Chemical composition of extracts
The compounds present in the broccoli, kale, and cauliflower extracts are shown in Supplementary material 2. Through analysis of variance (ANOVA), it was found that there were statistically significant differences (p > 0.05) between the extracts. The broccoli and kale extracts showed 3 glucosinolates in their composition, while in the cauliflower extract 4 glucosinolates were found. This result is important, as these compounds have several benefits to human health.
Analyzing the broccoli extract (Supplementary Material 2), significant differences were observed (p > 0.05) in relation to the concentrations of all compounds, being neoglucobrassicin, 5-Caffeoylquinic acid, and 1-O-sinapoyl-ß-D-glucose those with the highest concentration, while 4 -Mercaptobutyl glucosinolate, Kaempferol-triglucoside, Quercetin 3–(2-feruloylsophoroside)7-glucoside, Kaempferol-3-O-caffeoyldiglucoside-7-O-glucoside, p-Coumaric acid 4-glucoside, Kaempferol-3-Osininiglide 7-O-glucoside, Kaempferol-3-O-feruloyldiglucoside-7-O-glucoside, 4-p-Coumaroylquinic acid and Rutin which are in a lower concentration.
Statistically significant differences (p > 0.05) were observed between the compounds present in the kale extract (Supplementary material 2), with 5-Caffeoylquinic acid being the compound with the highest concentration, and 4-Mercaptobutyl glucosinolate, p-Coumaric acid 4-glucoside, Coumaric acid, Sinapic acid, and Ferulic acid in a lower concentration.
Cauliflower extract (Supplementary Material 2), in the same way as the others, showed a statistically significant difference (p > 0.05) between all the compounds present, with glucobrassicin being the major compound and 5-Caffeoylquinic acid, Kaempferol-triglucoside, 4-Hydroxybenzoic acid glucoside, and Quercetin 3-(2-feruloylsophoroside) 7-glucoside minorities.
In broccoli and kale extracts were found glucobrassicin, neoglucobrassicin, and 4-methoxyglucobrassicin, which are predominant glucosinolates, along with glucoraphanin and glucoiberin. Cauliflower extract contains glucobrassicin, neoglucobrassicin, 4-methoxyglucobrassicin, and 4-Mercaptobutyl glucosinolate, the first three are reported in the literature along with glucoiberin and sinigrin [18]. Among Brassicas, broccoli had the highest content of 4-methoxyglucobrassicin and neoglucobrassicin and the second-highest concentration of glucobrassicin, while kale extract had the lowest glucosinolate content.
The analysis of chemical data using the principal component analysis (PCA) technique allowed the compounds to be chemically grouped into groups, to express and show their similarities and differences. The data for the broccoli, kale, and cauliflower extracts represented by two axes present 99.59% of the total accumulated variance (Factor 1: 65.15% - factorial plane 1; Factor 2: 34.44% - factorial plane 2) (Fig. 1). It is observed that the extracts of broccoli and kale showed greater similarity to each other, due to the presence of 5-caffeoylquinic, glucobrassicin, and protocatechuic acid 4-glucoside, while the cauliflower extract showed a greater difference from the others.
Studies aiming at extracting glucosinolates from Brassicas use methanol as a solvent, however, due to the toxicity of this solvent that would make it impossible to apply the extracts in food matrices, it was decided to use non-toxic solvents. The differences between the profile and quantification of the compounds when compared with the literature, possibly due to the characteristics of the extraction solvent, as well as the associated edaphoclimatic characteristics and genetic factors [19].
The Brassicas extracts are also rich in phenolic compounds and organic acids, highlighting the high content of 5-caffeoylquinic acid in kale extract, which has an effect against cardiovascular disease by inhibiting enzyme expression of the cyclooxygenase which in turn suppresses p-selectin expression and acts to reduce the inflammatory process by suppressing interleukin and tumor necrosis factor (TNF-α) expression [20]. The cinnamic acid derivatives, such as 5-caffeoylquinic acid, high quantity in broccoli and kale, are good scavengers of peroxide free radicals [21].
According to studies, the compounds that stand out in brassicas are quercetin O-glycosides, kaempferol, ferulic acid, chlorogenic acid, gallic acid, caffeic acid, p-coumaric acid, and synapic acid, almost all found in our study [22, 23]. The presence of 1,2-Disinapoylgentiobiose and 1,2,2'-Trisinapoylgentiobiose, characteristic of Brassicas are also observed [23, 24].
It is noteworthy that the compounds found in Brassicas have several health benefits, such as antioxidant, antimicrobial, antihyperglycemic, anti-tumor, antihypertensive and anti-inflammatory activity and can be applied in the food and pharmaceutical industry [3, 25, 26]. The Phenolic and glucosinolates compound content (µg/g) in broccoli, kale, and cauliflower extracts shows in the Supplementary Material 2.
Antioxidant activity
The broccoli, kale, and cauliflower extracts showed high antioxidant activity compared to the DPPH radical at a concentration of 200mg/mL (Table 1). For the hydroxyl radical, moderate activities of broccoli and cauliflower extracts were observed, and the absence of activity of kale extract. The nitric oxide radical was moderately inhibited by the kale extract and low by the broccoli and cauliflower extracts at the tested concentration. The literature report results of inhibition of the DPPH radical for ethanolic and aqueous extracts of broccoli and cauliflower, ranging from 15.5 to 63.9% and between 3.7 to 65.5%, respectively [27–29]. These results are inferior to those found in our research, possibly due to edaphoclimatic factors, which directly influence the composition of plants, impacting their biological effects [4].
Cauliflower demonstrated the highest phenolic content with the strongest DPPH radical scavenging activity, ferric reducing antioxidant power and oxygen radical absorbance capacity when compared to Cabbage and Chinese cabbage, all of which also demonstrated properties to protect against glycation and protein oxidation. mediated by the glycation reaction in conditions involving the prevention of diabetic complications [30].
The ability to inhibit DPPH radical by the kale extract was slightly higher than the value found in the study by Armesto, Gómez-Limia, Carballo, and Martínez [31], who found 84.8% inhibition in fresh Spanish kale samples. Although the kale varieties in this research our study are different, it is observed that, differently from broccoli and cauliflower samples, the genotype does not influence so much the inhibition capacity of this radical.
Table 1
Antioxidant activity of broccoli, kale and cauliflower extracts against 2,2-diphenyl-1-picrylhydrazyl, hydroxyl and nitric oxide radicals.
| Extracts | Inhibition (%) |
| DPPH | Hydroxyl | Nitric oxide |
| Broccoli | 75.5cA | 54.8bB | 13.1bC |
| Kale | 89.2aA | Nd | 40.1aB |
| Cauliflower | 88.4bA | 62.8aB | 10.0cC |
DPPH − 2,2-diphenyl-1-picrylhydrazyl; Nd – not detected; Different lowercase letters in the same column differ statistically from each other by the Tukey test with 5% significance; Different capital letters on the same line differ statistically from each other by the Tukey test with 5% significance.
No data were found in the literature evaluating the inhibition of hydroxyl radicals and nitric oxide by extracts of broccoli, kale, and cauliflower, but it should be noted that these extracts showed a strong ability to inhibit the hydroxyl radical, the most produced reactive oxygen species in the body. Despite the low inhibition of the nitric oxide radical at the tested concentration, our study demonstrates that possibly at higher concentrations, the extracts can have a more positive effect on the inhibition of this reactive oxygen species. The inhibition of free radicals such as hydroxyl and nitric oxide is extremely important, as they are responsible for several damages to the human organism, such as lipoperoxidation and changes in DNA [32, 33].
Disk diffusion
The main bacteria that cause foodborne diseases in Brazil, between 2009 and 2018, were E. coli, Salmonella sp, and S. aureus, representing 44.1% of all reported cases [34]. Although L. monocytogenes does not appear on the list of 10 microorganisms with the highest number of notifications, this bacterium is of great importance because it has a high mortality rate [35].
The presence and diameter of the inhibition halos indicate the susceptibility of the microorganisms to the extracts, where halos smaller than 0.7 cm are considered non-active against bacteria, between 0.7 cm and 1.1 cm are active halos and halos greater than 1.2 cm are considered to have a satisfactory inhibitory effect [36].
The broccoli extract showed active halos against the bacteria S. Typhimurium, S. aureus, and E. coli, not effect L. monocytogenes. The kale extract showed active halos only for S. Typhimurium and S. aureus, and, finally, the cauliflower extract showed a satisfactory inhibitory effect against L. monocytogenes, active halos against S. aureus and E. coli, and no effect against S. Typhimurium (Table 2).
The diameter of the inhibition halos of S. aureus by the broccoli extract was greater than those reported by [36]who evaluated the antimicrobial potential of broccoli extracts produced with different solvents, and found halos of 0.6 cm for S. aureus when using ethanol and did not observe halos when the aqueous extract was applied. For E. coli and S. Typhimurium, the authors did not observe inhibition halos either using ethanol or using water as an extraction solvent.
Sousa et al. [37] evaluated the antimicrobial potential of Brassicas extracts, found zones of inhibition varying between 0.6 and 0.9 cm for samples of kale against S. aureus, a result similar to that found in our study. The antimicrobial activity of aqueous extracts and methanol-water of Brassicas (broccoli, kale, and cauliflower) against the microorganisms S. aureus, E. coli, S. Typhimurium, and L. monocytogenes, were in the aqueous extracts and water-methanol from kale obtained inhibition halos of 2.02 and 1.17 cm, respectively, against the microorganism E.coli, halos of 1.31 and 1.13 cm, for L. monocytogenes, and 2.04 and 1.24 cm halos, for S. Typhimurium [38]. The results found by Hu et al were superior to those found in this study, which is probably due to the solvents used in the extraction.
Table 2
Zone of inhibition of broccoli, kale and cauliflower extracts against the bacteria Salmonella Typhimurium, Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli
| Bacteria | Zone of inhibition (cm) |
| Extract |
| Broccoli | Kale | Cauliflower |
| Salmonella Typhimurium | 0.90aA | 0.78bB | Nd |
| Listeria monocytogenes | Nd | Nd | 1.30Aa |
| Staphylococcus aureus | 0.77cB | 0.86aA | 0.77Cb |
| Escherichia coli | 0.82bB | Nd | 0.90Ba |
Nd – not detected; Different lowercase letters in the same column differ statistically from each other by the Tukey test with 5% significance; Different capital letters on the same line differ statistically from each other by the Tukey test with 5% significance.
Prasad et al. [39] evaluated the antimicrobial potential of three important brassicas and found zones of inhibition by aqueous cauliflower extract ranging between 0.8 and 1 cm for E. coli, 1 cm for S. aureus, and on average 0.5 cm for S. Typhimurium. These results were similar in our research for the inhibition of E. coli, an important gram-negative bacterium that causes several cases of foodborne diseases in the world, while our results were inferior for S. aureus and S. Typhimurium. Possibly, these differences among the diameters of the inhibition halos occur due to the edaphoclimatic factors where the samples were grown. The cauliflower extract showed excellent zones of inhibition of L. monocytogenes, a gram-positive bacterium extremely resistant to temperature variations that can cause a disease called listeriosis, which can cause abortion in pregnant women and lead to severe septicemia and meningitis in susceptible individuals [40].
Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
Duarte et al. [41] classified the effect of plant extracts and essential oils when evaluated by MIC, where an extract that has MIC concentrations up to 0.5 mg/mL is considered a strong antimicrobial agent; between 0.6 to 1.5 mg/mL had a moderate effect and above 1.6 mg/mL it had a weak activity.
The kale extract showed an inhibitory effect against gram-positive bacteria at a concentration of 200 mg/mL, but it was not effective for gram-negative bacteria, possibly due to the greater resistance of these microorganisms that have an external phospholipid membrane that makes the wall most waterproof cell [42]. The cauliflower extract inhibited the growth of only S. aureus at a concentration of 100 mg/mL, while the broccoli extract at the tested concentrations did not affect any of the tested bacteria. None of the extracts had a bactericidal effect at the tested concentrations (Table 3).
The inhibitory effects of the growth of L. monocytogenes and S. aureus by the kale extract at a concentration of 200 mg/mL and the inhibition of S. aureus by the cauliflower extract at a concentration of 100 mg/mL demonstrate the potential of these extracts in the control of pathogenic microorganisms that cause foodborne diseases. Based on these results, these extracts can be applied in the industry for partial or total replacement of synthetic chemical preservatives after in situ studies in food.
Table 3
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of broccoli, kale and cauliflower extracts against bacteria Salmonella Typhimurium, Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli
| Bacteria | Extract concentrations (mg/mL) |
| MIC | MBC |
| Broccoli | Kale | Cauliflower | Broccoli | Kale | Cauliflower |
| Salmonella Typhimurium | Nd | Nd | Nd | Nd | Nd | Nd |
| Listeria monocytogenes | Nd | 200 | Nd | Nd | Nd | Nd |
| Staphylococcus aureus | Nd | 200 | 100 | Nd | Nd | Nd |
| Escherichia coli | Nd | Nd | Nd | Nd | Nd | Nd |
Nd: not detected; MIC: Minimum Inhibitory Concentration; MBC: Minimum Bactericidal Concentration.
None of the extracts had a bactericidal effect against the tested bacteria at the concentrations evaluated, although it promoted growth inhibition of these microorganisms, possibly the minimum bactericidal concentration of these extracts is absent, or higher than the tested concentrations.
The absence of inhibition and bacterial death of S. Typhimurium, L. monocytogenes, and E. coli by the action of broccoli extract has also been observed in the literature, while there are reports of bactericidal effect of S. aureus at concentrations of 0.32 mg/mL [37]. The results of bacteria inhibition by the kale extract were lower than those reported in the study by Sousa et al.[38], who observed growth inhibition of S. aureus at a concentration of 0.1 mg/mL. No research was found that evaluated the inhibitory and bactericidal potential due to the action of cauliflower extract. Probably these differences between concentrations with a bactericidal effect are due to edaphoclimatic factors, as well as differences between solvents and methods of extracting the compounds [4].
In conclusion, the broccoli, kale, and cauliflower extracts showed in their composition important glucosinolates and phenolic compounds that have biological activities in the human organism. High antioxidant activities were observed in vitro against the DPPH radical and moderate activities against the hydroxyl and nitric oxide radicals in all extracts, evidencing their possible application as antioxidant agents. Regarding the in vitro antimicrobial potential, although the tested concentrations do not promote bacterial death, the susceptibility of the microorganisms to the extracts by the analysis of diffusion disc and minimum inhibitory concentration stands out. Therefore, further studies are needed evaluating the potential in situ so that in the future these extracts can be applied to food. It is also considered that these extracts undergo complete and comprehensive investigations of their metabolic action in the body with in vivo studies or their action after passing through the digestive process to verify bioavailability.