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Note: The contraction induced by PGF2o, in the absence of gingerol derivatives was taken as control — 100 percent. The values are the mean percentages ± SEM (n — 3—5). Significant differences from the control values were determined by paired ¿-test at *P < .05 and **P < .01. Source: Kimura et al. (1989c).

Note: The contraction induced by PGF2o, in the absence of gingerol derivatives was taken as control — 100 percent. The values are the mean percentages ± SEM (n — 3—5). Significant differences from the control values were determined by paired ¿-test at *P < .05 and **P < .01. Source: Kimura et al. (1989c).

Table 13-3 Different effects of (6)-gingerdione in aqueous solution on prostaglandin-induced contractions

% Contraction

Compounds (mM) 0 2 hours 5 hours

(6)-Gingerdione (0.3) + PGF2„ 29 ± 3** 102 ± 2 124 ± 2**

Note: The contraction induced by PGF2o[ without (6)-gingerdione was taken as control — 100 percent. The values are the mean percentages ± SEM (n — 5). Significant differences from the control values were determined by paired ¿-test at **P < .01. Source: Kimura et al. (1989c).

incubation, it produced no marked effect, but after 5 hours of incubation, significant potentiation of PGF2a-induced contractions was observed.

To elucidate the correlations between the time-dependent effects and the change in the chemical structure of (6)-gingerdione, ferric chloride tests were performed, at first, to detect enol moiety. When the alcoholic solutions of benzoylacetone (0.15|M), (±)-(6)-gingerol (1.5 |M), and (6)-gingerdione (1.5 |M) were treated with an aqueous solution of ferric chloride (0.19 |M), benzoylacetone and (6)-gingerdione showed positive results for the presence of enol as indicated by the change in color of the solution (i.e., from colorless to red), whereas (±)-(6)-gingerol was negative.

Next, the pattern of ultraviolet (UV) absorption of these compounds was studied. The spectra for benzoylacetone (75 nM) showed maximum absorption bands at 250 and 310 nm. Similarly with (6)-gingerdione (1.5 |M), the absorption maxima obtained were at 235 nm and 280 nm. The same intensity of absorption was observed. Only one spectral band was noted with (±)-(6)-gingerol (1.5 |M) at a maximum of 245 nm.

The UV absorption spectra of (6)-gingerdione (0.3 ^M) in 10 percent ethanol under the same experimental conditions (i.e., incubation at 27°C) for different time intervals were compared (Figure 13.4). The measured absorbance at 235 |n decreased after several hours, whereas the absorbance at 280 nm increased.

Furthermore, the chemical structures of these compounds were studied using nuclear magnetic resonance (NMR). The 1H-NMR spectra (CDCl3, 8 ppm) of (6)-gingerdione showed proton signals of 1,2,4-substituted benzene ring (6.6 to 6.9, 3H, multiplet: m), methoxy signal (3.9, 3H, singlet: s), benzyl proton signal (2.9, 2H, m), ketone a-proton signal (2.6, 2H, m), an olefinic proton signal (5.5, 1H, s), signals for methylene groups (2.3, 2H, m; 1.2 to 1.4, 4H, m; 1.6, 2H, m), and a methyl signal (0.9, 3H, triplet: t). A proton signal (15.5, 1H, s) in deuterium chloroform (CDCl3), assigned to be an enol proton, disappeared by the addition of deuterium methanol (CD3OD) or deuterium oxide (D2O). The same was the case of a ketone a-proton signal to an olefinic proton signal. Several hours after solubilization, the ratio of the olefinic proton signal was increased.

Figure 13-4 UV absorption spectrum for (6)-gingerdione (3 ^M) just after preparation (0 hour) and after incubation for 2 and 5 hours at 27°C. (From Kimura et al., 1989c.)

Effects of (±)-(6)-Gingerol or Ethanol on Prostanoid-Induced Contractions in Mice Mesenteric Veins

Prostaglandin E2 (0.28 mM), I2-Na salt (0.27 mM), and a prostacyclin derivative TRK-100 (0.24 mM), also induced direct contraction of the mouse mesenteric vein, that is, 34 percent, 102 percent, and 25 percent of the maximal response to PGF2a (0.28 mM), respectively. (±)-(6)-Gingerol (0.3 mM) caused a transient relaxation of the smooth muscle that gradually recovered and then significantly potentiated the contraction induced by the above prostanoids (Figure 13.5A).

On the other hand, low concentrations of ethanol had no direct effect on the tissue, but higher concentrations (1.7 mM) significantly potentiated the PGF2a-induced contractions. A tendency to augment the contractions induced by PGE2 and TRK-100 was also observed, but was not significant (see Figure 13.5B). The potentiation effects of (±)-(6)-gingerol were reversible, but those induced by ethanol were irreversible.

The above result indicates that the extract contains other constituents that are more potent than ^-( + )-(6)-gingerol in enhancing the contractile effects of PGF2a. The potency of ^-( + )-(6)-gingerol was stronger than the synthetic compound, (±)-(6)-gingerol.

The potentiating effect of (±)-(8)-gingerol on PGF2a-induced contractions was greater than that of (±)-(6)-gingerol. The results showed that in gingerols shortening of the alkyl side chain decreases the activity (Hikino et al., 1985). The diarylheptanoid, (±)-hexahydrocurcumine (HHC), also potentiated the PGF2a-induced contractions but to a lesser extent compared to gingerols. Interestingly, shogaol produced significant inhibition of the PGF2a contractile response in contrast to gingerols. (6)-Dehydrogingerdione (DHG) and ^-( + )-(6)-gingerdiacetate (GDA) had no effect. Comparing the results and the chemical structures of the compounds tested, it shows that only those compounds containing the hydroxyl group at C-5 in the side chain potentiated the PGF2a-induced contractions. Further, alterations in the gingerol structure, such as elimination or substitution of the hydroxyl group at C-5 in the side chain (e.g., the presence of a double bond in shogaol or substitution of an acetate radical in ^-( + )-(6)-GDA) either inhibits or produces no effect on the PGF2a contractile response (see Figure 13.3).

Different effects of (6)-gingerdione on PGF2a-induced contraction were observed; that is, ranging from inhibition to potentiation after several hours of incubation at 27°C. The difference in responses of (6)-gingerdione is probably due to the diketones at C-3 and C-5 undergoing enolization.

Previous studies have shown that the activated hydrogen of the keto form migrates from the carbon by a, 7-shift to give an enol. The high enol content of 1,3 diketones such as acetylacetone and benzoylacetone showed that the activation by two carbonyl groups was more effective and favors enolization more readily than simple ketones such as acetone. Enols give a red color when treated with ferric chloride, forming colored ferric chloride complexes (Fieser, 1961).

Furthermore, absorption spectra determinations for benzoylacetone showed two absorption bands, with maximum near 247 and 310 nm, and their respective intensities differing from one solvent to another. These two maxima were associated with the ketonic and enolic forms. The intensity of the 247 nm peak in different solvents varies linearly with the total ketone content, and that at 310 nm with the enol content (Morton et al., 1934). The UV spectral data on benzoylacetone obtained in this study agree with the previous results.

Figure 13.5 Potentiation effects of 0.3 mM (±)-(6)-gingerol on PGF2„, PGE2, PGI2_Na salt, and TRK-100 (A) and 1.7 mM ethanol on PGF2a, PGE2, and TRK-100 (B)-induced contractions in mice mesenteric veins. The contraction in response to PGF2a without (±)-(6)-gingerol or ethanol was taken as control = 100 percent. The values are the mean percentages ± SEM (n = 3—4). Significant differences were determined by paired t-test at *P < .05 and **P < .01. (From Kimura et al., 1989c.)

The absorption maxima that occur with (6)-gingerdione at 235 and 280 nm may be associated with its ketone and enol contents, respectively. The 1H-NMR spectrum of (6)-gingerdione solution also showed a time-dependent increase of an enol type configuration. It is, therefore, presumed that in aqueous solution (6)-gingerdione changed its chemical structure after several hours of incubation forming enols, although the extent of conversion to enol and the resulting chemical structure need further elucidation. Figure 13.6 suggests one possible chemical structure of (6)-gingerdione in solution.

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