Food Phytochemicals for Cancer Prevention II - American Chemical

Chapter 25

Chemistry of Ginger Components and Inhibitory Factors of the Arachidonic Acid Cascade 1

Shunro Kawakishi, Y. Morimitsu , and Toshihiko Osawa

Downloaded by UNIV OF ARIZONA on November 23, 2012 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch025

Department of Food Science and Technology, Nagoya University, Chikusa, Nagoya 464-01, Japan

Ginger extracts exhibited a strong inhibitory effect on human plate let aggregation; the inhibitory factors were isolated and studied in detail. Six kinds of gingerol analogues from n-hexane extracts of gin ger were identified as the inhibitors of platelet aggregation, but their activities were very weak compared with that of eugenol analogues found in several spice species. Since the inhibitory activity of ginger could not be accounted for by the six gingerols, new inhibitors from the n-hexane extracts of ginger were investigated. Two labdane-type diterpene dialdehydes isolated from the extracts strongly inhibited the platelet aggregation as much as indomethacin, but these com pounds did not suppress the activity of prostaglandin endoperoxide (PGH) synthase in the arachidonic acid cascade. On the other hand, these diterpene dialdehydes also inhibited human 5-lipoxygenase as strongly as the α-sulfinyl disulfides found in onion.

Ginger (Zingiber officinale Roscoe) is widely used as a spice because of fragrant and pungent principles among its constituents and is well known as a crude drug with several pharmacological functions. Aqueous extract of ginger has exhibited in hibitory effects against the biosynthesis of thromboxane (TX) and prostaglandin (PG) (1,2) and the inhibitory principles have been reported to be gingerol analogues (3). Nutmeg oil also inhibited PG biosynthesis (4); its main inhibitor is eugenol, a related effect of which is the suppression of TXA formation in the arachidonic acid cascade (5). The methanol extracts of several other spices have been studied to characterize the inhibitors of human platelet aggregation, and the extracts of clove and allspice exhibited the strongest activities. It was determined that their active factors were also eugenol and its analogues, and o-methoxyphenol-containing components are essential for their activity. There are many omethoxyphenolcontaining compounds in ginger such as the gingerol analogues. 2

1

Current address: School of Food and Nutritional Sciences, University of Shizuoka, Yada 52-1, Shizuoka 422, Japan

0097-6156/94/0547-0244$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.

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245

Chemistry of Ginger Components

This review paper concerns the re-evaluation of ginger components as the inhibitors of platelet aggregation and their characterization. We have also isolated and identified two labdane type diterpenes as strong inhibitors of both platelet aggregation and 5-lipoxygenase of the arachidonic acid cascade.


Inhibitory Effects of Ginger Extracts Against Human Platelet Aggregation Sliced ginger (560 g) was extracted successively with «-hexane, chloroform, ethyl acetate and water at room temperature. The inhibitory activity of each fraction against human platelet aggregation was measured by the human platelet-rich plasma method described in (6). The results (Table I) showed the n-hexane extract, which gave four spots in T L C , to be the most active.

Table I. Inhibitory Effects of Ginger Extracts Against Human Platelet Aggregation Dose Oig) 100 40 20 10 a b

a

n-Hexane extract C H C h extract (105 mg) (683 mg) b

b

EtOAc extract Aqueous extract (3100 mg) (29 mg) b

b

± +++ ++ ++

++ + +

± ±

Amount added to 200 μΐ platelet rich plasma. Yield from 560 g sliced ginger.

From GC-MS analysis of the three higher R f spots, the major products were identified as zingiberene and α,β,γ-bisabolene, but these components did not exhibit any activity against platelet aggregation. The lowest R f spot was partially purified by silica gel column chromatography followed by preparative HPLC. Nine components were isolated from the HPLC (Figure 1), and among them, com ponents 1-5 and 7 were all gingerol analogues. Component 3 was identified as newly isolated 5-methoxy-[6]-gingerol. The activities (IC50 values) and structures of six gingerol analogues are shown in Table II. The inhibitory activities of [6]-gingerol, [6]-gingerdione and 5-methoxy-[6]-gingerol were medium level, but the others were very weak. Therefore, the strong activity of «-hexane extract could not be accounted for by only gingerol analogues, and the further investigations were undertaken to isolate other potent components from «-hexane extracts. Isolation and Characterization of New Inhibitors The components which were not gingerol analogues, 6, 8 and 9 in the HPLC chromatogram in Figure 1, were further isolated and purified by HPLC to give three pure materials termed compound 1, 2 and 3. Further investigation of this fraction identified galanolactone which has been already found and characterized as an anti5-hydroxytryptamine (serotonin) factor from ginger (7). Compounds 1 and 2 have


FOOD PHYTOCHEMICALS II: TEAS, SPICES, AND HERBS

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Table Π. Structures and Activity Against Human Platelet Aggregation of Gingerol Analogues _ _ ICsn V a l u e "

97.4 μ Μ

[6]-gingerol H H C 3

311.6 μ Μ


H H C 3

124.8 μ Μ

H H C 3

98.1 μ Μ

H H C 3

H

223.3 μ Μ

[6]-dehydrogingerdione ?CH

3

H C 3

H

81.9 μ Μ

5-methoxy-[6]-gingerol 4

3 >?,β

ι

7

r

Μ 10

20

30 R.T.(min)

Figure 1. H P L C of n-hexane extracts of ginger. Conditions: Develosil ODS-5 column (8x250 mm); M e O H / H 0 / A c O H (80/20/0.1, v/v) mobile phase at a flow rate of 3 ml/min; detection at 254 nm. 2


25. KAWAKISHI ET AL.


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been also isolated from Alpina galanga (Zingiberaceae) as antifungal diterpenes (8) and compound 3 was newly isolated from ginger by us. Compounds 1, 2, 3 and galanolactone were all labdane-type diterpenes and their spectroscopic data agreed with that of the references (7,8). The chemical structures of compounds 1 and 2 were determined as (£)-8(17),12-labd-diene-15,16-dial and (£)-8p(17)-epoxylabd12-ene-15,16-dial, respectively. Compound 3 was homologous to 2 and easily determined from its spectroscopic data to be 15-hydroxy-(£)-8p(17)-epoxylabd-12ene-16-al. The chemical structures of these four labdane-type diterpenes are shown in Figure 2.

3

galanolactone

Figure 2. Structures of labdane-type diterpenes isolated from ginger. The inhibitory activities of these compounds against platelet aggregation are compared to indomethacin in Table III. Compounds 1 and 2 exhibited strong activity, similar to indomethacin, but the activity of 3 was weak and galanolactone had none. These results suggested that the dialdehyde structure in labdane-type diterpenes is required for the developement of activity. This was also supported by the finding that when compound 2 is reduced with NaBHj in THF from dialdehyde to dicarbinol, the activity of compound 2 is completely destroyed. Inhibitory Effects of Ginger Constituents on the Arachidonic Acid Cascade The gingerol analogues we isolated had weak activities against platelet aggregation as previously described, so studies on whether gingerol inhibits the biosynthesis of TXA2 in the arachidonic acid cascade of platelets were performed. When TXA2 is formed from PGH2 in arachidonic acid metabolism, PGH2 is simultaneously



248


degraded to 12-(S)-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and malondialdehyde (MDA) (9,10). The suppressive effect of [6]-gingerol on TXA2 biosynthesis was examined by the determination of M D A . As shown in Table IV, gingerol exhibited weak inhibitory activity against P G biosynthesis compared with indomethacin. Other gingerol analogues might also suppress the formation of TXA2 like [6]-gingerol. Since labdane-type diterpene dialdehyde 1 and 2 strongly inhibited platelet aggregation, their suppressive actions against TXA2 biosynthesis and 5-lipoxygenase activity, which catalyzes leukotriene formation from arachidonic acid in leukocytes, were studied by using rabbit renal microsomes and human 5-lipoxygenase, respectively (6). Table ΠΙ shows the unexpected result that PG biosynthesis was not suppressed by the dialdehydes while 5-lipoxygenase was strongly inhibited. These results suggest that compounds 1 and 2 do not inhibit platelet aggregation by suppression of the arachidonic acid cascade.

Table ΙΠ. Inhibitory Activities of Labdane-type Diterpenes Against Human Platelet Aggregation, Prostaglandin Biosynthesis and 5-Lipoxygenase ΙΟ (μΜ) 50

Compound Platelet aggregation 1 2 3 Galanolactone Indomethacin AA861

P G biosynthesis

3.2 3.0 90.4 >1000 2.1 —

>100 >100 — — 0.75 —

5-lipoxygenase 18.9 4.0 >100 >100 — 0.3

— : not measured

Table IV. Inhibitory Activity of [6]-Gingerol Against Prostaglandin Biosynthesis MDA (nmol/10 platelets) 9

Control

5.75

Inhibition (%) —

[6]-Gingerol

200 μΜ 1 mM

4.26 2.62

25.9 60.7

Indomethacin

50 μΜ 200 μΜ

3.07 1.31

46.6 75.2

M D A values are proportional to P G H and T X A . 2

2


25.

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Proposed Mechanism for Inhibition of Platelet Aggregation by Labdane-type Diperpenes Since compounds 1 and 2 did not affect PG biosynthesis, the effects of many kinds of inducers on platelet aggregation were studied to make clear their mechanism of action. These compounds strongly inhibited the platelet aggregation induced by A D P , and moreover, the primary aggregation arising from low concentrations of A D P was also suppressed by compound 2. These results suggest that compounds 1 and 2 may block the A D P receptor site on platelets like ophthalaldehyde which is well known to inhibit ADP binding on platelets by reaction with thiol and amino groups of its binding site (77,72). This speculation is also supported by the fact that U V absorption maximum at 232 nm of compound 2 rapidly disappeared after the addition of platelets. This U V disappearance suggests that the chemical changes at the α,β-unsaturated carbonyl group of compound 2 occur according to the above reaction. Moreover, the inhibitory activity of compound 3 containing monoaldehyde was low (Table ΙΠ) and compound 2 completely lost its activity when the dialdehyde was reduced to dicarbinol. Conclusion The n-hexane extracts of ginger exhibited strong inhibitory action against human platelet aggregation. Among them, gingerol analogues, major pungent principles, had only a weak inhibitory activity against platelet aggregation depending on the formation of TXA2. Isolation of more active components identified two labdane-type diterpene dialdehydes having inhibitory activity against platelet aggregation and 5-lipoxygenase in leukocytes. It is noteworthy that these labdane dialdehydes differed from the active components of onion and garlic in that they inhibited platelet aggregation without inhibition of the arachidonic acid cascade. It is postulated that the A D P binding site on platelets may be masked by these labdane dialdehydes and as a result, the activation of platelets is suppressed, inhibiting platelet aggregation. Acknowledgments The authors wish to thank to Drs. T. Matsuzaki and T. Matsumoto of Japan Tobacco Inc. for kindly supplying rabbit renal microsomes and human 5-lipoxygenase and for their helpful discussion. Literature Cited 1. Srivastava, K. C. Prostaglandins Leukotrienes Med. 1984, 13, 227. 2. Srivastava, K. C. Prostaglandins Leukotrienes Med. 1986, 25, 187. 3. Kiuchi, F.; Shibuya, M.; Sankawa, U. Chem. Pharm. Bull. 1982, 30, 754. 4. Misra, V.; Misra, R. N.; Unger, W. G. Indian J.Med.Res. 1978, 67, 482. 5. Rasheed, Α.; Laekeman, G.; Totte, J.; Vlietinck, A. J.; Herman, A. G. New Engl.J.Med. 1984, 310, 50. 6. Kawakishi, S.; Morimitsu, Y. This Proceeding. 7. Huang, Q.; Iwamoto, M . ; Aoki, S.; Tanaka, N.; Tajima, K.; Yamahara, J.; Takaishi, Y.; Yoshida, M . ; Tomimatsu, T.; Tamai, Y. Chem. Pharm. Bull. 1991, 39, 397.


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8. Monta, H.; Itokawa, H. Planta Med. 1988, 54, 117. 9. Lassmann, G.; Odenwaller, R.; Curtis, J. F.; DeGray, J. Α.; Mason, R. P.; Marnett, L. J.; Eling, T. E. J. Biol. Chem. 1991, 266, 20045. 10. Okuma, M.; Steiner, M.; Baldini, M . J. Lab. Clin. Med. 1970, 75, 283. 11. Puri, R. N.; Colman, R. W. Arch. Biochem. Biophys. 1991, 286, 419. 12. Puri, R. N.; Roskoski, R., Jr. Anal. Biochem. 1988, 173, 26. R E C E I V E D April 14, 1993


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