Sulfur Chemistry of Onions and Inhibitory Factors of the Arachidonic

Chapter 8

Sulfur Chemistry of Onions and Inhibitory Factors of the Arachidonic Acid Cascade

Downloaded by UNIV OF PITTSBURGH on May 3, 2015 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0546.ch008

Shunro Kawakishi and Y. Morimitsu

1

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

Many volatile sulfur compounds and a lachrymatory factor are generated from S-alk(en)yl-L-cysteine sulfoxides by the action of cysteine sulfoxide lyase (CS-lyase) in onions that are cut or crushed. Ten kinds of α-sulfinyl disulfides (AC series) that were human platelet aggregation inhibitors have been isolated and their chemical structures and activities were determined. These products strongly inhibit prostaglandin endoperoxide synthase (PGH synthase) of the arachidonic acid cascade in platelets. Moreover, they have also exhibited a strong inhibitory effect on human 5-lipoxygenase which is concerned with the biosynthesis of leukotriene from arachidonic acid in leukocytes. The relationship between the structures of A C series compounds and their dual activities were studied in detail.

Allium species such as garlic, onion, rakkyo, leek, chive, etc., generate specific sulfur-containing flavors following the breakdown of their plant cells. This flavor generation is due to the formation of volatile sulfur compounds, alk(en)yl disulfides, from S-alk(en)yl-L-cysteine sulfoxides by CS-lyase (EC 4.4.1.4) (7). Four kinds of alkyl groups in disulfides specify for their odors in each Allium species. Among these species, garlic (A. sativum L.) and onion (A. cepa L.) have been widely investigated for their therapeutic effects on vascular diseases such as thrombosis, atherosclerosis, hyperlipidemia and rheumatic arthritis. Furthermore, the extracts and the essential oil of garlic and onion are well known to strongly inhibit human platelet aggregation in vitro (2-4). From garlic, (£,Z)-ajoene (4,5,9-trithiadodeca-1,6,11-trien 9-S-oxide), 2-vinyl-4//-l,3-dithiin, diallyltrisulfide (5,6) and methyl allyl trisulfide (7) have been identified as inhibitors of platelet aggregation. These compounds markedly suppress cyclooxygenase activity of PGH synthase in the arachidonic acid cascade of platelets and 5-lipoxygenase (EC 1.13.11.12) in leukocytes (8-11). On the other 1

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

0097-6156/94/0546-0120$06.00/0 © 1994 American Chemical Society In Food Phytochemicals for Cancer Prevention I; Huang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

8. KAWAKISHI AND MORIMITSU

121

Sulfur Chemistry of Onions


hand, adenosine (72) and alliin (13) have been isolated as platelet aggregation inhibitors from onion. Even though the oily extracts of onion also exhibit a strong antiplatelet action, the active compounds were not characterized until now. Recently, we have isolated and identified several antiplatelet compounds, oc-sulfinyl disulfides (named A C series), from the methanol extracts of crushed onion (14,15) and their inhibitory action of cyclooxygenase in platelets and human 5-lipoxygenase have been revealed. Moreover, the active A C series were formed through a lachrymatory factor, thiopropanal S-oxide, derived from S-l-propenyl-Lcysteine sulfoxide by CS-lyase in crushed onion (16). In this review paper, we will describe the characterization of A C series antiplatelet factors, their inhibitory activities against platelet aggregation, cyclooxygenase in arachidonic acid cascade of platelets and human 5-lipoxygenase, and their structure-activity relationship. Formation of Flavors and Lachrymatory Factor in Onion Four kinds of S-alk(en)yl-L-cysteine sulfoxides exist in all Allium species and their degradation by CS-lyase generates a characteristic flavor component, dialk(en)yl disulfide (7). Their alk(en)yl groups, methyl, η-propyl, 1-propenyl and 2-propenyl(allyl) are present in natural parent compounds. Onion contains three kinds of cysteine sulfoxides having methyl, propyl and 1-propenyl groups. Among them, 1-propenyl is a major group, but the propyl group mainly contributes to the form ation of onion flavor. Thus, while the major flavor of onion, dipropyl disulfide, is formed from S-n-propyl-L-cysteine sulfoxide through n-propanesulfenic acid and propyl propanethiosulfinate, the main sulfoxide in onion, S-l-propenyl-L-cysteine sulfoxide, is degraded to form a lachrymatory factor, thiopropanal S-oxide, by the isomerization of 1-propenesulfenic acid (77). As shown in Figure 1, sulfenic acid and thiopropanal S-oxide are rapidly decomposed by desulfuring to propanal which is transformed to 2-methyl-2-pentenal by Aldol condensation and dehydration. When onion tissues are homogenized with water, however, the lachrymatory factor is stabilized in tissue suspension (18). This phenomenon is closely related to the for mation of antiplatelet factors from the lachrymatory compound as described below.

1-propenyl cysteine sulfoxide C - S lyase

ο desulfurization

^ V

0

H

+

S

^ 1-propenyl sulfenic acid

thiopropanal S-oxide (sulfine)

2-methyl pentenal

Figure 1. Formation and degradation of lachrymatory factor in onion.

In Food Phytochemicals for Cancer Prevention I; Huang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

122

FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES


Characterization of Antiplatelet Factors and Their Inhibitory Effects for Human Platelet Aggregation For the determination of platelet aggregation, platelet-rich plasma (PRP) was prepared from human blood containing citrate by centrifugation at 120 χ g for 10 min. Platelet aggregation was measured by turbidimetric method of the reaction mixture of PRP (200 μΐ) containing 0.2 μg collagen as an aggregation inducer and 1 μΐ methanolic sample solution using a dual-channel aggregometer. The inhibitory activities were expressed as IC50 values, that is, the sample concentration that resulted in 50% inhibition of platelet aggregation (75). The methanol extract of crushed onion was fractionated by silica gel column chromatography with n-hexane, benzene, chloroform and 20% methanol/ chloroform as eluents. The chloroform fraction showed the strongest inhibitory effects, so this fraction was rechromatographed on a silica gel column with step wise gradients of chloroform in benzene. The fractions eluted with 50 and 75% chloroform in benzene exhibited the highest activities for platelet aggregation. These eluates were further fractionated by HPLC using a Develosil SI-60-5 column to give 10 active substances named the A C series. Their chemical structures were determined by analyses of their H and C - N M R , and EI mass spectra, and all A C series were constituted from a common structural unit of oc-sulfinyl disulfide. The structures and inhibitory activities (IC50 values) against platelet aggregation of A C series are shown in Table I. The letters a, b, and c represent the respective diastereoisomers. A C series bore a common structural skeleton with cepaenes (19,20), which were found to be antiasthmatic, antiallergic and anti platelet compounds in crushed onion. A C series exhibited a strong activity com pared to the anti-inflammatory drug aspirin, besides A C - l a and AC-11 a, AC-12b was the most effective, as potent as indomethacin. Chiralcel OB column HPLC was used to separate the diastereoisomer from the respective enantioisomer of A C - l b , AC-11a and A C - l i b . Separation was con firmed by the measurement of their CD spectra (data not shown) (27). As shown in Table II, the differences in IC50 values between enantioisomers were not so large. These antiplatelet compounds were probably formed by the interaction of lachrymatory factor, thiopropanal S-oxide and other sulfenic acids. The addition of methyl sulfenic acid to the C\ position of the lachrymatory, followed by the condensation with another sulfenic acid and dehydration, may transform to oc-sulfinyl disulfide (Figure 2). The lachrymatory factor is ordinarily very labile and rapidly decomposed in sliced onion. When onion is rapidly homogenized, it may be stabilized a little to generate oc-sulfinyl disulfide. l

13

"carbophilic" addition thiopropanal S - o x i d e (sulfine)

AC-series/cepaenes

Figure 2. Proposed formation mechanism of oc-sulfinyl disulfides (AC series) from lachrymatory factor.




123

Table I. Inhibitory Activities of AC Series Against Human Platelet Aggregation


R

Κ: (μΜ) 50

AC-la AC-lb

methyl

67.6 18.4

AC-10

propyl

12.8

AC-lla AC-llb

trans-1-propenyl

48.9 11.7

AC-12a AC-12b

cis- 1-propenyl

AC-13a AC-13b AC-13c

( 1 -methoxy)-propyl 26.4 13.1 13.1

6.1 1.4

2.1

Indomethacin Aspirin

41.2

SOURCE: Reproduced with permission from reference 15. Copyright 1990 Pergamon. Table II. Inhibitory Activities Of AC Series Enantioisomers Against Human Platelet Aggregation

IC50 (μΜ) AC-lb AC-lb-(l) AC-lb-(2) AC-lla AC-lla-(l) AC-lla-(2) AC-llb AC-llb-(l) AC-llb-(2)

18.4 23.2 16.4 48.9 49.4 23.7 11.7 9.1 22.9

SOURCE: Reproduced with permission from ref erence 21. Copyright 1991 Japan Society for Bioscience, Biotechnology, and Biochemistry. Inhibitory Effects of A C Series on Cyclooxygenase and 5-Lipoxygenase in the Arachidonic Acid Cascade Inhibition of platelet aggregation by the A C series may be due to the suppression of the enzymes related to the arachidonic acid cascade in platelets, similarly to the inhibitors isolated from garlic. Pharmacological actions of aspirin and indomethIn Food Phytochemicals for Cancer Prevention I; Huang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


124


acin depend on the inhibition of cyclooxygenase in PGH synthase (Figure 3). The P G H synthase pathway possesses both cyclooxygenase and hydroperoxidase activities, which generate prostaglandins (PGs) and thromboxanes (TXs) from arachidonic acid via P G H2 and G2. The enzyme 5-lipoxygenase participates in biosynthesis of leukotriene, a chemical mediator of inflammatory diseases in leukocytes. We have examined the suppressive effects on prostaglandin biosynthesis of A C series by using rabbit renal microsomes. Furthermore, the inhibitory activities of A C series on 5-lipoxygenase were studied by using an enzyme preparation obtained from gene cloning of human 5-lipoxygenase. The results of our studies on six kinds of A C series — la and b, 1 l a and b, and 12a and b, are shown in Table III. These compounds clearly inhibited PGH synthase and 5-lipoxygenase in the arachidonic acid cascade. Moreover, A C - l l a and b were comparable to indomethacin in their inhibition of PGH synthase, and AC-1 la and b and 12a and b also inhibited 5-lipoxygenase in a manner similar to AA861, a potent inhibitory drug.

Table III. Inhibitory Effects of Several α-Sulfinyl Disulfides (AC Series) on PG Biosynthesis and 5-Lipoxygenase IC50O1M)

Platelet aggregation

Prostaglandin biosynthesis

5-Lipoxygenase

AC-la AC-lb

67.6 18.4

—

27.3 19.6

AC-lla AC-llb

48.9 11.7

1.2 1.6

1.4 1.5

AC-12a AC-12b

6.1 1.4

ND ND

2.5 2.8

Indomethacin

2.1

0.75

—

AA861

—

—

0.3

NDGA

—

—

16.5

ND: not determined

Relationships between Structure and Activity of α-Sulfinyl Disulfide The a-sulfinyl disulfide structure in A C series is related to ajoene from garlic in possessing sulfinyl and disulfide groups. Many analogues of α-sulfinyl monosulfide and disulfide were prepared to study their structure-activity relationship. α-Sulfinyl monosulfide analogues synthesized in this experiment did not inhibit either platelet aggregation or 5-lipoxygenase. α-Sulfinyl disulfide analogues,


8.

KAWAKISHI AND MORIMITSU

125


COOH

.COOH

OH

COOH

OH .COOH


Glu-Cys-Gly

LTB4

LTC4

.COOH. LTA4

I

OH .COOH r=^^^OQ

OOH /=v^r-^^COOH

5-HETE

5-HPETE ^124ipo^genase

15-HPETE-

15-lipoxygenase

arachidonic acid

t

gCQ^S-

/^-^COOH

OOH

A-/

PGG2

OH TXA2

^

Λ

OOH

? < 3 ζ ^ ^ £ 0 0 Η ^ OH °'

OH PGI2

ÔH PGH2

OH

/

OH

ι

\

\

v

COOH

OH

PGF20

Figure 3. The formation of prostaglandin (PG), thromboxane (TX) and leukotriene (LT) in the arachidonic acid cascade. HETE: Hydroxyeicosatetraenoic acid; HPETE: hydroperoxyeicosatetraenoic acid.


126



however, showed a clear relationship between their structures and both inhibitory activities as shown in Table IV. α-Alkylthiodisulfide (n=0) did not have inhibitory activity against platelet aggregation or 5-lipoxygenase, but both oc-sulfinyl disulfide (n=l) and oc-sulfonyl disulfide (n=2) exhibited strong inhibitory activities in both systems. These data suggest that both sulfinyl and disulfide groups in A C series are essential for the development of both activities. Moreover, their activities against platelet aggregation were markedly increased with the chain length of their alkyl groups. In the case of 5-lipoxygenase, no clear relation was observed between inhibitory activity and alkyl chain length.

Table IV. Structure-Activity Relationship of oc-Sulfinyl Disulfide Inhibition of Human Platelet Aggregation and Human 5-Lipoxygenase

R

ICsoiMM)

n =0 Human platelet aggregation methyl 465 ethyl

>1000

propyl

>1000

Human 5-lipoxygenase methyl ND ethyl

>100

propyl

>100

n= 1

n =2

67.6 18.4 26.8 12.0 5.1 3.0

126.2

27.3 19.6 ND 10.0 ND 32.1

ND

40.6 13.0

50.6 88.0

Conclusion Ten kinds of oc-sulfinyl disulfides (AC series) were isolated and characterized as active components from onion for the inhibition of platelet aggregation. These active components were derived from the lachrymatory factor, thiopropanal S-oxide, and sulfenic acid, and were structurally similar to ajoene, an antiplatelet factor in garlic, regarding its sulfinyl and disulfide groups. Both sulfur containing groups were essential for development of the activity as antiplatelet factors. The activity of oc-sulfinyl disulfides was due to inhibition of cyclooxygenase in P G H synthase of platelets. Moreover, these components were also potent inhibitors of 5-lipoxygenase in leukocytes. Since onion is a widely consumed vegetable, physio logical characteristics of onion components as inhibitors of these enzymes have important meaning for human health from the viewpoint of the possible prevention of thrombosis and asthmatic and inflammatory diseases.




127

Acknowledgments The authors sincerely thank Drs. T. Matsuzaki and T. Matsumoto of Japan Tobacco Inc. for their kind supply of rabbit renal microsomes and human 5-lipoxygenase.


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April 14, 1993


Sulfur Chemistry of Onions and Inhibitory Factors of the Arachidonic

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