The Chemical Signatures of Water Extract of Zingiber officinale Rosc

Background: Ginger (Z. officinale Rosc.) is a common herb and is widely used as a diet-based or home therapy in traditional medicine worldwide. However, fresh ginger turns into dried ginger after kiln drying and shows a different treatment effect in clinical practice. Objective: To characterize the changes of major bioactive constituents in dried ginger after the processing of fresh ginger. Methods: A novel, ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC–QTOF/MS) method was established to characterize the changes in the bioactive constituents of dried ginger. The novel strategy was split into two steps: firstly, the MS selected the most intense precursor ions of tandem MS; then, target MS/MS acquisition with different collision energies (10, 20, and 40 eV) was used to characterize the compound’s accurate MS/MS spectra and compare the MS/MS spectrum with the building MS reference library and reference standards. Result: Fifty-three compounds, including diarylheptanoids, gingerols, gingerodiols, gingerdiones, and shogaol-related compounds, were identified based on summarized fragmentation patterns. Fifteen out of fifty-three compounds were diarylheptanoids, which was different from fresh ginger. Conclusion: These identified compounds could be used to characterize the quality of dried ginger, pharmacologic studies should focus on diarylheptanoids explaining the different treatment effects between fresh ginger and dried ginger.


Introduction
Ginger (Z. Rosc.) is a common spice that is widely used as a diet-based or home therapy in various traditional systems of medicine around the world. It has been used in traditional medicine practice for the treatment of arthritis [1][2][3]; rheumatological diseases; gastrointestinal disorders such as distress symptoms, digestive disorders, and pain [4][5][6]. Clinical pharmacology research has shown that the active compounds in ginger are responsible for antioxidant and cancer-preventive properties [7][8][9], the prevention of chemotherapy-induced toxicity [10], and the improvement of inflammatory bowel disease and colitis [11,12]. However, the chemicals responsible for ginger's pharmacology vary considerably.
Gingerols, shogaols, and their homologues are the major volatile components of Zingiber officinale Rosc. The percentage of volatile components varies based on the plantation region and harvesting season. Non-volatile components include paradols and their derivatives; zingerone; monoterpenoids; organic acids; and flavonoids [13][14][15][16][17]. In clinical practice, Molecules 2022, 27, 7818 2 of 14 the decoction of dried ginger is a common practice in traditional medicine and shows different health effects from fresh ginger against chronic diseases [18]. One reason is that the major compounds in fresh ginger are liable to dehydrate and convert when exposed to heat and/or acidic conditions. The other is that during the decoction process, the active components vary sharply with changes in temperature and boiling time [19,20]. Therefore, in the current study, we explored the bioactive constituents in the aqueous extract of dried ginger.
The components of fresh ginger has been chiefly studied by high-performance liquid chromatographyor with a gas chromatography-mass spectrometer (GC-MS), because volatile oil is believed to be its main effective constituent [21]. These are also the usual ways to conduct quality assessments of ginger [22][23][24]. However, due to the characteristics of volatile oil, the quality control and bioactive components of ginger are still under investigation.
Mass spectrometry is currently one of the most robust and sensitive instrumental methods applied to the structural characterization of the secondary metabolite of ginger [25,26]. Therefore, we will use UHPLC-QTOF/MS to tentatively identify and characterize the chemical signature of the dried ginger. In addition, the gingerols and shogaols ' homologues usually have the same skeletons as the parents. Therefore, the fragmentation of the parents molecules will provide specific structural information about the functionality of different compounds and is necessary and helpful for the characterization of the gingerols and shogaols ' analogues in the tandem mass spectrometry (MS/MS) model.

LC/MS Conditions
In this study, tandem mass spectrometry (MS/MS) was used to identify the molecular ions of different components. The results were shown in Figures 1 and 2 and Table 1. Most of the ingredients in dried ginger were identified within 40 min. Both positive and negative ionization modes were used to detect the compounds (Figures 1 and 2). It was found that the signal in the QTOF/MS positive mode was much more sensitive than that in the negative mode. Different polarity compounds are detected in the two ionization modes. It looks like the gingerol and shogaol derivatives ionize better in the negative mode. In the tandem MS negative mode, the intensity of the compound fragments was too weak to analyze. About 90 different ions were presented in the positive MS mode. In total, we characterized 53 compounds.

Establishment of Fragmentation Patterns
Reference standards (zingerone, 6-gingerol, and 6-shogaol) were used in this study to demonstrate identity ( Figure 3). These compounds were dissolved in 50% methanol at a final concentration of 1 µM for the UHPLC-ESI QTOF MS/MS analyses. Figure 3 shows the reference standards' base peak chromatograms (BPC) in both positive and negative ionization modes. All constituents were identified by comparing the UHPLC retention time, accurate mass, and mass spectrum with those standards ( Table 1). The abnormal peak in Figure 3 was the background contamination ion in the MS, and it did not impact our analysis.

Establishment of Fragmentation Patterns
Reference standards (zingerone, 6-gingerol, and 6-shogaol) were used in this study to demonstrate identity ( Figure 3). These compounds were dissolved in 50% methanol at a final concentration of 1 μM for the UHPLC-ESI QTOF MS/MS analyses. Figure 3 shows the reference standards' base peak chromatograms (BPC) in both positive and negative ionization modes. All constituents were identified by comparing the UHPLC retention time, accurate mass, and mass spectrum with those standards ( Table 1). The abnormal peak in Figure 3 was the background contamination ion in the MS, and it did not impact our analysis.

Gingerol-Related Compounds
Gingerol-related compounds are the most important components in fresh ginger. Gingerol, gingerdione, gingerdiol, and shogaol contribute the predominant peppery taste in Z. officinale Rosc. Additionally, they all have a 4-hydroxy-3-methoxyphenylmoiety with different hydrocarbon chains. In our study, only a few gingerol-related compounds were

Gingerol-Related Compounds
Gingerol-related compounds are the most important components in fresh ginger. Gingerol, gingerdione, gingerdiol, and shogaol contribute the predominant peppery taste in Z. officinale Rosc. Additionally, they all have a 4-hydroxy-3-methoxyphenylmoiety with different hydrocarbon chains. In our study, only a few gingerol-related compounds were identified in the extraction of dried ginger, including compound 33([6]-gingerol),

Gingerdione-Related Compounds
Gingerdione, a gingerol derivative, is one of the major constituents of dried ginger.  [27].

Gingerdione-Related Compounds
Gingerdione, a gingerol derivative, is one of the major constituents of dried ginger.

Gingerdiol-Related Compounds
The precursor ion of compound 48 was sodium adduct ions in (+) ESI-MS, which is different from the standard compound, [6]-gingerol (Table 1). However, the fragmentation pattern of [6]-gingerdiol was similar to that of [6]-gingerol, and they both broke into m/z 177.0899 and 137.0589. According to the (+) ESI-MS/MS spectra for compound 43, we know that the parent ion fragments into 321.2052[M+H-CH3COO] + due to the loss of 60 Da (AcOH). This information suggests that the acetoxy group, and not the hydroxy group, is present on the aliphatic side chain of compound 48.

Gingerdiol-Related Compounds
The precursor ion of compound 48 was sodium adduct ions in (+) ESI-MS, which is different from the standard compound, [6]-gingerol (Table 1). However, the fragmentation pattern of [6]-gingerdiol was similar to that of [6]-gingerol, and they both broke into m/z 177.0899 and 137.0589. According to the (+) ESI-MS/MS spectra for compound 43, we know that the parent ion fragments into 321.2052[M+H-CH 3 COO] + due to the loss of 60 Da (AcOH). This information suggests that the acetoxy group, and not the hydroxy group, is present on the aliphatic side chain of compound 48. Compound 43 was identified as diacetoxy-

Diarylheptanoids
More than 15 compounds were identified as diarylheptanoids, which are primarily responsible for cytotoxicity and apoptosis in ginger [9,29,30]. These diarylheptanoids were characterized by the presence of 5-hydroxy and 3-oxo groups on the heptane skeleton ( Figure 5A,B).

Diarylheptanoids
More than 15 compounds were identified as diarylheptanoids, which are primarily responsible for cytotoxicity and apoptosis in ginger [9,29,30]. These diarylheptanoids were characterized by the presence of 5-hydroxy and 3-oxo groups on the heptane skeleton ( Figure 5A, B).   Figure 5B) at different collision energies (Figure 6). We identified these compounds based on the characteristics of the compounds shown in research conducted by Riethmüller [31,32] and Svarc-Gajic [33]. The typical scheme of diarylheptanoid fragmentation was shown in Figure 6.  24, 30, 32, 35, 36, and 38 have CH3CO-at both the 3 and 5 positions of the carbon chain. Most of them can fragment into m/z 177.0690 ( Figure 5A) or 137.0588 ( Figure 5B) at different collision energies ( Figure 6). We identified these compounds based on the characteristics of the compounds shown in research conducted by Riethmüller [31,32] and Svarc-Gajic [33]. The typical scheme of diarylheptanoid fragmentation was shown in Figure 6.  Table 1, indicating that it may be a homolog of compound 6 with a difference in the carbon-carbon double bond. The protonated ion compound 15 (387.1729) is 14 Da larger than compound 7 (373.1578), which indicates that compound 15 has a methoxy group. Compounds 9 and 14 displayed the same fragmentation behavior; however, protonated ion compound 14 (405.1885) is 14 Da larger than compound 9 (391.1731), differing in a methoxy group. The fragmentation pattern of compound 23 is unique and was identified as 3-acetoxy-5-hydroxy-1-(3,4-dihydroxy-5-methoxyphenyl)heptane by referring to the literature [34]. In the precursor ion scan spectrum, m/z of compound 3 [M+H] + was 153.1282. It can fragment into m/z 93.0332[M+H-CH 2 COO] + and 65.0383[M+H-CH 2 COO-CO] + in the positive ionization mode. Therefore, compound 3 was identified as citral [35]. Compounds 28,39, and 52 are fatty acids and were tentatively identified by comparison with the literature [36].

Two Amino Acids, Four Fatty Acids, and Others
Compounds 4 and 29 were also identified in the extractions of Z. mioga and Z. officinale, and their daughter ions matched galanganol C and curcumadiol's daughter ions [37].

Plant-Material and Sample Preparation
Dried ginger materials appeared as yellow primrose pieces of wood and were purchased from Kangmei Chinese Traditional Medicine. Co. Ltd. (Guangdong, China). Original herbs were produced from Sichuan in 2017 (MAN: 2017.04.01)-10 g in each package (LOP: 170400541). The dried ginger was immersed in eight-fold volumes of water (1:8, w/v) for 30 min and boiled for 1 h. The decoction was filtered through 8 layers of gauze and was lyophilized to dried powder. We evaluated the amount of solvent (1:2, 1:4, and 1:8) and the extraction time (0.25, 0.5, 1, and 2 h) impact on the extraction efficiency, and we found that the ginger was boiled for a long time (2 h) or short time (0.5 h), removing valuable components from the extractions. Therefore, we closed 8-fold water and only boiled 1 h to acquire more peaks in the mass spectrum.
The LC-MS sample preparation: 1 mL of deionized water was added to 50 mg of the freeze-dried powder, vortexed for 10 min, and then sonicated for 15 min at 40 • C. The mixture was then vortexed vigorously for 3 min, after centrifugation at 15,000 rpm for 30 min. Five microliters of supernatant was injected into the UPLC-MS/MS system for qualitative analysis.

Mass Spectrometry
Full acquisition MS, auto MS/MS, and targeted MS/MS were performed with a 6540 QTOF Mass Spectrometer in both positive and negative ionization modes. Full acquisition MS spectra were collected over a mass range of m/z 100-1700, and the acquisition rate was 1 Hz at 1000 ms/scan. In the auto MS/MS and targeted MS/MS modes, the precursor MS spectrum was from m/z 100 to 1500, and the acquisition rate was 2 Hz with 500 ms/scan; the MS/MS spectrum was from m/z 50 to 1000, and the acquisition rate was 3 Hz with 333 ms/scan. The conditions of the ESI source were as follows: a drying gas (N2) flow rate of 11 (L/min); a drying gas temperature of 300 • C; a sheath gas temperature of 350 • C; a nebulizer at 40 psi; a capillary voltage of 3.5 kV (negative mode) or 4 kV (positive mode); a fragmentor at 175 V; a skimmer voltage of 60 V; and an octopole RF of 250 V. Every day, prior to the analyses of the samples, the mass axis was calibrated. All of the operations, acquisitions, and analyses of data were controlled by Mass Hunter software version B.06.00 (Agilent Technologies, Santa Clara, CA, USA).

Building the Chemical Database of Ginger
The ginger database was created using the Agilent software, Personal Compound Database Library (PCDL). The database contained the formulas, accurate masses, compound names, and original plants. The ginger database is available in Supplementary  Table S1. The records of 236 compounds were input into the database by comprehensively searching databases such as Sci Finder, PubMed, TCM Database@Taiwan, Chinese National Knowledge Infrastructure of Tsinghua University, and KNApSAcK for all of the compounds reported in the literature for ginger (Supplementary Table S1).

Preparation of the Reference Standard
The stock solutions of the reference standard (gingerol, zingerone, and 6-shogaol) were prepared in ethanol/DMSO (4:1, v:v) at final concentrations of 10 mM. Then, each stock solution was diluted by 50% methanol to 1 µM for analysis.

Conclusions
A novel UHPLC-ESI-QTOF/MS approach was developed to identify chemical profiles of Z. officinale Rosc.. Many studies have demonstrated that the major function of the drying process is to reduce the gingerol concentration, increase the terpene hydrocarbon level, and convert some monoterpene alcohols into their corresponding acetates [13,24,38,39].
Our study results showed that the major components of dried ginger are diarylheptanoids. However, the main compounds in fresh ginger, gingerols, and shogaols, which are responsible for the bioactivity and spicy taste, were present in very low concentrations. This result may support the fact that dried ginger has different chemical constituents and pharmacological activities when compared with fresh herbs in clinical practice. Therefore, future pharmacologic studies should focus on these diarylheptanoids.
Most of the gingerol-related compounds have -OH and -OCH 3 groups on their benzene ring. All of them fragment into the basement product ion m/z 137 (2-hydroxy-3methoxyphenyl-CH 2 + and 2,3-methoxyphenyl + ). This fragmentation pattern was useful for diagnosing fragmentation behavior in positive and negative ESI-QTOF/MS, and analyzing the structures of homologs and allowed us to classify compounds by group and identify them based on key structural features. Overall, our novel strategy only requires 1-2 h to complete each peak, with each compound feature guided by the in-house database.
Author Contributions: H.C. and F.L. wrote the initial manuscript with major input from W.X.; R.S. H.C., F.L. and W.X. performed the experiments and analyzed the results; S.L. carried out the data interpretation; and S.L., W.X. and R.S. designed the study and obtained funding for the studies. All authors have read and agreed to the published version of the manuscript.