germplasm which is pre-requisite of any breeding/improvement programme. The extent of variability in (Table 2) present among different culivars was estimated in terms of phenotypic and genotypioc (Vp and Vg) and phenotypic and genotypic coefficients of variation. The Vg was highest in total phenolics content followed by ascorbic acid, total carotenoids, CUPRAC, FRAP, β-carotene, lycopene and anthocyanin. The magnitude of phenotypic coefficient of variation was slightly higher than the corresponding genotypic coefficient of variation for all the 8 traits. The respective phenotypic coefficient of variation and genotypic coefficient of variation were high for total carotenoids (229.23 and 229.14%) and followed by lycopene (189.71 and 178.27%), while it was lowest for ascorbic acid (47.99 and 47.82%). Heritable portion of variation can be explained by computing the heritability. High broad sense heritability (>80.0%) estimates were computed for all eight antioxidant related activities, i.e. CUPRAC (99.2%), FRAP (99.9%), total phenolics (99.4%), anthocyanin (99.1%), ascorbic acid (99.3%), lycopene (88.2%), total carotenoids (99.9%) and β-carotene (99.8%) indicating least effect of the environment in the expression of these traits. Genetic advance expressed as per cent of mean was observed in the range of 97.9% to 471.7% with a maximum for total carotenoids (471.7%) followed by lycopene (344.7%) and anthocyanin (309.3%). High genetic advance coupled with high heritability observed for all the traits is attributed to considerable additive genetic effects (Panse and Sukhatme, 1957) and selection for these traits will be most effective.


Table 2 Estimates of variance, coefficient of variation, heritability and genetic advance for CUPRAC, FRAP, anthocyanin, ascorbic acid, total phenolics and carotenoids contents in 5 brassica vegetables

3.3 Correlation analysis
Correlation refers to magnitude and direction of association between two traits, and determines the component traits on which effective selection can be made. The genotypic coefficients of correlation were in general higher in magnitude than the corresponding phenotypic ones (Table 3). The antioxidant CUPRAC has shown significant and positive correlation at both genotypic and phenotypic levels with all the other 7 traits whereas FRAP was significantly correlated with total phenolics and anthocyanin only as also reported by Kaulmann et al. (2014). Total phenolics was positively correlated with anthocyanins, ascorbic acid and the 3 carotenoids. Similarly, anthocyanin contents showed positive correlation with ascorbic acid and carotenoids. Ascorbic acid also showed positive association with the carotenoids. The 3 carotenoids (lycopene, total carotenoids and β- carotene) were in positive association with each other.


Table 3 Estimates of correlation coefficients (Genotypic and Phenotypic) for antioxidant enzymes among ten varieties of 5 different brassica vegetables

3.4 Grouping of Genotypes into Various Clusters based on D2 analysis
Based on D2 analysis the 10 genotypes were classified into 4 clusters (Table 4). The cluster IV had maximum 4 genotypes and cluster II had only 1 genotype. Intra cluster distance was highest in cluster I with 2 genotypes and lowest in cluster II (Table 5). Highest inter cluster distance, however, was between III vs I (22828289.4607) followed by the same IV vs I (18068045.8627), II vs I (11447292.3664), II vs I (4120162.0206). The lowest inter cluster distance was between IV vs IV (29853.9254) followed by III vs III (115834.9027), III vs IV (340204.5986). According to the Arunachalam et al. (1984), the optimum level of genetic divergence between the parents gives the best heterosis and better segregants. The study of cluster mean value of 4 clusters indicated considerable differences for the character studied (Table 6). A dendrogram constructed based on the hierarchical cluster analysis has been shown as Figure 1. The variation observed in cluster means also point out the degree of variability. The characters viz., CUPRAC, FRAP, total phenolics, anthocyanin, ascorbic acid, lycopene, total carotenoids and β-carotene were highest in cluster I.


Table 4 Cluster classification of 10 genotypes cabbage, cauliflower, knol-khol, broccoli and kale on D2 analysis


Table 5 Inter and Intra –cluster distances among 4 clusters based on D2 analysis


Table 6 Cluster means of 10 genotypes for 8 characters based on D2 analysis


Figure 1 Dendrogram based on the hierarchical cluster analysis

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