BioMed Research International
Volume 2015 (2015), Article ID 671058, 7 pages
http://dx.doi.org/10.1155/2015/671058

Research Article

Analysis of Chemical Properties of Edible and Medicinal Ginger by Metabolomics Approach

Ken Tanaka,¹ Masanori Arita,2,3 Hiroaki Sakurai,⁴ Naoaki Ono,⁵ and Yasuhiro Tezuka⁶

¹College of Pharmaceutical Science, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Shiga 525-8577, Japan
²Center for Information Biology, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
³RIKEN Center for Sustainable Resource Science, Yokohama, Tsurumi 230-0045, Japan
⁴Department of Cancer Cell Biology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan
⁵Graduate School of Information Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan
⁶Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920-1181, Japan

Received 27 April 2015; Revised 1 July 2015; Accepted 1 July 2015

Academic Editor: Paul M. Tulkens

Copyright © 2015 Ken Tanaka et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

In traditional herbal medicine, comprehensive understanding of bioactive constituent is important in order to analyze its true medicinal function. We investigated the chemical properties of medicinal and edible ginger cultivars using a liquid-chromatography mass spectrometry (LC-MS) approach. Our PCA results indicate the importance of acetylated derivatives of gingerol, not gingerol or shogaol, as the medicinal indicator. A newly developed ginger cultivar, Z. officinale cv. Ogawa Umare or “Ogawa Umare” (OG), contains more active ingredients, showing properties as a new resource for the production of herbal medicines derived from ginger in terms of its chemical constituents and rhizome yield.

1. Introduction

Traditional medicine embodies accumulation of knowledge, skills, and practices on the maintenance of health as well as the prevention, diagnosis, improvement, or treatment of physical and mental illness. World Health Organization reported that, even today, more than 80% of the world’s population utilizes traditional medicine for primary health care [1]. Such medicinal system prescribes a combination of herbal, animal, and mineral parts, collectively known as crude drug, whose core materials are derived from plants including seeds, berries, roots, leaves, bark, or flowers [2]. The chemical constituents of crude drug are therefore considered a “chemical system,” which consists of a complex mixture of primary and secondary metabolites such as saponins, flavonoids, and alkaloids. The system is represented as a matrix in which rows and columns represent natural species and their chemical ingredients, respectively. This matrix works on another matrix representing the human body system, in which rows and columns represent interactive biomolecules (e.g., genes, proteins, and metabolites) and their tissue distribution, respectively. Thus, the research on traditional medicine deals with this “system to system” methodology, instead of the “point to point” methodology of western medicines (e.g., one particular chemical and its receptor gene). To understand the total function of traditional medicine, the knowledge of the interactions between matrices representing “chemical system” and “body system” is crucial. The matrix representing the human body system has gradually been made clear through several omics approaches, whereas knowledge on chemical system is not enough since almost all studies were done based on “point to point” or “point to system” methodology. Thus, we are accumulating the knowledge on chemical system with metabolomics approach [3–6].

Following our previous report on a newly registered turmeric (Curcuma longa cv. Okinawa Ougon) [3], we recently here investigate the chemical system of ginger cultivars. Ginger, the rhizome of the plant Zingiber officinale Roscoe, is widely used as a spice and herbal medicine for the treatment of catarrh, rheumatism, nervous diseases, gingivitis, toothache, asthma, stroke, constipation, and diabetes [7]. The genus Zingiber is distributed in tropical and subtropical Asia, Far East Asia, and Africa and is under cultivation mostly in India and China. The global consumption of ginger has been increasing rapidly, and the recent, growing demand for natural products as additives for functional food and beverages makes ginger an ideal candidate for development. Thus, attempts at crop improvement for ginger have been performed in order to increase the yield and enhance the concentration of its active constituents. Traditionally, crop improvement involves controlled crosses (hybridization) between selected cultivars with desirable properties.

The target of our metabolomic approach is three medicinal ginger types, “Shokyo” (dried rhizome of Z. officinale var. rubens), “Kankyo” (steamed and dried rhizome of Z. officinale var. rubens) from Kampo (traditional Japanese) medicine, and “Red ginger” (rhizome of Z. officinale var. rubra) from Indonesian traditional medicine (Jamu) [8, 9], and two edible ginger types, “Shoga” (fresh rhizome of Z. officinale var. rubens) from Japan and “White ginger” (rhizome of Z. officinale var. amarum) from Indonesia. From the comparison of five cultivars, we evaluate a new cultivar, Z. officinale cv. Ogawa Umare or “Ogawa Umare” (OG), and show its effectiveness as crude drug. OG was recently registered in the Japanese Plant Variety Protection (Ministry of Agriculture, Forestry and Fisheries, Japan) [10] and is characterized by its bold rhizome (3 times bigger than ordinary ginger) and a more pungent taste than standard medicinal ginger. All assays were conducted in a metabolomics platform with LC-MS and our results are consistent with the ginger taste.

2. Experimental

2.1. Specimens

The specimens of OG and “Shoga” used in this study were obtained from an official breeder. Fresh rhizomes of OG and “Shoga” were sliced and air-dried. Two specimens of Indonesian ginger, “Red ginger” and “White ginger,” were purchased from Oryza Oil & the Fat Chemical Co., Ltd. (Nagoya, Japan). Two Japanese herbal medicines, “Shokyo” and “Kankyo,” were bought from Tochimoto Tenkaido (Osaka, Japan). All specimens were deposited in the Museum of Materia Medica, College of Pharmaceutical Science, Ritsumeikan University (RIN).

2.2. Analytical Instruments

LC-MS analyses were performed using a Shimadzu LC-IT-TOF mass spectrometer equipped with an ESI interface. The ESI parameters were as follows: source voltage 4.5 kV, capillary temperature 200°C, and nebulizer gas 1.5 L/min. The LC-MS mass spectrometer was operated in the negative ion mode, scanning from m/z 50 to 2000. In the LC-MS analysis, a Waters Atlantis T3 column (2.1 mm i.d. × 150 mm) was used and the column temperature was maintained at 40°C. The mobile phase was a binary eluent of (A) 0.1% HCOOH solution and (B) CH₃CN under the following gradient conditions: 0–30 min linear gradient from 20% to 100% B, 30–40 min isocratic maintained at 100% B. The flow rate was 0.2 mL/min.

2.3. LC-MS Sample Preparation

Individual specimens were homogenized to a fine powder using a multibeads shocker (Model MB755U, Yasui Kikai Co., Osaka, Japan). Two grams of the fine powder was accurately weighted and extracted four times with 50 mL of methanol under reflux conditions for 30 min. After centrifugation, the methanol layers were combined and evaporated in vacuo to give an extract. The extract was dissolved in 10 mL of methanol and filtrated through 0.2 μm Millipore filter (polytetrafluoroethylene (PTFE) filter). Two milliliters of this solution was injected into LC-MS.

2.4. Standard Samples and Reagents

The isolated compounds (-gingerol, -shogaol, -gingerdiol, and diacetoxy--gingerdiol) were identified by comparing their ¹H- and ¹³C-NMR spectra with those reported in the literature [11, 12]. All chemicals were of analytical grade, and chromatographic solvents were of HPLC grade.

2.5. Data Analysis

All statistical analyses were carried out using Pirouette software (GL Science Inc., Tokyo).

2.6. Cell Proliferation Assay

HT-29 human colon cancer cells were seeded in 96-well plates (1 × 10³ cells/well). Cells were allowed to adhere to overnight culture and then treated with metabolites at the final concentration of 3–100 μM. After a 72 h incubation, cell viability was determined with a WST-1 reagent (DOJINDO, Kumamoto, Japan).

3. Results and Discussion

The major pungent principles of ginger are gingerols and shogaols (dehydrated form of gingerols). The conversion of gingerols to shogaols is favored at higher temperature [7], and shogaols show stronger activity than gingerols [13]. As Japanese “Shoga” contains lower amount shogaol than Chinese one, heat processing is used for the production of herbal medicines derived from ginger. In this study, the oleoresins and their derivatives such as gingerdiols, acetoxy gingerdiols, and diacetoxy gingerdiols [7] were identified based on mass spectral fragmentations with high-resolution mass data (Table 1). The fragmentation processes for []-gingerol, []-shogaol, []-gingerdiol, and diacetoxy-[]-gingerdiol were determined from their mass spectra shown in Figure 1. []-Shogaol gave the (M+H)⁺ ion at m/z 277.1807, whereas []-gingerol did not provide the (M+H)⁺ ion and showed (M+Na)⁺ and (M+K)⁺ ions at m/z 317.1717 and 333.1469, respectively, together with [(M+H)–H₂O]⁺ ion at m/z 277.1798. []-Gingerdiol predominantly provided the [(M+H)–2H₂O]⁺ ion at m/z 261.1849 together with the weak (M+H)⁺ and [(M+H)–H₂O]⁺ ions. In the case of diacetoxy-[]-gingerdiol, intense signals for the [(M+H)–CH₃COOH]⁺ and [(M+H)–2CH₃COOH]⁺ ions were observed at m/z 321.2046 and 261.1843, respectively. Furthermore, three characteristic adduct ions, (M+NH₄)⁺, (M+Na)⁺, and (M+K)⁺, were detected. These results indicate that gingerol, shogaol, and their related compounds could be annotated by ESI mass spectral patterns together with high-resolution mass data.

Table 1: Compounds in ginger, their compositions, and expected weight of (M+H)⁺ ions.

Compounds
C
H
O
MW
M+H⁺

6-Paradol
17
26
3
278.1882
279.1960

7-Paradol
18
28
3
292.2038
293.2117

8-Paradol
19
30
3
306.2195
307.2273

9-Paradol
20
32
3
320.2351
321.2430

10-Paradol
21
34
3
334.2508
335.2586

11-Paradol
22
36
3
348.2664
349.2743

13-Paradol
24
40
3
376.2977
377.3056

Methyl 6-paradol
18
28
3
292.2038
293.2117

4-Gingerol
15
22
4
266.1518
267.1596

6-Gingerol
17
26
4
294.1831
295.1909

7-Gingerol
18
28
4
308.1988
309.2066

8-Gingerol
19
30
4
322.2144
323.2222

10-Gingerol
21
34
4
350.2457
351.2535

Methyl 4-gingerol
16
24
4
280.1675
281.1753

Methyl 6-gingerol
18
28
4
308.1988
309.2066

4-Shogaol
15
20
3
248.1412
249.1491

6-Shogaol
17
24
3
276.1725
277.1804

8-Shogaol
19
28
3
304.2038
305.2117

10-Shogaol
21
32
3
332.2351
333.2430

12-Shogaol
23
36
3
360.2664
361.2743

Methyl 6-shogaol
18
26
3
290.1882
291.1960

Methyl 8-shogaol
20
30
3
318.2195
319.2273

Acetoxy-4-gingerol
17
24
5
308.1624
309.1702

Acetoxy-6-gingerol
19
28
5
336.1937
337.2015

Acetoxy-8-gingerol
21
32
5
364.2250
365.2328

Acetoxy-10-gingerol
23
36
5
392.2563
393.2641

Methyl acetoxy-6-gingerol
20
30
5
350.2093
351.2172

1-Dehydro-3-gingerdione
14
16
4
248.1049
249.1127

1-Dehydro-6-gingerdione
17
22
4
290.1518
291.1596

1-Dehydro-8-gingerdione
19
26
4
318.1831
319.1909

1-Dehydro-10-gingerdione
21
30
4
346.2144
347.2222

4-Gingerdiol
15
24
4
268.1675
269.1753

6-Gingerdiol
17
28
4
296.1988
297.2066

8-Gingerdiol
19
32
4
324.2301
325.2379

10-Gingerdiol
21
36
4
352.2614
353.2692

5-Acetoxy-4-gingerdiol
17
26
5
310.1780
311.1859

5-Acetoxy-6-gingerdiol
19
30
5
338.2093
339.2172

5-Acetoxy-7-gingerdiol
20
32
5
352.2250
353.2328

Methyl 5-acetoxy-4-gingerdiol
18
28
5
324.1937
325.2015

Methyl 5-acetoxy-6-gingerdiol
20
32
5
352.2250
353.2328

Diacetoxy-4-gingerdiol
19
28
6
352.1886
353.1964

Diacetoxy-6-gingerdiol
21
32
6
380.2199
381.2277

Methyl diacetoxy-4-gingerdiol
20
30
6
366.2042
367.2121

Methyl diacetoxy-6-gingerdiol
22
34
6
394.2355
395.2434

Methyl diacetoxy-10-gingerdiol
26
42
6
450.2981
451.3060

(a)

(b)

(c)

(d)

Figure 1: Mass spectra of (a) []-shogaol, (b) []-gingerol, (c) []-gingerdiol, and (d) diacetoxy-[]-gingerdiol.

LC-MS chromatograms of OG and “Shoga” are shown in Figure 2. Intense peaks in the respective chromatogram were annotated by detailed analysis of their mass spectral data. Comparison of the chromatographic data shows that OG contains larger amounts of diacetoxy-[]-gingerdiol and methyl diacetoxy-[]-gingerdiol than “Shoga.”

Figure 2: LC-MS chromatograms of (a) OG and (b) “Shoga.”

In order to clarify the medicinal properties of ginger, LC-MS chromatograms of the extracts of all six ginger types are shown in Figure 3. Although there are clear visual differences between the chromatograms of the upper three and lower three samples in Figure 3, this classification does not match their medicinal usage or tastes. For more unbiased interpretation and to reduce the dimensionality of the multivariate data, we analyzed the LC-MS chromatographic data using principal component analysis (PCA).

Figure 3: LC-MS chromatograms of the extracts of (a) dry ginger (“Shokyo”), (b) “Shoga” (Z. officinale var. rubens), (c) “White ginger” (Z. officinale var. amarum), (d) steamed and dried ginger (“Kankyo”), (e) “Ogawa Umare” (Z. officinale cv. Ogawa Umare, OG), and (f) “Red ginger” (Z. officinale var. rubens).

PCA is an unsupervised method of multivariate data analysis and is used for clarifying the characteristic properties of the metabolomic profiles of complex mixtures, such as plant extracts. The annotated peaks and relative intensities detected in the chromatograms of the extracts (Table 2) were normalized and subjected to the PCA analysis. In Figure 4, the PCA scores plot and loading plot were shown. The first two PCs accounted for 90.4% of total variance (PC1, 71.1%; PC2, 19.3%). The scores plot clearly indicated that the chemical content patterns of the medicinal and edible ginger were different. In the chemometric analysis, the peaks having big loading values could be considered as the makers strongly contributing to the classification of the samples by PCA. In the present results, “Shokyo” and “Kankyo” showed similar properties, which were higher concentrations of acetoxy--gingerdiol and diacetoxy-[]-gingerdiol. “Red ginger” was also characterized by its higher content of acetylated compounds, but low methyl diacetoxy-[]-gingerdiol content. The new cultivar, OG, was also grouped with medicinal ginger. On the other hand, two edible ginger types (raw Z. officinale var. rubens and Z. officinale var. amarum) showed higher contents of []-gingerol and lower contents of acetylated compounds. Although Z. officinale var. rubens is used as the raw material in the production of Kampo medicine (“Shokyo” and “Kankyo”), only the most pungent fresh ginger is selected and utilized [14], which suggests the importance of shogaols and gingerols, the pungent and active constituents, for medicinal purpose [8]. So far, []-gingerol and []-shogaol were described as main bioactive constituents of ginger with “point to point” methodology [15–17], whereas []-gingerol was reported to be metabolized to (3R,5S)- and (3S,5S)-6-gingerdiols in mice to induce cell death toward H-1299 cancer cells [11]. On the other hand, our metabolomics approach to chemical system of medicinal ginger is based on “system to system” methodology and has suggested the importance of acetylated compounds, diacetoxy-[]-gingerdiol. Thus, we examined the cytotoxicity of diacetoxy-[]-gingerdiol, a main constituent of OG. As shown in Figure 5, diacetoxy-[]-gingerdiol exhibited stronger cytotoxicity to HT-29 human colon cancer cells than []-gingerol. These results should indicate the importance of acetylated compounds such as diacetoxy-[]-gingerdiol for the use as Kampo medicine and for the classification of medicinal and edible ginger. In addition, from the viewpoint of its chemical constituents and rhizome yield, OG has valuable properties as a new resource for the production of herbal medicines derived from ginger.

Table 2: Annotated peaks and relative intensities detected in the chromatograms of the ginger extracts.

Compounds
Retention time (min)
Relative intensity

Red ginger
OG
Kankyo
White ginger
Shoga
Shokyo

-Gingerdiol
20.13
63181888
34195478
35356476
29468319
38266151
40782586

-Gingerol
20.93
167039972
46057215
20037041
163492434
78274022
136439541

Methyl -gingerol
22.97
9115912
7474131
10056226
40450873
31652131
30072261

5-Acetoxy--gingerdiol
22.97
41701072
14320787
8371635
2022737
1469088
10809735

Diacetoxy--gingerdiol
23.58
0
3764215
3927640
773810
0
4670940

-Gingerdiol
23.78
14502585
2161811
1983957
4419134
3260478
5932284

-Gingerol
24.48
53406657
13470644
3991218
76799848
26551957
63339810

Acetoxy--gingerol
24.60
0
5066601
2597449
0
3259442
5569505

Methyl 5-acetoxy--Gingerdiol
24.95
13575229
17357866
18372059
5087324
3214607
18486380

-Shogaol
25.37
55671256
0
34992211
44191998
2677276
33490436

Methyl -shogaol
26.33
11794505
1309059
1624192
1752366
0
1482872

Methyl acetoxy--gingerol
26.33
17107183
7053201
5050693
4379036
1820760
3071943

Diacetoxy--gingerdiol
26.70
368541323
117403970
88966249
47412894
4212568
96827979

1-Dehydro--gingerdione
27.13
9896109
80710864
63993719
131863825
109603237
113232143

-Gingerdiol
27.21
18056089
1755611
1728210
10924634
4379361
10721515

-Gingerol
27.79
31802196
19048130
5682838
140215493
45855844
27.825

-Shogaol
28.62
20999783
0
7432153
20607889
0
0

Methyl diacetoxy--gingerdiol
28.62
190863115
136670779
166419472
114138767
64094728
164358531

-Shogaol
31.52
7237938
0
4268883
24167568
0
16005930

(a)

(b)

Figure 4: The score plot (a) and the loading plot (b) of PCA.

Figure 5: Cell viability of HT-29 human colon cancer cells after treatment with []-shogaol, diacetoxy-[]-gingerdiol, []-gingerol, and []-gingerdiol.

4. Conclusion

Up to now, several studies reported on the contribution of []-gingerol and []-shogaol to many biological activities of ginger. Prasad and Tyagi summarized many molecular targets of the compounds [15]. However, medicinal activities of ginger are not attributable to only []-gingerol and []-shogaol. Their derivatives have been actively investigated for novel bioactivities such as antihaemolysis by longer chain oleoresins [18], quorum sensing inhibition by []-azashogaol [19], and antiplatelet aggregation by []-paradol [20]. Synergistic bioactivity of []-gingerol with another metabolite is also reported [21]. Our observation that acetoxy derivatives are relatively abundant in medicinal ginger and the compound possesses biological activities may provide additional clues to find more bioactivities of ginger. On the other hand, scarcity of []-gingerol, []-gingerol, or gingerdiols in both medicinal and edible ginger indicates that these bioactive components [22] play fewer roles in the medication of traditional medicines.

The molecular targets of the certain compounds have gradually been made clear through several omics approaches, whereas knowledge on chemical system is still limited. Integration of the knowledge of  “chemical system” as described in this paper may help understand the action between “chemical system” and “body system” in traditional medicines.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

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