Context: There have been no reports of the extraction of essential oil (EO) from white cabbage [Brassica oleracea L. var. capitata (L.) Alef. f. alba DC. (Brassicaceae)] (Bocfal) or its chemical composition, antioxidant activity, or hepatoprotective effects. Objective: To extract Bocfal EO, to identify and quantify its chemical components, to assess their antioxidant capacity, and to evaluate the hepatoprotective properties of Bocfal EO. Materials and methods: Bocfal EO was obtained using hydrodistillation (200 mm Hg/58 °C). The chemical composition was analyzed using GC-MS and was quantified using GC-FID. The antioxidant activity of Bocfal EO and its main constituents was evaluated using TBARS in rat brain homogenates. A Bocfal EO hepatoprotective effect (192 mg/kg) on acute carbon tetrachloride (CT)-induced liver damage was determined in rats using biochemical markers and histological analysis. Diallyl disulphide (DADS) (1 mmol/kg) was used as a control for comparison. Results: Bocfal EO contained organic polysulphides (OPSs), such as dimethyl trisulphide (DMTS) 65.43 ± 4.92% and dimethyl disulphide (DMDS) 19.29 ± 2.16% as major constituents. Bocfal EO and DMTS were found to be potent TBARS inhibitors with IC50 values of 0.51 and 3 mg/L, respectively. Bocfal EO demonstrated better hepatoprotective properties than did DADS (p < 0.05), although both slightly affected the hepatic parenchyma per se, as observed using histopathology. Discussion and conclusion: The antioxidant properties of Bocfal EO and DMTS may be the mechanism of hepatoprotective action; the parenchymal disturbances by Bocfal EO or DADS alone may be related to the high doses used.
Keywords: Brassica oleracea var. capitata f. alba; organic polysulphides; hepatoprotection
The liver is one of the largest internal organs in the human body, and it controls the flow and safety of substances absorbed from the digestive system into the systemic circulation. The main liver functions include the production of energy by protein, carbohydrate and lipid metabolism, the synthesis of bile salts, the storage of vitamins, and the production of lipoproteins, angiotensin and coagulation factors among others. Therefore, hepatic diseases such as fatty liver disease, hepatitis and cirrhosis highly impact the homeostasis of the body (Arias et al. [
The research on the aromas from cruciferous vegetables (i.e., cabbages and related vegetables, Brassica oleracea Linn. spp.) and, in consequence, their bioactive compounds, represents a real challenge because of the scarce yields of these aroma compounds, which have been described to contain isothiocyanates (ITCs) and organic polysulphides (OPSs) as major components (Stoewsand [
Currently, there are no published reports concerning the extraction, identification and quantification of the chemical constituents of white cabbage essential oil (B. oleracea var. capitata f. alba, Bocfal EO), its antioxidant capacity or its hepatoprotective activity. Therefore, the aim of this study was to obtain Bocfal EO via hydrodistillation (at low pressure/temperature), to identify and quantify its chemical constituents (OPSs content), to assess their antioxidant capacity through an in vitro assay, and to evaluate Bocfal EO hepatoprotective properties by comparing them with those of DADS in an acute, CT-induced liver damage rat model.
All reagents were of analytical grade. Dichloromethane of chromatographic grade and diallyl disulphide (DADS) were purchased from Sigma-Aldrich (St Louis, MO). Dimethyl trisulphide (DMTS) and dimethyl disulphide (DMDS) (purity 98%) were supplied by Natural Advantage (Oakdale, LA).
The aerial parts of fresh Bocfal were collected in parcels at the 'Ejido de San Vicente Chicoloapan, Estado de México' in August 2015. The Bocfal showed edible maturity and was harvested within 24 h period. The identity of the plant was confirmed by Dr N. Ivalú Cacho and a voucher specimen (1420213MEXU) was deposited at the Herbario Nacional de México (MEXU).
Distillation is the traditional method for EO extraction (Amorati et al. [
GC-MS analyses of the EO samples was performed using an AutoSystem XL-GC equipped with a polar capillary column coated with free fatty acid phase (HP-FFAP, 30 m × 0.32 mm ID ×0.25 μm film thickness) and coupled to a selective quadrupole TurboMass-MS detector with an electron impact ionization system at 70 eV and 215 °C (Perkin-Elmer, Norwalk, CT). The oven temperature started at 50 °C and was gradually increased up to 130 °C at a rate of 6 °C/min, where it was maintained for 3 min. A second program was used, where the temperature was increased to 200 °C at a rate of 8 °C/min, where it was maintained for 8 min. The injector temperature was 180 °C, and the flow rate of helium was 1.0 mL/min at 8 psi. The EO samples were diluted (1.0 μL) in dichloromethane 1:25 v/v, and they were then manually injected in split mode.
The linear retention indices (RI) of the volatile compounds were calculated with a series of n-alkanes (C
The quantification of the major EO constituents was performed using a GC-FID on an Agilent Technologies 6890 plus GC (Agilent Technologies, Santa Clara, CA) equipped with a single injector, the same HP-FFAP polar capillary column was used. The flow of the carrier gas (N
Male Wistar rats weighing approximately 180–200 g were housed in standard plastic cages at a temperature of 22–24 °C, under a 12 h light–dark cycle. They had free access to food (standard Purina chow diet, USA) and purified water. All the animals received humane care according to the respective institutional guidelines, the Mexican Official Norm (NOM-062-ZOO-999) regarding technical specifications for the production, care and use of laboratory animals, and the criteria outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health 1985).
The capacity to inhibit the products of lipid peroxidation was determined using thiobarbituric acid reactive substance (TBARS) quantification in homogenized rat brain samples according to the procedure described in the literature, with minor modifications (Ohkawa et al. [
Lipid peroxidation was initiated by the addition of 50 μL of 100 μM FeSO
IR (%) = [(C–E)/C] × 100, where C is the absorbance of the control sample, and E is the absorbance of the test sample.
To assess the hepatoprotective properties of Bocfal EO on CT-induced liver damage, the rats were divided into six groups with n = 7 animals in each group. The first group was the normal control group (VE + VCT) that received two p.o. doses that were 12 h apart of 1 mL of olive oil as the vehicle (VE) for the Bocfal EO or DADS, which was used as a positive control because its hepatoprotective properties have been previously demonstrated and because it is a sulfane sulphur, or OPS, compound. In addition, 2 h after the first administration of VE, the rats were orally given 1 mL of mineral oil as the vehicle for CT (VCT). All the remaining groups underwent the same administration schedule. The second (Bocfal EO + VCT) and third (DADS + VCT) groups received Bocfal EO and DADS, respectively, twice orally dissolved in their vehicles and also received the VTC p.o.; thus, both were control groups intended to show any adverse effects of Bocfal EO or DADS per se on normal rats. The fourth group was the damaged control (VE + CT), wherein rats received the VE twice orally and a single dose of 4 g/kg CT dissolved in its vehicle (1:1 v/v). Finally, the fifth (Bocfal EO + CT) and sixth (DADS + CT) groups were administered their respective substances and were injured by CT.
Regarding the Bocfal EO dose, it was considered an amount of EO that contained 1 mmol/kg of DMTS (192 mg/kg of EO), which was later determined to be its main constituent at 65.43 ± 4.92%, as well as 1 mmol/kg of DADS (146 mg/kg). These doses were based on a previous report wherein this moderate dose of pure OPSs, including DADS, was used in Wistar rats (Siess et al. [
All the animals were sacrificed by exsanguination under light ether anaesthesia 24 h after CT administration; hence, blood was collected by cardiac puncture using a syringe containing sodium heparin as an anticoagulant. The liver was rapidly removed and rinsed in saline. All the samples were either kept on ice for immediate use or frozen at −70 °C until analyzed.
Plasma was obtained for the determination of the canalicular membrane enzyme activities of cholestasis markers, such as alkaline phosphatase (AP) and γ-glutamyl transpeptidase (GGTP), and for the cytosolic activity of the necrosis indicator alanine aminotransferase (ALT) (Fernández-Martínez et al. [
Small liver pieces (0.5 g) were separated for glycogen measurement using anthrone–sulphuric acid reagent (Seifter, et al. [
The extent of lipid peroxidation (LP) was estimated in the liver homogenates by measuring malondialdehyde (MDA) formation using the thiobarbituric acid method (Ohkawa et al. [
For hepatic catalase (CAT) activity, H
Liver samples were taken from all the animals and fixed with 10% formaldehyde in phosphate-buffered-saline for 24 h. Then, they were washed with tap water, dehydrated in alcohols and embedded in paraffin. Sections of 6–7 μm were mounted on glass slides covered with silane; the paraffin was previously eliminated. They consequently were used for haematoxylin/eosin staining for histological examinations using light microscopy.
TBARS data underwent a one-way variance analysis (ANOVA), and the significant differences were obtained using Dunnett's test. For the hepatoprotective assessment, an ANOVA with the Student–Newman–Keuls test was used to compare groups. The resulting data are expressed as the means ± SEM and were analyzed using Sigma Stat software version 3.1 (Systat Software Inc., San Jose, CA). In all cases, a difference was considered significant when p < 0.05.
The Bocfal EO extraction yield was 37.56 ± 3.71 mg/kg (33.33 ± 3.29 μL/kg) (n = 3). The extraction process was repeated until the amount of EO was enough to carry out the biological tests. The GC-MS permitted the identification of 28 volatile compounds in the Bocfal EO, accounting for approximately 99% of the total amount (Table 1). They were categorized into six chemical families, which are listed according to their relative abundance. The first family includes OPS compounds (R-S
Table 1. Major components of B. oleracea var. capitata F. alba essential oil (Bocfal EO) obtained by hydrodistillation at low pressure (200–150 mmHg) and low temperature (55–58 °C) conditions.
Peak(#) RT (Min) RI Compound Content (%) (1) 2.26 1224 Dimethyl disulphide (DMDS) 19.29 ± 2.16 (11) 6.24 1544 Dimethyl trisulphide (DMTS) 65.43 ± 4.92 (13) 7.12 1595 Methyl pentyl disulphide 0.19 ± 0.04 (17) 10.07 1750 Allyl methyl trisulphide 0.22 ± 0.07 (19) 11.39 1818 Methyl methylthiomethyl disulphide 1.39 ± 0.24 (20) 12.89 1893 Dimethyl tetrasulphide 1.50 ± 1.01 (10) 5.94 1527 Allyl isothiocyanate (AITC) 4.31 ± 0.70 (14) 7.59 1621 3-Butenyl isothiocyanate 0.85 ± 0.16 (22) 17.64 2109 Indole 0.10 ± 0.01 (23) 18.04 2125 1-Methoxyindole 0.01 ± 0.00 (24) 19.38 2181 3-(Methylthio)propyl isothiocyanate 0.47 ± 0.13 (26) 25.62 2511 Butyl isothiocyanate 0.19 ± 0.05 (28) 27.67 2731 Phenylethyl isothiocyanate 0.61 ± 0.15 (3) 3.21 1336 3-Butenenitrile 1.50 ± 0.23 (6) 4.03 1415 4-Methylpentanenitrile 0.16 ± 0.03 (7) 4.48 1441 2-Pentenenitrile 0.41 ± 0.06 (8) 4.83 1462 Hexanenitrile 0.14 ± 0.01 (25) 22.04 2352 Benzene;propanenitrile 0.08 ± 0.02 (2) 2.94 1306 Penten-3-ol 0.51 ± 0.07 (5) 3.84 1404 2-Pentenol 0.23 ± 0.04 (9) 5.76 1516 1-Hexanol 1.32 ± 0.34 (12) 6.73 1556 2-Hexen1-ol 0.39 ± 0.10 (4) 3.67 1388 2-Hexenal 0.30 ± 0.05 (18) 10.71 1783 Heptanal 0.07 ± 0.01 (21) 14.43 1970 2,4-Decadienal 0.05 ± 0.02 (15) 8.00 1642 3,3-Dimethylallylbromide 0.23 ± 0.03 (16) 8.60 1673 Camphor 0.04 ± 0.01 (27) 26.93 2601 Eugenol 0.03 ± 0.02
1 RI: retention index; RT: retention time.
The quantifications of DMTS, DMDS and AITC amounts, the three most abundant compounds in the Bocfal EO, were calculated using the linearity of the responses. This was conducted by preparing standard curves in a concentration range of pure volatile controls for comparison and using the equation from the linear regression obtained from each curve with the GC-FID equipment. The results indicate that each 1000 μL of Bocfal EO contains 575 ± 125 μL of DMTS, 483 ± 88 μL of DMDS and 50 ± 39 μL of AITC. The correlation coefficients of the three standard curves were values higher than 0.95.
Table 2 presents the antioxidant activity of Bocfal EO through a TBARS assay with rat brain homogenates; which showed an IC
Table 2. Antioxidant activity by TBARS assay.
Tested compound TBARS – IC50 (mg/L) Bocfal EO 0.51 ± 0.03 DMTS 3.00 ± 0.2 DMDS No activity AITC 3.16 ± 0.13 DADS 444.64 ± 49.57 BHT 0.27 ± 0.10 α-Tocopherol 2.92 ± 0.93
- 2 Results are expressed as the mean value ± SEM of experiments performed in duplicate with samples of rat brain homogenates from at least five animals.
- 3 Different from Bocfal EO, p < 0.05, ANOVA, Dunnett's test.
Figure 1 depicts the plasma enzyme activities of AP and GGTP as well as TB concentration. Due to CT-provoked injury, the AP activity was increased twofold in the damaged group (VE + CT) when compared with that of the normal control group (VE + VCT). In addition, the administration of Bocfal EO and DADS elevated that marker, though neither resulted in a significant difference in contrast with VE + VCT. However, the treatment with Bocfal EO significantly decreased the enzyme activity (p < 0.05) during the liver injury compared with VE + CT, which shows an anticholestatic effect. In addition, DADS did not modify the CT-induced AP elevation.
Graph: Figure 1. Plasma cholestasis markers: enzyme activities of alkaline phosphatase (AP) and γ-glutamyl transpeptidase (GGTP); total bilirubin determined in the plasma from control rats treated with their respective vehicles (VE + VTC), with Bocfal EO (Bocfal EO + VCT) or with DADS (DADS + VCT) as well as those administered the hepatotoxicant CT (VE + CT) but treated with the same compound, respectively (Bocfal EO + CT and DADS + CT). Each bar represents the mean value of experiments performed in duplicate with samples from at least four animals ± SEM. (a) Difference from the VE + VCT group and (b) from the VE + CT group, p < 0.05, Student–Newman–Keuls test.
GGTP enzyme activity was increased almost fivefold by CT administration (p < 0.05) with respect to that in VE + VTC control group; conversely, the Bocfal EO or DADS administrations slightly reduced the normal levels, but not in a significant manner. Regarding the CT-injured groups treated with the respective compounds, their GGTP activity increases were completely prevented; again, Bocfal EO was more effective as an anticholestatic agent than was DADS.
TB in the experimental groups showed a pattern very similar to that of AP because CT-induced liver damage increased the TB concentration, but in a statistically significant way; furthermore, the Bocfal EO and DADS control groups also exhibited a small degree of elevated TB concentration. However, although DADS lowered this marker, this change was not statistically significant. In contrast, Bocfal EO completely lowered the TB level (p < 0.05).
Figure 2 shows the markers of liver injury: ALT, glycogen, LP and CAT. ALT is a cytoplasmic enzyme marker of necrosis; its activity was slightly diminished by Bocfal EO but significantly lowered by DADS administration. As expected, CT induced a notable increment in ALT activity (p < 0.05), while treatments with both Bocfal EO and DADS abolished such elevation of activity, indicating their antinecrotic properties during oxidative damage, particularly for Bocfal EO.
Graph: Figure 2. Plasma and liver markers of necrosis, functionality and oxidative stress: alanine aminotransferase enzyme activity (ALT) determined in the plasma, hepatic levels of lipid peroxidation (LP) and glycogen, and catalase activity (CAT) quantified in the livers of control rats treated with their respective vehicles (VE + VTC), with Bocfal EO (Bocfal EO + VCT) or with DADS (DADS + VCT) as well as those administered the hepatotoxicant CT (VE + CT) but treated with the same compound, respectively (Bocfal EO + CT and DADS + CT). Each bar represents the mean value of experiments performed in duplicate with samples from at least four animals ± SEM. (a) Difference from the VE + VCT group and (b) from the VE + CT group, p < 0.05, Student–Newman–Keuls test.
The Bocfal EO + VCT and DADS + VCT groups showed normal levels of glycogen with respect to the control group, while the CT-damaged group exhibited depleted glycogen content (p < 0.05). However, Bocfal EO treatment completely prevented the depletion of glycogen; DADS did as well but to a much lesser extent.
LP represents the oxidative stress of cellular membranes. This hepatic indicator was augmented several-fold through CT administration in contrast with the VE + VCT and the control groups of Bocfal EO and DADS, which did not alter the normal level of LP per se. In contrast, Bocfal EO treatment partially prevented the CT-induced oxidative injury by lowering the LP level approximately 70%; in addition, DADS maintained this marker at normal levels.
The normal liver CAT activity was augmented by Bocfal EO administration; conversely, DADS administration lowered it, and both of these results were significant (p < 0.05). In addition, treatment with either Bocfal EO or DADS completely prevented the CT-induced CAT activity elevation in a similar way.
Liver damage was also evaluated by a histological approach through haematoxylin/eosin staining, which stains nuclei a black/dark blue colour and parenchymal hepatocyte cytoplasm a pink/magenta colour (Figure 3). A normal hepatic cell population and tissue homogeneity are shown in a representative liver sample from the VE + VCT normal control group (Figure 3(a)). In contrast, type CT-injured liver sample showed important damage zones, diffuse ballooning necrosis, pyknotic nuclei and high hepatic steatosis with hepatocyte vacuolization (Figure 3(b)). Bocfal EO administration provoked diffuse hepatocyte ballooning by itself (Figure 3(c)); thus, when Bocfal EO was administered concomitantly with CT, the liver presented an increased diffuse ballooning degeneration (Figure 3(d)). The treatment with the OPS standard resulted in a similar case, as the DADS + VCT group demonstrated focal swollen hepatocytes (Figure 3(e)). Furthermore, liver samples from animals damaged by CT and treated with DADS showed individual and zonal necrosis accompanied by high neutrophil infiltration (Figure 3(f)). Moreover, despite the apparent hepatoprotective properties, the mortality in groups treated with the substances was higher (up to 30%) than the regular mortality observed for the acute CT model was (less than 10%); thus, perhaps hepatoprotection was achieved in the stronger rats. Additionally, the administration of Bocfal EO or DADS caused weight loss (data not shown), piloerection and diminished activity in the rats.
Graph: Figure 3. Haematoxylin/eosin staining of representative liver sections from: (A) normal control rats administered vehicle (VE + VCT); (B) damaged control rats administered CT (VE + CT); (C) control rats administered Bocfal EO (Bocfal EO + VCT); (D) damaged rats administered Bocfal EO (Bocfal EO + CT); (E) control rats administered DADS (DADS + VCT) and (F) damaged rats administered DADS (DADS + CT). Central vein (CV), original magnification: 10×.
The Bocfal EO extraction yield was limited (37.56 ± 3.71 mg/kg), considering that there are few studies on the extraction of volatile compounds from Brassica spp. using a Likens–Nickerson-type apparatus for hydrodistillation (Hifnawy et al. [
The volatile compounds in Bocfal EO were identified and classified into six chemical families, listed according to their relative abundance. DMTS (65.43%) was the most abundant OPS, in agreement with many works on Brassica species (Maruyama [
The antioxidant activity of Bocfal EO assessed using a TBARS assay showed an antioxidant capacity very similar to that of the synthetic controls, BHT and α-tocopherol. Similarly, DMTS and AITC exhibited IC
DADS, diallyl sulphide (DAS), and diallyl trisulphide (DATS) are the principal constituents of garlic EO (Calvo-Gómez et al. [
Cholestasis is defined as the mechanical or functional stoppage of bile flow in the intrahepatic or extrahepatic bile ducts, with bile components passing into the blood (Kuntz & Kuntz [
CT induced a notable increase of the necrosis marker ALT (Hosono-Fukao et al. [
Several hepatotoxic chemicals cause liver damage by inducing free radicals that, in turn, provoke oxidative stress, which is defined as a strong imbalance between antioxidant defences and an excessive production of oxidative species. The liver is an important source of free radicals, as these reactive species are metabolic products of exogenous or endogenous antioxidants that involve a variety of enzymatic and non-enzymatic mechanisms, which induce oxidation and electron uncoupling (Zhu et al. [
CAT is an endogenous antioxidant enzyme involved in the elimination of ROS, particularly hydrogen peroxide. CAT activity was significantly augmented by CT and Bocfal EO, while it was lowered by DADS; nevertheless, the treatment with either Bocfal EO or DADS during the CT-induced injury diminished CAT activity elevation. In contrast, most reports have found a CT-induced decrease in the activity of CAT that reflects an impaired antioxidant defence system, whereas the administration of DADS or Brassica extracts promotes its recovery and inhibits the LP (Sankhari et al. [
A histopathological analysis was performed; despite the hepatoprotective properties demonstrated by the biochemical markers for Bocfal EO and DADS, both substances produced hepatic parenchymal damage. These deleterious effects may be related to the 1 mmol/kg dose of Bocfal EO or DADS, which is higher than the majority of pharmacological doses used by others. The LC
Bocfal EO or DADS administration resulted in inhibitory effects on the enzyme activities of liver damage markers, particularly ALT, and ALT activity in rats treated with Bocfal EO was even lower during CT-induced injury; the effects on GGTP activity exhibited a similar pattern. These inhibitory properties on the enzymatic liver damage markers may mask adverse hepatic outcomes and mislead the true biochemical diagnosis. OPSs are perhaps capable of either inhibiting the intrinsic enzyme activity or the de novo synthesis, considering that diverse OPSs suppress CYP2E1 activity in the rat liver (Melega et al. [
This is the first report regarding the extraction, chemical constituent analysis, and evaluation of the antioxidant and hepatoprotective activities of the EO obtained from Bocfal. Thus, at the right dose, the edible OPSs in white cabbage are suggested as hydrogen sulphide donors that may serve as useful tools for the research of a variety of diseases. In addition, they might be economical and promising drug candidates for restoring endogenous organic sulphur compounds.
The authors would like to express gratitude to the following individuals: Mrs Vivian Esquivo-Rechy for providing facilities; Brian Byrne, Ph.D. (Natural Advantage, NJ, USA), for supplying DMTS and DMDS; N. Ivalú Cacho-González, Ph.D., for identifying taxonomies; Verónica Juárez-Jaimes, M.Sc., for guiding the preparation of the herbarium specimen (MEXU, Instituto de Biología, UNAM, Mexico); Claudia Rivera-Cerecedo and Héctor Malagón-Rivero (Instituto de Fisiología Celular, UNAM, Mexico) for providing the rats; and Alma Lilia García-Ortiz, Ph.D, for analyzing the GC-FID. The authors thank the Instituto Politécnico Nacional (IPN), Mexico, for the financial support (Proyectos SIP 20120526 and 20141449) and M.C.E. Luis Carlos Romero-Quezada for the administrative support.
The authors declare no conflicts of interest.
By Javier Morales-López; Mónica Centeno-Álvarez; Antonio Nieto-Camacho; Mercedes G. López; Elizabeth Pérez-Hernández; Nury Pérez-Hernández and Eduardo Fernández-Martínez
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