4α-methylvernosterol and other sterols from Vernonia anthelmintica seeds

Phytochemrstry,Vol.3 I, No. 5, pp. 1759 1763, 1992 Printedin Great Britain.

0031 9422f92$5.00+ 0.00 1992 PergamonPressplc

4ceMETHYLVERNOSTEROL AND OTHER STEROLS FROM VERNONIA ANTHELMINTKA SEEDS TOSHIHIRO AKIHISA, YOSHIHIROHAYASHI,GLENN W. PATTERSON,*NAOTO SHIMIZU~ and TOSHITAKETAMURA College of Science and Technology, Nihon University, 1-8, Kanda Surugadai, Chiyoda-ku, Tokyo 101, Japan; *Department of Botany, University of Maryland, College Park, MD 20742,U.S.A.; fibaraki Research Laboratory, Hitachi Chemical Co., Hitachi, Ibaraki 317, Japan (Received in revised form8 Umber

1991)

Key Word Index-- Vemonia ~nt~e~~i~ticu; Compositae; seeds; sterols; 4amethylvernostetol; 4a-methyl-%sti~asta-8,14,2qZ4~Z-t~en-3~-01; vernosterol; S~-~ti~asta-8,14~q2~)Z-t~en-3~-01.

Abstract-A novel 4c+methylsterol isolated from the seeds of Yernonia u~~~e~~~~~~c~ was shown to have the structure ~-methyl-5~-sti~asta-8,i4,Zq2~)Z-t~en-3~-ol (4a-methylvernosterol) based on spectroscopic methods. The 4demethylsterol and 4,4-dimethylsterol fractions from the seed material were also investigated. The 4-demethylsterol fraction contained 5a-stigmasta-8,14,24(24’)2-trien-3fi-ol (vernosterol) and 5a-stigmasta-7,24(24’)2-dien-38-01 (avenasterol) as the dominant sterols. 4a-Methylvernosterol is the possible intermediate in the biosynthesis of vernosterol in K anthelmintica seeds.

I[NTRODtJCTION Vernmia u~~~e~~j~~~c~ (purple fleabane) is a native plant of India, and its seed fat is known to contain high proportions (64-72% ) of an unusual vernolic acid (12,13epoxyoctadec-9-enoic acid) [l]. The seeds of Y. anthelmintica contain two 24Zethylidenesterols as the dominant steroIs, viz., avenasterol (3j) [2-5-J (see Table 1 for the systematic names of sterols) and vernosterol(4j) 15). The latter represented a rare example of naturally occurring stero’ls containing a A**14-conjugated diene system in the skeleton [6,7) which prompted us to undertake further investigation on the sterol constituents of the seeds. This paper describes our study on the sterol constituents of Y. antheCmirttica seeds which led to the isolation and characterization of a novel 4a-methylsterol, 4a-methylvernosterol (i;j), besides several other known 4-demethyl-, 4a-methyl- and 4,4-dimethylsterols.

RESULTSANDDISCUSSION Seventeen 4-demethylsterols, five 4tx-methylsterols and eight 4,4-dimethylsterols (triterpene alcohols) identified in the seeds of V: ~~~~e~~~~~ic~are listed in Table 1. Table 1 also gives the abundance, MP and chromatographic data of each compound. Standard procedures were followed for the separation of sterol mixtures from the seeds and for the isolation of the components from these mixtures (see Experimental). Acetates of individual sterols (and triterpene alcohols) were identified on the basis of their chromatographic data which was then confirmed by 400 MHz ‘K NMR. Determination of C-24 epimeric composition of the 24-alkylsterols possessing a chiral eentre at C-24 was aided by the “H NMR data. Details of the characterization of the novel 4c+methyl-

sterol, viz., 4~-m~hyl-5a-sti~asta-8,14,2q2~)Z-trien3B-01 [4c+methylvernosterol, 6jJ acetate, follow. The mass spectrum of 6j-acetate showed a molecular ion at m/z 466, corresponding to C,, H5002, and diagnostic fragments at m/z 451 [M -Me] +, 391 [M-Me(side chain) - HOAc] * HOAc] +, 267 [M -CroH19 and 265 (267 -2H) indicating that it was an acetate of a C,,-sterol with a monounsaturated C,,-side chain, and a disunsaturated skeleton with an additional methyl group. Occurrence of further fragments at m/z 368 [M -C7H14 (part of side chain)] +, 353 (368 -Me) and 293 (353-HOAc), formed by the allylic cleavage of the C-22/C-23 bond due to the McLafferty-type rearrangement, limited the possibilities for the position of the double bond in the side chain to A24(28)or A24(2$)[8] of which the latter could be excluded by the presence of the prominent ion at m/z 423 fM -C3H,]+ formed most probably by the loss of the terminal isopropyl group (C-25/C-27) of the side chain. The UV spectrum of tijacetate showed absorption peak at 250 nm, suggesting the presence of a A**l4 -conjugated diene system [S, 9-J. The ‘HNMR spectrum of 6j-acetate displayed only one skeletal olefinic proton deshielded to 65.373 (H-15), the H,-18 and H,-19 singlets at 60.820 and 1.039, respectively, and other skeletal proton signals at 60.878 (3H, d, H,-30), 2.059 (3H, s, OAc-3fl) and 4.377 (lH, dt, H-3@ which were in agreement with the corresponding signals of a 4ol-methyl-A 8*14-diunsaturated sterol, &-methyl-fiacholesta-8,14-dien-3P-ol (locereol, 6a) acetate [9]. This suggested that sterol6j possesses a 3fl-hydroxy-5a-Asv 14cholestadiene skeleton. The other four methyl doublets observed at SO.969 (f-I,-21), 0.979 and 0.983 (H,-26, H,27) and 1.596 (I-f,-29) and two methine signals at 62.839 (Sept., H-25) and 5.124 (q, H-28) were very close to the corresponding signals of vernosterol(4j) acetate possessing a 24Zethylidene side chain. Based on the spectral

1759

T.

7

AKIHISA et al.

8

9

Side chains (R)

=p/)/

-y/y/ a

.

=&y ..& f (22E,24/3/R)

..&

..+ b

. c (24ulR)

([MI’, m/z 424,Cx,HuO).

..A

...A h (24alR)

evidence cited above, 4a-methylsterol 6j was thus considered to have the structure 4a-methyl-5cr-stigmasta8,14,24(24’)Z-trien-3/I-ol (4a-methylvernosterol). Hydrolysis of 6j-acetate yielded free 4a-methylvernosterol 4a-Methylsterol6j, thus isolated from V. anthelmintica seeds in this study, is considered to be a novel natural product. There has so far been only one report for the natural occurrence of 4a-methylated Aa, i4-sterol, oiz., 4r-methyl-5a-cholesta-8,14-dien-3/I-o1 (locereol, 6a), isolated from a senita cactus, Lophocereus schottii [9]. In addition, only a few reports have so far appeared for the natural occurrence of 4-demethyl As, 14-sterols, viz., 5acholesta-8,14-dien-3/I-ol (4a) [9], vernosterol (4j) [2-51, and (24&Sa-stigmasta-8,14-dien-3#?-ol (249-411) [lo]. The As* 14-sterols are well known as transitory intermediates during cholesterol @a) and phytosterol biosyntheses [ll], and these compounds have been shown to

d (248/S)

i (22E,24a/S)

...* e (ZZE, 24a/S)

...& j [24(24vl

accumulate dramatically in plant cells treated with an inhibitor of the sterol A14-reductase [l l- 13].4a-Methyl5a-ergosta-8,14,24(24’)-trien-3/I-ol(6g) has been shown to be the intermediate demethylated metabolite of obtusifoliol (7g) by a cytochrome P-450 dependent obtusofiliol 14a-demethylase isolated from Zea mays microsomes [14]. Taking this into account, 4a-methylvernosterol (6j) in V. anthelmintica seeds can have at least two roles as the intermediate in the biosynthesis of the other sterols occur in the seeds: one to yield citrostadienol (5j) by A14-bond reduction followed by As-A’ isomerization, and the other to form vernosterol (4j) by demethylation at C-4. Two 24Z_ethylidenesterols, avenasterol (3j) and vernosterol (4j), were the dominant sterols in the 4-demethylsterol fraction from V. anthelmintica seeds in agreement with the previous findings [2-51. In addition to these, we have identified 10 As-sterols and five A’-

Sterols of Vernonia anthelmintica Table 1. Mp, chromatographic

1761

data and compositions (%) of 4-demethyl-, 4u-methyl- and 4&dimethylsterols anthelmintica seeds*

Sterol

4,CDimethylsterol (triterpene alcohol) fraction: 9b,l9-Cyclolanost-24-enol (cycloartenol) (9b) 24-Methyl-9~,19-cyclolanost-24(24’)-enol(24-methylenecycloartanol) Tirucalla-7,24-dienol (10) Urs-12-en01 (a-amyrin) (11) Olean-12-en01 (P-amyrin) (12) D-friedo-Olean-14-enol (taraxerol) (13) Lup20(29)-en01 (lupeol) (14) Fern-9( 11)-enol (fernenol) (IS) Others, unidentified

RR,t (GC)

1.45 1.00 1.12 1.12 0.88 0.95 0.81 1.27 1.11 1.01 1.10 1.10 0.82 1.27 1.07 1.01 0.72

1.55 1.00 1.27 1.27 1.13 1.13 1.33 1.54 1.37 1.71 1.50 1.50 1.57 1.81 1.60 2.02 1.86

0.1 0.1 0.6 0.5 0.2 0.1 0.4 3.9 6.3 2.3 0.2 0.2 0.7 5.7 2.5 36.4 39.2 0.6

123-125”

0.91 1.17 0.82 0.88 0.97

1.71 2.22 1.99 1.46 1.71

12.9 44.8 20.6 0.9 6.9 13.9

120-123” 116-118” 120-123” 230-232” 225-228” 218-220” 224-226”

0.99 1.08 0.92 1.06 0.97 0.94 0.79 1.40

1.81 1.99 1.87 1.91 1.68 1.64 1.99 2.29

5.3 21.8 14.5 8.7 8.2 1.5 23.8 5.8 10.4

-

119-121” 144-146” 133-135”

141-144” 150-152” 180-184 156-157” -

4a-methylsterol fraction: 4a-Methylergosta-7,24(24’)-dienol (gramiserol) (Sg) 4a-Methylstigmata-7,24(24’)Z-dienol (citrostadienol) (Sj) 4c(-Methylstigmasta-8,14,24(24’)Z-trienol(4a-methylvernosterol) (6j) 24-Methyl-31-norlanosta-8,24(24’)-dienol (obtusifoliol) (70) 24-Methyl-9/?,19-cyclo-31-norlanost-24(24’)-dienol (cycloeucalenol) (8g) Others, unidentified

(9~)

Composition of each fraction (%)

RR,t (HPLC)

Mpt

4-Demethylsterol fraction: (24t)-Stigmastanol (24<-lh) Cholest-5-enol (cholesterol) (2a) (24a/R)-Ergost-5-enol (campesterol) (Zc)$ (248/S)-Ergost-5-enol (22-dihydrobrassicasterol) &I)$ (24a/S)-Ergosta-5,22-dienol (crinosterol) (2e) (24fiIR )-Ergosta-5,22-dienol (brassicasterol) (2f) Ergosta-5,24(24’)-dienol (24-methylenecholesterol) (2g) (24a/R)-Stigmast-5-enol (sitosterol) (2h) (24a/S)-Stigmasta-5,22-dienol (stigmasterol) (2i) Stigmasta-5,24(24’) Z-dienol (isofucosterol) (2j) (24a/R)-Ergost-7-enol (3c)S (248/S)-Ergost-7-enol (fungisterol) (3d)S Ergosta-7,24(24’)-dienol (episterol) (3g) (24a/R)-Stigmast-7-enol (schottenol) (3h) (24c(/S)-Sigmasta-7,22-dienol (spinasterol) (3i) Stigmasta-7,24(24’)Z-dienol (avenasterol) (3j) Stigmasta-8,14,24(24’)Z-trienol (vernosterol) (4j) Others, unidentified

from Vernonia

134-135” 155-157”

*All sterols and triterpene alcohols possess the C-38 hydroxyl group. All compounds, with the exception of As-sterols, are Sa-compounds. All C-22/C-23 double bonds are trans (E)-orientated. tData for the acetyl derivatives. RR,: retention time relative to cholesterol (2a) acetate. $Isolated as the C-24 epimeric mixture.

sterols as the minor 4-demethylsterols. The stereochemical configuration at C-24 of the 24-alkylsterols thus identified was found to be consistent with those observed in the great majority of higher plants [6,7]. Thus, the 24-methylsterols consisted of the C-24 epimeric mixtures (2c/2d, 2e/2f, 3c/3d) whereas the 24-ethylsterols (lacking a Az5-bond) occur only as the 24aqimers (2h, 2i, 3h, 3i). The four 4a-methylsterols, 5g, 5j, 7g and 8g, and two 4,4-dimethylsterols, 9b and 9g, as well as several pentacyclic triterpene alcohols, identified in K anthelmintica seeds in this study, are widespread in higher plants [6,7]. In contrast to these, it is worth noting that Sa-tirucalla7,24-dien-3/Sol(lO) has so far been detected only in a few seed-bearing plants [15-171.

EXPERIMENTAL Crystallizations were. performed in Me&O-MeOH. Mp: uncorr. Prep. TLC silica gel, developed x 2 with hexane_EtOAc (4: 1). Argentation prep. TLC: silica gel-AgNO, (4: 1) developed x 3 with Ccl,-CH,CII (2: 1). HPLC: Altex Ultrasphere ODS 5 p column (Beckman Altex: 25 cm x 10 mm i.d.), MeOH as mobile phase (flow rate, 4 mlmin-I); GC: DB-17 fused silica capillary column (30 m x 0.3 mm i.d.), column temp. 275”. RR, on HPLC and GC expressed relative to cholesteryl(2a) acetate. UV spectra were recorded in EtOH. EIMS (70 eV): probe. The MS data do not include peaks at m/z ~200. ‘H NMR (400 MHz): CDCl, with TMS at int. standard. Acetylation: ActO-pyridine at room temp. overnight. Acetates of the following sterols and triterpene alcohols were used as reference com-

1762

T. AKIHISAet al.

pounds: lb, Za. mixt. of 2c and 26, 2h and 2i [18]; 2e, 2f. 2g, Zj, and mixt. of 3c and 36 [ 191; 3g, 3h. 3i and 3j [20]; Sg, Sj, 7g and 8g [21], 9b, 9g and lo-13 [17]; and 4j, 14 and 15 [22]. The seeds of k’ernonra anthelmintica were obtained courtesy of Dr Robert Klemmann. Northern Reg. Res. Ctr., U.S.D.A., Peoria, Ill., U.S.A. ~so~~?fun of 4-de~lef~~lsterols, 4~-meth~lsterols and 4,4dimethykterols. The seeds (460 g) of V. anthelmintlca were ovendried, ground (425 gf and then extracted with CHzClz in a Soxhlet extractor giving hpids (57 g). The liprds were refluxed with 5% KOH in 90% MeOH for 3 hr followed by diisopropyl ether extractton to yield unsaponifiable lipid (3.18 g) which was then subjected to CC over silica gel (130 g). Elution m order of mcreasing polarity: hexane (0.6 l), hexane-EtOAc (9: 1, 0.8 1) and hexane_EtOAc (4: 1.1.6 1).The eluate of the hexane-EtOAc (9: 1) gave the 4,4-drmethyl- (280 mg, fr. A) and 4~-methylsterol fr. (62 mg: fr B) whereas a portion of the eluate of the hexane--EtOAc (4: 1) yielded the 4-demethylsterol fr. (1745 mg; fr. C). The three frs were subjected to further TLC on silica gel yielding purified frs A (129 mg), B (25 mg) and C (1130 mg) which. upon acetytation, gave the acetylated frs A (120 mg), B (22 mg) and C (1050 mg). The compositions of individual sterol frs hsted in Table 1 were determined based on the argentatton TLC. GC. HPLC and ‘H NMR data. 4,4-Dimerhplsterals (trirerpene alcohols). Argentatron TLC of the acetate fr. A gave five bands (bands Al-AS in order of polarity, beginning with the least polar). The frs recovered from mdividual bands were further subJected to HPLC fractionation giving the following compounds as the acetyl derivatives: band Al (R, 0.63.-0.79; 59mg): 11,12 and 15; band A2 (R, 0.37-0.54; 5mg)’ 13; band A3 (R, 0.30-0.37; 3mg): 9b: band A4 (R, 0.22-0.30; 8 mg): 14; and band A5 (Rf 0.12-0.22; 12 mg): Qg and 10. 4a-Methylsterols. HPLC fractionation of the acetate fr. B yielded five 4~-methyisterols, Sg, 4, 6j, 7g and Sg, as the acetyl derivatives. Hydrolysis of 6j-acetate in 5% KOH in MeOH at room temp. overnight yielded free sterol 6j. 4a-~eth~l-24-erhpl-Sa-ckolesta-8,14.24(24’)Z-trien-3~-ol (4amethykernosterol, 6j) acetate. UV E.,,,: 250 nm. MS m/z (rel.

mt.): 466.3819 [M] + (100, C32HS002, requires 46638OQ), 451.3541 (32, C3, H&,0,), 437.3383 (3, &H,,O,), 423.3295 (33, C,,H,,O,), 406.3587 (3, C3,,H4e), 391.3384 (25, C,,H& 368.2746 (4, C25H3C02), 363.3035 (14, C,,H,,), 353.2475 (54, 341.2517 (14, C 23H 330 2)- 327.2342 (8, C,,H,,G,). C 22H 310 z ), 326.2271 (29, C,,H,,O,), 311.2044 (18, C,,H,,O,), 293.2280 (10, C,,H,,), 274.1926 (22. C,,H,,O,). 267.2154 (C,,H,,, 10). 265 1953 (13, C,,H,,k 255.2130 (12, C,,H2,), 253.1953 (20, C,,H,,), 251.1804 (20, C,,H,,). ‘H NMR. 64.377 (dt, J ~5.1, 11.0 Hz, H-3%),2.059 ts, OAc-3@), 5 373 (brs, H-15), 0.820 (s, He-18), 1.039 (s, H,-19), 0.969 (d, 5~5.9 Hz, Ha-21), 2.839 (sept.,J==66Hz, H-25), 0.979 and 0.983 (each d, J = 6.6 Hz. H,-26 and Ha-27), 5.124 (q, J =6.6 Hz, H-24’), 1.596 (d, J =6.6 Hz, H&4’), 0.878 (d, Jx6.6 Hz, Ha-30). 4r-Metlt~t-5cr-srzgmasta-8,14,24(24’)Z-trien-3~-ol (4a-me~~y~ternosterol, 6j). RR, (HPLC)=0.40. MS m/z (rel. int.). 424.3693 [M]’ (100, C,,H,,O. requires 424.3702). 409.3484 (31, CZsHa50), 381.3180 (31, Ca7H4, 0), 327.2663 (25, C,,H,,O), 311.2367 (62, C,,H,,O), 299.2417 (49, C,,H,, 0), 285.2304 (31, C,,H,,O), 283.2101 (24, C,,H,,O), 271.2139 (31, C,,H,,O), 269.1955 (25, C,,H,,O). 267.2123 (12, C,,H,,), 255.2143 (25, C,,H,,), 245.1934 (23, C,,H,,O), 213.1720 (43, ClbH2i). ‘HNMR: 3.115 (dc, .J=5.1, ll.OHz, H-3a). 5.360 (brs, H-15), 0.821 (s, H,-18), 1.014 (s, H,-19). 0.968 (d, J=5.9 Hz, H,-21), 2.840(sept.,J=7.3 Hz, H-25).0.980and0.983 (each d. J=6.2 Hz, HA-26 and H,-27), 5.124 (4, .I=?.7 Hz, H-24’), 1.595 (d, 5x6.6 Hz, H,-24”). 1.014 (d, J=6.2 Hz, H,-30).

~~e~e~~~~s~e~o~s.The acetate fr. C was crystallized to gave crystals (370 mg) and residue (680 mg) from the mother liquor. Argentation TLC of a portion (100 mg) of the crystals gave the acetates of 3j (R,. 0.30.-0.50, 48 mg) and 4j (R, 0.04-0.30: 7 mg). Low recovery of 4j-acetate after argentation TLC might be ascribed to its high susceptibility to decomposition owing to the presence of a conjugated-diene system (A8.14) in the skeleton. The residual portron was hydrolysed (5% KOH in MeOH, room temp., overnight) to give free sterol mixtures (455 mg) which on silica gel TLC gave two frs. one, recovered from the less polar and, consisting mainly of As-sterols (fr. Cl, 60 mg) and another, recovered from more polar band. consisting mainly of A’-sterols (fr. C2, 194 mg). The frs Cl and C2 were separately acetylated to give the acetylated fr. Cl (55 mg) and C2 (159 mg). Argentation TLC of fr. Cl gave five bands (bands Cl-l-Cl-5 m order of polarity, beginnmg with the least polar). Individual frs recovered were then subjected to HPLC which afforded the following compounds as the acetates: band Cl-t (Rf 0.65-0.75; 15mg): 24(‘-lh, 2a, mixt. of 2s and 2d, and 2h; band Cl-2 (R, 0.45.-0.65; 24 mg): 2i; band Cl-3 (RI 0.39-0.45: 3 mg): 2e and 2f: band Cl-4 (R,0.08-0.31; 8 mg): 2j; and band Cl-5 (R,0.03-0.08; 2 mg): Zg. The fr. C2, upon argentation TLC, afforded four bands (bands C2-l-CZ-4 in order of polarity. beginning with the least polar). HPLC of the individual frs recovered gave the following compounds as the acetyl derivatives: band C2-1 (RI, 0.72-0.77: 6mg): mrxt of 3e and 3d, and 3h, band C2-2 (Rr 0.68-0.72; 10 mg): 3i; band C2-3 (RI 0.22-0.59; 90 mg): 3j; and band C2-4 (RI 0.07-0.22; 5 mg): 3g. Sa-Stigmasta-8,1424(24’)Z-trien-38_ot (wrnosterol, 4j) acetare. UV &,X: 250 nm. MS m/z (rel. mt.): 452.3662 LMJ’ (100, C,, H,sO,, requires 452.3652), 437 (36). 423 (S),409 (43). 392 (4f, 377 (9), 354 (5). 349 (12), 339 (18), 327 (16), 313 (lo), 312 (33), 299 (12), 297 (21), 279 (12), 260 (Z6), 251 (15), 239 (22). 238 (22), 211 (11). ‘H NMR 4.709 (rt, J=S.l, 11.7 Hz, H-31). 2.033 (s, OAc3/J), 5.371 (bts, H-15), 0.822 (s, Ha-18). 1.006 (s, H,-19), 0.971 (d, J = 5.9 Hz, H,-21), 2.841 (Sept., .I =7.3 Hz, H-25), 0.980 and0.984 (each d, J = 7.3 Hz, Ha-26 and H,-27), 5.125(4. J = 7.3 Hz, H-24’), 1.597 (d, J = 6.6 Hz. He-24“).

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Matsumoto, T. (1992) Lipids 26, 660. 19. Akihisa, T., Matsubara, Y., Ghosh, P., Thakur, S., Tamura, T. and Matsumoto, T. (1989) Steroids 53, 625. 20. Akihisa, T., Shim& N., Ghosh, P., Thakur, S., Rosenstein, F. U., Tamura, T. and Matsumoto, T. (1987) Phytochemistry 26, 1693. 21. Akihisa, T., Yokota, T., Takahashi. N., Tamura, T. and Matsumoto, T. (1989) Phytochemistry 28, 1219. 22. Itoh, T., Tani, H., Fukushima, K., Tamura, T. and Matsumoto, T. (1982) J. Chromatogr. 234, 65.