Abstract
6-Gingerol and 6-shogaol are the most abundant gingerols and shogaols in ginger root and have been shown to reduce the asthmatic phenotype in murine models of asthma. Several studies have described the pharmacokinetics of gingerols and shogaols in humans following the oral ingestion of ginger, while little was known about the metabolism of these components in humans, particularly in patients with asthma. In this study, a dietary supplement of 1.0 g of ginger root extract was administered to asthma patients twice daily for 56 days and serum samples were drawn at 0.5–8 h on days 0, 28, and 56. The metabolic profiles of gingerols and shogaols in human plasma and the kinetic changes of gingerols, shogaols, and their metabolites in asthma patients collected on the three different visits were analyzed using liquid chromatography–mass spectrometry (LC–MS). Ketone reduction was the major metabolic pathway of both gingerols and shogaols. Gingerdiols were identified as the major metabolites of 6-, 8-, and 10-gingerols. M11 and M9 were identified as the double-bond reduction and both the double-bond and ketone reduction metabolites of 6-shogaol, respectively. Cysteine conjugation was another major metabolic pathway of 6-shogaol in asthma patients, and two cysteine-conjugated 6-shogaol, M1 and M2, were identified as the major metabolites of 6-shogaol. Furthermore, gingerols, shogaols, and their metabolites were quantitated in the human serum collected at different time points during each of the three visits using a very sensitive high-resolution LC–MS method. The results showed that one-third of 6-gingerol was metabolized to produce its reduction metabolites, 6-gingerdiols, and more than 90% of 6-shogaol was metabolized to its phase I and cysteine-conjugated metabolites, suggesting the importance of considering the contribution of these metabolites to the bioavailability and health beneficial effects of gingerols and shogaols. All gingerols, shogaols, and their metabolites reached their peak concentrations in less than 2 h, and their half-lives (t1/2) were from 0.6 to 2.4 h. Furthermore, long-term treatment of ginger supplements, especially after 56 days of treatment, increases the absorption of ginger compounds and their metabolites in asthma patients.
Keywords: ginger supplement, gingerols and shogaols, metabolic profile, pharmacokinetics, asthma patients
1. INTRODUCTION
The ginger root (Zingiber officinale Roscoe, Zingiberaceae) is a worldwide used dietary substance. Fresh or dried ginger is commonly used in cooking, while ginger supplements are also consumed for their potential health benefits, for example, for easing the symptoms of a cold or flu and for treating nausea and vomiting. Scientifically, many medicinal properties of ginger have been identified, such as anti-arthritis, anti-inflammatory, anti-diabetic, anti-bacterial, anti-fungal, and anti-cancer.1–6
The nutraceutical and medicinal values of ginger were ascribed to the major phytochemicals in ginger, gingerols, and shogaols. In fresh ginger, gingerols are the major polyphenols, which are responsible for the pungent taste of fresh ginger rhizome. Among these gingerols, 6-gingerol (6G) is the major one, while 4-, 8-, 10-, and 12-gingerol (4, 8, 10, and 12G) are present in lower amounts. After heat treatment or storage for a long time, the gingerols can be transformed into their corresponding shogaols, and 6-shogaol (6S) is the major product among them, along with 4-, 8-, 10-, and 12-shogaol (4, 8, 10, and 12S) as minor components.7–9
An increasing number of studies have reported a variety of medicinal activities of gingerols and shogaols including those for the treatment of asthma.7,8 In isolated human and guinea pig tracheas, 6G, 8G, and 6S induced rapid relaxation of the precontracted airway smooth muscle (ASM).10 In human ASM, Townsend et al. observed that 6G, 8G, and 6S could significantly potentiate the relaxation induced by β-agonists.11
Similar to many other dietary compounds, gingerols and shogaols undergo a wide range of metabolic reactions, including phase I and II metabolism. 6G is metabolized to 6-gingerdiols in cells and mice.12 Shogaols are extensively metabolized to form thiol-conjugated metabolites in mice and humans.13–15 The double bond and the carbonyl group of 6S are able to be reduced to generate the corresponding metabolites.16 More importantly, these metabolites retained the bioactivities of gingerols and shogaols.17–19 Similar to the parent compound 6S, its metabolites were proved capable of preventing G protein receptor-coupled stimulation of intracellular calcium [Ca2+]i and inositol trisphosphate (IP3) synthesis by inhibiting phospholipase C, and these metabolites also relax muscarinic receptor-induced contractions in upper (human) and lower (murine) airways.20 These ginger compounds and their metabolites may provide a therapeutic option alone or in combination with accepted therapeutics in asthma.
Although a couple of studies have measured the pharmacokinetics of the ginger phytochemicals in healthy humans,21–26 these studies only measured kinetic changes of the ginger compounds and their glucuronides and sulfates. It is important to detect the presence and measure the pharmacokinetics of the phase I metabolites of gingerols and shogaols and the thiol conjugates of shogaols in the plasma of asthma patients. In this present study, we investigated the metabolic profile of gingerols and shogaols and measured the pharmacokinetics of gingerols, shogaols, and their metabolites in the plasma of asthma patients by high-resolution liquid chromatography–mass spectrometry (LC–MS).
2. MATERIALS AND METHODS
2.1. Chemicals and Reagents.
The ginger capsules and the corresponding placebos were a gift provided and manufactured by Pure Encapsulations LLC (Sudbury, MA). The ginger capsules contain 6.55% 6G, 0.94% 8G, 1.88% 10G, 2.21% 6S, 0.37% 8S, and 0.95% 10S. β-Glucuronidase from bovine liver, sulfatase from Helix pomatia, d-saccharic acid 1,4-lactone monohydrate (β-glucuronidase inhibitor), ascorbic acid, and N-vanillylnonanamide (internal standard, IS) were purchased from Sigma-Aldrich Inc. (St Louis, MO, USA). Sodium acetate was purchased from Fisher Scientific. All ginger standard compounds were previously purified or synthesized in our lab.27,28 Their purities were as follows: 6G, 97.0%; 8G, 96.2%; 10G, 97.0%; 6S, 97.6%; 8S, 96.3%; 10S, 95.5%; 6G-diol-2, 95.7%; M2, 99.1%; M9, 99.1%; M11, 96.2%. Acetonitrile, methanol, and water (LC–MS grade) were purchased from Thermo Fisher Scientific (Waltham, MA). Other general chemicals were purchased from VWR (Radnor, PA).
2.2. Clinical Trial Design.
The study protocol (IRB# AAAR8427) was approved by the institutional review board at Columbia University, New York, NY. Study subjects with mild to moderate asthma with inadequate control of their asthma symptoms (defined by an asthma control test value of ≤19), despite maintenance therapy on inhaled corticosteroids ± a long-acting β-agonist, were recruited through the asthma program at Columbia University Medical Center between October 2019 and February 2021. Inclusion criteria were (1) age > 18, (2) treatment with inhaled corticosteroids ± long acting β-agonists, (3) physician-diagnosed asthma, (4) forced expiratory volume (FEV1) ≥ 50% of predicted, (5) Δ in FEV1 of at least 12% or 200 mL 15 min after inhalation of two puffs of albuterol using a metered dose inhaler, (6) non-smoker for ≥1 yr, and (7) ≤10 pack-year smoking history. Exclusion criteria included (1) other major chronic illnesses; (2) physician diagnosis of chronic bronchitis, emphysema or COPD; (3) current consumption or known adverse reactions to ginger products; (4) oral corticosteroid use with the past 6 weeks; and (5) pregnant or lactating patients. Following a screening visit, on the randomization visit (day 0) study, subjects had baseline pulmonary function tests, assessments of daily asthma symptoms using validated questionnaires, exhaled nitric oxide measurements, and blood draws for measurements of eosinophilia and cytokine measurements. Subjects were randomly assigned (by an algorithm within REDCap software) to consume an identical appearing placebo capsule or 1.0 g of ginger extract twice daily for 56 days. In a subset of study subjects (n = 8), blood was drawn before (time 0) and at 0.5, 1, 2, 3, 4, and 8 h after study drug consumption on days 0, 28, and 56. The study subjects and investigators were blinded to the assigned groups. In addition to blood measurements for pharmacokinetic measurements of metabolites, study subjects underwent repeated pulmonary function tests and measurements of inflammation at days 0, 28, and 56. Blood samples for metabolite analysis were collected in BD Vacutainer serum separator tubes (SST) on ice and subjected to centrifugation at 2000g for 10 min at 4 °C. Aliquots of serum were stored at −80 °C until analysis.
2.3. LC–MS/MS Analysis.
The qualitative and quantitative LC–MS/MS analyses were performed on a Thermo Q Exactive plus mass spectrometer coupled with a Vanquish LC system (Thermo Scientific, San Jose, CA, U.S.A.) incorporated with an electrospray ionization (ESI) interface.
2.4. Liquid Chromatography Parameters.
Liquid chromatography was performed using a 3.0 μm Phenomenex Luna C18(2) 100 Å column (2.0 × 50 mm i.d.). The injection volume was 10 μL, and the flow rate was 200 μL/min. The column temperature was controlled at 40 °C. The mobile phases include water with 0.1% formic acid (v/v) as phase A and acetonitrile with 0.1% formic acid (v/v) as phase B. The flow gradient was initially at 5% (v/v) of B for 2 min, linearly ramped to 40% of B over 1.0 min, held at 40% for 2.0 min, linearly ramped to 85% of B over 4 min and held for 1.0 min, linearly ramped to 95% of B over 1.0 min and held for 1.0 min, and then returned to 5% of B over 1.0 min and held for 2.0 min. This condition was held for a further 2 min prior to the injection of another sample.
2.5. MS/MS Parameters.
The mass spectrometer was operated in the ESI positive ion mode. For mass spectrometric parameter optimization, the mixture of standards in 50% methanol solution (500 ng/mL) were infused directly into the ESI source and analyzed in the positive ion mode. Then, the following optimized parameters were obtained: spray voltage, 3.5 kV; sheath gas (nitrogen) flow rate, 30 (arbitrary units); auxiliary gas, 30; spare gas, 5; probe heater temperature, 350 °C; and S-lens radio frequency level, 60. The following settings were used for higher energy C-trap dissociation fragmentation: maximum injection time, 100 ms; automatic gain control (AGC), 1 × 105; and resolution, 17,500. Data acquisition and analysis were performed using Xcalibur 3.0 (Thermo Electron; San Jose, CA). The serum samples from seven time points were analyzed with the full mass/dd-MS2 [data-dependent analysis (DDA)] mode to get the metabolic profile and the parallel reaction monitoring (PRM) mode to do quantification. The parameters of DDA were shown as follows: Full MS: microscans, 1; resolution, 70,000; AGC target, 3 × 106; maximum IT, 100 ms. dd-MS2: microscans, 1; resolution, 17,500; AGC target, 1 × 105; maximum IT, 50 ms; loop count, 10; isolation window, 4.0 m/z; stepped NCE, 10, 20, 30. The parameters of PRM analysis were listed as follows: microscans, 1; resolution, 17,500; AGC target, 2 × 105; maximum IT, 200 ms; loop count, 1; isolation window, 1.0 m/z; NCE, 10.
To set up the PRM experiment, targeted precursor ions for each analyte were added to the inclusion list with the accurate mass and molecular formula and the corresponding detection time window-based on DDA results and standards. After data acquisition, the MS/MS transitions listed in Table 1 were used to extract the analytes.
Table 1.
MS/MS Transitions Used to Quantitate Gingerols, Shogaols, and Their Metabolitesa
| compounds | parent ion (m/z) | product ion (m/z) |
|---|---|---|
| 4G | 249.1485 | 177.0909 |
| 6G | 277.1798 | 177.0909 |
| 8G | 305.2111 | 177.0909 |
| 10G | 333.2424 | 177.0909 |
| 6S | 277.1798 | 137.0595 |
| 8S | 305.2111 | 137.0595 |
| 10S | 333.2424 | 137.0595 |
| M1 | 400.2152 | 137.0595 |
| M2 | 398.1996 | 137.0595 |
| M9 | 263.2006 | 137.0595 |
| M11 | 279.1955 | 137.0595 |
| 6G-diols | 261.1849 | 163.0750 |
| 8G-diols | 289.2162 | 163.0750 |
| 10G-diols | 317.2475 | 163.0750 |
| IS | 294.2064 | 137.0595 |
(6G: 6-gingerol; 8G: 8-gingerol; 10G: 10-gingerol; 6S: 6-shogaol; 8S: 8-shogaol; 10S: 10-shogaol; 6G-diols: 6-gingerdiols; 8G-diols: 8-gingerdiols; 10G-diols: 10-gingerdiols; M1, M2, M9, and M11 are the metabolites of 6S).
2.6. Preparation of the Stock Solution, Calibration Standard, and Quality Control Samples.
The stock solutions of 6G, 8G, 10G, 6G-diol-2, 6S, 8S, 10S, M2, M9, M11, and IS were prepared in methanol with the final concentration of 1 mg/mL. Then all the working standards of the analyte mixture containing 0.12 to 8000 ng/mL of these analytes were prepared by dilution from the stock solutions with 80% methanol. The internal standard working solution was prepared by diluting the stock solution to the final concentration of 2 μg/mL with 80% methanol. Low-, medium-, and high-concentration quality control working stock solutions were prepared in 80% methanol using separately weighed stock solutions of these analytes. Calibration standard solutions were prepared by spiking blank serum with appropriate amounts of working standards. Quality control serum samples were prepared in the same way as the calibration standard solutions. The blank serum sample without analytes and the internal standard was also prepared and analyzed.
2.7. Sample Preparation for the Metabolic Profile of Ginger Compounds in Human Serum.
The serum samples from one subject at 0, 0.5, 1, 2, 3, 4, and 8 h were hydrolyzed with a mixture of β-glucuronidase and sulfatase and analyzed by LC–MS with data-dependent acquisition. The profiles of ginger compounds in human serum after taking the ginger capsules were analyzed and compared with the control serum after taking the placebo using Qual Browser in Thermo Scientific Xcalibur 4.2. The result of serum collected at 1.0 h was shown in Figure 1.
Figure 1.
Selected ion chromatograms of gingerols (blue), shogaols (blue), and their major metabolites (red) in human serum detected by LC–MS and their chemical structures. Human serum samples were collected 1.0 h after taking 1 g of ginger extract capsules (GE-treated) or after taking the placebo (blank) from one of the subjects in each group.
2.8. Sample Preparation for Quantification.
To detect the free form of gingerols, shogaols, and their phase I- and cysteine-conjugated metabolites, serum samples (100 μL) were mixed with 450 μL of water, 5 μL of IS (2 μg/mL), and 1 mL of MeOH and vortexed for 30 s. Then 1.5 mL of ethyl acetate was added to the above solution and vortexed for another 30 s. After centrifugation for 5 min at 4000 rpm, the supernatant was transferred to a 5 mL tube. The pellet was extracted again with 1.5 mL of the methanol–ethyl acetate mixture (1:2) by sonicating for 10 min. After centrifugation, the supernatant was transferred to the same 5 mL tube. The combined extract was dried by streaming nitrogen. After drying, the extract was reconstituted with 50 μL of 80% MeOH for LC–MS analysis.
To analyze the glucuronide conjugates of gingerols, shogaols, and their metabolites, the serum samples (100 μL) were pre-incubated with β-glucuronidase from bovine liver (500 U) in NaOAc buffer (50 mM, pH 5.0) for 2 h at 37 °C. After incubation, the solution was extracted according to the above-mentioned procedure. Finally, the extract was reconstituted with 50 μL 80% MeOH for LC–MS analysis.
To analyze the sulfate conjugates of gingerols, shogaols, and their metabolites, the serum samples (100 μL) were pre-incubated with sulfatase from H. pomatia (17 U) and d-saccharic acid 1,4-lactone (24 mM, as an inhibitor of β-glucuronidase29) in NaOAc buffer (50 mM, pH 5.0) for 2 h at 37 °C. After incubation, the solution was extracted according to the above-mentioned procedure. Finally, the extract was reconstituted with 50 μL of 80% MeOH for LC–MS analysis.
2.9. Method Validation.
2.9.1. Extraction Recovery.
Three levels of quality control working solutions were spiked into the blank serum before and after extraction and extracted using the above procedure in triplicate. Extraction recovery was determined by comparing the peak areas of each analyte in the extracts from spiking before and after extraction.
2.9.2. Linearity and the Lower Limit of Quantification.
Linearity was evaluated in a concentration range from 0.12 to 8000 ng/mL. The lower limit of quantification (LLOQ) was determined as the lowest concentration of the analyte which had a signal-to-noise ratio (S/N) over 10. The calibration curves of the standards were built up using human blank serum spiked with various concentrations of standards with consistent IS and expressed the peak area ratios of standards to IS versus concentrations.
2.9.3. Accuracy and Precision.
Accuracy was calculated as the mean percentage deviation of the measured concentrations of the three quality control samples from their nominal concentrations. Precision was calculated as the coefficient of variation of multiple determinations. Both the inter-day and intra-day results were determined for the accuracy and precision.
2.10. Pharmacokinetic and Statistical Analysis.
Pharmacokinetic analysis of the serum analyte concentration–time profile was carried out by PK solution 2.0 (Summit research services, Montrose, CO). All values are expressed as means ± standard error (SE). One-way ANOVA with Tukey’s test was performed to analyze the difference of ginger compounds and their metabolites between the three visits using GraphPad Prism 8. Student’s t-test was used for comparing the concentrations of gingerols, shogaols, and their metabolites in Figure 5 between V5 and V3. A p-value < 0.05 was considered significant.
Figure 5.
Comparison of the total amount of gingerols, shogaols, and their metabolites on visit 3 (V3), visit 4 (V4), and visit 5 (V5) at baseline and 8 h. The concentrations were averaged (n = 3 except V4 at 8 h for n = 2 because one subject did not provide the 8 h sample) at each time point. V3, the randomization visit; V4, the second visit at 28 days after the randomization visit; V5, the final visit at 56 days after the randomization visit. The amounts of ginger compounds at baseline on visit 3 were 0. Student’s t-test was used for comparing the concentrations of gingerols, shogaols, and their metabolites between V5 and V3. *p < 0.05; **p < 0.01. (6G: 6-gingerol; 8G: 8-gingerol; 10G: 10-gingerol; 6S: 6-shogaol; 8S: 8-shogaol; 10S: 10-shogaol; 6G-diols: 6-gingerdiols; 8G-diols: 8-gingerdiols; 10G-diols: 10-gingerdiols; M1, M2, M9, and M11 are the metabolites of 6S).
3. RESULTS AND DISCUSSION
3.1. Metabolic Profile of Ginger Compounds in Human Serum.
To determine the metabolic profile of ginger compounds in human serum collected from subjects taking 1 g of ginger capsules, gingerols, shogaols, and their potential metabolites were searched in human serum samples incubated with glucuronidase and sulfatase using high-resolution LC–MS/MS. As shown in Figure 1, six major ginger components including three gingerols, 6G, 8G, and 10G, and three shogaols, 6S, 8S, and 10S, were identified by comparing with authentic standards (data not shown). Another ginger compound detected was 4G, which was tentatively identified based on its molecular formula (C15H22O4) and the typical gingerol tandem mass fragments at m/z 137.0595 and 177.0909 with 177.0909 as the major fragment (6G as an example in Figure 2). Gingerdiols, the ketone bond-reduced products of gingerols, were identified as the major metabolites of gingerols (Figure 1).30,31 All gingerdiols (6G-diols, 8G-diols, and 10G-diols) showed three major tandem mass fragments at m/z 137.0595, 163.0750, and 177.0909 with 163.0750 as the major fragment (6G-diol as an example in Figure 2). For shogaols, only the metabolites of 6S, the double-bond reduction metabolite (M11), both double-bond and ketone bond reduction metabolite (M9), and two cysteine conjugates (M1 and M2)32 were detected and identified by comparing their LC–MS spectra with those of the authentic standards (M9 as an example in Figure 2), and their major tandem mass fragments were at m/z 137.0595.17 Their structures were shown in Figure 1.
Figure 2.
Ion chromatograms of gingerols, shogaols, and their metabolites under the PRM mode and representative mass spectra of 6G, 6G-diol, 6S, and M9 by LC–MS in serum collected at 1.0 h of one subject after taking 1 g of ginger extract capsules. (6G: 6-gingerol; 8G: 8-gingerol; 10G: 10-gingerol; 6S: 6-shogaol; 8S: 8-shogaol; 10S: 10-shogaol; 6G-diols: 6-gingerdiols; 8G-diols: 8-gingerdiols; 10G-diols: 10-gingerdiols; M1, M2, M9, and M11 are the metabolites of 6S).
These results indicate that gingerols and shogaols are metabolized similarly in humans as in mice with reduction and thiol conjugation as the major metabolic pathways. This is the first study to report that gingerols and shogaols and their metabolites are absorbed in the circulatory system of asthma patients.
3.2. Quantification of Gingerols, Shogaols, and Their Metabolites in Human Serum.
3.2.1. Validation of the Analytical Method.
In order to quantitate the identified gingerols and shogaols (4G, 6G, 8G, 10G, 6S, 8S, and 10S) and their metabolites (6G-diol-1, 6G-diol-2, 8G-diols, 10G-diols, M1, M2, M9, and M11) in human serum, the LC–MS analytical method was optimized to be selective for all these analytes without interference at their retention times and mass transitions (Figure 2). As diastereomers, two 6G-diols could be baseline-separated, but the two 8G-diols and 10G-diols could not be completely separated. Therefore, the two 6G-diols were quantitated separately, and the two 8G-diols and the two 10G-diols were quantitated as one peak. 6G, 8G, 10G, 6S, 8G, 10S, 6G-diol-2, M2, M9, and M11 were quantitated based on their corresponding authentic standards. 4G was expressed as the equivalent of 6G; 6G-diol-1, 8G-diols, and 10G-diols were expressed as the equivalent of 6G-diol-2; and M1 was expressed as the equivalent of M2.
Table 2 shows the linear ranges, coefficients, and LLOQs. The LLOQs were 0.97 ng/mL for 6G and M11, 0.49 ng/mL for 8G and 10G, and 0.24 ng/mL for all other compounds, which is 5–10 times more sensitive than that of the LC–MS method used in a previous pharmacokinetic study.22 Table 3 shows that the extraction recovery of ginger compounds ranged from 84 to 97%, except for 6G, where the range was from 105 to 114%, indicating that the extraction was efficient and consistent. The precision and accuracy of all standards were within 15% (Table 4).
Table 2.
Calibration Curve Parameters of Gingerols, Shogaols, and Their Metabolites in Human Seruma
| compounds | R 2 | linear range (ng/mL) | LLOQ (ng/mL) |
|---|---|---|---|
| 6G | 0.999 | 0.97–4000 | 0.97 |
| 8G | 0.999 | 0.49–4000 | 0.49 |
| 10G | 0.997 | 0.49–4000 | 0.49 |
| 6S | 0.999 | 0.24–2000 | 0.24 |
| 8S | 0.998 | 0.24–2000 | 0.24 |
| 10S | 0.999 | 0.24–2000 | 0.24 |
| M2 | 0.999 | 0.24–2000 | 0.24 |
| M9 | 0.996 | 0.24–2000 | 0.24 |
| M11 | 0.998 | 0.97–1000 | 0.97 |
| 6G-diol-2 | 0.998 | 0.24–2000 | 0.24 |
(6G: 6-gingerol; 8G: 8-gingerol; 10G: 10-gingerol; 6S: 6-shogaol; 8S: 8-shogaol; 10S: 10-shogaol; 6G-diol-2: 6-gingerdiol-2; M2, M9, and M11 are the metabolites of 6S).
Table 3.
Extraction Recovery for Gingerols, Shogaols, and Their Metabolites in Human Serum, Expressed as Mean ± RSD (%)a
| QC (ng/mL) | 6S | 8S | 10S | M2 | M9 | 8G | 10G | 6G-diol-2 |
|---|---|---|---|---|---|---|---|---|
|
| ||||||||
| 6.25 | 87.6 ± 0.9 | 89.4 ± 3.2 | 89.3 ± 6.4 | 90.6 ± 5.3 | 90.9 ± 6.4 | 91.8 ± 3.9 | 88.0 ± 3.7 | 90.4 ± 7.8 |
| 100 | 88.8 ± 5.1 | 87.7 ± 6.2 | 86.2 ± 6.6 | 90.5 ± 1.3 | 90.5 ± 3.3 | 89.3 ± 6.5 | 88.4 ± 5.6 | 87.5 ± 2.2 |
| 400 | 91.8 ± 5.8 | 92.3 ± 3.2 | 91.3 ± 6.8 | 96.8 ± 1.4 | 91.8 ± 2.4 | 96.4 ± 2.7 | 93.5 ± 5.5 | 95.8 ± 3.0 |
|
| ||||||||
| QC (ng/mL) | 6G | QC (ng/mL) | M11 | |||||
|
| ||||||||
| 15.6 | 114.0 ± 4.6 | 3.9 | 87.7 ± 8.6 | |||||
| 250 | 105.3 ± 4.1 | 31.2 | 85.8 ± 0.7 | |||||
| 1000 | 112.3 ± 4.4 | 250 | 91.0 ± 3.7 | |||||
(6G: 6-gingerol; 8G: 8-gingerol; 10G: 10-gingerol; 6S: 6-shogaol; 8S: 8-shogaol; 10S: 10-shogaol; 6G-diol-2: 6-gingerdiol-2; M2, M9, and M11 are the metabolites of 6S).
Table 4.
Precision and Accuracy of LC–MS/MS Analysis of Gingerols, Shogaols, and Their Metabolitesa
| intra-day |
inter-day |
||||||
|---|---|---|---|---|---|---|---|
| compounds | nominal concentration (ng/mL) | measured concentration (ng/mL) | precision (% RSD) | accuracy (% bias) | measured concentration (ng/mL) | precision (% RSD) | accuracy (% bias) |
| 6G | 1.9 | 2.0 | 2.8 | 2.4 | 2.0 | 3.0 | 2.7 |
| 125 | 127.3 | 5.0 | 1.9 | 118.5 | 7.5 | −5.2 | |
| 1000 | 964.3 | 5.4 | −3.6 | 878.1 | 11.4 | −12.2 | |
| 8G | 1.0 | 1.0 | 9.2 | 2.6 | 1.0 | 2.7 | 0.4 |
| 62.5 | 60.7 | 4.0 | −2.9 | 61.8 | 1.3 | −1.2 | |
| 500 | 474.0 | 4.3 | −5.2 | 469.5 | 6.1 | −6.1 | |
| 10G | 1.0 | 1.0 | 12.6 | −3.1 | 0.9 | 2.3 | −5.6 |
| 62.5 | 60.3 | 6.0 | −3.6 | 61.2 | 2.1 | −2.1 | |
| 500 | 456.0 | 7.6 | −8.8 | 479.1 | 5.4 | −4.2 | |
| 6S | 0.5 | 0.5 | 8.3 | 2.8 | 0.5 | 2.8 | 5.5 |
| 31.2 | 30.3 | 4.9 | −3.2 | 31.5 | 3.8 | 0.8 | |
| 250 | 243.6 | 8.1 | −2.6 | 258.8 | 4.1 | 3.5 | |
| 8S | 0.5 | 0.5 | 3.6 | −1.7 | 0.5 | 3.4 | 1.7 |
| 31.2 | 30.3 | 10.4 | −3.2 | 31.7 | 4.4 | 1.3 | |
| 250 | 229.2 | 10.9 | −8.3 | 249.7 | 7.1 | −0.1 | |
| 10S | 0.5 | 0.5 | 9.4 | −0.5 | 0.5 | 4.6 | 4.2 |
| 31.2 | 28.6 | 12.2 | −8.4 | 31.4 | 6.2 | 0.5 | |
| 250 | 243.0 | 13.0 | −2.8 | 266.4 | 7.1 | 6.6 | |
| M2 | 0.5 | 0.5 | 5.8 | 1.9 | 0.5 | 0.1 | 1.9 |
| 31.2 | 31.0 | 8.9 | −0.8 | 31.4 | 2.4 | 0.5 | |
| 250 | 257.7 | 4.9 | 3.1 | 256.6 | 1.0 | 2.6 | |
| M11 | 3.9 | 4.0 | 7.1 | 1.5 | 4.0 | 1.3 | 0.0 |
| 31.2 | 31.7 | 2.0 | 1.3 | 31.5 | 0.8 | 0.1 | |
| 250 | 251.0 | 2.6 | 0.4 | 252.6 | 0.8 | 1.1 | |
| M9 | 0.5 | 0.5 | 3.2 | 3.7 | 0.5 | 2.3 | 5.1 |
| 31.2 | 32.3 | 7.8 | 3.3 | 31.0 | 5.4 | −0.7 | |
| 250 | 243.3 | 9.8 | −2.7 | 252.6 | 4.8 | 1.1 | |
| 6G-diol-2 | 0.5 | 0.5 | 11.7 | 4.8 | 0.5 | 0.4 | 5.1 |
| 31.2 | 31.2 | 5.6 | −0.3 | 27.9 | 13.7 | −10.7 | |
| 250 | 255.4 | 3.9 | 2.1 | 231.3 | 12.0 | −7.5 | |
(6G: 6-gingerol; 8G: 8-gingerol; 10G: 10-gingerol; 6S: 6-shogaol; 8S: 8-shogaol; 10S: 10-shogaol; 6G-diol-2: 6-gingerdiol-2; M2, M9, and M11 are the metabolites of 6S).
3.2.2. Phase I- and Cysteine-Conjugated Metabolites Make Significant Contribution to the Total Absorption of Gingerols and Shogaols in Asthma Patients.
In this study, eight subjects were treated with ginger extract or placebo. To identify which subjects were in the ginger-treated group, gingerols, shogaols, and their metabolites were searched from serum samples collected at 1 h of each subject by LC–MS, and four of the eight randomized subjects were in the ginger treatment group (data not shown). Among the four subjects, the concentrations of gingerols, shogaols, and their metabolites in one subject are less than 5% of those in the other three subjects, which might be due to the fact that this subject was not in compliance with the study protocol or has a very poor absorption of ginger compounds. Therefore, this subject was not included in the further quantification analysis.
As shown in Figure 3 and Table 5, gingerols, shogaols, and their metabolites reached their peak concentrations (Cmax) from 0.67 to 1.33 h (Tmax) after taking 1 g of ginger extract and were almost undetectable after 8 h of treatment. Their peak concentrations (Cmax) were observed at 586.4 ng/mL for 6G, 74.5 ng/mL for 8G, 86.8 ng/mL for 10G, 9.1 ng/mL for 4G, 58.7 ng/mL for 6G-diol-1, 192.1 ng/mL for 6G-diol-2, 6.3 ng/mL for 8G-diols, 5.5 ng/mL for 10G-diols, 26.4 ng/mL for 6S, 11.0 ng/mL for 8S, 60.4 ng/mL for 10S, 127.8 ng/mL for M1, 40.0 ng/mL for M2, 40.3 ng/mL for M9, and 201.1 ng/mL for M11. The half-lives of gingerols, shogaols, and their metabolites were between 1 and 3 h in human serum, except for 8S, which had a half-life of 0.6 h, which is very similar to the finding from a pharmacokinetic study in human subjects after taking 2 g of ginger supplement.22
Figure 3.
Pharmacokinetics of gingerols, shogaols, and their metabolites in human serum collected on the randomization visit (visit 3, V3). The concentrations (TOTAL means the sum of three forms: free, glucuronide, and sulfate; GLU, glucuronide conjugate; SUL, sulfate conjugate; FREE, free form) were averaged (n = 3) at each time point, and the variation was expressed as standard error. The last chart illustrates the ratio of the AUCs of 6G or 6S and its corresponding metabolites. (6G: 6-gingerol; 8G: 8-gingerol; 10G: 10-gingerol; 6S: 6-shogaol; 8S: 8-shogaol; 10S: 10-shogaol; 6G-diols: 6-gingerdiols; 8G-diols: 8-gingerdiols; 10G-diols: 10-gingerdiols; M1, M2, M9, and M11 are the metabolites of 6S).
Table 5.
Pharmacokinetic Parameters of the Total Amount of Gingerols, Shogaols, and Their Metabolites in Human Serum after Oral Administration of Ginger Extract Capsules (1.0 g)a
| compound | AUC(0–t) (ng/mL/h) | t1/2 (h) | Tmax (h) | Cmax (ng/mL) |
|---|---|---|---|---|
| 6G | 830.7 ± 166.5 | 1.7 ± 0.4 | 0.7 ± 0.2 | 586.4 ± 169.7 |
| 8G | 111.9 ± 20.1 | 2.1 ± 0.1 | 0.7 ± 0.2 | 74.5 ± 24.0 |
| 10G | 139.6 ± 7.1 | 1.4 ± 0.1 | 1.3 ± 0.3 | 86.8 ± 10.6 |
| 4G | 16 ± 3.2 | 1.5 ± 0.4 | 0.8 ± 0.2 | 9.1 ± 1.3 |
| 6G-diol-1 | 107.1 ± 26.7 | 1.3 ± 0.3 | 1.0 ± 0 | 58.7 ± 15.1 |
| 6G-diol-2 | 338 ± 110.8 | 1.1 ± 0.2 | 0.7 ± 0.2 | 192.1 ± 73.9 |
| 8G-diols | 14.2 ± 2.6 | 2.4 ± 0.5 | 1.3 ± 0.3 | 6.3 ± 1.6 |
| 10G-diols | 10.8 ± 1.4 | 1.7 ± 0.3 | 1.0 ± 0 | 5.5 ± 0.8 |
| 6S | 32.8 ± 7.6 | 1.3 ± 0.2 | 0.7 ± 0.2 | 26.4 ± 10.0 |
| 8S | 14.5 ± 2.6 | 0.6 ± 0.2 | 0.8 ± 0.2 | 11.0 ± 3.4 |
| 10S | 101 ± 12.5 | 1.3 ± 0.1 | 1.3 ± 0.3 | 60.4 ± 13.8 |
| M1 | 253.5 ± 37.6 | 1.1 ± 0.1 | 1.0 ± 0 | 127.8 ± 21.3 |
| M2 | 89.9 ± 8.1 | 1.6 ± 0.7 | 0.7 ± 0.2 | 40.0 ± 16.2 |
| M9 | 73.6 ± 23.1 | 1.9 ± 0.2 | 1.3 ± 0.323 | 40.3 ± 15.8 |
| M11 | 292.8 ± 78.2 | 0.1 ± 0.2 | 1.0 ± 0.5 | 201.1 ± 98.9 |
(6G: 6-gingerol; 8G: 8-gingerol; 10G: 10-gingerol; 6S: 6-shogaol; 8S: 8-shogaol; 10S: 10-shogaol; 6G-diol: 6-gingerdiol; 8G-diols: 8-gingerdiols; 10G-diols: 10-gingerdiols; M1, M2, M9, and M11 are the metabolites of 6S).
When calculating the area under curves (AUCs) and comparing between the parent ginger compounds (6G and 6S) and their metabolites, as shown in the last chart of Figure 3, the reduction metabolites of 6G (6G-diol-1 and -2) were detected as over half the amount of 6G in serum after taking 1 g of ginger extract. The amounts of the reduction metabolites of 6S including M9 and M11 were detected to be 10.5 times higher than that of 6S, and the cysteine conjugates of 6S including M1 and M2 were detected to be 11.2 times higher than 6S, indicating that more than 90% of 6S is metabolized to the phase I- and cysteine-conjugated metabolites. These findings suggest that the absorption of gingerols and shogaols was underestimated by previous ginger pharmacokinetic studies, which did not include the phase I- and thiol-conjugated ginger metabolites.22,24,25,33
We have reported that the metabolites of gingerols and shogaols remain biologically active to inhibit cancer cell growth,12,17 activate the Nrf2 pathway,18 and modulate intracellular calcium and relax the airway smooth muscle.20 Therefore, it is critical to measure the concentrations of the phase I- and thiol-conjugated metabolites of gingerols and shogaols and consider their contributions to the health benefits of ginger, for example, for asthma patients after ingestion.
3.2.3. Gingerols, Shogaols, and Their Phase I- and Cysteine-Conjugated Metabolites are Extensively Glucuroni-dated and Sulfated in Asthma Patients.
In order to determine the proportions of the free, glucuronidated, and sulfated gingerols, shogaols, and their phase I- and cysteine-conjugated metabolites, the serum samples were hydrolyzed with pure β-glucuronidase or sulfatase separately. The free forms were also quantitated without enzymatic hydrolysis. The amount of each glucuronidated metabolite was expressed as the subtraction from the amount after hydrolysis with pure β-glucuronidase by the free form, and the amount of each sulfated metabolite was expressed as the subtraction from the amount after hydrolysis with sulfatase by the free form.
As shown in Figure 3, all four gingerols were predominately present in their glucuronidated forms and free gingerols were barely detectable. The sulfated form of 6G was detected as half of its glucuronidated form, while the sulfated forms of other gingerols including 4G, 8G, and 10G were below 20% of their corresponding glucuronidated forms. The reduction metabolites of gingerols were also predominately presented in their glucuronidated forms, except for the minor reduction metabolite of 6G, 6G-diol-1, which had a slightly higher amount of the sulfated form than the glucuronidated form. Interestingly, a significant amount of free shogaols were detected. The amount of free 6S was similar to that of the sulfated 6S and about two-third of the glucuronidated 6S. The amounts of free 8S and 10S were even higher than that of the glucuronidated or sulfated 8S and 10S, respectively. For the metabolites of 6S, M1, M2, and M11 were present mainly in their glucuronidated forms and their free forms were barely detectable. The amounts of their sulfated forms were 30–50% of their glucuronidated forms. For M9, the reduction metabolite of 6S, the amount of its sulfated form was slightly higher than that of its glucuronidated form and its free form was not detectable. These data were comparable to those in healthy subjects after taking 2 g of ginger extract.22 Since gingerols, shogaols, and their metabolites are mainly in phase II-conjugated forms, it is important to further compare the bioactivities of these conjugated metabolites with those of their corresponding free forms.
3.2.4. Accumulation Effects of Gingerols, Shogaols, and Their Metabolites in Asthma Patients after 1 and 2 Months of Treatment.
In this study, participants took 1.0 g of ginger supplement in the morning and 1.0 g in the evening for 56 days, which began on their third study visit (V3; randomization visit: day 0), with an interim visit at day 28 (visit 4, V4) and the final visit at day 56 coinciding with the last day of study drug treatment (V5). Kinetic samples were collected from participants after the morning dose of 1.0 g of ginger supplement on day 1 (V3), day 28 (V4), and day 56 (V5). To measure whether there was an accumulation effect of ginger compounds after 28 and 56 days of treatments, the sum of the AUCs of free, glucuronidated, and sulfated forms of each of the gingerols, shogaols, and their metabolites was calculated and used for the comparison among the three visits. As shown in Figure 4, the AUCs of three major gingerols, 6G, 8G, and 10G, showed a gradient ascent trend from the randomization visit (V3; day 0) through the final visit (V5; day 56). The AUC of V5 for 4G was higher than that of V3 and V4, while no difference was observed for V3 and V4. The reduction metabolites of gingerols showed the same trend with gingerols. The AUCs of 6G-diol-2 and 10G-diols showed a gradient ascent from the randomization visit (V3) through the final visit (V5). The AUCs of V4 and V5 of 8G-diols were higher than that of V3 with V4 being the highest one. For 6G-diol-1, no difference was observed for V3 and V4 and the AUC of V5 was higher than those of V3 and V4. For shogaols, only 10S showed a gradient ascent trend from V3 to V5, the AUCs of V4 and V5 of 6S were even lower than that of V3, and no obvious change of 8S was observed among the three visits. Two metabolites of 6S, M9 and M11, showed a gradient ascent from V3 to V5. However, the two cysteine-conjugated metabolites, M1 an M2, did not show any accumulation effect. Finally, after counting the total amounts of all gingerols, shogaols, and their metabolites together in each subject, a gradient ascent trend was observed from the randomization visit (V3) through the third visit (V5) (the last chart in Figure 4). However, no statistical difference was observed because this is a pilot project with samples from only three participants.
Figure 4.
AUCs of gingerols, shogaols, and their metabolites in human serum collected on the three visits. The AUCs were averaged (n = 3) for each compound as the total amount of three forms at each time point, and the variation was expressed as standard error. V3, the randomization visit; V4, the second visit at 28 days after the randomization visit; V5, the final visit at 56 days after the randomization visit. The last graph shows the sum of AUCs of all compounds in one subject on each visit, and the variation was expressed as standard error from three subjects. (6G: 6-gingerol; 8G: 8-gingerol; 10G: 10-gingerol; 6S: 6-shogaol; 8S: 8-shogaol; 10S: 10-shogaol; 6G-diols: 6-gingerdiols; 8G-diols: 8-gingerdiols; 10G-diols: 10-gingerdiols; M1, M2, M9, and M11 are the metabolites of 6S).
To further determine whether long-term treatment has any accumulation effect on the absorption and metabolism of gingerols and shogaols in asthma patients, we compared the concentrations of gingerols, shogaols, and their metabolites at baseline (0 h) and 8 h time point of the three pharmacokinetic studies. As shown in Figure 5A,B, no gingerols, shogaols, and their metabolites were detectable at baseline for V3. However, after continuously consuming ginger extract capsules for 28 and 56 days, the ginger compounds were obviously detectable at baseline on V4 and V5 with the highest amounts on V5 for most of these compounds. When comparing the concentration of each compound at 8 h on each visit (Figure 5C,D), V4 and V5 also exhibited higher concentrations of ginger compounds and their metabolites than V3 with V5 showing the highest concentrations for all compounds. Although large individual variations were observed and we only have data from three participants, we were still able to get statistical differences for 8G, 10G, 6S, 8S, 10S, and M9 between V5 and V3. Because one out of the three participants for V4 did not provide an 8 h sample, we were not able to conduct statistical analysis between V4 and V3.
In a previous study, no accumulation effect was observed from human subjects who received 2.0 g of ginger supplement daily for 28 days.22 However, that study collected only one sample within 24 h of the last dose, which cannot accurately reflect the accumulation effect of ginger compounds and their metabolites after long-term treatment. Using AUCs, baseline levels, and data from 8 h time point, our results provide more convincing evidence for the accumulation effects from long-term ginger treatment at least for some of the metabolites. However, we do realize that this is a pilot study with data from only three participants. It is worthwhile to conduct a larger study with enough sample power to get a more clear-cut conclusion on the accumulation effects from long-term treatment.
In summary, this is the first study to report the major phase I- and cysteine-conjugated metabolites of gingerols and shogaols and the pharmacokinetics of these metabolites in human serum. It is also the first pharmacokinetic study of gingerols and shogaols in asthma patients. Our pharmacokinetic data showed that one-third of 6G, the most abundant gingerol in ginger supplement, was metabolized to the reduction metabolites, 6G-diols, and more than 90% of 6S, the most abundant shogaol in ginger supplement, was metabolized to its reduction and cysteine-conjugated metabolites. This finding clearly demonstrates the importance of investigating the metabolism of ginger compounds, considering the contributions of these phase I- and cysteine-conjugated metabolites to the total absorption of ginger compounds and associating their amounts to the changes of the measured biomarkers. Another important finding of this study is that long-term ginger consumption enhances the accumulation of gingerols, shogaols, and their metabolites, which should be validated in a larger study.
ACKNOWLEDGMENTS
We gratefully acknowledge financial support from the National Institutes of Health grant R61 AT009989 and the donation of the ginger and placebo capsules from Pure Encapsulations, LLC. We thank all patients enrolled in the study.
ABBREVIATIONS
- 4G
4-gingerol
- 6G
6-gingerol
- 8G
8-gingerol
- 10G
10-gingerol
- 6S
6-shogaol
- 8S
8-shogaol
- 10S
10-shogaol
- 6G-diols
6-gingerdiols
- 8G-diols
8-gingerdiols
- 10G-diols
10-gingerdiols
- M1, M2, M9, and M11
the metabolites of 6-shogaol
- PRM
parallel reaction monitoring
Footnotes
Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jafc.2c03150
The authors declare no competing financial interest.
Contributor Information
Shuwei Zhang, Laboratory for Functional Foods and Human Health, Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, Kannapolis, North Carolina 28081, United States.
Emily DiMango, Department of Medicine (Pulmonology, Allergy and Critical Care), Columbia University, New York, New York 10027-6902, United States.
Yingdong Zhu, Laboratory for Functional Foods and Human Health, Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, Kannapolis, North Carolina 28081, United States.
Tarnjot K. Saroya, Department of Medicine (Pulmonology, Allergy and Critical Care), Columbia University, New York, New York 10027-6902, United States
Charles W. Emala, Department of Anesthesiology, Columbia University, New York, New York 10027-6902, United States
Shengmin Sang, Laboratory for Functional Foods and Human Health, Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, Kannapolis, North Carolina 28081, United States.
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