Filipendula ulmaria, also known as meadowsweet, is an herb; its extract was examined for the prevention of histamine production, primarily that caused by contaminated fish. The efficacy of meadowsweet was assessed using two parameters: inhibition of Morganella morganii histidine decarboxylase (HDC) and inhibition of histamine accumulation in mackerel. Ellagitannins from F. ulmaria (rugosin D, rugosin A methyl ester, tellimagrandin II, and rugosin A) were previously shown to be potent inhibitors of human HDC; and in the present work, these compounds inhibited M. morganii HDC, with half maximal inhibitory concentration values of 1.5, 4.4, 6.1, and 6.8 μM, respectively. Application of the extracts (at 2 wt%) to mackerel meat yielded significantly decreased histamine accumulation compared with treatment with phosphate-buffered saline as a control. Hence, F. ulmaria exhibits inhibitory activity against bacterial HDC and might be effective for preventing food poisoning caused by histamine.
Many cases of food poisoning can be traced to histamine, a bioactive amine generated by bacterial contaminants of foods. In fish, Morganella morganii is a well-known bacterial source of histamine production; histamine is generated by the activity of this organism's histidine decarboxylase (HDC) activity. Generally histamine content of freshly caught fish is less than ~2 ppm (1, 14). When freshly caught sea fish such as mackerel and tuna are stored under inappropriate conditions (e.g., at room temperature for longer than 24 h), histamine accumulates. In most cases, histamine levels in illness-causing fish have been above 200 ppm, and often above 500 ppm (13). Once histamine has accumulated, there is a danger of food poisoning, regardless of whether the fish, bacteria, or both are alive. Hence, it is desirable to prevent HDC activity.
In previous work performed by Wendakoon and Sakaguchi (15, 16), screens were performed to identify effective inhibitors of bacterial HDC. Testing included selected plant extracts such as black pepper, nutmeg, allspice, cinnamon, mustard, cardamom, cumin, sage, and clove. The active compounds, which were identified by application to fish, were unappealing due to the undesirable flavors at the effective concentrations (15, 16), and brown algae extracts that showed inhibitory effects on bacterial HDC exhibited weak effectiveness even at higher concentrations (4). Thus, more effective and appropriate components are still being sought.
Subsequent screens for HDC inhibitors used recombinant human HDC; use of the overexpressed and purified protein permitted screening using the enzyme assay as well as an X-ray crystallographic study (6, 7, 9, 10, 11). In fact, four ellagitannins from Filipendula ulmaria, also known as meadowsweet, were identified as potent inhibitors of human HDC, with Ki values in the micromolar range (8). Ellagitannins are effective inhibitors of human HDC, but it remained to be shown that these compounds would work on bacterial HDCs (12). In the present study, ellagitannins from meadowsweet were examined for activity against recombinant HDC from M. morganii and for efficacy in attenuating histamine accumulation in mackerel.
MATERIALS AND METHODS
Purification of recombinant M. morganii HDC.
To obtain purified M. morganii HDC, the protocol that was used for recombinant human HDC was applied. Methods for the expression and purification of human HDC have been described previously (5). M. morganii HDC was expressed in Escherichia coli BL21 (DE3) as a glutathione transferase–tagged fusion protein. The bacterial strain of M. morganii was JCM1672 and was obtained from the Microbe Division of the RIKEN BioResource Center (Ibaraki, Japan). The cDNA encoding HDC was amplified by PCR using PrimeSTAR Max DNA polymerase (Takara, Shiga, Japan). The primers used for the amplification were HDC-F, 5′-ACGCGGATCCATGACTCTGTCTATCAATG-3′, where the BamHI restriction site is shown in bold, and HDC-R, 5′-ACGCGAATTCTTATGCCGCGTGTAAG-3′, where the EcoRI restriction site is shown in bold. After the PCR amplification, amplified product was cloned into pGEX-6p-1 plasmid between its BamHI and EcoRI sites. Nucleotide sequence of the constructs was confirmed by DNA sequencing (Hokkaido System Science Co. Ltd., Sapporo, Japan).
Transformed E. coli BL21 (DE3) cells were grown at 37°C in Luria-Bertani medium supplemented with ampicillin at the concentration of 50 mg ml−1. At logarithmic growth phase, isopropyl β-d-thiogalactoside was added into the media to give a final concentration of 0.1 mM. At this stage, the incubation temperature was lowered to 25°C. The cells were incubated for ~20 h and then collected by centrifugation and resuspended in 50 mM phosphate buffer (pH 6.8) containing 50 mM NaCl and protease inhibitor cocktail (Roche, Indianapolis, IN). After cell disruption by the sonication, the lysates were centrifuged at 16,000 × g for 60 min. The supernatant was collected and loaded onto a glutathione affinity column. Glutathione transferase–tagged protein was eluted from the column with 10 mM glutathione in 50 mM phosphate buffer (pH 6.8) containing 50 mM NaCl. Then, the glutathione transferase tag was cleaved with PreScission protease (GE Healthcare, England, UK) treatment for 16 h at 4°C. Subsequently, the PreScission-treated sample was applied onto a second glutathione column to sequester uncleaved protein. The flowthrough fraction was collected and loaded onto a Resource Q column (GE Healthcare) that was preequilibrated with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.0) containing 10 mM NaCl. The column was washed with the same buffer, and the desired protein fraction was eluted with a linear gradient of NaCl from 10 to 250 mM at a flow rate of 1 ml min−1. Tris(2-carboxyethyl) phosphine hydrochloride was added in the fractions containing M. morganii HDC to give a final concentration of 10 mM. Fractions containing M. morganii HDC were concentrated by using a Pierce Concentrator 9K MWCO (Thermo Fisher Scientific Inc., Waltham, MA). Purity of the final protein preparation was evaluated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).
Inhibitory assay of HDC.
Tellimagrandin II, rugosin A, rugosin D, and rugosin A methyl ester were isolated from meadowsweet flowers as described previously (8) and tested in the present study (Fig. 1) by dissolving them in 50% (vol/vol) ethanol to obtain solutions with concentrations from 0.002 to 2 mM. The HDC inhibition assay mixture contained 0.1 mM dithiothreitol, 0.01 mM pyridoxal 5′-phosphate (PLP), a test meadowsweet sample, and enzyme in 50 mM potassium phosphate buffer, pH 6.5 or 6.0, and the reaction was initiated by the addition of l-histidine at 37°C. Histidine concentration was chosen to be 50 mM since histidine-rich fish was reported to contain histidine at concentrations from 40 to 120 mM (2). The pHs of the assay condition were chosen to be 6.5 and 6.0 since pH of the fish muscle after death was reported to be 5.5 to 6.0 (2), with the optimal pH of M. morganii HDC of 6.5 (12). The final volume of the assays was 200 μl, including 10 μl of ellagitannin solutions. For the control, 10 μl of 50% (vol/vol) ethanol was contained in the final volume of the assay mixture. After 5 min of incubation, the reaction was terminated by adding 10 μl of 60% perchloric acid. The histamine produced in the assays was measured by injecting the aliquot of the assay mixture onto a high-performance liquid chromatography (HPLC) system equipped with a histamine Pak column (Tosoh, Tokyo, Japan). Separated histamine was fluorometrically measured by using the o-phthalaldehyde method as described previously (9). Quantification of histamine was made based on the peak area. The HDC activity (%) of each sample was calculated by the following equation: [(histamine peak area of sample) − (histamine peak area of blank)]/[(histamine peak area of control) − (histamine peak area of blank)] × 100. Duplicated analysis was carried out for each experiment.
Preparation of the meadowsweet extract and quantitative analysis of ellagitannins in the extract.
The meadowsweet extract was prepared as follows. Hot distilled water containing 2 or 10 wt% meadowsweet flowers was kept at temperature above 90°C for 5 min and then filtered. The 10 wt% extract was used for HDC inhibition assay. The volume of the assays was 200 μl, including 10 μl of the extracts in which the final concentration of the extract was from 0.0005 to 0.5%. For the control, 10 μl of distilled water was contained in the final volume of assay mixture. The 2 wt% extract was used for mackerel meat. The solids content of 2 wt% extract was determined by freeze drying to be 0.44% ± 0.14%. The pH of the extract was 5.3. The amount of rugosin A, tellimagrandin II, and rugosin D in the 2 wt% meadowsweet extract was estimated by HPLC to be 465.1, 322.7, and 388.0 μg/ml, respectively. The amount of rugosin A methyl ester was too low to be quantified. The HPLC separation of rugosin A and tellimagrandin II was carried out with a PU-2080 Plus Intelligent HPLC pump equipped with an MD-4015 photodiode array detector (Jasco, Tokyo, Japan). The column for HPLC was a Mightysil RP-18GP (5 μm, 250 by 4.6 mm; Kanto Chemical, Tokyo, Japan). Solvent A was 0.3% (vol/vol) formic acid in water and solvent B was 0.3% (vol/vol) formic acid in methanol. The linear gradient elution used was as follows: 20 to 30% B in A over 30 min, 30 to 35% B in A over 5 min, 35% B in A held for 10 min, 35 to 100% B in A over 10 min, and 100% B in A held for 10 min. The flow rate was 0.6 ml/min and the wavelength was 280 nm. The HPLC separation of rugosin D was carried out with a PU-2080 Plus Intelligent HPLC pump (Jasco) equipped with an MD-4015 photodiode array detector (Jasco). The column for HPLC was a Migthysil RP-18GP (5 μm, 250 by 4.6 mm; Kanto Chemical). Solvent A was 0.2% (vol/vol) formic acid in water and solvent B was 0.2% (vol/vol) formic acid in acetonitrile. The linear gradient elution used was as follows: 10 to 15% B in A over 15 min, 15 to 20% B in A over 40 min, 20 to 50% B in A over 20 min, and 50% B in A held for 5 min. The flow rate was 0.6 ml/min and the wavelength was 280 nm.
Histamine content of mackerel meat with or without meadowsweet treatment.
Histamine content of mackerel was determined by using a histamine test kit (Kikkoman, Tokyo, Japan), a colorimetric enzyme assay for the quantitative analysis of histamine in fish. Histamine dehydrogenase catalyzes the oxidation of histamine. In the presence of 1-methoxy-5-methylphenazinium methylsulfate, this reaction can produce a colored tetrazolium salt that can be measured at 460 nm. One-gram portions of mackerel muscle were added to 24 ml of 0.1 M EDTA buffer (pH 8.0), mixed by shaking for 1 min, and then boiled for 20 min. After cooling, the supernatant was used for histamine detection. The histamine concentration (mg/liter = ppm) of the sample was determined by the following calculation:
where Es is absorbance of the sample, Eb is absorbance of sample blank, Estd is the absorbance of standard solution, Ec is the absorbance of reagent blank, and df is the dilution factor of the sample solution. The number 4 and 25 in the formula mean that the histamine concentration of the standard solution is 4 ppm and that sample has been diluted 25-fold by extraction procedure, respectively. The recovery test was performed in the sample containing meadowsweet extract, and recovery rate of added histamine was 92%. The mackerel fillet obtained from nearby fish store, which was kept at below 10°C, was used at the final day of the expiration (2 days after processing). Blocks of mackerel meat weighing ~1 g were immersed in the extract or phosphate-buffered saline (PBS) at 4°C for 1 h. Then, the extract or PBS was removed and each 1-g block was kept in the 50-ml tube at room temperature (22 ± 3°C) during experimental storage time. Eleven individual filleted mackerel, which were distributed in the marketplace, were examined.
RESULTS AND DISCUSSION
Inhibitory effects on HDC activity.
HDC from M. morganii was expressed in E. coli and purified using the method previously established for human HDC. Purified M. morganii HDC migrated on SDS-PAGE with an apparent size of 43 kDa, in agreement with the molecular weight based on the predicted amino acid sequence (Fig. 2a). The purified enzyme, when examined for UV-visible spectrum, yielded bands at ~415 and 330 nm (Fig. 2b), consistent with the spectrum obtained with the native HDC of M. morganii (12). The presence of two absorption bands at ~420 and 330 nm is typical for PLP-dependent enzymes. Table 1 shows catalytic parameters of Km and Vmax for recombinant M. morganai HDC at pHs 6.0 and 6.5. These values were close to the values for native M. morganii HDC (12). These results suggest that the recombinant HDC obtained in this study represents enzymatic properties of the native M. morganii HDC.
Figure 3 shows the effect of ellagitannins on HDC activity. Enzyme activity became almost zero when it was incubated with 100 μM ellagitannins (Fig. 3). At concentrations below 100 μM, the order of inhibition strength of ellagitannins was rugosin D > rugosin A methyl ester > rugosin A ≈ tellimagrandin II, matching the pattern observed for human HDC (8). The half maximal inhibitory concentration values were estimated to be 1.5, 4.4, 6.1, and 6.8 μM for rugosin D, rugosin A methyl ester, tellimagrandin II, and rugosin A, respectively. Thus, the tested ellagitannins were potent inhibitors of M. morganii HDC. The inhibition profile seen at pH 6.5 (Fig. 3) was similar to that seen at pH 6.0 (data not shown).
The extract of meadowsweet was tested at the range between 0.0005 and 0.5%. As shown in Figure 4, HDC activity decreased with an increasing concentration of meadowsweet extract. The concentration of rugosin A, tellimagrandin II, and rugosin D at the final concentration of 0.5% was estimated to be 105, 86, and 52 μM, respectively, based on their amounts in the 2 wt% extract measured by analytical HPLC. This suggested that the inhibitory activity of the extract presumably was attributable to those of the ellagitannins. Thus, meadowsweet extracts were expected to prevent histamine production in fish by inhibiting HDC activity of M. morganii.
Inhibitory effects on histamine accumulation in mackerel.
To examine the inhibitory effect on histamine accumulation in mackerel, a 2% extract of meadowsweet was prepared and applied to mackerel meat. In mackerel treated with the 2% extract of meadowsweet flowers and stored for 1 day, histamine levels remained lower than those in control fish treated with PBS, with the histamine level in the latter exceeding 100 ppm (Table 2). Hence, the extracts of meadowsweet seemed to prevent histamine accumulation in mackerel.
It is promising to use herbs for the prevention of fish-associated food poisoning; agents targeting PLP-dependent HDC activity provide one such route. Meadowsweet, identified (among 122 plant samples) as an effective herbal inhibitor of human HDC (7), was also shown to serve as an inhibitor of recombinant M. morganii HDC. Furthermore, four ellagitannins, originally identified as inhibitors of human HDC, also acted as micromolar inhibitors of the recombinant M. morganii HDC. PLP-dependent HDCs from other microbial species include those from Raoultella planticola, Photbacterium damselae, and Photobacterium phosphoreum; all of these HDCs are histamine-producing bacteria detected in fish. The corresponding enzymes use PLP at the active site and exhibit 76 to 84% amino acid sequence homology to the M. morganii HDC (3). Therefore, we will investigate whether meadowsweet can inhibit the HDC activity of a wide variety of bacteria in the next study.
Application of meadowsweet extract to fish is better than spice extracts that were reported to be effective previously (15, 16) since there is no undesirable flavors at effective concentrations. The 2 wt% concentration corresponds to that of tea for drinking. Therefore, it will be easy to try at home or in food services that offer meadowsweet tea.
This work was supported by Japan Society for the Promotion of Science Grants-in Aid for Scientific Research no. 26350101 and 15K00783.