Lettuces, family Asteraceae (Composite), are the most popular vegetables either used as ingredients for salads or consumed fresh owing to beneficial health effects [1
]. Iceberg and butterhead lettuce are the most popular cultivars predominantly used for preparing salads, and the demand for green and red oak-leaf lettuces has considerably increased in recent years [3
]. Lettuce of three different colors can be obtained: dark-red, red, and green; in recent years, red lettuce is commonly used in salads or regularly consumed raw because of its attractive red color and appealing taste [4
]. Red lettuce has strong antioxidant activity owing to its higher anthocyanin content than that of green lettuce; moreover, red vegetables and fruits usually have greater health benefits in humans [5
]. The increased demand of “health food” associated with high antioxidant potential and reduced risk of diseases has resulted in increased consumerism worldwide; moreover, the quality, quantity, and wide variety of healthy food products are essential for the market. Various approaches involving cultivars, including cultural and management related practices are important to enhance the quality of lettuce; in particular, a wide variety would broaden the phytochemical spectrum and other health-promoting attributes [9
Anthocyanins are water-soluble phenolic glycosides derived from anthocyanidins; many fruits, vegetables, and pigments have color owing to different types of anthocyanidins such as cyanidin, delphinidin, pelargonidin, and malvidin [12
]. The free radical-scavenging potential of anthocyanidins and its anti-inflammatory effects have been reported. Cyanidin-3-O
-glucoside has been reported to display anti-tumor activity in breast cancer [14
]. In addition to being rich in anthocyanidins, red lettuce is also a good source of different classes of phenolic compounds, primarily hydroxycinnamic acid, usually from caffeic acid derivatives and flavonols [10
]. Dark vegetables have higher nutrient composition, anthocyanin content, vitamin C, and mineral content than green vegetables, especially purple cabbage, red pepper, and red lettuce [16
]. The nutrient composition of vegetables is an important index for studying their health benefits, and studies on red lettuce as a new cultivar are increasing [19
]. Biological functions of nutrients have increased the current interest in developing healthy food products, and consumers have rendered vegetables as an important source of antioxidants in their daily diet. Simultaneously, studies have emphasized that the choice of cultivar determines the nutritive index and health benefits; hence, strategizing the selection of the appropriate cultivar is critical to maximally increase levels of bioactive nutrients [20
In this study, based on previous findings obtained from breeding red lettuces in our laboratory and a comprehensive evaluation of vegetable shape, taste, and yield, we investigated the nutrient profiles of six red lettuce cultivars and investigated their nutritional quality and biological function. We attempted to identify the important metabolites in red lettuce and mineral elements in different cultivars, and compared the antioxidant capacity of different cultivars based on the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and total antioxidant capacity (T-AOC-FRAP) antioxidant indices. Furthermore, we used a 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the effect of red lettuce on cancer cell growth. This study aimed to compare polyphenol levels in lettuce cultivars and identify suitable cultivars for the development and utilization of healthy vegetables. Our results may help understand the nutrient profile of red lettuce and provide a technical theoretical support for cultivators and consumers to choose lettuce varieties with higher nutritional value.
3. Materials and Methods
3.1. Plant Materials
The leaves of leaf red-lettuce (Lactuca sativa L.) cultivars marked S-1, S-2, B-1, B-2, S-3, and B-3 were grown in the Changping District of Beijing Seed Management Experimental Station under standard greenhouse conditions. S-3 and B-3 are the control varieties, as they are widely available in the market and other varieties were bred in our laboratory. Peat control and water management were performed in accordance with standard practice, with temperature control at 20–25 °C in the daytime and 10–15 °C at night. The six cultivars were planted on August 2017 and harvested on November 2017. The experiment was performed using a randomized block design of six cultivars with three biological replicates, and 5–10 were used in each plot. After collection of their leaves per plot, freezing them in liquid nitrogen, and storing them at −80 °C, mass spectrometry (MS) analysis and analysis of in vitro anticancer effects activity were performed. The remaining leaves were stored at −20 °C for enzyme analysis.
3.2. UPLC–QTOF-MS Analysis Phytochemical Composition
Some fresh lettuces were placed in a ModulyoD-230 freeze dryer (Thermo Fisher, New York, NY, USA) to obtain a powder at −80 °C, 1.0 g of dry powder was collected, and 10 mL of methanol/water/formic acid (80:19:1, v/v/v) was added to the sample, followed by 70 min of bath sonication at 12,000 Hz at 45 °C to extract phytochemicals. The compounds were filtered through a 0.22 μm membrane (Shanghai ANPEL, Shanghai, China) before ultra-performance liquid chromatography (UPLC)-MS analyses.
An ACQUITY UPLC I-Class with FTN Sample Manger instrument (Waters, Milford, MA, USA) was used in our experiments. For chromatographic separation, Eluent A was 0.01% aqueous formic acid, and Eluent B was 100% acetonitrile. Separation was performed using the following elution gradient: 90% A at 0 min, 90% A to 81% A from 0 to 3 min, 81% A to 70% A from 3 to 4 min, 70% A to 60% A from 4 to 5 min, 60% A to 5% A from 5 to 7 min, and 5% A to 90% A from 7 to 9 min. The flow rate was 0.4 mL/min, and 2 μL aliquots of the analytes were injected. The column temperature was maintained at 30 °C for all analyses. The photodiode array detector functioned between 200 and 600 nm.
Xevo G2-S QTOF (Waters MS Technologies, Milford, MA, USA), a quadrupole and orthogonal acceleration time-of-flight tandem mass spectrometer, was used with an electrospray ionization source. Both positive and negative ion modes were used for compound ionization. Mean square error data were collected. At one sample injection, the mode could collect precise mass data of quasi-molecular ions and fragment ions by alternating the low and high collision energy rapidly. The detection conditions were as follows: capillary voltage, 0.45 kV; cone voltage, 40 V; source temperature, 120 °C; desolvation temperature, 500 °C; cone gas flow, 50 L/h; desolvation gas flow, 700 L/h; low energy, 6 V; high energy ramp, 20–40 V. Time-of-flight (TOF)-MS ranged from 100 to 1200 m/z. The scan time was 0.2 s. All analyses were obtained using the Lockspray to ensure accuracy and reproducibility. Leucine-enkephalin was used as the lockmass at a concentration of 200 ng/mL and a flow rate of 10 μL/min. Data were acquired in real time (scan time, 0.5 s, interval, 15 s). The UPLC-QTOF-MS data of samples were acquired and analyzed using Waters UNIFI 1.7 software (V1.7, Waters Corporation, Milford, CT, USA).
Mass spectrometry can be carried out using mass analyzers with a range of mass resolution, triple quadrupoles was capable of measuring metabolite masses with unit resolution. Triple quadrupole instruments can be used to carry out tandem mass analysis (MS/MS). Here, each quadrupole has a separate function; the first quadrupole (Q1) scans across a preset m/z range for selection of one or more ions of interest, with fragmentation in the second quadrupole (Q2) using a collision gas (argon). Q2 is typically an octapole in modern triple quadrupole instruments. According to the subsequent selected reaction monitoring (SRM) experiment, fragment ions generated in Q2 can be subjected to further selection, and this SRM capability of triple quadrupole instruments constitutes a highly sensitive approach for quantifying known metabolites.
3.3. Analysis of Mineral Content
Of the total freeze-dried samples, 0.2 g was used for analysis with the microwave muffle digestion system (Multiwave 3000, Anton Paar GmbH, Graz, Austria) to prepare the test solution. Thereafter, by using inductively coupled plasma atomic emission spectroscopy (ICPE-9000, Shimadzu, Kyoto, Japan), the mineral content was quantified. A total of nine elements (Ca, Cu, Fe, K, Mg, Mn, Na, P, and Zn) were analyzed. The same specimen was tested four times, with high reproducibility. For mineral quantity, the diluted standard solution was prepared by purchasing a 100 ppm standard solution (An Apex Co., Seoul, Korea), and high-purity argon gas was used. Every tool used in the experiment resulted in no contamination.
3.4. Analysis of Antioxidant Activity
Each sample was extracted using a methanol/water/formic acid (80:19:1, v/v/v) solution and a material-to-solvent ratio of 1:10 under sonication at a frequency of 12,000 Hz at 45 °C. Extraction was performed three times in total for 2.0, 1.5, and 1.0 h. Subsequently, the solutions were filtered and concentrated with a rotary evaporator at 40 °C and finally freeze-dried at −80 °C in a ModulyoD-230 freeze dryer (Thermo Fisher) to obtain sample powder. We accurately weighed 25.00 mg of the sample powder in a 25 mL brown volumetric flask separately, dissolved it in 80% methanol and adjusted the final volume to 25 mL to obtain a standard stock solution (1.00 mg/mL). A stepwise dilution method was used to configure a series of standard solutions.
An antioxidant assay was analyzed using the method of Maria John et al. and Lee et al. [40
] with some modifications. ABTS and DPPH powder were purchased from Sigma-Aldrich (St. Louis, MO, USA) and DPPH powder (0.0196 g) was dissolved in 500 mL of methanol and then set to 0.10 mM. Potassium peroxodisulfate solution (140 mM) was mixed with 7 mM ABTS solution (1:1, v
) to react overnight at 25 °C in the dark to obtain ABTS radical working solution. Two concentrations of working solution were prepared, and the absorbance value was 0.70 ± 0.02 at 414 nm. The fresh ABTS radical working solutions were prepared for each assay. First, 50 μL of 0.5 mg/mL sample extracts were added to 3950 μL of radical solution (ABTS and DPPH). After 15 min, the absorbance was measured spectrophotometrically at 517 nm (DPPH)/414 nm (ABTS). DPPH/ABTS radical scavenging activity was determined using the following equation:
DPPH/ABTS scavenging effect (%) = [(A0 − At)/A0] × 100%
where At implies the absorbance in the presence of the compounds, and A0
is the absorbance without those compounds.
All enzymes were extracted at 4 °C. Each sample (0.1 g fresh weight) was thoroughly homogenized in 1.0 mL of 50 mM phosphate buffer (pH 7.8) containing 0.1 mM ethylene diamine tetraacetic acid (EDTA) and 0.05 g of quartz for homogenization. The homogenate for T-AOC-FRAP analysis was centrifuged at 10,000× g for 10 min, and T-AOC-FRAP was measured using the ferric reducing activity of plasma (FRAP) assay and commercial kits (Product Codes: FG5, Suzhou Comin Biotechnology Co., Ltd., Suzhou, China).
3.5. Extraction of Phytochemical Composition in Red-Lettuce B-2 Cultivar
Ten kilograms of fresh red-lettuce leaves were ground into a slurry, using a soya-bean milk machine (Wallmate Corporation, Suzhou, China, No, TAOB-40236093317) and extracted with a hot refluxing extraction method (an 80% ethanol solution was used for extraction and extracted three times; each extraction time was 2, 1.5, and 1 h; extraction temperature was 80 °C; the solid-to-liquid ratio was 1:10, implying that 1 kg of sample were extracted with 10 L of 80% ethanol solution). Thereafter, we used cotton for vacuum filtration of the extraction solution, the flavonoids and phenolic acid were separated via AB-8 macroporous resin and, using a 95% ethanol elution, spin steaming enrichment (until the screw ethanol was evaporated out). The extracts were then dissolved with purified water, and the concentrated solutions were frozen at −80 °C with a ModulyoD-230 freeze dryer (Thermo Fisher) and reduced to a powder.
3.6. Analysis of Tumor Cell Growth Inhibition In Vitro
An MTT cell viability assay was performed. Mitochondrial succinate dehydrogenase can convert the insoluble violet crystalline formazan produced from MTT and deposit it in the cells, but dead cells cannot. Dimethyl sulfoxide (DMSO) could dissolve the formazan in cells, and its light absorption value was measured by ELISA at 490 nm. The number of living cells can be determined from the measured absorbance value (OD value).
Human lung adenocarcinoma cell line A549, human hepatoma cell line Bel7402, human colorectal cancer cell line HepG2, and colon cancer cell HT29 (ATCC, Manassas, VA, USA) were used in our assay [42
]. These four tumor cell lines were selected in their logarithmic growth phase with trypsin digestion and cultured in RPMI1640 Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (GIBCO BRL, Carlsbad, CA, USA) and obtained a cell suspension of 15,000 cells/mL. Thereafter, these cells were seeded in 96-well plates at 190 μL/plate at 37 °C for 24 h and 5% CO2
The samples were diluted to 10, 30, 50, 100, 200 μg/mL by normal saline in an MTT assay. A 10 μL aliquot of the test samples was added to cells and cultured at 37 °C for 3 days at 5% CO2
. The colorimetric MTT assay was referred to as Dai [43
]. The A549, Bel7402, and HepG2 cells were treated with MTT solution (a final concentration of 0.5 mg/mL in DMEM) for 4 h at 37 °C in a 96-well plate, the supernatant was carefully removed, and DMSO (150 μL) was added to each well to dissolve the precipitate. The absorbance at 570 nm was measured using a Model 680 microplate reader (BIO-RAD, Hercules, CA, USA).
The loss of membrane integrity of non-viable cells allows for the permeation of dyes such as trypan blue (TB) into the cell. Based on this principle, it is possible to determine cell viability by the capacity of a viable cell to exclude TB, and a dead cell to incorporate it [44
]. Cell viability was detected with Trypan Blue staining previously reported by Sean D.A. [45
cell viability (%) = (total number of viable cells/total number of viable cells + total number of dead cells) × 100%
3.7. Statistical Analysis
The MS data were pre-processed using SIEVE 2.2 software (Thermo Scientific, Waltham, MA, USA) and used for peak extraction, alignment, filtration, normalization and feature identification. OPLS-DA was performed to analyze differences in the metabolite profiles among cultivars and a VIP score greater than 1.0 was chosen to select the most discriminant features. Only features with VIP > 1 were assigned.
The data are presented as the mean ± standard deviation (SD). Analysis of variance (ANOVA, SPSS 17.0 software, SPSS Inc., Chicago, IL, USA) of all values was performed to assess differences in the means among different samples (p < 0.05 indicated statistical significance). Duncan’s multiple analysis and a Student’s t-test were used to identify significant differences among groups (p < 0.05, p < 0.01). Graphs were prepared in Origin Pro 8.0 SR4 (Origin Lab, Northampton, MA, USA) and Microsoft Office PowerPoint 2007.