Quantitation of Gingerols in Human Plasma by Newly Developed

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Quantitation of Gingerols in Human Plasma by Newly Developed Stable Isotope Dilution Assays and Assessment of Their Immunomodulatory Potential Carola Schoenknecht, Gaby Andersen, Ines Schmidts, and Peter Schieberle* Deutsche Forschungsanstalt für Lebensmittelchemie, Lise-Meitner-Straße 34, 85354 Freising, Germany ABSTRACT: In a pilot study with two volunteers, the main pungent and bioactive ginger (Zingiber officinale Roscoe) compounds, the gingerols, were quantitated in human plasma after ginger tea consumption using a newly established HPLC-MS/ MS(ESI) method on the basis of stable isotope dilution assays. Limits of quantitation for [6]-, [8]-, and [10]-gingerols were determined as 7.6, 3.1, and 4.0 nmol/L, respectively. The highest plasma concentrations of [6]-, [8]-, and [10]-gingerols (42.0, 5.3, and 4.8 nmol/L, respectively) were reached 30−60 min after ginger tea intake. Incubation of activated human T lymphocytes with gingerols increased the intracellular Ca2+ concentration as well as the IFN-γ secretion by about 20−30%. This gingerolinduced increase of IFN-γ secretion could be blocked by the specific TRPV1 antagonist SB-366791. The results of the present study point to an interaction of gingerols with TRPV1 in activated T lymphocytes leading to an augmentation of IFN-γ secretion. KEYWORDS: intracellular Ca2+ concentration, cytokine secretion, ginger (Zingiber officiale Roscoe), gingerols, IFN-γ, T lymphocytes, TRPV1

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INTRODUCTION As part of a healthy and balanced lifestyle, increased knowledge of the beneficial properties of certain bioactive food ingredients is of great interest. However, although many plants and products thereof have been used in traditional medicine for centuries, data on the active principles as well as their mode of action are scarce. Due to its flavor and pungency, Zingiber officinale Roscoe, commonly known as ginger, is an important spice and has also been used for medicinal purposes and cuisine in Asia for thousands of years.1 For example, ginger is used for the treatment of diseases caused by chronic inflammation such as rheumatoid arthritis and atherosclerosis. Furthermore, it is thought to relieve common cold as well as flu-like symptoms.2 The characteristic pungency of fresh ginger is mainly attributed to the gingerols,3 a homologous series of β-hydroxy phenolic ketones (Figure 1), as well as to other minor components such as shogaols, zingiberen and paradols, all located in the nonvolatile yellow, oily oleoresin.4 Approximately 25% of the

oleoresin consists of (S)-gingerols, with [6]-gingerol being the most abundant, followed by [8]- and [10]-gingerols with the increased number of carbon atoms in the gingerol structure leading to a decrease in pungency.5 In addition, gingerols are assumed to be the main bioactive compounds in ginger due to their antiemetic,6 anticancer,7 antioxidative,8 and immunomodulating activities.8,9 The anti-inflammatory activity of [6]gingerol has been shown in murine LPS-stimulated macrophages. Here, the secretion of pro-inflammatory cytokines and antigen presentation as well as the LPS-induced NOS and COX-2 was inhibited by [6]-gingerol.10 These effects were associated with a suppression of I-κBα phosphorylation, NF-κB nuclear activation, and PKC-α translocation.11 However, the impact of gingerols on cells of the adaptive human immune system has not yet been investigated adequately. One of the most important cell type of the adaptive immune system is the T lymphocytes. These cells are antigen-specifically activated via the major histocompatibility complex-II (MHC-II) and the T cell receptor (TCR). Because the downstream signaling of T cell activation requires calcium (Ca2+) influx from the extracellular space into the cytosol,12 ion channels located in the plasma membrane might contribute to this process.13 In particular, members of the transient potential receptor (TRP) family are discussed to affect T cell activation and thus the immune response.14 On the basis of the similarity in amino acid sequence, the 27 mammalian members of the TRP ion channels are classified in six subfamilies.15 Each TRP channel consists of six transmembrane domains, and it is proposed that TRP proteins assemble to a tetrameric structure, of which the transmembrane domains 5 and 6 of each protein Received: January 4, 2016 Revised: March 2, 2016 Accepted: March 4, 2016

Figure 1. Structures of (S)-[6]-gingerol (1), (S)-[8]-gingerol (2), and (S)-[10]-gingerol (3). © XXXX American Chemical Society

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DOI: 10.1021/acs.jafc.6b00030 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. (A) Synthesis route for isotopically labeled [6]-gingerol ([2H4]-1) used in the stable isotope dilution assays as an example for the synthesis of all [2H4]-gingerols. (B) Structures of [2H4]-[6]-gingerol ([2H4]-1), [2H4]-[8]-gingerol ([2H4]-2), and [2H4]-[10]-gingerol ([2H4]-3). used, operating with Merck LiChroPrep RP-18-material (25−40 μm) as stationary phase at a flow rate of 15 mL/min and acetonitrile/ water/acetic acid (50:50:1; v/v/v) as solvent A and acetonitrile/ methanol/acetic acid (80:19:1; v/v/v) as solvent B. The resulting fractions were purified by semipreparative chromatography using an HPLC-UV system equipped with a Jasco PU 2089 Plus gradient pump, a Jasco UV-2075 UV-detector, and a Phenomenex Synergi Fusion RP18 column, 250 × 10 mm, 4 μm (Torrance, CA, USA) as stationary phase. The solvent system was composed of acetonitrile/water/acetic acid (50:50:1; by vol) (A) and acetonitrile/methanol/acetic acid (80:19:1; by vol) (B). A linear gradient was applied by increasing the concentration of B from 0 to 100% within 30 min, returning to 100% A within 5 min, followed by a preconditioning step at 100% A for 5 min. The purified gingerols were then subjected to HPLC-MS and NMR analysis. NMR data of isolated (S)-gingerols were in accordance with those published earlier.22 High-Performance Liquid Chromatography−Mass Spectrometry (HPLC-MS). Mass spectra were recorded with a Finnigan MAT ion trap LCQ-MS (Bremen, Germany) operating in the positive electrospray ionization mode (ESI+). The stationary phase was a Phenomenex Synergi Fusion RP-18 column (250 × 4.6 mm, 5 μm) equipped with a C18 guard column (4.0 × 20 mm, 5 μm). For gradient elution, 0.1% aqueous formic acid (A) and 0.1% formic acid in acetonitrile (B) were used at a flow rate of 0.2 mL/min, starting at a ratio of 70:30 (A/B, by vol), changing to 100% B within 15 min, holding for 8 min, and then returning to 70:30 (A/B, by vol). Synthesis of [2H4]-Gingerols. [2H4]-Gingerols were synthesized in a three-step synthesis (Figure 2A). Initially, alkynols were reduced to the corresponding [2H4]-alcohols by homocatalytic hydrogenation with tris(triphenylphosphine) rhodium(I) chloride (Wilkinson catalyst) and deuterium gas,23 followed by oxidation to the corresponding [2H4]-aldehydes24 and by aldol addition to vanillyl acetate as previously described.25 Synthesis of [2H4]-Hexanol. For catalyst activation, Wilkinson catalyst (160 mg, 0.17 mmol) was dissolved in 15 mL of toluene in a deuterium atmosphere and stirred until a crude orange solution was obtained. 5-Hexynol (5.6 mmol), dissolved in 15 mL of toluene, was dropwise added to the solution. After 2−4 h, the solution was diluted with pentane (100 mL), and toluene was removed by column

form the ion channel, which is permeable for cations including Ca2+.16,17 A specific transcript for the first member of the vanilloid subfamily, the transient receptor potential vanilloid 1 (TRPV1) channel, could be detected previously in human T lymphocytes.14 However, with regard to the potential immunomodulating effect of gingerols, the important question was at which plasma concentrations do these substances appear in biological fluids and reach the potential target molecules after the consumption of ginger? So far, plasma concentrations of gingerols in human subjects have only been quantitated after the intake of encapsulated ginger extracts at doses ranging from 0.5 to 2 g.18,19 Only [10]-gingerol and [6]-shogaol, but not nonmetabolized [6]- and [8]-gingerols, could be detected in human plasma after the intake of 2 g of ginger extract. To assess the bioactivity of gingerols, it is necessary to quantify even low concentrations in plasma. Thus, a more sensitive method based on stable isotope dilution assays was established. Additionally, because TRPV1 is expressed in human T lymphocytes and gingerols are TRPV1 agonists,20 it can be hypothesized that gingerols might be potential candidates to modulate immune responses by enabling a TCR-independent, non-store-operated Ca 2+ influx in T lymphocytes mediated by TRPV1.

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MATERIALS AND METHODS

Chemicals. Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany). Solvents used as mobile phases in liquid chromatography were from Merck Millipore (Merck Chemicals, Darmstadt, Germany). Extraction of Gingerols from Fresh Ginger Rhizome. For cell culture experiments, gingerols were isolated from fresh Chinese ginger rhizome. For this purpose, fresh ginger rhizome was peeled, chopped, and extracted with methanol as described before.21 For purification of the crude extract, a flash chromatography system consisting of a BUECHI Sepacore Control Chromatography C-620, an UV photometer C-635, and an autosampler C-660 (Flawil, Switzerland) was B

DOI: 10.1021/acs.jafc.6b00030 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

brewed with 1 L of boiling water, steeped for 15 min, filtered to remove nonsoluble ingredients, and stored at −20 °C until analysis. For quantitation an aliquot (1 mL) was spiked with [2H4]-1 (60.4 nmol), [2H4]-2 (2.78 nmol), and [2H4]-3 (2.64 nmol) and equilibrated for 45 min at room temperature. Gingerols were isolated using solid phase extraction (SPE), according to the method of Gagné et al.26 For this, tea samples (1 mL) were applied to a mixed phase anion exchange SPE cartridge (Oasis Max 500 mg, 6 cm3) (Waters, Eschborn, Germany) conditioned with 4 × 5 mL of 2% formic acid in methanol, 4 × 5 mL of methanol, 3 × 5 mL of 5% aqueous ammonium hydroxide, and finally 3 × 5 mL of water. The samples were washed with 5% ammonium hydroxide solution (15 mL total) and dried for 10 min, before sequential elution with 4 × 5 mL of methanol. The methanol fractions were combined, the solvent was evaporated under nitrogen, and the residue was redissolved in water/acetonitrile (70:30; v/v, 500 μL) prior to LC-MS/MS analysis. Quantitation of Gingerols in Human Plasma after Ginger Tea Intake. All procedures carried out were in accordance with the Helsinki Declaration of 1975, as revised in 2013. Because the samples in question were used and donated by two of the authors (C.S. and G.A.), no informed consent documents had to be signed. Two healthy, female volunteers, aged 27 and 40 years (BMI 20.9 and 21.3) were the subjects of this study. The volunteers consumed no ginger-containing food 3 days prior to the study. After an overnight fast, 1 L of ginger tea (75 °C) was prepared in the morning as described above and consumed within 20 min, respectively. Blood samples (15 mL) were taken through a nonpermanent port at baseline, as well as 30, 60, 90, and 120 min after tea intake. During the study time no food was consumed by the volunteers. The plasma was then separated immediately from erythrocytes by centrifugation (1950g, 15 min) and stored at −80 °C until use. For the determination of the gingerol concentrations in plasma, 0.21 nmol of [2H4]-1, 0.007 nmol of [2H4]-2, and 0.01 nmol of [2H4]-3 were added to 1 mL of plasma and stirred for 45 min at room temperature. Then, proteins were precipitated using 200 μL of ice-cold acetonitrile. After centrifugation (9500g, 10 min), the supernatant was applied onto the SPE cartridge, and the gingerols were isolated as described above. Liquid Chromatography−Tandem Mass Spectrometry (LC-MS/ MS). Quantitation of gingerols in ginger tea and in human plasma was performed using a Surveyor Plus HPLC system coupled to a Thermo Finnigan TSQ Quantum Discovery mass spectrometer (Dreieich, Germany). A Phenomenex Synergi Fusion RP-18, 150 × 2 mm, 4 μm, with a SecurityGuard cartridge C18 served as stationary phase and 0.1% aqueous formic acid (solvent A) and 0.1% formic acid in acetonitrile (solvent B) as mobile phases. The gradient started at 70:30 (A/B) at a constant flow of 0.2 mL/min, and the column was equilibrated for 1 min before injection of the sample (full loop mode, 10 μL) and changing to 100% B within 15 min. These conditions were held for 8 min before the column was reconditioned to 70:30 (A/B, by vol) for 2 min. The acquisition parameters in ESI+ mode were adjusted to a spray needle voltage of 4.0 kV and to sheath and auxiliary gas pressures of 30 and 10 arbitrary units. The capillary temperature was set to 290 °C, and the source CID was adjusted to 10 V. The most intense product ion of each labeled and unlabeled gingerol was used as quantifier, and the second most intense served as qualifier (Table 1). The peak width was adjusted to 0.8, and the scan time for each transition was 0.2 s. All samples were analyzed in triplicates. Response curves were established for each gingerol in water/ acetonitrile (70:30; by vol) by analyzing seven different known molar ratios of unlabeled and labeled gingerol, from 1:10 to 10:1 for 1 and 3 and from 1:20 to 20:1 for 2 (0.005−2.0 nmol) and plotted against the peak area ratios of unlabeled and labeled gingerols. The response curves for all analytes showed a good linearity (r2 = 0.99) in the applied range, and on the basis of the appropriate linear equation, the molar amounts of gingerols could be calculated. Detection and quantitation limits were determined according to the method published by Vogelgesang and Haedrich.27 Analyte-free plasma was spiked with five different analyte concentrations (1, 0.42−4.2 ng/mL; 2, 0.19−1.9 ng/mL; 3, 0.38−3.8 ng/mL) as well as the equivalent amount of isotopically labeled standard. The recovered

chromatography on silica gel (silica 60, 0.063−0.2 mm, containing 7% H2O) (Merck Chemicals) with n-pentane as eluent. The alcohol was then eluted with diethyl ether and purified using high-vacuum distillation. [2H4]-octanol and [2H4]-decanol were synthesized in the same way but using 3-octynol (4.4 mmol) and 5-decynol (3.6 mmol) instead of 5-hexynol. Synthesis of [2H4]-Hexanal. [2H4]-Hexanol (6.38 mmol) was oxidized with a 1.5-fold amount of 1,1,1-triacetoxy-1,1-dihydro-1,2benziodoxol-3(1H)-one (Dess−Martin periodinane) in 10 mL of dichloromethane. The resulting 1-acetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one was reduced with sodium thiosulfate (1 mol/L; saturated on sodium hydrogen carbonate) by vigorous stirring for 10 min. The organic layer was subsequently extracted with sodium thiosulfate, saturated sodium hydrogen carbonate solution, and water before drying over anhydrous Na2SO4. Further separation was performed by column chromatography using a water-cooled glass column filled with silica gel 60 (0.043−0.06 mm, containing 7% H2O) conditioned with 100 mL of n-pentane. Stepwise elution was performed with n-pentane (100 mL), n-pentane/diethyl ether (90:10, by vol; 100 mL), n-pentane/diethyl ether (80:20, by vol; 100 mL), n-pentane/diethyl ether (70:30, by vol; 100 mL), n-pentane/ diethyl ether (60:40, by vol; 100 mL), and finally n-pentane/diethyl ether (50:50, by vol; 100 mL). [2H4]-octanol (3.22 mmol) and [2H4]decanol (1.23 mmol) were oxidized in the same way to generate [2H4]-octanal or [2H4]-decanal, respectively. The synthesis yields were between 50 and 91%. Synthesis of [2H4]-[6]-Gingerol ([2H4]-1). For the synthesis of [2H4]-1 (Figure 2B) 1 equiv of vanillyl acetate (zingeron) was dissolved in dry tetrahydrofuran (0.7 mol/L) and cooled to −78 °C under an argon atmosphere. One equivalent of n-butyllithium in hexane was added, and the mixture was stirred for 30 min at −78 °C. Then, 1 equiv of lithium diisopropylamide was gradually added to the solution. After 3 h of stirring, 1 equiv of [2H4]-hexanal (6.34 mmol) dissolved in tetrahydrofuran (0.7 mol/L) was added. The mixture was stirred for 3 h at −78 °C followed by 1 h at 0 °C. The solution was adjusted to pH ∼6.5 with 20% hydrochloric acid and was extracted two times with diethyl ether. Purification of [2H4]-1 was performed by column chromatography on silica gel (silica 60, 0.040−0.063 mm, containing 7% H2O) using methylene chloride/ethyl acetate as the eluent. The synthesis yield was 71%. For NMR analysis [2H4]-1 was dissolved in deuterated chloroform using tetramethylsilane as the internal standard. For the synthesis of [2H4]-[8]-gingerol ([2H4]-2) and [2H4]-[10]-gingerol ([2H4]-3) 4.54 mmol of [2H4]-octanal or 3.13 mmol of [2H4]-decanal, respectively, was treated as described for [2H4]-1. The synthesis yields were 41% for [2H4]-2 and 5% for [2H4]3. [2H4]-1 (Figure 2B): LC-MS (ESI+) m/z 299 [M + 1]+, 281 [M − H2O]+; 1H NMR (400 MHz, CDCl3, 297 K) δ 7.28 (m, 1H, J = 7.9 Hz, H−C6′), 6.69 (m, 2H, J = 7.9 Hz, H−C2′;5′), 4.05 (m, 1H, H−C5), 3.9 (s, 3H, H−C7′), 2.86 (m, 2H, H−C1), 2.75 (m, 2H, H−C2), 2.51 (m, 2H, H−C4), 1.28 (m, 6H, H−C6−8), 0.85 (s, 1H, H−C10). [2H4]-2 (Figure 2B): LC-MS (ESI+) m/z 327 [M + 1]+, 309 [M − H2O]+; 1H NMR (400 MHz, CDCl3, 297 K) δ 6.85 (d, 1H, H−C6′), 6.69 (m, 2H, H−C2′;5′), 4.04 (m, 1H, H−C5), 3.89 (s, 3H, H−C7′), 2.86 (m, 2H, H−C1), 2.75 (m, 2H, H−C2), 2.54 (m, 2H, H−C4), 1.50 (d, 2H, H−C6), 1.3 (m, 6H, H−C9−11), 0.88 (t, 3H, J = 6.8 Hz, H− C12). [2H4]-3 (Figure 2B): LC-MS (ESI+) m/z 337 [M − H2O]+; 177 [M − 178]+, 137 [M − 218]+. Because of the small amount of [2H4]-3 obtained, NMR data could not be measured. Nuclear Magnetic Resonance Spectroscopy (NMR). NMR spectroscopy was performed by means of a Bruker Avance III (400 MHz) (Rheinstetten, Germany). Gingerols and [2H4]-gingerols were dissolved in deuterated chloroform using TMS as internal standard. Data processing was performed by using Topspin version 1.3 (Bruker, Rheinstetten, Germany) and MestReNova software (Mestrelab Research, Santiago de Compostela, Spain). Development of Stable Isotope Dilution Assays (SIDA) for the Analysis of Gingerols. Quantitation of Gingerols in Ginger Tea. Fresh Chinese ginger rhizome (100 g) was peeled and chopped, C

DOI: 10.1021/acs.jafc.6b00030 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

7-aminoactinomycin (7-AAD) (BD Biosciences, Heidelberg, Germany) for 10 min in the dark at ∼2 °C. Afterward, 400 μL of PBS was added and cell viability was determined by flow cytometry (FACS Calibur, BD Biosciences, Heidelberg, Germany). The T-cell viability was checked for each applied gingerol concentration. Measurement of Intracellular Calcium Concentration. The impact of gingerols on the intracellular calcium concentration of human T lymphocytes was measured by means of a Pathway 855 fluorescence microscopy (Becton Dickinson, Heidelberg, Germany). For this, human T lymphocytes were isolated and activated for 48 h as described above. Each well of a multiwell plate (μClear 96 well plate, Greiner BioOne, Kremsmünser, Austria) was coated with 100 μL of Cell-Tak (26.2 μg/mL in 0.1 mol/L sodium hydrogen carbonate, BD Biosciences) 24 h prior to fluorescence microscopy analysis at 4 °C in the dark. After activation, T lymphocytes were transferred to the coated microscope plate and then incubated for 30 min at room temperature to allow adhesion and were subsequently loaded (1 h, 37 °C, in the dark) with Fluo-4 AM (100 μL, 2 μmol/L in HEPES buffered saline (HBS): 140 mmol/L sodium chloride, 5 mmol/L potassium chloride, 20 mmol/L HEPES, 1 mmol/L calcium chloride, 10 mmol/L glucose, and 74 mg/mL probenecid in 1 mol/L sodium hydroxide; the pH was adjusted to 7.4 (Life Technologies, Braunschweig, Germany). Afterward, cells were washed once with HBS (5 mmol/L calcium chloride), incubated for another 45 min at 4 °C in the dark, and subjected to fluorescence analysis. The fluorescence intensity of each cell was determined in 10 s intervals at an emission wavelength of 515 nm, after excitation at 488 nm, and rationed (F340/F380). After recording baseline for 20 s, 50 μL of a 3 times concentrated gingerol solution in HBS with 0.1% methanol was applied to the cells, leading to final concentrations of 82, 68, and 23 μmol/L of 1, 2, and 3, respectively, in the well. Capsaicin (60 μmol/L) served as positive control and HBS with 0.1% methanol as negative control. Data processing was performed using BD AttoVision software (BD Biosciences). Only responding cells showing a rate of rise of ≤0.5 under basal conditions and a rate of rise ≥0.75 after treatment were included in the calculation of gingerol-induced fluorescence intensity. Amplitudes of gingerol- or methanol-induced fluorescence signals at each time point were baseline corrected. Furthermore, the gingerolinduced fluorescence intensity was expressed as difference from the negative control. Statistical Analysis. Unless otherwise stated, data are expressed as mean values ± standard deviation (SD) or standard error of the mean (SEM) calculated by Microsoft Excel 2010. Differences of IFN-γ secretion with and without SB-366791 were analyzed using a onetailed Student’s t test for nonpaired samples. A p value of 1) of all tested substances was interferon-γ (IFNγ). No cytokine could be detected in nonactivated T cells. According to the cytokine production profile, the naı̈ve T cells investigated differentiated into Th1 cells. It is known that activating T cells via CD3/CD28 generates Th1 cells.33 Thus, none of the gingerols tested induced a shift of this differentiation route toward Th2 cells. Impact of Gingerols on IFN-γ Secretion of Human in Vitro Activated T Lymphocytes. Because IFN-γ is one of the most important cytokines and also showed the highest extinctions in the cytokine screen, it was decided to analyze the influence of gingerols on the amount of secreted IFN-γ in a concentration-dependent way. Throughout activation by CD3/ CD28, cells were incubated with different concentrations (0.03−300 μmol/L) of [6]-, [8]-, and [10]-gingerols. Because other studies showed higher plasma concentrations of gingerols after intake of ginger-containing nutraceuticals,34 the plasma concentrations determined in our pilot study were chosen as the lowest concentrations for the in vitro experiments. The treatment with [6]-gingerol did not lead to elevated IFN-γ levels (Figure 6A), whereas [8]-gingerol in concentration ranges between 0.03 and 2.7 μmol/L led to a statistically significant increase of IFN-γ secretion of about 20−30% compared to the control (Figure 6B). Additionally, [10]gingerol concentrations between 0.03 and 1.2 μmol/L revealed

volunteers consumed 1 L of ginger tea prepared from fresh Chinese ginger rhizome. The quantitative measurements showed that 1 L of ginger tea contained 96.6 μmol of [6]hingerol, 4.4 μmol of [8]-gingerol, and 2.2 μmol of [10]gingerol. Thus, each volunteer consumed 28 mg of [6]gingerol, 1.4 mg of [8]-gingerol, and 0.8 mg of [10]-gingerol. Compared to the determined gingerol amounts in ginger tea by means of HPLC by Schwertner and Rios30 (1.42 mg/g 1, 0.78 mg/g 2, and 0.22 mg/g 3), the ones obtained in the present study were much lower. This can be mainly attributed to different solvents used for gingerol extraction and the fact that the ginger tea analyzed by Schwertner and Rios was prepared from dried ginger. Blood samples were taken at baseline and 30, 60, 90, and 120 min after ginger tea intake. The maximum plasma concentration of [6]-gingerol was detected 30 min after ginger tea intake, with a concentration of 42.0 ± 16.3 nmol/L, whereas, as expected, [8]-gingerol and [10]-gingerol reached lower concentrations of 5.3 ± 0.8 and 4.8 ± 0.08 nmol/L, respectively, after 60 min (Figure 5). Thus, [6]-gingerol

Figure 5. Concentration−time curves (nmol/L) of gingerols in human plasma after ginger tea intake analyzed by means of SIDA: (solid curve) [6]-gingerol; (dotted curve) [8]-gingerol; (dashed curve) [10]gingerol. Data are shown as the average of two individual donors ± SD.

showed the highest maximum plasma concentration, whereas [8]- and [10]-gingerol concentrations were close to the LoQs, which were approximately 50% lower than those reported in the LC-MS/MS method described previously.19 In this study Yu et al. determined nonmetabolized [10]-gingerol and [6]shogaol after ingestion of 2.0 g of ginger extract, but no free [6]- and [8]-gingerol. Therefore, the present study is the first report of nonmetabolized [6]- and [8]-gingerols in human plasma after ginger tea consumption and shows that these substances are potentially bioactive substances.

Table 2. Six of 14 Screened Cytokines Secreted by Activated T Lymphocytes Incubated with 0.15 μmol/L [6]-Gingerol, 0.14 μmol/L [8]-Gingerol, and 0.12 μmol/L [10]-Gingerola

a

expt with

IL-2

IL-8

IL-10

IFN-γ

TNF-α

GM-CSF

[6]-gingerol [8]-gingerol [10]-gingerol nonactivated T lymphocytes

0.1−0.5 0.1−0.5 0.1−0.5 1