Structure of Antioxidative Compounds in Ginger - ACS Symposium

Chapter 24

Structure of Antioxidative Compounds in Ginger Hiroe Kikuzaki, Yoko Kawasaki, and Nobuji Nakatani

Downloaded by UNIV OF MINNESOTA on October 14, 2014 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch024

Department of Food and Nutrition, Osaka City University, Sumiyoshi, Osaka 558, Japan

Fourteen gingerol related compounds and seven diarylheptanoids were isolated from ginger rhizomes. Their structures were elucidated on the basis of chemical and spectroscopic evidence. Most of the isolated compounds exhibited stronger antioxidative effect than α-tocopherol. The antioxidant activity depended upon substituents on the side chain and benzene ring, and the length of side chain.

The fact that ancient people empirically used spices and herbs as preservatives of foods suggests they have antioxidative or antimicrobial properties. Accordingly, many scientists have given their attention to spices and herbs as a source of natural antioxidants with the idea of finding safe and effective compounds. The rhizome of ginger (Zingiber officinale Roscoe) is currently widely used as a spice and a food seasoning due to its sweet aroma and pungent taste. It is known from several investigators that ginger has antioxidative effects (1-2), but there have been few reports on its antioxidative constituents (3-4). Therefore, we studied the chemical constituents of ginger and their antioxidant activity. Structures of the Constituents of Ginger Extraction and Isolation of the Constituents of Ginger. Ground rhizomes of dried steamed ginger were successively extracted with dichloromethane and methanol. The former was separated by steam distillation to give a volatile and a nonvolatile fraction. The antioxidant effect of each extract and fraction was measured by the ferric thiocyanate method and thiobarbituric acid method (TBA) using linoleic acid as a substrate in ethanol-water system. The dichloromethane extract showed stronger activity than α-tocopherol, whereas the methanol extract exhibited the same activity as α-tocopherol. Between two fractions of the dichloro methane extract, only the nonvolatile one showed strong activity. This fraction was purified by column chromatography on silica gel and Sephadex LH-20, and by prep arative HPLC to give fourteen gingerol related compounds (Figure 1, compounds 1-14) and seven diarylheptanoids (15-21). We have already reported the structure

0097-6156/94/0547-0237$06.00/0 © 1994 American Chemical Society

In Food Phytochemicals for Cancer Prevention II; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

238

FOOD PHYTOCHEMICALS II: TEAS, SPICES, AND HERBS

Gingerol related compounds

Diarylheptanoids

5-hydroxy-3-one type Ο OH CH 0, (CH ) CH

5-hydroxy-3-one type Ο OH

3

2


J?

n

3

1: η = 8 2: η = 6 3: η = 4 4: η = 2 Ο OH

HO

XJ

,OCH

15

3

Κλ,

OH

4-en-3-one type 0CH3

CH3O.

OH

3,5-diol type

4-en-3-one type

OH OH ^OCH

CH 0. 3

CH3O

(CH )nCH 2

^

7: η = 8 8:n = 6 9: η = 4 10: η = 2

3

HO'

17

3

*OH

OH OH OCH3

OCH3

,OCH

Figure 1. Structures of the isolated compounds of ginger.


3

24. KIKUZAKI ET AL.

Structure of Antioxidative Compounds in Ginger

elucidation of compounds 13-21 (5-7). In this paper we describe the structure determination of compounds 1-12 and their antioxidant activity. Structure Elucidation of the Isolated Compounds. The structures of the isolated compounds were determined by the methods described below. Compound 3. Compound 3 was obtained as one of the major components of ginger rhizome. The spectral data (IR, H - and C - N M R and MS) and specific rotation ([α]^\ +23.9°) showed this compound to be [6]-gingerol (8). Downloaded by UNIV OF MINNESOTA on October 14, 2014 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch024

1

13

Compounds 1,2 and 4. The spectral characteristics of compounds 1, 2 and 4 were very similar to those of 3 except for their molecular ion peaks. A [M] peak of compound 1 at m/z 350 was 56 mass larger than that of 3, which indicated 1 to be [10]-gingerol, having a side chain composed of fourteen carbon atoms. Compounds 2 and 4 were determined to be [8]- and [4]-gingerol, respectively, from their mass fragmentation patterns (Figure 2) (9). +

Ο -H 0 2

1:332 2:304 3:276

Ο

4:248 5:290 6:246

V^R, *CH + 2

1:R (CH ) CH R = OCH R = Η [ M , 350] 2: R ( C H ) C H R = O C H , R = Η [ M , 322] 3: R (CH ) CH , R = OCH R = Η [ M , 294] 4: R i C H , R = O C H R = Η [ M , 2 6 6 ] 5: R i ( C H ) C H , R = O C H , R = C H [ M , 3 0 8 ] 6: R i ( C H ) C H , R = R = Η [ M , 2 6 4 ] +

1

2

6

3 I

2

3 >

3

+

2

2

4

3 I

2

3

3

+

1

2

2

3

2

3 >

1,2,3,4 : 151 5:165 6: 121

3

+

3

2

3 J

3

+

2

2

3

2

3

3

3

+

2

2

3

2

3

McLafferty rearrangement OH

+

1,2,3,4 : 1 3 7 5: 151 6: 1 0 7

1,2,3,4 : 1 9 4 5:208 6: 1 6 4

Figure 2. Mass spectral fragmentation pattern of gingerols. In Food Phytochemicals for Cancer Prevention II; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

239


240

Compound 5. Compound 5 was obtained as colorless needles. The spectral data were very close to those of [6]-gingerol. The EI mass spectrum exhibited a [ M ] peak at m/z 308 and [M-H20] at m/z 290, which are 14 mass larger than those of 3. The presence of a 3,4-dimethoxyphenyl group was supported by aromatic proton signals [δ6.73 (2H, m) and 6.79 (1H, d, J=8.6 Hz)] accompanied with arylic methoxyl groups [δ3.85 (3H, s) and 3.86 (3H, s)] in the ' H - N M R , aromatic carbon signals (5148.6, 147.3, 133.9, 120.2, 111.7 and 111.4) in the C N M R spectrum and a typical fragment ion peak at m/z 151 [CH2C6H3(OCH3)2] in the mass spectrum. The C - N M R spectrum revealed ten more carbons, corresponding to the straight chain carbons, in which a carbonyl (5211.3) and a hydroxyl methine (567.7) carbons were found. In the H - N M R spectrum, the signal at 52.73 (2H, t), corresponding to methylene protons at C-2, indicated that C-3 was substituted by a carbonyl group. When the signal of a methine proton adjacent to a hydroxyl group at 54.02 was irradiated, two double doublets of geminal methylene protons at 52.48 and 2.57 changed into two doublets with a coupling constant of 17.4 Hz each. This fact showed a hydroxyl group to be at C-5. The substitution pattern of the decane was supported by the presence of a characteristic fragment ion peak at m/z 208 derived from McLafferty rearrangement between C-4 and C-5 (Figure 2). These data indicated compound 5 to be a methylated derivative of [6]gingerol. To confirm the structure of 5, [6]-gingerol was methylated with methyl iodide in the presence of dry K 2 C O 3 A l l spectral data and specific rotation of 5 were identical with those of the synthetic compound. Consequently, the structure of 5 was determined to be 5-hydroxy-l-(3,4-dimethoxyphenyl)decan-3-one. The presence of this compound was speculated to occur only by GC-MS and H P L C in ginger (10,11), and has not been isolated previously. +

+

1 3

+

1 3


!

+

Compound 6. Compound 6 gave a [M] peak at m/z 264.1703 in the H R mass spectrum, corresponding to a molecular formula of C 1 6 H 2 4 O 3 . The spectral characteristics indicated that compound 6 had an analogous structure to [6]gingerol. In comparison with the *H-NMR spectrum of [6]-gingerol, a pair of two 6>ri/i6>-coupling protons were observed at 56.74 and 7.03 in 6 in place of the signals due to a 4-hydroxy-3-methoxyphenyl group. This finding denoted compound 6 possessed a 1,4-disubstituted aromatic ring. In addition, the presence of aromatic carbon signals [5154.0, 132.9, 129.4 (2C) and 115.4 (2C)] in the C - N M R spectrum and a stable ion peak at m/z 107 [ C ^ C é H ^ O H ) ] * confirmed that a 4hydroxybenzyl moiety was present in the molecule. This fact indicated that compound 6 is a demethoxylated derivative of [6]-gingerol, which was supported by a typical mass ion peak at m/z 164 [ Q o H ^ C ^ (Figure 2). Consequently, compound 6 is concluded to be l-(4-hydroxyphenyl)-5-hydroxydecan-3-one, which was isolated for the first time. 1 3

Compounds 7-10. Compounds 7-10 were identical with homologues of shogaol from their spectral data and were determined to be [10]-, [8]-, [6]- and [4]shogaol, respectively. Among them, compound 10, [4]-shogaol, was isolated for the first time, though it had been detected in ginger by GC-MS before (12,13). Compound 11. Compound 11 had a molecular formula of C18H26O3 ([M] , m/z 290.1880). The spectral data were very close to those of [6]-shogaol, except for the presence of another methoxyl group instead of the hydroxyl one. In +


24. KIKUZAKI ET AL.

Structure of Antioxidative Compounds in Ginger241

the IR spectrum, no hydroxyl absorption band was observed in 11. The ^ - N M R spectrum showed the signals of two methoxyl groups as each 3H singlet at 63.85 and 3.86. The pattern of signals due to an aromatic ring was similar to that of compound 5 in the H - and C - N M R spectra. The signals at 56.09 (1H, brd, J=15.9 Hz) and 6.82 (1H, td, J=7.0, 15.9 Hz) in the ^ - N M R spectrum showed the presence of an α,β-ira/w-unsaturated carbonyl group, which was supported by the signals at δ199.7, 147.8 and 130.3 in the C - N M R spectrum. A fragment ion at m/z 219 [C^H^C^]" " produced by cleavage at α position of double bond indicated that a carbonyl group was located on C-3 and a conjugated double bond on C-4. Furthermore, the methylated compound of [6]-shogaol with methyl iodide gave the same compound as 11. Accordingly, compound 11 was determined to be l-(3,4-dimethoxyphenyl)-4-decen-3-one, which is newly isolated from a natural source. 1

13

13


1

+

Compound 12. Compound 12 gave a [ M ] peak at m/z 246.1608, corresponding to a molecular formula of C16H22O2. The signals due to an aromatic ring of 12 resembled to those of compound 6 in the H - and C - N M R spectra, which indicated compound 12 to have a 4-hydroxyphenyl group. The presence of an a,§-trans-unsaturated double bond was supported by the signals at 56.09 (1H, td, J=1.2, 15.9 Hz) and 6.82 (1H, td, J=6.7, 15.9 Hz) in the Ή - N M R and at 5200.0, 148.0, 130.3 in the C - N M R spectrum. Furthermore, the mass spectrum exhibited characteristic fragment ion peaks at m/z 175, produced by a cleavage at a-position of a double bond, and at m/z 107, corresponding to a benzylic cation. Consequently, compound 12 was concluded to be l-(4-hydroxyphenyl)-4-decen-3one. This is the first time that compound 12 was isolated from a natural source. 1

13

13

Structures of the Isolated Compounds of Ginger. Gingerol related compounds and diarylheptanoids which we have isolated until now can be classified into four groups according to the substitution pattern of side chain as follows: (1) 5-hydroxy3- one type, (2) 4-en-3-one type, (3) 3,5-diol type and (4) 3,5-diacetate type as shown in Figure 1. Taking note of the substituents on benzene ring, compounds having five types of phenyl group such as 4-hydroxyphenyl, 3,4-dihydroxyphenyl, 4- hydroxy-3-methoxyphenyl, 3,4-dimethoxyphenyl and 4-hydroxy-3,5-dimethoxyphenyl group were found in ginger. Among them, there were the greatest number of compounds with 4-hydroxy-3-methoxyphenyl group in the isolated ones. In addition, many gingerol related compounds had a side chain with ten carbons. Antioxidant Activity of the Isolated Compounds The antioxidant activity of the isolated compounds was examined by the ferric thiocyanate method. Gingerol related compounds with the same length of side chain and with a 4-hydroxy-3-methoxyphenyl group (3, 9, 13 and 14) had a higher activity than α-tocopherol when they were added at a concentration of 200 μΜ to a substrate solution. The activity increased in the order 3 (type 1), 9 (type 2), 13 (type 3), 14 (type 4). Diarylheptanoids having two 4-hydroxy-3-methoxyphenyl groups (15-19) were also more active than α-tocopherol at the same concentration. The activity increased in the order 18, 15, 16,17,19, which indicated the similar result at gingerol related compounds. Furthermore, these five compounds showed a stronger activity than curcumin, a known antioxidative compound from turmeric (Curcuma domestica). These results suggested that the substitution pattern of side chain has an influence on the antioxidant activity.


242


Ο

l:n=8

3: n=4

OH

2: n=6 4: n=2


ΙΟΟμΜ

7: n=8 9:n=4

8: n=6 10: n=2

ΙΟΟμΜ

Figure 3. Antioxidant activity of gingerols and shogaols with different length side chains (ferric thiocyanate method: concentration 100 μΜ). In Food Phytochemicals for Cancer Prevention II; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

24. KIKUZAKI ET AL.

Structure of Antioxidative Compounds in Ginger 243


Concerning the substitution pattern on the benzene ring, compound 3 showed a remarkable activity, while compound 5 had no activity and 6 exhibited slight activity. Similar results were obtained in the case of shogaols (9,11 and 12). In addition, compound 19 had a higher activity than 20 or 21. These results indicated that the substitution pattern of the benzene ring definitely played an important role in the antioxidant activity. Figure 3 shows the effects on the activity of gingerol related compounds with different length side chains when they were added at a concentration of 100 μΜ. All these compounds were effective and higher activity is correlated with longer side chains. Accordingly, it is suggested that antioxidant activity also depends upon the length of side chain. Literature Cited 1. Hirosue, T.; Kawai, H.; Hosogai, Y. Nippon Shokuhin Kogyo Gakkaishi 1978, 25, 691. 2. Lee, C. Y.; Chiou, J. W.; Chang, W. H. J. Chinese Agric. Chem. Soc. 1982, 20, 61. 3. Fujio, H.; Hiyoshi, Α.; Asari, T.; Suminoe, K. Nippon Shokuhin Kogyo Gakkaishi 1969, 16, 241. 4. Lee, I. K.; Ahn, S. Y. Korean J. Food Sci. Technol. 1985, 17, 55. 5. Kikuzaki, H.; Usuguchi, J.; Nakatani, N. Chem. Pharm. Bull. 1991, 39, 120. 6. Kikuzaki, H.; Kobayashi, M.; Nakatani, N. Phytochemistry 1991, 30, 3647. 7. Kikuzaki, H.; Tsai, S. M.; Nakatani, N. Phytochemistry 1992, 31, 1783. 8. Connell, D. W.; Sctherland, M. D. Aust. J. Chem. 1969, 221, 1033. 9. Shoji, N.; Iwasa, Α.; Takemoto, T.; Ishida, Y.; Ohizumi, Y. J. Pharm. Sci. 1982, 71, 1174. 10. Chen, C. C.; Ho, C. T. J. Chromatogr. 1986, 360, 163. 11. Masada, Y.; Inoue, T.; Hashimoto, K.; Fujioka, M.; Uchino,C. Yakugaku Zasshi 1974, 94, 735. 12. Masada, Y.; Inoue,T.; Hashimoto, K.; Fujioka, M.; Siraki, K. Yakugaku Zasshi 1973, 93, 318. 13. Harvey, D. J. J. Chromatogr. 1981, 212, 75. RECEIVED

April 4, 1993


Structure of Antioxidative Compounds in Ginger - ACS Symposium

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