Article pubs.acs.org/JAFC
The Nutritive Value of Organic and Conventional White Cabbage (Brassica Oleracea L. Var. Capitata) and Anti-Apoptotic Activity in Gastric Adenocarcinoma Cells of Sauerkraut Juice Produced Therof Ewelina Hallmann,*,† Renata Kazimierczak,† Krystian Marszałek,‡ Nadzieja Drela,§ Ewelina Kiernozek,§ Peeter Toomik,∥ Darja Matt,⊥ Anne Luik,⊥ and Ewa Rembiałkowska† †
Department of Functional, Organic Food and Commodities, Faculty of Human Nutrition and Consumer Sciences, WULS-SGGW, Nowoursynowska 159c, 02-776 Warsaw, Poland ‡ Department of Fruit and Vegetable Product Technology, Institute of Agricultural and Food Biotechnology, Rakowiecka 36, 02-532, Warsaw, Poland § Department of Immunology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096, Warsaw, Poland ∥ Department of Food Sciences and Hygiene, Estonian University of Life Sciences, Kreutzwaldi 58A, 51014, Tartu, Estonia ⊥ Department of Plant Protection, Estonian University of Life Sciences, Kreutzwaldi 1, 51014, Tartu, Estonia S Supporting Information *
ABSTRACT: White cabbage is one of the most important vegetables grown both in Poland and worldwide. Cabbage contains considerable amounts of bioactive compounds such as glucosinolates, vitamin C, carotenoids, and polyphenols. Some experiments indicate that vegetables from organic production contain more bioactive compounds than those from conventional production, however, only a few studies have been conducted on cruciferous plants. The presented study has proved that organic fresh cabbage, compared to the conventional one, contained significantly less total flavonoids in both years of experiments (3.95 ± 0.21 mg/100 g FW and 3.71 ± 0.33 mg/100 g FW), several flavonoid compounds, total chlorophylls (1.51 ± 0.17 mg/100 g FW and 1.30 ± 0.22 mg/100 g FW) carotenoids, nitrites (0.55 ± 0.04 mg/kg FW and 0.45 ± 0.02 mg/kg FW), and nitrates (0.50 ± 0.13 g/kg FW and 0.47 ± 0.11 g/kg FW). The organic sauerkraut juice, compared to the conventional one, contained significantly more total polyphenols (5.39 ± 0.22 mg/100 g FW and 9.05 ± 1.10 mg/100 g FW) as well as several flavonoids. Only CONV sauerkraut juice produced with the highest N level of fertilization induced a statistical significant increase of the level of necrosis of human stomach gastric adenocarcinoma cell line AGS. KEYWORDS: organic cabbage, conventional cabbage, sauerkraut juice, polyphenols, cancer cells
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INTRODUCTION Cabbage is the most widely grown vegetable worldwide, ahead of carrot, onion, and beetroot, therefore it plays an important role in vegetable production and marketing. White cabbage is one of the most important vegetables grown in Poland. According to data from 2015, cabbage was cultivated in Poland on 24.9 thousand ha of land in the conventional (CONV) system, and the total yield was more than 875 thousand tonnes. Organic (ORG) cabbage was grown in Poland in 2015 on 443 ha, and the total production rate was 2.6 thousand tonnes.1 Cabbage is used not only raw but also as sauerkraut, i.e., after a natural lactic acid fermentation process. Sauerkraut is very popular not only in Poland but also in Germany and Eastern Europe. Statistically, an average Polish consumer eats 11 kg of raw cabbage and 6 kg of sauerkraut per year.1 Fresh cabbage is a source of many antioxidants: glucosinolates, polyphenols, carotenoids, and vitamins. They all protect the human body against free radicals and reduce the risk of many chronic diseases.2 Epidemiological and in vitro studies clearly indicate that antioxidant compounds have a strong inhibiting effect on many cell cancer types3,4 There is today a big interest in whether organic production methods can increase the level of antioxidants in plants. © 2017 American Chemical Society
According to three recent meta-analyses, the concentration of phenolic compounds is significantly (on average about 20%) higher in organic compared to conventional crops. It was also found in our earlier studies.5,6 There are some concepts trying to explain this phenomenon; they will be discussed later in the paper.7 Only a few studies have been conducted on organic vs conventional cabbage (Brassica oleracea L. var. Capitata), showing that organically produced cabbage contained a higher level of total polyphenols as compared to the conventional one.8−11 There is very limited scientific data on the apoptotic and necrotic properties of the organically vs conventionally produced cabbage. However, there are recently published data documenting that sauerkraut juice obtained from cabbage grown in organic farming exhibited more potent inhibitory activity on the key enzyme of estrogen synthesis, aromatase, and expression in MCF10A cells compared to the sauerkraut juice from conventional cabbage.12,13 Received: Revised: Accepted: Published: 8171
March 8, 2017 August 23, 2017 August 24, 2017 August 24, 2017 DOI: 10.1021/acs.jafc.7b01078 J. Agric. Food Chem. 2017, 65, 8171−8183
Article
8172
170 100 150 120 190
100 50 90 60 100
Amistar 250 SC Pirimor 500 WG Fusilade Forte Topsin M 500 SC Sarfun 500 SC Pirimor 500 WG
160 110 140 80 190 Grevit 200 SL
80 60 120 70 120 Bioczos BR Grevit 200 SL
super phosphate granulated. amonium nitrate. potassium sulfate
2−3 weeks after planting. Six weeks later and in time of heads formatting in both experimental years 2−3 weeks after planting. Six weeks later and in time of heads formatting in both experimental years polyphosphate. magnesium sulfate. amonium nitrate
(60 t ha−1) in autumn a year before planting in both experimental years green manure
(30 t ha−1) in autumn a year before planting in both experimental years green manure
(30 t ha−1) in autumn a year before planting in both experimental years cow manure
Mierzyn (53°11′ N 18°88′ E)
Pokrzydowo (53°19′ N 19°27′ E)
Płonne (53°11′ N 19°18′ E)
Płonne (53°12′ N 19°17′ E)
Ca
120 100 160 120 180 Biosept 33 SL Grevit 200 SL
80
Mg K
Zgniłobłoty (53°16′ N 19°14′ E)
Pokrzydowo (53°19′ N 19°25′ E)
organic farm no. 1 (low N-fertilization) organic farm no. 2 (high N-fertilization) organic farm no. 3 (low N-fertilization) organic farm no. 4 (high N-fertilization) conventional farm no. 1 (low N-fertilization) conventional farm no. 2 (high N-fertilization)
kg ha−1
120 90
P N
110 Grevit 200 SL
red clover and onion (in 2009 and 2010) carrots and alfalfa (in 2009 and 2010) red clover and onion (in 2009 and 2010) alfalfa and carrots (in 2009 and 2010) alfalfa and wheat (in 2009 and 2010) alfalfa and wheat (in 2009 and 2010) (20 t ha−1) in autumn a year before planting in both experimental years cow manure
sandy middle soil IV category 15% (floatable particles) pH 6.6 sandy- loamy slight soil III category 15% (floatable particles) pH 6.7 sandy middle soil III category 10% (floatable particles) pH 6.9 sandy- loamy middle soil III category 25% (floatable particles) pH 7.1 sandy middle soil IV category 20% (floatable particles) pH 6.5 sandy-clay soil II category 25% (floatable particles) pH 6.8
plant protection system precrop dose of fertilizers and time of given kind of fertilizer type of soil
Chemicals. Chemicals came from the following sources: Acetonitryl (Chempur, Poland), anhydrous sodium carbonate (POCH, Poland), annexin V-PE (Diag-Med, Poland); carotenoids and chlorophylls β-carotene, lutein, zeaxanthin, α-carotene, Carrez I (15% potassium ferrocyanide Alchem, Poland) and Carrez II (30% potassium sulfate liquid, Alchem, Poland), chlorophyll a, chlorophyll b (Sigma-Aldrich, Poland); citric acid (Alchem, Poland), copper sulfate (Alchem, Poland), 2.6-dichlorophenyloindophenol (Sigma-Aldrich, Poland), ethyl alcohol (70%), formic acid (Alchem, Poland), gentamicin FACSFlow, siringin (Sigma-Aldrich, Estonia), n-hexane (POCH, Poland), hydrochloric acid (36%, Chempur, Poland), Luff− Schoorl liquid (Chempur, Poland), magnesium carbonate (SigmaAldrich, Poland), methyl orange (Chempur, Poland), oxalic acid (2% POCH, Poland), penicillin, potassium iodide (30% Alchem, Poland); polyphenols gallic, chlorogenic, caffeic, synaptic, p-coumaric, ferulic acids, quercetin-3-O-rutinoside, myrycetin, quercetin, and kaempferol (Sigma-Aldrich, Poland); and RPMI 1640 medium with glutamax-1, HEPES buffer solution 1 M, fetal bovine serum, sodium pyruvate MEM (100 mM), 2-mercaptoetanol, F-12K medium for AGS cell line, sodium thiosulfate (Alchem, Poland), sodium hydroxide (0.1 M, POCH, Poland), sodium benzoate (2.0 Mm Sigma-Aldrich, Poland), sodium nitrite and sodium nitrate (standard solutions Sigma-Aldrich, Poland), starch (30%) (Alchem, Poland), stomach gastric adenocarciroma cell line AGS (Life Technologies, Poland), sulfur acid (25%) (Chempur, Poland), streptomycin, CellWash (Diag-Med, Poland), trypsin-EDTA (Invitrogen, Poland). Cabbage Cultivation. White head cabbage “Sufama F1” was selected for the experiment as a very popular cultivar used to prepare sauerkraut. According to Rijk Zwan Company information, “Sufama F1” is a hybrid variety. The cabbage was grown in 2010 and 2011 in four certified organic farms (ORG) and two conventional farms (CONV) located nearby, all in the Kujawsko-Pomorskie region. The neighboring farms (ORG and CONV ones) were chosen in such a way as to ensure similar agrotechnical conditions. The locations of the farms, the levels and types of fertilizers used and their doses, soil types, cropping, management history, as well as the detailed content of the nutrients applied in the cultivation of the cabbage are given in Table 1. For experiment purposes, two levels of nitrogen fertilization have been chosen: low N-fertilization and high N-fertilization: in two experimental farms cow manure and in two green manure have been used. The amounts of N, P, Mg, Ca, and K in the given manure (Table 1) have been calculated based on Siebeneicher (1997), who provided the system of such calculations. It is possible to calculate the content of macro elements by knowing dry matter content, dose, and composition of every type of manure.14 The cabbage was harvested at the same time (mid-October) at all of the farms (40 kg from each experimental plot) and delivered to the laboratory. In every experimental farm (field), three plots were appointed. In general, 120 kg of cabbage from individual farm were taken for all analysis purposes. In Table 2, detailed information about weather conditions (average rain range in mm, temperature in °C as well as sum of sun radiation in kWh/m2) have been presented. The collected data are representative for both experimental years in the time of cabbage
localization
MATERIALS AND METHODS
cultivation system
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Table 1. Characteristic of Localization: Fertilizers Regime and Plant Protection Used for Organic and Conventional Cultivation of Cabbage (Average Value for 2010−2011)
The aim of the present study was to examine the effect of two cultivation systems (organic and conventional) and two different N-fertilization levels on the content of bioactive compounds in cabbage and sauerkraut juice and their apoptotic and necrotic activity. Sauerkraut juice is a popular product in some countries, therefore it has been decided to study its apoptotic and necrotic properties as an interesting scientific aspect. The hypothesis was that organically grown cabbage should contain more bioactive compounds, primarily polyphenols, compared to the conventionally grown one. An additional hypothesis was that organic sauerkraut juice should induce stronger apoptosis of stomach cancer cells than the conventional one.
100
Journal of Agricultural and Food Chemistry
DOI: 10.1021/acs.jafc.7b01078 J. Agric. Food Chem. 2017, 65, 8171−8183
Article
Journal of Agricultural and Food Chemistry Table 2. Weather Conditions and Cultivation Schedule of Cabbage in Both Experimental Years (2010 and 2011) months weather conditions
III
IV
Average Rain Range (in mm) 2010 33 28 2011 12 8 Average Temperature (in °C) 2010 3.4 8.1 2011 2.9 10.5 Sum of Total Sun Radiation (in kWh/m2) 2010 77 118 2011 80 130 III cabbage cultivation schedule
seeds sowing
V
VI
VII
VIII
IX
X
XI
120 36
62 96
145 139
154 33
74 46
4 13
125 3
12.1 14.1
16.8 18.3
21.7 24.3
18.8 18.5
12.4 14.8
6.1 9.2
4.6 3.3
145 158
160 175 IV
158 178
140 162 VI
98 110
58 60 IX
20 23
V
seedling production in greenhouse
planting
VII
VIII cultivation
X
XI harvest
phenol. This dye is reduced to the leuco color (colorless) and at pH 4.2 is pink; the reaction proceeds quantitatively. The weighed 20 mg of freeze-dried cabbage and 5 mL of sauerkraut juice samples were extracted in 2% oxalic acid. The solution was filtered. The filtrate was collected and then titrated with the dye (2.6-dichlorophenyloindophenol) until reaching a permanent pink color. The result was read from the curve and calculated using the formula described in the methodology. Nitrates and Nitrites. The content of nitrites and nitrates was evaluated using the method described in Polish Norm PN-EN 120142:2001.19 The freeze-dried samples of raw cabbage and sauerkraut juice were weighed to 50 mg. Proteins were removed from the samples using two Carrez solutions. Next, the samples were filtrated and a small amount of extract (1 mL) was used for the HPLC analysis. The isocratic gradient was applied. The mobile phase was 2.0 mM sodium benzoate (pH 6.5), and a Hamilton column 150 mm × 4.1 mm PRPX100 was used. Oven temperature was set at 21 °C. The wavelength used was 260 nm and injection volume −100 μL. The contents of nitrates and nitrites were calculated on the standard solution base. Carotenoids and Chlorophylls. Carotenoids and chlorophylls were determined by the HPLC method.20 The examined freeze-dried cabbage samples were weighed (100 mg) and put into plastic test tubes, then MgCO3 (5 mg) was added. The samples were incubated in a cold ultrasonic bath (15 min at 0 °C) with ice. Then hexane was added (5 mL), and the samples were incubated in the bath again. After saponification, the samples were centrifuged (3780g, 2 °C, 10 min). From the test tube, 1 mL of supernatant was collected and recentrifuged (41574.4g, 3 °C, 5 min). Then 900 μL of supernatant was placed in HPLC vials and analyzed. To determine carotenoids, the Shimadzu HPLC was used, consisting of two LC-20AD pumps, a CMB-20A system controller, SIL-20AC autosampler, UV/vis SPD20AV detector, CTD-20AC controller, and Max-RP 80A column (250 mm × 4.60 mm) with stationary phase: ether-linked phenyl phase with polar end-capping. The gradient solvents (deionized water, acetonitrile, ethyl acetate −10:90:100) were selected, with a flow of 1 mL min−1. The wavelength used was 445−450 nm and injection volume −100 μL. To identify compounds the external standards in the form of chlorophyll a and b (Sigma-Aldrich), β-carotene, zeaxanthin, and lutein (Fluka) with purity of 99.98% were used. Each measurement was performed in triplicate. Polyphenols. Phenolic compounds were determined by the HPLC method modified by Hallmann and Rembiałkowska.9 A weighed amount of powdered freeze-dried cabbage and sauerkraut juice sample (100 mg) were placed in plastic test tubes, then 1 mL of methanol with 1% ascorbic acid were added, mixed thoroughly by vortex, and incubated in an ultrasonic bath (20 min at 20 °C). Then the samples were centrifuged (3780 g, 10 °C, 15 min). From the test tube, 1 mL of extract was collected and recentrifuged (31180.8g, 8 °C, 6 min). Then 500 μL of extract was placed in HPLC vials and analyzed. To analyze the phenolic compounds, a Synergi Fusion-RP 80i column (250 mm × 4.60 mm) was used; the stationary phase was ether-linked phenyl phase with polar end-capping. The mobile phase was acetonitrile and
cultivation from when seeds were sowed through planting and cultivation time until harvest. Preparation of Plant Materials. The heads of cabbage were cut off, and two wedges (located on the opposite sides of the head) were cut out. Wedges were cut into tiny pieces, and the entire material was freeze-dried using a LabconCo freeze-drier. After freeze-drying, the plant material was ground in a laboratory mill (A-11). The ground samples were poured into plastic tubes and stored at −80 °C for subsequent analysis. Sauerkraut Juice Preparation. First, 60 kg of cabbage from each experimental farm were divided into three samples and every sample was used to produce sauerkraut juice. A spontaneous fermentation process was selected without implantation of lactic acid bacteria. The cabbages, together with stumps, were cut using a commercial shredder. Cabbage for fermentation was prepared using table salt (NaCl), 3 g per kg of cabbage, and incubated at a temperature of 20−23 °C to obtain a pH of approximately 3.5. To control the fermentation process, the pH was measured: the initial pH was 4.35, and after the fermentation process (2 weeks) the pH decreased to 3.5. Sauerkraut was squeezed using a layer hydraulic press TPZ 7 (Bucher-Guyer), and the juice was used for chemical and apoptotic and necrotic studies. Then 0.5 L of sauerkraut juice from each experimental combination was frozen in −80 °C, (24h). The samples were then transferred to a LabconCo freeze-drier and kept there for 120 h. The parameters of freeze-drying were: −40 °C and 0.080 mbar. Freeze-dried sauerkraut juice samples was kept in −80 °C to protect it against uncontrolled conditions. Dry Matter. Dry matter in cabbage had been determined before the heads of cabbage were subjected to freeze-drying. The cabbage was cut into thin pieces with a sharp knife. Small samples of cabbage tissue and sauerkraut juice (1g or 2 mL) were subjected to a drying process in the following conditions: temperature 105 °C, constant pressure, time 24 h, equipment Dryer KC-65 with free air circulation. After 24 h, partially dried samples were cooled in a desiccator and weighed, then dried again; this operation was repeated three times to achieve a constant weight. Then dry matter content was calculated in grams per 100 g of material. The analytical method is described by Ohene and Maalekuu15 Total and Reducing Augars. Total and reducing sugars were determined by the Luff−Schoorl method. A powdered freeze-dried cabbage and sauerkraut juice samples (0.2 g) were weighed and analyzed according to the cited method.16 Total Acidity. The method is based on neutralization of the total acids content in the water solution. Electrodes were placed in the solution, which was then titrated with sodium hydroxide (0.1 M) until reaching a pH of 8.0. Total acidity was converted into a corresponding acid, in this case, lactic acid. The result was calculated using the formula described by Hoehn et al.17 Vitamin C. The content of vitamin C was measured according to method described by Rekha et al.18 The method consists in the oxidation of L-ascorbic acid (ASC) to dehydroxyascorbic acid (DHA) in an acidic environment using the blue dye 2.6-dichlorophenyloindo8173
DOI: 10.1021/acs.jafc.7b01078 J. Agric. Food Chem. 2017, 65, 8171−8183
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Journal of Agricultural and Food Chemistry Table 3. LC-MS/MS Conditions for Quantification of Glucosinolates by Negative Ion MRM retention time (min)
transmision m/z
fragment ion
1.8 0.9 0.7 0.8
371.82 357.82 421.82 387.79
96.70 96.70 96.70 96.70
glucoraphanin glucobrassicin 4-methoxyglucobrassicin
1.0 7.5 11.7
435.80 449.80 477.10
96.70 96.80 96.90
neoglucobrasicin glucoiberverin
15.1 2.9
477.20 406.10
96.80 96.70
glucosinolate gluconapin siringin glucoiberin progoitrin
collision energy (eV)
molecular formula
241, 227, 210,
23 28 26 28
C11H19NO9S2 C35H42N2O11 C11H20NO10S3 C11H19NO9S2
235, 235, 227,
33 25 25
C12H23NO10S3 C16H20N2O9S2 C17H22N2O10S2
25 26
C16H20N2O9S2 C11H21NO10S3
MS2 (spectrum ions); Base ion in bold 292, 275, 259, 227, 195, 194, 278, 275, 259, 227, 196, 180, 358, 259 332, 308, 301, 298, 275, 259, 195 136 372, 259 275, 259, 251, 205 463, 397, 299, 291, 275, 259, 195 447, 466, 284, 259 326, 275, 259, 228, 195
MiliQ water (55% and 10%) and brought to pH 3.0 with orthophosphoric acid (85%), flow rate 1 mL min−1, detection 280−370 nm, injection volume 50 μL. Polyphenols were identified based on Fluka and Sigma-Aldrich external standards with purity of 99.5%. Glucosinolates Analysis. Glucosinolates were analyzed according to the method described by Hong et al.,21 in particular 50 mg of powdered freeze-dried plant material was extracted with 1.5 mL of 70% boiling EtOH in a water bath at 70 °C for 5 min. After centrifugation (13000g, 4 °C, 20 min), the supernatant was collected into a plastic tube. The instrument used for analysis: Agilent 1290 Infinity UPLC system connected to 6450 UHD Accurate Mass LC/QTOF, column, Agilent Eclipse Plus C18 RRHD 1.8 mkm, 2.1 mm × 50 mm, heated to 40 °C; eluent A, 0.1% aqueous formic acid; eluent B, 0.1% formic acid in acetonitrile; gradient, from 0.1% B to 10% B in 15 min followed by column regeneration with 99% B during 10 min. Spectra were registered in Auto MS/MS mode in the m/z range 100− 800 in negative ion mode. Compounds were identified by their accurate mass and, if the compound was fragmented and MS/MS spectra available, with Agilent MassHunter molecular structure correlator. To build a calibration curve for individual GLs, quantification the external standard siringin (siringin hydrate potassium salt mg/L) was used; HPLC area and response factor as well as molar weights were calculated (ISO 9167-1, 1992).22 For the identification of the individual GLs, the MS analysis was carried out with an ESI interface operated in the positive ion mode. The MS operating conditions were as follows: ion spray voltage, 5.5 kV; curtain gas (20 Pa), nebulizing gas (50 Pa), and heating gas (50 Pa), high purity nitrogen (N2); heating gas temperature, 550 °C; spectra range, m/z 100−800 (scan time 4.8 s). For calibration with sinigrin, a negative ion −m/z 358.03 was used, the injection volume was 20 μL, and the Q-TOF settings were as follows: gas temp 300 °C; drying gas 8 L min−1; nebulizer 35 psig; sheath gas temp 350 °C; sheath gas flow 11 L min−1; fragmentor 175 V; skimmer 65 V Oct 1RF Vpp 750 V; 8 spectra/s were recorded. All other Q-TOF parameters were set automatically by autotuning procedure and were not controlled by the operator; All chromatograms were run in the same way. LC-MS/MS conditions for quantification of glucosinolates by negative ion MRM are presented in (Table 3). Apoptotic and Necrotic Activity Measuring Method. Preparation of Extracts for Cell Line Experiments. Freeze-dried samples of sauerkraut juice were used to prepare three different concentrations of juice extracts used in the studies on cancer cell line: I-0.68%, II-0.34%, III-0.17%.23 The concentrations of ethanol in the cultures with all used dilutions of extracts were 0.05%, 0.025%, and 0.0125%. All of them were not toxic for cultured cells. Cancer Cell Culture Conditions. Human stomach gastric adenocarcinoma cell line AGS (ATCC CRL-1739, American Type Culture Collection) was used. The cells were propagated in Ham’s F12K medium supplemented with 10% FBS, 2 mM glutamine and 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma), in the conditions of 37 °C and 5% CO2, in humidified atmosphere. Cultures were maintained in the log phase of growth (70−80% confluent). Cell detachment were performed using 0.25% trypsin with EDTA. Cell
176 162
viability was assessed by trypan blue exclusion. A 1:4 split ratio was used for further experiments. Treatment of Gastric Adenocarcinoma Cells with Sauerkraut Extracts. For experiments that incorporated the incubation of AGS cells with sauerkraut extracts, RPMI1640 medium was used supplemented with 10% of inactivated FBS, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL gentamycin. Cells (1.0 × 105) were plated in 24-well culture plates in a total volume of 1 mL and 20 μL of appropriate concentrations of extracts were added in each well. The experiment for each concentration of extracts was repeated three times. Each culture was performed in triplicate. Flow Cytometry Analysis of Apoptosis. The percentage of apoptotic or necrotic AGS cells from each culture were analyzed after 24 and 72 h of incubation. Cells were stained with annexin V/ FITC and propidium iodide (PI) using an Annexin V Apoptosis Detection Kit, BD Pharmingen, according to standard protocol.24 According to this method, four populations of cells can be distinguished: AnV−PI− (living cells), AnV+PI− (early apoptotic cells), AnV+PI+ (late apoptotic cells), and AnV-PI+ (necrotic cells) as shown in Figure 5. Because 24 h incubation of AGS cells with sauerkraut juice extracts did not reveal changes in the distribution of living, apoptotic, and necrotic populations, we decided to extend the time of incubation, and the results of the 72 h experiments are presented in this paper. The results of the extract effect was compared to controls: AGS cells incubated in the medium alone and in the medium containing the highest concentration of EtOH (0.05%). Statistical Analysis. The results for fresh cabbage and sauerkraut juice are expressed as the mean for the ORG and conventional cabbages and also for the low and high level of N-fertilization and for the year of the experiment. In the case of apoptotic and necrotic activity, the fourth factor was the concentration of the sauerkraut juice extract in the cancer cells culture. The mean value for the ORG cabbage was obtained from 24 individual measurements (four farms, two levels of N-fertilizations, and three independent samples) each year (2010, 2011). The value for the conventional cabbage was obtained from 12 individual measurements (two farms, two levels of N-fertilization, two experimental years, and three independent samples). The statistical calculations were based on three- and fourway analysis of variance with the use of Tukey’s test (p = 0.05) using Statgraphics 5.1 software (StatPoint Technologies, Inc., Warranton, VA, USA). A lack of statistically significant differences between the examined groups was marked with the same letters. A standard deviation was given at the mean value. To obtain a more clear picture of the interrelations between different compounds, the principal component analysis (PCA) was used. Principal component analysis (PCA) is a statistical tool which applies an orthogonal transformation to process a set of data of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components. PCA figures were made using Statistica 10.0 (Statsoft, Tulsa, USA). PCA was conducted on the basis of correlation matrix not covariance matrix, which corresponds to the analysis on the standardized data. 8174
DOI: 10.1021/acs.jafc.7b01078 J. Agric. Food Chem. 2017, 65, 8171−8183
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Journal of Agricultural and Food Chemistry
Table 4. Effect of Cultivation System (Organic, ORG; Conventional, CONV) and Nitrogen Fertilization (Low, LNF; High, HNF) on the Chemical Composition of Fresh Cabbage Sufama in year 2010 dry matter (g/100 g FW) ORG (n = 6) 9.22 ± 0.25 aa,b CONV 8.81 ± 0.25 b (n = 6) LNF (n = 9) 9.07 ± 0.31 a HNF (n = 9) 8.97 ± 0.33 a ANOVA p-value cultivation 0.0075 (C) fertilization NS (F) C×F NSc
total sugars (g/100 g FW)
total flavonoids (mg/100 g FW)
total carotenoids (mg/100 g FW)
total chlorophyll (mg/100 g FW)
nitrates ( g/kg FW)
nitrites (mg/kg FW)
total glucosinolates (mg/g FW)
6.59 ± 1.14 a 6.63 ± 0.30 a
3.95 ± 0.21 b 4.36 ± 0.25 a
0.40 ± 0.02 a 0.37 ± 0.01 b
1.51 ± 0.17 b 2.19 ± 0.22 a
0.50 ± 0.13 b 0.91 ± 0.03 a
0.55 ± 0.04 b 0.78 ± 0.12 a
4.98 ± 0.81 a 4.12 ± 0.96 b
6.12 ± 0.66 b 7.10 ± 0.69 a
4.10 ± 0.21 a 4.21 ± 0.38 a
0.38 ± 0.03 a 0.40 ± 0.02 a
1.79 ± 0.25 a 1.92 ± 0.49 a
0.68 ± 0.22 a 0.72 ± 0.24 a
0.61 ± 0.08 b 0.72 ± 0.17 a
4.43 ± 1.32 a 4.67 ± 1.92 a
NS
0.0005
0.0034
