512

Sesamin Is a Potent and Specific Inhibitor of A5 Desaturase in Polyunsaturated Fatty Acid Biosynthesis Sakayu Shimlzu a,~ Kengo Akimolo a,1, Yoshifumi Shlnmen a,1, Hlroshi Kawashima a,2, Michlhiro Sugano b and Hldeaki Yamada a aDepartment of Agricultural Chemistry, Kyoto University, Sakyo-ku, Kyoto 606 and bDepartment of Food Science and Technology, Kyushu University, Higashi-ku, Fukuoka 812, Japan

Incubation with sesame oil increases the mycelial dihomo- Japan). [1-14C]Stearic acid (18:0) (2.07 GBq/mmol), [1-14C]y-linolenlc acid content of an arachidonic acid-producing oleic acid (18:1) (1.92 GBq/mmol), [1-14C]linoleic acid fungus, Mortierella alpina, but decreases its arachidonic (18:2) (1.89 GBq/mmol) and [2-14C]DGLA (2.13 GBq/ acid content [Sbimizu, S., K. Aidmoto, H. Kawashlm~ Y. retool) were purchased from Amersham (Buckingham, Shlnmen and H. Yamada (1989) J. Am. Oil Chem. Soc. 66, U.K.). (+)-Episesamin and (+)-sesamlnol were prepared 237-241]. The factor causing these effects was isolated from refined sesame oil as described previously (3). and identified to be (-b)-sesamln. The results obtained in Sesamol was obtained from Aldrich (Milwauke~ WI). All experiments with both a cell-free extract of the fungus other reagents used in this work were obtained as deand with rat liver microsomes demonstrated that (-F)- scribed previously (4,5,9). sesamin specifically inhibits A5 desaturase at low concenMicroorganism and cultivation. The fungus MortiereUa trations, but does not inhibit A6, A9 and A12 desaturases. alpina 1~4 (AKU 3998) (4,5) was grown aerobically for Kinetic analysis showed that (-F)-sesAmln is a noncom- 7 days at 28~ in a 10-mL Erlenmeyer flask containing petitive inhibitor (Ki for rat liver A5 desaturase, 155 ~aM). 2 mL of a basal medium (4% glucose and 1% yeast ex(-b)-Sesamolin, (+)-sesamiuol and (-b)-episesRmlu also in- tract, pH 6.0), unless otherwise stated. hibited only A5 desaturases of the fungus and liver. These Extraction and determination of mycelial fatty acids. results demonstrate that (-F)-sesamin and related lignan Fungal mycelia were harvested by suction filtration, compounds present in sesame seeds or its oil are specific washed with 50 mL water and then dried overnight at lnhlbitors of A5 desaturase in polyunsaturated fatty acid 100~ Transmethylation of the fatty acids in the mycelia, biosynthesis in both microorganisms and animals. extraction of the fatty acid methyl esters and analysis by Lipids 26, 512-516 (1991). gas-liquid chromatography were carried out as described previously (5,6,9). Sesame seed oil, which has long been used as an edible Fractionation of sesame oil by high-performance liquid oil contains considerable amounts of sesarnin and related chromatography (HPLC). An acetone-extractable non-oil liguan compounds (1,2). Little is known about the biologi- fraction of crude oil was prepared as described previously cal activity of these compouncL~ except that some of them (7). The non-oil fraction (1.5 g) was dissolved in chloroform show potent antioxidative activity (3). During our studies (ca. 3 mL) and the resultant solution (150/~L each) was on the biosynthesis of C20 polyunsaturated fatty acids subjected to HPLC on a reversed phase 10C18 Cosmosil (PUFA) in Mortierella alpina and related filamentous packed column (20 X 250 m~; Nakarai Tesqu~ Kyot~ fungi (4-11), we found that the mycelial dihomo-y-linolenic Japan). The chromatograph was operated with a mobile acid (DGLA or 20:3) content of M. alpina drastically in- phase of methanol/water (67:33, v/v) at a flow rate of creases, while arachidonic acid (AA or 20:4) decreases 9.9 mL/min and the effluent was monitored at 205 nnL Six upon incubation with sesame oil. This phenomenon was fractions of 100 mL each were collected and concentrated explained by specific inhibition of the conversion of DGLA under reduced pressur~ The same separation was repeated to AA, i.e., inhibition of A5 desaturation (7,8). To clarify 20 times. the mechanism by which sesame oil exerts this inhibitory Assay of inhibitors of A5 desaturation reaction with effect, we searched for factors that could be responsible M. alpina culture Fractions from i of the 20 HPLC runs for this effect. We report here that (+)-sesamin and related were used for the assay. Each of the concentrated fraclignan compounds, which are commonly present in sesame tions was dissolved separately in 150 pL of chloroform, oil, are potent and apparently specific inhibitors of mam- and 40 pL of the resultant chloroform solution was addmalian and microbial A5 desaturases involved in PUFA ed to the sterilized basal medium. The assay of inhibitors biosynthesis. of the A5 desaturation reaction was carried out by monitoring the changes in the fatty acid profile of M. MATERIALS AND METHODS alpina grown in the basal medium supplemented with each the concentrated fractions as described above (see also Chemicals. Crude and refined oils from unroasted sesame of Fig. 1). seeds were obtained from Kitamura Shokai Ca (Osaka, Isolation of A5 desaturase inhibitors. The other fractions obtained from the remaining 19 HPLC runs were used for *To whom correspondence should be addressed. the isolation of the inhibitors. (+)-Sesamin was obtained 1Present address: Laboratory of Microbial Science, Institute for from the fractions corresponding to fraction 4 in Figure 1 Fundamental Research, Suntory Ltd., Mishima-gun, Osaka 618, by repeated crystallization from ethanol until the comJapan. pound was homogenous as judged by thin-layer chrom2On leave from Suntory Ltd. tatography (TLC) (yield from 1.5 g of the non-oil fraction, Abbreviations: AA or 20:4, arachidonic acid; DGLA or 20:3,dihomoy-linolenic acid; HPLC, high-performance liquid chromatography; 137 rag;, Anal. Calcd for C~0H1806: C, 67.79; H, 5.12%; MS, mass spectrometry; NMR, nuclear magnetic resonance; PUFA, Found: C, 67.86; H, 5.20%; mp, 123.2-123.9~ MS (direct polyunsaturated fatty acids; TLC, thin-layer chromatography; 18:0, inlet, 200~ m/z (%): 354 (45, M+), 323(5), 203(19), stearic acid; 18:1, oleic acid; 18:2, linoleic acid. 178(12), 162(8), 149(100), 135(42), 122(25), 103(13), 89(6), LIPIDS,Vol. 26, Na 7 (1991)

513 INHIBITION O F A5 D E S A T U R A S E B Y S E S A M I N

65(15), 39(10); [a]26 (c=l, CHCI3), +64.7~ 1H NMR d (CDC13): 3.05 ppm (m, 2H), 3.87 (dd, J=4.0 and 8.SHz, 2H), 4.24 (dd, J=6.0 and 8.5Hz, 2H), 4.74 (d, J=4.0I-Iz, 2H), 5.96 (s, 4H), 6.85 (m, 6H). In a similar manner, (+)sesamolin was isolated from the fractions corresponding to fraction 5 in Figure 1 (yield, 70 mg; Anal Calcd for C20H1sO7: C, 64.86; H, 4.90%; Found: C, 64.93; H, 4.97%; rap: 93.5~ MS (direct inlet, 200~ m/z (%): 370 (12, M+), 233(36), 203(43), 173(3), 149(24), 135(100), 115(12), 81(41), 65(11), 52(7), 39(7); [a]2s (c=1, CHC13), +213~ 1H NMR d (CDCI3): 2.95 ppm (m, 1H), 3.30 (dd, J=9.0 and 16.5Hz, 1H), 3.64 (dd, J=7.5 and 9.0Hz, 1H), 3.96 (dd, J=l.0 and 9.0Hz, 1H), 4.13 (dd, J=6.0 and 9.0Hz, 1H), 4.40 (d, J=7.SHz, 1H), 4.44 (t, J=9.0Hz, 1H), 5.50 (s, 1H), 5.94 (d, J=18.5Hz, 4H), 6.50 (dd, J=3.5 and 8.5Hz, 1H), 6.62 (d, J=2.5Hz, 1H), 6.71 (d, J=8.5Hz, 1H), 6.78 (dd, J=8.0Hz, 1H), 6.82 (dd, J=1.5 and 8.0Hz, 1H), 6.88 (d, J=l.SHz, 1H). Preparation of ceU-free extract of M. alpina. This was done as described previously (5). Preparation of rat liver microsomes. Male Wistar rats maintained on a standard pelleted diet for eight weeks were killed and the livers were immediately removed and rinsed in cold homogenizing medium (12). They were homogenized in four volumes of the same medium in a Teflon pestle homogenizer and then centrifuged at 10,000 X g for 20 rain at 4~ Microsomes were recovered by centrifuging the superuatant at 100,000 X g for I hr at 4~ The pellet was resuspended in the homogenizing medium to give a protein concentration of 30 mg/mL. The sample was stored at - 8 0 ~ till u s e Assay of desaturase activities. The ceil-free extract of M. alpina was assayed for A9, A12, A6 and A5 desaturase activities using labeled 18:0, 18:1, 18:2 and 20:3 as substrates, respectively. The standard reaction mixture contained, in 1.0 mL, 250 pmol of sucrose, 100 ~xnol of potassium phosphate buffer (pH 7.4), 40/maol of NaF, 150 vmol of KC1, 4 ~anol of N-acetyl-L-cysteine, 0.3 pmol of nicotinamide, 15 ~mol of MgC12, 7.5 ~cmol of ATP, 0.4/~mol of CoA, 1.5/zmol of NADH, 100 ~tmol of fatty acid (18:0, 18:1, 18:2 or 20:3 containing 3.7 KBq of the respective labeled fatty acid) and the cell-free extract (1.0 mg protein). The reactions were carried out at 28~ for 30 min and terminated by the addition of 5 mL of ethanol and I mL of 4N KOH. Each of the resultant mixtures was saponified at 60~ for 30 min. After addition of 3 mL of water, 100 pg of the corresponding unlabeled substrate and product fatty acids, and I mL of 6N HC1, the fatty acids were extracted twice with n-hexane (5 mL each). The procedures for the preparation of fatty acid methyl esters and silica gel/silver nitrate TLC, and the quantitative analysis of radioactive fatty acid methyl esters were as described previously (13). The liver microsomes were assayed for A9, &6 and A5 desaturases. Each incubation mixture contained, in 1 mL, 250 vmol of sucrose, 100~maol of potassium phosphate buffer (pH 7.4), 45/~mol of NaF, 150 ~anol of KC1, 1.5 vmol of glutathione, 0.5 vmol of nicotinamide, 5 ~anol of MgC19, 7.5 ~aol of ATP, 0.4 ~mol of CoA, 1.5 ~mol of NADH, microsomal suspension (1.2 mg protein) and 100 vmol of the desired labeled fatty acid as described above The incubations were carried out for 10 min (except as stated) at 37~ Other conditions were the same as those for the assays with the M. alpina cell-fres extract.

Other methods. TLC on lignan compounds was per ~ formed on a 60F245 silica gel plate (Merck, Darmstadt, Germany) with chioroform]diethyl ether (9:1, v/v) as a solvent. Fractions were localized under an ultraviolet lamp. Protein concentrations were determined by the method of Bradford (14). Mass spectra were recorded on a Hitachi M-80B instrument at 70eV; 1H NMR spectra were recorded on a Nicolet 360-MHz instrument with tetramethylsilane as internal standard. RESULTS

Isolation and iden~fica~on of ~ desatur~e i.hibi~rs. Trio major fractions (Rf=0.65 and 0.8D were detected by TLC of the non-oil fraction together with several minor ones. The separation of the non-oil fraction into six fractions by preparative HPLC (see Fig. 1) and TLC of each of the six fractions demonstrated that the compounds corresponding to Rf--0.65 and 0.81 were present in fractions 4 and 5, respectively. The inhibitor assays as judged by the change of mycelial fatty acid profile of M. alpina showed that only fraction 4 caused an increase in mycelial DGLA content and a decrease in AA content, as shown in Figure 1. The mycelial DGLA/AA ratio was 0.51. By contrast, no significant change in mycelial fatty acid profile was observed on cultivation with the remaining five fractions. In each case, the ratio was only about 0.09. The compound isolated from fraction 4 was identified as (+)sesamin based on the data of MS, 1H NMR, optical rotation and melting point in comparison to literature values

2o0 . {B)

3 4 m

150

1 100

50

,
sesamolin > sesaminol > episesamin. tracts of the fungus and rat liver microsomes as enzymes. However, no significant inhibition was observed with Figure 1 shows that sesamin specifically inhibits only A5 sesamol, which does not carry the 3,7-dioxabicyclo[3.3.0]desaturase in both the microbial and rat systems. The octane ring. concentrations inducing 50% inhibition were 5.6 (0.002 mg/mL) and 75 ~ (0.002 mg/mL) for microbial and DISCUSSION rat enzymes, respectively. By contrast, no significant decrease with sesamin was observed for A6, A9 and A12 The present study demonstrated that (+)-sesamin and desaturases in the fungal extract (Fig. 2A). Rat liver A6 related lignans specificallyinhibit A5 desaturase in P U F A and A9 deeaturases also were not sensitive to sesamin biosynthesis. This inhibitory activity seems to be unrelated to the known antioxidative activity of sesamin (Fig. 2B). The rat enzyme was studied in more detail to clarify the related lignans because only the compound carrying a sesamin-induced inhibition of A5 desaturation. Double hydroxyl group at the 6 position of the methylenediox-

(A)

(B)

(C)

xl0

30

100

Y

8O O ..O

6O

r" r"

--

I 3

40

I

!

Sesamin 84.7 p.M &5-desaturase ~ 2 ~M

E

AS-desaturase

20

9 A J ~ b i t

0r

A6-desaturase

2o

~9-desaturase~ 9-desaturase

0 - - ~

0

'

i

20

Sesamin (I~M)

40

0

100

,

200

Sesamin (tIM)

i

300

,

t

0.02

,

I

t

0.04

I

0.06

I

I

0.08

1/DGLA (1/p.M)

FIG. 2. The effect of sesamin concentration on various desaturase activities of M. alpina cell-free extract (A) and rat liver microsomes (B and C). (C) shows double reciprocal plots for A5 desaturase activities in the presence ( 9 ,A) or absence (O) of sesamin. A s s a y of desaturases were carried out under the conditions described in Materials and Methods, except that the indicated amount of sesamin was present in the incubation mixtures in (A) and (B) and concentrations of substrate (DGLA) and sesamin were changed as indicated in (C). In (A) and (B), the values without sesamin were taken as 0% inhibition (see Table 1 for absolute values) and each point represents percentage of inhibition. All values are means of three independent assays (standard deviation, within ___10%).

LIPIDS,Vol. 26, NQ 7 (1991)

515

INHIBITION OF A5 DESATURASE BY SESAMIN TABLE 1 Specific Inhibition of Fungal and Rat Liver A5 Desaturases by Sesamin-Related Compoundsa

Desaturase activity(pmol/min/mg protein)

M. alpina

Componnd b

added None Sesamin EpisesRmin Sesaminol Sesamolin Sesaraol

A9

AI2

10.1 10.5 10.3 10.1 9.93 9.86

8.16 9.27 8.40 8.37 8.77 8.10

A6

Rat liver A5

1 8 . 2 21.2 19.1 2.80 18.2 5.20 18.4 4.67 17.7 3.97 1 7 . 2 16.8

A9

37.2 34.9 36.1 36.0 37.7 34.3

A6

A5

Sesamin ~EpiSesamin~Sesaminol

1 5 . 0 116 16.4 71.8 17.5 92.1 15.4 87.9 18.6 86.6 14.7 114 o

a Desaturase activities were measured as described under Materials and Methods except that each of the indicated compounds were added and incubations with the liver microsomes were carried out for 30 min. All the compounds were present at 28 or 85 WMin the reaction mixtures with M. alpina extract or rat liver microsomes, respectively. Values are means of three independent assays {standard deviation, within • bSee Figure 3 for chemical structures.

yphenyl moiety (i.~, sesaminol, episesaminol and sesamol) exhibit antioxidative activity, and sesAmln itself is inactive as an antioxidant (1-3). The synergistic activity of sesamin for some insecticides, such as pyrethrin, has been suggested to be due to inhibition of the cytochrome P-450 oxygenase system catalyzing degradation of the insecticides (2). Since f a t t y acid desaturases are also of the oxygenase family, there may be some relation between the inhibitory activity on A5 desaturase and the synergistic activity. The finding t h a t sesamol, which lacks the dioxabicyclo[3.3.0]octane ring, does not show any inhibitory activity suggests t h a t this ring (or some other bulky substituent equivalent to the ring) is necessary for the activity. The inability of sesamolin to cause the increase in D G L A production and the decrease in AA production in the fungus M. alpina might be due to degradation of the lignan during the growth of the fungus. 8 Our s t u d y is the first to demonstrate the occurrence of desaturase inhibitors in n a t u r e Several unnatural desaturase inhibitors have been synthesized; most of them are analogs of f a t t y acids and they rather nonspecificany in. hibit any of the desaturases involved in PUFA biosynthesis. For example, several trans-octadecenoic acids are competitive inhibitors of A9, 46 and 45 desaturases of rat liver microsomes (13,19). B y contrast, sesamin or related lignans inhibit only A5 desaturase noncompetitively. This suggests t h a t the inhibition induced by sesamin differs from those previously reported (13,19). The enzyme m a y have a site different from the substrate binding sit~ which shows high affinity toward sesamin. The enzyme assay system used here involves acylation of the substrate with CoA, and the rates of the desaturation reactions measured here are the results of at least two sequential reactione, acylation and desaturatiom Since the

o

Sesamol FIG. 3. Chemical structures of sesamin and related compounds.

acylation step is not the rate-limiting step in rat liver microsomes and a single enzyme is responsible for the activation of an desaturase substrates used here (13,20), we can exclude the possibility t h a t sesamin inhibited the acylation reaction. The present results suggest t h a t sesamin alters in v i v o metabolism and composition of PUFA. Nutritional experiments are in progress in our laboratories. REFERENCES 1, Budowski, P. (1964)J. Am. Oil Chem. Soa 41, 280-285. 2. MacRae, W.D., and Towers, G.H.N. (1984) Phytoehemistry 23, 1207-1220. 3. Fukuda, Y., Nagata, T., Osawa, T., and Namiki, M. (1986)J. Am. Oil Chem. Soc 63, 1027-1031. 4. Yamada, H., Sblmizu, S., and Shinmen, Y. (1987)Agria BioL Cher~ 51, 785-790. 5. Sbimizu, S., Sbinmen, Y., Kawashima, H., Akimoto, K., and Yamada, H. (1988) Biocher~ Biophys. Res. Commun. 150, 335-341. 6. Sbimizu, S., Kawashima, H., Shinmen, Y., Akimoto, K., and Yamada, H. (1988) J. Am. Oil Chem. Soa 65, 1455-1459. 7. Sblmizu, S., Akimoto, K., Kawashima, H., Shinmen, Y., and Yamada, H. (1989)J. Am. Oil Chem. Soa 66, 237-241. 8. Shimizu, S,, and Yamada, H. (1990)Comments Agria Food Chem. 2, 211-235. 9. Shimizu, S., Kawashima, H., Shinmen, Y., Akimoto, K., and Yamada, H. (1989) J. Am. Oil Chem. Soc. 66, 341-347. 10. Shinmen, Y., Shimizu, S., Akimoto, K., Kawashima, H., and Yamada, H. (1989)AppL MicrobioL Biotechnol. 31, 11-16. 11. Shimizu, ~, Kawashima, H., Akimoto, K., Shinmen, Y., and Yamada, H. {1991)J. Am. Oil Chem. Soc 68, 254-258. 12. de Gometz Dumn, I.N.T.,de Alaniz, M.J.T.,and Brenner, R.R. {1970) J. Lipid Res. 11, 96-101. 3{+)-Sesamolin was hydrolyzed to sesamol and probably samin on incubation with mycelia of M. alpiana (unpublished observation).

LIPIDS, Vol. 26, Na 7 0991)

516 S, SHIMIZU E T AL. 13. Mahfouz, M.M., Johnson, S., and Holman, R.T. (1980) Lipids 15, 100-107. 14. Bradford, M. (1976)Anal. Biochem. 72, 248-254. 15. Pelter, A., Ward, R.S., Rao, E.V., and Sastry, K.V. (1976) ~trahedron 32, 2783-2788. 16. Beroza, M. (1956)J. Am. Chem. Soc. 7& 5082-5084. 17. Haslam, E. (1970)J. Chem. So~ (C), 2332-2334.

LIPIDS,Vol. 26, No. 7 0991)

18. Cornish-Bowden, A. (1974) Biochem. J. 137, 143-144. 19. Chang, H.-C., Kanke, J., Pusch, F., and Holman, R.T. (1973) Biochim. Biophys. Acta 306, 21-25. 20. Svensson, L. (19831 Lipids 18, 171-178. [Received August 11, 1990, and in revised form January 29, 1991; Revision accepted May 18, 1991]