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Pharmacokinetics of 10-gingerol and 6-shogaol in the plasma of healthy subjects treated with red ginger (Zingiber officinale var. Rubrum) suspension

Red ginger (Zingiber officinale var. Rubrum) is among the most widely consumed medicinal herbs in Indonesia. Ginger rhizome contains phenol compounds including gingerol and shogaol. 10-gingerol has been reported to exhibit the greatest anti-inflammatory and anti-oxidant activities compared with those of other gingerols. Pharmacokinetic studies on ginger have been reported, but there is a lack of such study on red ginger. The present work studied the pharmacokinetics of 10-gingerol and 6-shogaol in the plasma of healthy subjects treated with a single dose of red ginger suspension. Healthy subjects (n=19) were given a single dose of red ginger suspension (2 g/15 ml), and blood samples were taken at baseline (0 min), 30, 60, 90, 120, and 180 min. Analysis of 10-gingerol and 6-shogaol was performed by dissolving 200 µl of the subjects' plasma in 800 µl acetonitrile. The mixture was vortexed and centrifuged at 20,440 × g for 15 min at room temperature. The supernatant was filtered using Millipore membrane (pore size 0.2 µm) and injected into an RP-C18 column for liquid chromatography-mass spectrometry. A mixture of 0.1% (v/v) formic acid in water and acetonitrile (38:62) was used as the mobile phase. The maximum plasma concentration (Cmax) and time to reach Cmax of 10-gingerol and 6-shogaol were 160.49 ng/ml (38 min) and 453.40 ng/ml (30 min), respectively. The elimination half-lives were 336 and 149 min for 10-gingerol and 6-shogaol, respectively. Thus, 10-gingerol and 6-shogaol were absorbed after per oral single dose of red ginger suspension and could be quantified in the plasma of the healthy subjects. Additionally, the red ginger analytes exhibited relatively slow elimination half-lives.

A comparative study of Z. officinale var. Roscoe pulp and peel demonstrated that the hydro-alcoholic extract of the peel exhibited marked inhibition of the growth of colon cancer cells on MTT assay, while the pulp extract exhibited high anti-inflammatory and antioxidant activities, allegedly due to differing polyphenolic content and lipophilic composition (8). Data on the effects of Z. officinale var. Roscoe have been reviewed, and it has been concluded that the rhizome of this plant has potential as an anti-inflammatory and anti-oxidative Pharmacokinetics of 10-gingerol and 6-shogaol in the plasma of healthy subjects treated with red ginger (Zingiber officinale var. Rubrum) suspension source, nonetheless further research has been suggested prior to confirming its efficacy (9). The medicinal properties of Z. officinale var. Roscoe may be due to the presence of gingerol, paradol and shogaols, among other compounds (10). Gingerols are homologues of 1-(3-methoxy-4-hydroxy phenyl)-3-keto-5-hydroxyhexane, shogaols are dehydration products of the gingerols, and paradols are β-ketone hydroxyl deoxygenation products of gingerols (11). In previous studies Z. officinale var. Roscoe has exhibited anti-inflammatory activity (8,(12)(13)(14)(15)(16). 10-gingerol (Fig. 1A) has been reported to exhibit the highest anti-inflammatory and anti-oxidant activities compared with those of other gingerols (17). Other studies on 6-gingerol have confirmed its inhibition of nitric oxide production in activated J774.1 mouse macrophages and prevention of peroxynitrite-induced oxidation and nitration reactions (18). This compound may also inhibit cyclooxygenase (COX)-2 expression by blocking the activation of p38 mitogen-activated protein kinase and nuclear factor-κB in phorbol ester-stimulated mouse skin (19). However, studies on the effects of red ginger (Z. officinale var. Rubrum) are limited.
Previous study by Fikri et al (20) concluded that the hot water extract of Z. officinale var. Rubrum rhizome inhibited the rate of prostaglandin production in vitro. Furthermore, a computational study of gingerol and shogaol against COX enzymes reported that 6-gingerol and 6-shogaol were preferential COX-2 inhibitors and therefore potential candidates for development into anti-inflammatory drugs (21). Pharmacokinetic studies of the dry extract of ginger rhizomes (at doses of 100 mg to 2 g) in the plasma of healthy American volunteers have been conducted (22,23). However, there is a lack of reports on red ginger. Thus, the present work studied the pharmacokinetics of 10-gingerol (Fig. 1A) and 6-shogaol ( Fig. 1B) in the plasma of healthy Indonesian volunteers treated with a single dose (2 g) of red ginger suspension. The aims of this study were to: i) Determine if red ginger suspension is absorbed and biotransformed in humans; and ii) assess the human pharmacokinetics of 10-gingerol and 6-shogaol.

Materials and methods
Instruments. Instruments used in the study were a liquid chromatography-mass spectrometry (LC-MS) XEVO Preparation of red ginger (Zingiber officinale var. Rubrum) suspension. The 1 kg of fresh rhizome of Z. officinale var. Rubrum was thin-sliced (1-2 mm) and dried in a dehydrator at 50˚C for 4 h. The dried rhizome was ground and each portion per subject contained 2 g of the powder dissolved in 15 ml hot distilled water (70˚C).
Identification of 10-gingerol and 6-shogaol in Z. officinale var. Rubrum suspension. Identification of 10-gingerol and 6-shogaol in Z. officinale var. Rubrum suspension was performed by dissolving the suspension in 15 ml methanol hypergrade. The mixture was vortexed and centrifuged at 20,440 x g at room temperature for 15 min. The supernatant was scanned at 200-380 nm in a double beam UV spectrophotometer against the methanol hypergrade its maximum wavelength (λmax) was compared with those of 10-gingerol and 6-shogaol.
Preparation of 10-gingerol and 6-shogaol standard solutions. A total of 2.5 mg of each of the 10-gingerol and 6-shogaol standards was dissolved in 100 ml methanol hypergrade in a volumetric flask (25 µg/ml). The solutions were scanned at 200-380 nm in the double beam UV spectrophotometer against the methanol hypergrade to obtain the λmax's of 10-gingerol and 6-shogaol. Various concentrations were prepared by diluting the standard solution (Tables I and II Table III.

Validation of analytical method
Selectivity. The selectivity of the method was investigated by comparing the chromatogram of extracted blank plasma (3 ml) obtained from six randomly selected participants described below, each spiked with 25.0 ng/ml 6-shogaol.
Linearity. Linearity of the standard curves was calculated using human plasma spiked with various concentrations Accuracy and precision. The accuracy and precision of the method were evaluated by analyzing 10-gingerol and 6-shogaol in three sets of quality control samples [lower concentration of quality control (LCQC)=2.5 ng/ml; medium concentration of quality control (MCQC)=15 ng/ml; and higher concentration of quality control (HCQC)=25 ng/ml each] within the same day. Each solution was injected into the column in three replicates. The percentage recovery and standard deviation (SD) were calculated to ascertain accuracy and precision of the analytical method, respectively.

Limit of detection (LOD) and limit of quantification (LOQ).
The LOD and LOQ were calculated by using: LOD=(3xSD)/b; and LOQ=(10xSD)/b where b is the y-intercept of the linear regression curve (25).
Subjects and treatment. A total of 21 participants (8 females and 13 males) aged 21-30 years, with body mass indices (BMIs) of 18-25, non-smokers and/or alcohol drinkers, were solicited by advertisement from May to September 2017. All participants were confirmed to be healthy by physical examination at Padjadjaran Clinic, Padjadjaran University (Jatinangor, Indonesia; Table IV). Two of the participants (R14 and R20) had low BP (90/60). Initially both were included in the study, but one of them (R14) vomited during the treatment, and was excluded. One of the participants (R21) was menstruating on the day of preliminary health screening and was excluded. The remaining 19 subjects continued the study. All study procedures were administered at the Faculty of Pharmacy, Padjadjaran University following collection of signed written informed consent according to the principles of the Declaration of Helsinki. The subject protocols and treatment were approved by the Research Ethics Committee of Padjadjaran University (approval nos. 1211/UN6.C.10/PN/2017 for 10-gingerol and 924/UN6.C.10/PN/2017 for 6-shogaol). The subjects were asked to avoid all foods containing ginger within 7 days prior to the project and completed a food checklist to verify that they were not consuming any ginger-rich food or beverages. All subjects received the Sundanese standard meal (one portion of white rice equivalent to 240 cal, 100 g of fried chicken/tofu equivalent to 110-120 cal, hot tea equivalent to 70 cal, and a banana/orange 3 times/day at 24 h pre-study and for breakfast on the day of the study. The red ginger suspension was administered the subsequent morning after the standard breakfast had been received. Each subject was given one portion of red ginger suspension (2 g in 15 ml of water) as a single oral dose, and 3 ml blood samples were taken from the subjects at baseline (0 min), 30, 60, 90, 120 and 180 min. The blood was put into the K2EDTA vacutainer tubes. Plasma was separated by centrifuging at 20,440 x g for 15 min at room temperature and kept at -20˚C until assayed.
Pharmacokinetics of 10-gingerol and 6-shogaol. Analysis of 10-gingerol and 6-shogaol was performed by dissolving 200 µl of the subjects' plasma in 800 µl acetonitrile. The mixture was vortexed and centrifuged at 20,440 x g for 15 min at room temperature. The supernatant was filtered using Millipore membrane (pore size 0.2 µm) and injected into the RP-C18 column embedded in the LC-MS XEVO-QTOFMS.

Results and Discussion
Identification of 10-gingerol and 6-shogaol in Z. officinale var. Rubrum suspension. Gingerol and/or shogaol were indi-  cated to be present in the red ginger suspension by the λmax of red ginger suspension in methanol (λmax=286 nm) which was similar to those of 10-gingerol (λmax=284 nm) and 6-shogaol (λmax=282 nm; Fig. 2). Ethanol can extract gingerols more effectively than water; moreover, hot water is reportedly more effective than cold water (26). Ghasemzadeh et al (27) when working on the optimization procedure for the extraction of 6-gingerol and 6-shogaol (focusing on temperature 50-80˚C and time 2-4 h) reported that increasing the extraction temperature (up to 76.9˚C) and time (3. h) induced the highest yield of 6-gingerol (2.74 mg/g dry weight) and 6-shogaol (1.59 mg/g dry weight) from Z. officinale var. Rubrum Theilade (27). The present work employed hot distilled water (70˚C) to extract 2 g of the red ginger (Z. officinale var. Rubrum) powder.
Validation of analytical method. The LC-MS analytical method was selective for both 6-shogaol (Fig. 3) and 10-gingerol (Fig. 4) as proven by the respective chromatogram peaks that were free of interference at the retention time of 6-shogaol (6.70 min) and 10-gingerol (8.26 min). The MS Table III. LC-MS optimum analytical conditions for 10-gingerol and 6-shogaol (N 2 gas temperature 350˚C; drying N 2 gas flow rate 10 l/min; nebulizer pressure 50 psi).    Linearity of the standard curves was constructed using human plasma spiked with various concentrations (2.5, 5.0, 10.0, 15.0, 20.0 and 25.0 ng/ml) of 10-gingerol and 6-shogaol (Fig. 5). The LCQC (2.5 ng/ml), MCQC (15.0 ng/ml) and HCQC (25.0 ng/ml) of the ginger analytes were assayed on an intra-day basis to determine the accuracy and precision of the method. The intra-day values for the SD ranged from 0.16-0.69 for 10-gingerol and 0.74-1.49 for 6-shogaol, whereas the recovery of the analytes was 100.64-104.44% for 10-gingerol and 103.40-104.22% for 6-shogaol. The data (plot of AUC of the chromatogram peak against the concentration   (Table V).

LC-MS parameter
The LODs in human plasma for 10-gingerol and 6-shogaol were 1.31 and 1.51 ng/ml, respectively, and the LOQs were 4.36 and 5.03 ng/ml, respectively.
Pharmacokinetics of 10-gingerol and 6-shogaol. 10-gingerol and 6-shogaol could be quantified in the plasma of healthy  subjects treated with a single dose of red ginger suspension. They were eluted at 6.80 min (6-shogaol) and 8.0 min (10-gingerol; Fig. 6), compared with the pure compounds (for 6-shogaol, 6.70 min and for 8.26 min;Figs. 3 and 4), respectively. There was a slight difference of elution time between the standards (pure 6-shogaol and 10-gingerol compounds) and that of red ginger suspension due to different conditions; the standards were spiked in vitro into human plasma, while the red ginger suspension was administered orally and taken ex vivo from the subjects' blood. The pharmacokinetic profile of each of the red ginger compounds is presented in Table VI.
The present study used a single dose (2 g) of red ginger (Z. officinale var. Rubrum) suspension, administered orally to 19 healthy Indonesian participants (13 males and 6 females). Both 10-gingerol and 6-shogaol could be detected and quantified. In the plasma of the healthy Indonesians, 10-gingerol was quantified to a lower level than 6-shogaol.
10-gingerol and 6-shogaol exhibited slower elimination in Indonesian subjects compared with that in Caucasians (T½ elimination <120 min). In the current study, no serious adverse effects were reported following ingestion of the red ginger suspension, though several female subjects complained of the potent pungent odor and spicy taste that caused stomach discomfort. These mild adverse effects were consistent with those reported by Zick et al (22) and in a previous review study by Chrubasik et al (28), who documented that the majority of the disadvantages were transient gastrointestinal reactions, such as gas and bloating.
In conclusion, 10-gingerol and 6-shogaol were absorbed after per oral single dose of red ginger suspension and could be quantified in the plasma of healthy Indonesian subjects. The Cmax of 10-gingerol (160.49 ng/ml) was lower than that of 6-shogaol (453.40 ng/ml). Notably, the two red ginger analytes exhibited relatively slow elimination half-lives. In the present trial, no serious adverse effects were reported following ingestion of the red ginger suspension, but several female subjects complained about the potent pungent odor and spicy taste that caused stomach discomfort. Overall, the present pharmacokinetic findings of 10-gingerol and 6-shogaol in Indonesian subjects confirmed that different ethnicities may contribute to different pharmacokinetic profiles. Identification of these differences may lead to personalized-medicine, and thus contribute in a clinical context, particularly in the discovery and development of anti-inflammatory drugs. of Mathematics and Natural Sciences, Padjadjaran University, for identifying the plant specimen.

Availability of data and materials
The datasets used and/or analyzed in the current work are available from the corresponding author on reasonable request.

Authors' contributions
JL and AD were principally responsible for conception and design of the study. JL, MM and AD participated in the acquisition of the reported data. DMS, RDS, SM and MF participated in the processing, analysis and interpretation of the reported data. JL and SM contributed to the writing and revising of the manuscript. All authors read and approved the final manuscript to be published.

Patient consent for publication
Informed consent was obtained for publication of the participants' data.