Original papers
Anti-allergic Effects of the Alkaline Hydrolysis of Rapeseed Cake in a Rat Basophilic Leukemia Cell Line (RBL-2H3)
2018 Volume 24 Issue 5 Pages 935-942
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2018 Volume 24 Issue 5 Pages 935-942
We investigated the anti-allergic activities of rapeseed cake extract (RCEx) using RBL-2H3 cells. RCEx was produced using the solvent methanol:acetone:water (7:7:6, v/v/v) and was also alkaline-hydrolyzed (AH-RCEx) using HP-20 column and the ethanol stepwise method. AH-RCEx showed a concentration-dependent decrease in degranulation in RBL-2H3 cells, whereas RCEx did not. The water-extracted fraction (WEx) from AH-RCEx mildly inhibited degranulation, and the 40% and 60% ethanol-eluted (EtEx) fractions of AH-RCEx significantly inhibited degranulation compared with that in the control. In HPLC and TLC, the main peak of WEx was consistent with sinapinic acid (SA). In addition, we found a characteristic m/z 473.15 peak in the 40% and 60% EtEx, which was not SA. Therefore, it was suggested that the anti-allergic constituents in rapeseed cake were successfully extracted by alkaline hydrolysis, and their activities were attributed to SA and other unknown compounds.
The “Rapeseed Eco Project” is carried out in various Japanese cities such as Sumoto-shi (Hyogo), Higashiomi-shi (Shiga), and Tahara-shi (Aichi), in which rapeseed (Brassica napus), an oil plant, is cropped in non-cultivated farms. The production of rapeseed oil, which accounts for merely 20–30% of the plant's weight, has generated tons of processing wastes called the rapeseed cakes. The rapeseed cake is used as fertilizer and livestock feed due to its content of various plant nutrients such as nitrogen and phosphorus (Rymer and Short, 2003). Based on the fact that canola (Brassica napus) cake was reported to contain polyphenols and exhibit antioxidant activity (Teh and Birch, 2014), we considered a new potential use of rapeseed cake.
Recently, the number of patients suffering from allergic diseases, such as rhinitis, conjunctivitis, and food and pollen allergies, has been increasing worldwide (Alessandro et al., 2011). It is very important to inhibit allergic reactions. Many plants and herbs have anti-allergic compounds (Horigome et al., 2008; Asano et al., 2011). Generally, these are low-molecular compounds, such as polyphenols, alkaloids, and peptides (Itoh et al., 2012; Nakamura et al., 1992; Tanaka et al., 2012). In addition, the screening of anti-allergic foods and herbs is often conducted in vitro using a rat basophilic leukemia cell line (RBL-2H3) (Horigome et al., 2008; Asano et al., 2011), which can initiate IgE–FcεRI interactions and subsequently induce a model reaction of type I allergic responses, such as the release of β-hexosaminidase (β-hex) and various allergic mediators through intracellular signaling (Passante and Frankish, 2009). Therefore, we investigated the anti-allergic activity of rapeseed cake extract using RBL-2H3 cells.
Sample preparation The rapeseed cakes (kizakinonatane), provided by the agricultural division of Sumoto-shi in Hyogo, were unroasted, compressed (supplemental Fig 1A), and extracted according to a previous method (Teh and Birch, 2014). 200.38 g of the rapeseed cake were mixed with 2 L methanol:acetone:water (7:7:6, v/v/v) in a glass beaker in darkness, stirred with a magnetic stirrer overnight at room temperature, and left to stand for 90 mins after. To decant, the mixture was then centrifuged for 30 min at 10,000 rpm at room temperature (Hi mac CR20G, Hitachi Ltd., Tokyo, Japan), and the supernatant were filtered through filter paper (No.2, Advantech Co., Ltd, Tokyo, Japan). The filtered solution was evaporated in vacuo to dryness and centrifuged for 10 min at 3,000 rpm and room temperature to further decant. The supernatant was lyophilized to produce the final rapeseed cake extract (RCEx) powder, which was then dissolved in ultra-pure water (10 mg/mL).
The alkaline hydrolysis of RCEx (AH-RCEx) were carried out according to a previously described method (Shrestha et al., 2012), in which 2.004 g RCEx were dissolved in 20 mL 35% methanol and 5 mL 10 M NaOH. The solution was covered with aluminum foil and kept on a shaker for 4 h at room temperature. The pH of the solution was adjusted to 2 using 12 M HCl and the phenolic compounds were extracted 3 times each with 20 mL ethyl acetate. The layer of ethyl acetate was filtered through filter paper (No.2S, Advantech co Ltd, Tokyo, Japan), evaporated in vacuo to dryness, and lyophilized to produce the AH-RCEx. Subsequently, 0.259 g AH-RCEx was dissolved in 2.59 mL 1 M NaOH, adjusted to pH 9.8 by 1 M HCl, diluted to the appropriate volume with ultra-pure water (10 mg/mL).
Next, we fractioned AH-RCEx following a method previously reported (Itoh et al., 2012; Maeta et al., 2017). First, we re-extracted AH-RCEx from 5.0679 g of RCEx. The re-extracted AH-RCEx (0.6685 g) was dissolved in 6.69 mL of 1 M NaOH. The solution was fractioned using a glass column packed with Diaion® HP-20 (Mitsubishi Chemical Co., Tokyo, Japan) and eluted with a gradient of polar solvents {ultra-pure water:99% ethanol (EtOH) (100:0, 80:20, 40:60, 60:40, and 0:100)}. The five fractions obtained (WEx, 20% EtEx, 40% EtEx, 60% EtEx, and 99% EtEx) were evaporated in vacuo to dryness, lyophilized, and dissolved in ultra-pure water (10 mg/mL). The 99% EtEx solution was only suspension.
Cell culture RBL-2H3 cells were obtained from the Health Science Research Resource Bank (Tokyo, Japan) and grown in Eagle's minimum essential medium (Nissui Pharmaceutical Co., Ltd. Tokyo, Japan) containing 0.2% NaHCO3, 1.0% penicillin-streptomycin (Nacalai Tesque, Inc. Kyoto, Japan), 2 mM L-glutamine (Nacalai Tesque, Inc.), and 10% fetal calf serum (Corning, Inc., New York, NY, USA) in a humidified atmosphere of 5% CO2.
β-hex and MTT assays in RBL-2H3 cells. We used β-hex as index of degranulation (Passante and Frankish, 2009). The β-hex assay was performed using a previously published method (Maeta et al., 2017; Asano et al., 2011; Yamamoto et al., 2011). RBL-2H3 cells were seeded in a 96 well plate at a density of 1.0 × 106 cells/mL in 100 µL of medium, incubated and sensitized for 16–18 h at 37 °C and 5% CO2 with 0.3 µg/mL mouse monoclonal anti-dinitrophenyl (DNP) IgE (Sigma-Aldrich, St. Louis, USA). The cells were washed twice with a releasing mixture (RM; 116.9 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4·7H2O, 5.6 mM glucose, 25 mM HEPES, 2.0 mM CaCl2, and 1.0 mg/mL BSA, pH 7.7) to eliminate free IgE. The cells were incubated at 37 °C for 10 min in 140 µL/well of RM-containing samples. Next, 10 µL/well of 4.0 µg/mL DNP-BSA in PBS was added, and incubated at 37 °C for 50 min. A 10 µL aliquot of PBS was used as a negative control (Cont) and 10 µL of 3.0 mM ketotifen fumarate (Keto; LKT Laboratories, Inc., St. Paul, MN, USA) was used as a positive control. The plates were put on ice to terminate the reaction and 70 µL of supernatant was transferred to another 96 well plate. Next, 70 µL of the substrate solution (2.5 mM 4-nitrophenyl N-acetyl-β-D-glucosaminide in a 100 mM citrate buffer, pH 4.5) was added to the supernatant, and the plates were incubated at 37 °C for 30 min. Finally, 100 µL/well of a stop buffer (2.0 M glycine buffer, pH 10.4) was added, and absorbance at 405 nm was determined using the microplate reader (SUNRISE Thermo RC-R, TECAN Ltd., Männedorf, Switzerland).
Cell viability was measured using MTT assay. After the supernatant from the β-hex assay was removed and washed, 100 µL of 0.5 mg/mL MTT dissolved in medium without phenol red was added to the wells, and samples were incubated for 1 h at 37 °C. After incubation, the medium was removed. After 200 µL of dimethyl sulfoxide was added to the wells, the absorbance at 570 nm (650 nm reference absorbance) was determined using a microplate reader (Infinite M200, TECAN Ltd, Männedorf, Switzerland).
Thin-layer chromatography (TLC) of the WEx We used 15 × 3 cm glass TLC plates pre-coated with silica gel 60 F254 (Merck Millipore, Billerica, MA, USA). The solvent comprised heptane:diethyl ether:acetic acid (3:6:1, v/v/v). The TLC plates were completely dried before use. A 1 µL aliquot of 10 mg/mL WEx was spotted on the TLC plate. To detect phenol in the WEx, the plates were sprayed with 1% FeCl3 in 50% methanol and dried at 100 °C for 5 min. We spotted a 3 µL aliquot of 1 mg/mL sinapinic acid (SA, Nacalai Tesque, Inc. Kyoto, Japan) in acetonitrile as standard.
High performance liquid chromatography and ultraviolet detection (HPLC-UV) of the WEx The HPLC-UV system used was comprised of JASCO PU-2089 Plus pump, JASCO LC-Net II/ADC automated gradient controller, JASCO UV-2075 Plus detector, and a computerized data station equipped with JASCO software (JASCO Co., Tokyo, Japan). We used a Crest Pak C18T-5 column (250 mm × 4.6 mm, i.d.; average particle size, 5 µm; JASCO Co.). The mobile phase was 0.1% trifluoroacetic acid in 15% acetonitrile. The flow rate was 1.0 mL/min, the wavelength was 320 nm, and the amount of injection was 20 µL. We injected a 20 µg/mL SA in the mobile phase as standard.
HPLC-PDA-MS of the 20% EtEx, 40%EtEx and 60% EtEx Analyses were performed on a Shimadzu prominence series HPLC system (Shimadzu, Kyoto, Japan) consisting of a binary pump, an online degasser, and a photodiode array detector (set to scan 190-800 nm) coupled with an Orbitrap Elite Fourier transform (FT) mass spectrometer (Thermo Fisher Scientific, Inc., USA). Both Thermo Xcalibur 2.2 (Thermo Fisher Scientific, Inc., USA) and LabSolutions version 5.53 (Shimadzu) were used in the spectral analyses.
The HPLC separation of 20% EtEx, 40% EtEx and 60% EtEx. was carried out using a Scherzo SM-C18 column (150 mm × 2.0 mm i.d., 3 µm, Imtakt, Kyoto, Japan) and the binary gradient elution was conducted with 0.1% formic acid in H2O (eluent A) and 0.1% formic acid in acetonitrile (eluent B) as the mobile phases at a flow rate of 0.2 mL/min. The column temperature was kept at 40 °C. The linear gradient program was performed as follows: the initial condition was 90% A/10% B; 0–0.5 min, 10% A/90% B; 0.5–7.5 min, 0% A/100% B; 7.5–9.5 min, maintaining this proportion for 11.5 min. The three faction samples were prepared as 0.5 mg/mL in H2O.
The analytical condition of the mass spectrometer was in the range of m/z 150–1000 with mass resolution of 60,000 at m/z 400 in ESI negative ion mode.
Statistical analysis The statistically significant differences in the β-hex assay and MTT assay were determined using a one-way ANOVA followed by the Dunnett's multiple comparison test. Differences were considered significant at P values less than 0.05. GraphPad Prism version 5.0 (GraphPad Software, San Diego, USA) was used for all analyses.
The extraction from rapeseed cake, fractionation In this experiment, 16.36 g (8.2%). of RCEx powder was yielded from 200.38 g of rapeseed cake (supplemental Fig 1B). The RCEx was then twice alkaline-hydrolyzed, yielding 12.9% and 13.2% of viscous AH-RCEx in the first and second instances, respectively (data not shown). Therefore, the recovered percentage of AH-RCEx from the rapeseed cake was 1%.
We subjected AH-RCEx to HP-20 column chromatography using a stepwise EtOH elution method and obtained five fractions: WEx, 20% EtEx, 40% EtEx, 60% EtEx, and 99% EtEx. The weights of the eluted fractions from 0.6685 g AH-RCEx were 0.6514 g (WEx), 0.0497 g (20% EtEx), 0.0107 g (40% EtEx), 0.0063 g (60% EtEx), and 0.0070 g (99% EtEx), respectively. The constituents of AH-RCEx almost eluted to the WEx, which was powder (supplemental Fig 1C). Based on the sodium ion contained, the total weight of the five factions exceeded the initial weight of AH-RCEx.
β-hex and MTT assays We examined the inhibitory effects of the extracted fractions in the degranulation of RBL-2H3 cells in vitro. RCEx showed a concentration-dependent increase of degranulation (Fig 1A). However, AH-RCEx showed a concentration-dependent decrease of degranulation and an even significantly lower decrease compared with cont at 5 µL of 10 mg/mL concentration (Fig 1B). In addition, RCEx and AH-RCEx did not exhibit cell toxicity (Fig 1D and 1E). Among the five fractions of AH-RCEx, WEx showed mild inhibition of degranulation (Fig 1C), and 40% EtEx and 60% EtEx showed significant inhibition (Fig 1C). These three factions did not exhibit cell toxicity (Fig 1F). Furthermore, the percentage of degranulation inhibition of 30 mg/mL WEx-water solution (m/v) at 10 µL was 60.7 ± 5.9% (Mean ± SD, n = 3). The degranulation by 10 mg/mL 99% EtEx at 10 µL was significantly lower than the cont (Fig 1C). However, the addition of this fraction also significantly lowered cell viability (Fig 1F).
The inhibitory effects (A–C) and cell viabilities (D–F) of RCEx (A and D), AH-RCEx (B and E), and HP-20 separated fractions (C and F) on antigen-stimulated β-hex release.
The values are expressed as mean ± SD, n = 3–5. The statistically significant differences in the β-hex and MTT assays were determined using a one-way ANOVA followed by Dunnett's multiple comparison test. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. cont.
Analysis of WEx In this experiment, the constituents of AH-RCEx were almost eluted to the WEx, which exhibited mild inhibition of cell degranulation. WEx was subsequently analyzed by TLC and HPLC-UV. The RF value of WEx was consistent with SA on the TLC plates after the spraying of 1% FeCl3 in 50% methanol (Fig 2A), whereas the other factions did not move on the TLC plates (data not shown). Furthermore, in the HPLC-UV analysis, the elution time of SA was approximately 23 mins (Fig 2B), and the same elution time of the main peak in WEx (Fig 2C). These chromatograms results suggested that approximately 35% of the weight of WEx was SA. In vitro, SA showed a concentration-dependent decrease of degranulation (Fig 2D), and did not exhibit cell toxicity (Fig 2E).
TLC spots of sinapinic acid (SA) and WEx (A), HPLC-UV chromatograms of SA (B) and WEx (C), and the inhibitory effects (D) and cell viability (E) of SA on antigen-stimulated β-hex release.
The values of β-hex assay are expressed as mean ± SD, n = 4. The statistically significant differences in the β-hex assay was determined using a one-way ANOVA followed by Dunnett's multiple comparison test. * p < 0.05, ** p < 0.01, vs. cont.
Analysis of 20% EtEx, 40%EtEx and 60% EtEx 40% EtEx and 60% EtEx showed strong inhibition of cell degranulation. The retention time (RT) of the characteristic peaks in 40% EtEx and 60% EtEx at 320 nm was 7.03, which was not present in 20% EtEx (Fig. 3A–C). The MS molecular ion data displayed the [M-H]− between RT 7.00 and 7.50 of 20% EtEx, 40% EtEx and 60% EtEx (Fig 4A–C) and a m/z 473.15 compound was present in the 40% EtEx and 60% EtEx (Fig 4A–C). In addition, the MS2 ion fragments in this peak were m/z 399.11, 443.10 and 458.12 (Fig 4D).
HPLC-PDA chromatograms of 20% EtEx (A), 40% EtEx (B), and 99% EtEx (C) at 320 nm.
The injection volume was 5 µL. The wavelength was 320 nm by PDA. The three fraction samples were prepared in 0.5 mg/mL in H2O.
HPLC-MS molecular ion data of 20% EtEx (A), 40% EtEx (B) and 60% EtEx (C) between the retention time 7.00–7.50, and MS2 ions data of m/z 473.15 (D).
The analytical condition of the mass spectrometer was in the range of m/z 150–1000 with the mass resolution of 60,000 at m/z 400 in ESI negative ion mode.
In this experiments, RCEx showed a concentration-dependent increase of degranulation. We examined the concentration-dependent increase of absorbance in the no cell (not seeded cell), the blank (not sensitized by DNP-IgE), and the test (sensitized by DNP-IgE). The slope of the test was higher than that of the no cell and blank (supplemental Fig 2A). Moreover, RCEx did not exhibit cell toxicity. Therefore, it was suggested that RCEx promoted the degranulation by allergic response. The protein of rapeseed cake contains about 400 g/kg dry matter (Rymer and Short, 2003), and the half of total polyphenol in canola meal is sinapine, which is one of the alkaloid amine and a choline ester of SA (Khattab et al., 2010). The ethyl acetate fraction from RCEx, which largely contains polyphenols such as sinapine and SA (supplemental Fig 2B), showed concentration-dependent decrease of degranulation, and did not exhibit cell toxicity (supplemental Fig 2C and2D). Therefore, we considered that high polarity ingredients such as proteins and water soluble carbohydrates in RCEx may relate the promotion of degranulation.
AH-RCEx showed a concentration-dependent decrease of cell degranulation in spite that RCEx promoted the degranulation. SA is present in the extracts of Australian canola meal (Obied et al., 2013) and B. juncea var. oriental after alkaline hydrolysis (Shrestha et al., 2012). Therefore, we considered that SA is responsible for the anti-allergic activity of AH-RCEx. AH-RCEx contained SA (supplemental Fig 3A and 3B), and we found that SA showed a concentration-dependent decrease of cell degranulation in vitro. We could confirm that the extract of Japanese rapeseed cake after alkaline hydrolysis also contained SA, and that SA showed anti-allergic activity in vitro.
The alkaline-hydrolyzed, hydrophobic rapeseed extract reportedly also has potential as a food additive for the quality management of mayonnaise dressing during storage (Kim and Lee, 2017). Although AH-RCEx in the present study was also hydrophobic and not powdered, in order to widely utilize rapeseed extract as a food additive or functional food, the water solubility and powderization are important considerations. SA dissolves alkaline solutions such as NaOH because it is considered that the carboxyl group of SA bonds to a cation in alkaline solutions such as Na+. Therefore, we attempted to convert the SA in AH-RCEx to SA-sodium (SA-Na), and to develop a simplified separation method using HP-20 column. The eluted percentage of WEx from the initial weight of AH-RCEx was about 97%, and the constituents of AH-RCEx were almost eluted to the WEx, in which exhibited mild inhibition of cell degranulation. It was found that WEx had high water solubility and contained SA-Na, and that SA-Na converted SA in acidic solution (supplemental Fig 4A–C). In vivo, it is estimated that the physiological effect of SA-Na is similar to that of SA because it is predicted that gastric acid converts SA-Na to SA. Based on HPLC-UV analysis, it was suggested that the recovered percentage of SA in the WEx from rapeseed cake was approximately 0.35%. SA was reported antihyperglycemic action in type 1-like diabetic rats using streptozotocin (Chemg et al., 2013), and the prevention of hyper ten s ion and car d iovas cular remodel ing in pharmacological model rats (Silambarasan et al., 2014). Further, SA was one of the candidates for a cerebral-protective and cognition-improving medicine (Karakida 2008), and had the neuroprotective potential in the 6-hydroxydopamine-induced hemi-parkinsonian rat (Zaew et al., 2015). Therefore, we considered that this separation method is important to develop new method for manufacturing rapeseed cake for purposes other than as fertilizer or livestock feed, and we expect that WEx which contains SA sodium is available as a functional food.
In this experiment, 40% EtEx and 60% EtEx showed strong inhibition of cell degranulation, and exhibited very low toxicity. It was reported that alkaline-hydrolyzed Australian canola meal contained not only SA, but also kaempferol (Obied et al., 2013). Flavonoids such as apigenin, luteolin and quercetin showed anti-allergic activities in vitro (Kawai et al., 2007). The molecular weights of kaempferol, apigenin, luteolin and quercetin are 286.23, 270.05, 286.24 and 302.24, respectively. However, the molecular weight of the characteristic peak in 40% EtEx and 60% EtEx was 474.15, which did not correspond with quercetin and kaempferol (supplemental Fig 5A–C). Thus, in this experiment, we could not identify the main compounds of 40% EtEx and 60% EtEx. The recovered percentages of 40% EtEx and 60% EtEx from rapeseed cake were approximately 0.016% and 0.009%, respectively, much lower than that of WEx. Therefore, we considered that it was difficult to utilize 40% EtEx and 60% EtEx in the manufacture of rapeseed cake, despite these fractions having notable anti-allergic effects and were very low toxicity.
In conclusion, RCEx did not have in vitro anti-allergic effect, whereas AH-RCEx showed a concentration-dependent decrease of cell degranulation. The WEx fractioned in high percentage (97%) from AH-RCEx had mild anti-allergic activity and contained SA. Despite the fact that 40% EtEx and 60% EtEx showed strong anti-allergic activities, their eluted weights from AH-RCEx were low. Therefore, it was suggested that the anti-allergic constituents in rapeseed cake were successfully extracted by alkaline hydrolysis, and their activities were attributed to SA and other unknown compound. Future, we expected that WEx which contains SA-Na could be available as a functional food.
Acknowledgments This work was supported by the Education and Research Fund from the Mukogawa Women's University. We are grateful to Yurie Hiramatsu, Misa Yasukawa, Emi Kitayama and Yuko Kuwano for their support on the work. The rapeseed cake used in this work was provided by the agricultural division of Sumoto-shi in Hyogo, the members of which we gratefully acknowledge. The HPLC-PDA-MS analysis was supported by the Suntory Foundation for Life Sciences. We express our sincere thanks to Takehiro Watanabe and other members of Suntory Foundation for Life Sciences. Finally, we would like to thank Editage for English language editing.
α-hexosaminidase
HPLChigh performance liquid chromatography
RBL-2H3rat basophilic leukemia cell line
SAsinapinic acid
TLCthin-layer chromatography
