Sterols of Cajanus cajan and three other leguminosae seeds

Phytochemrstry, Vol. 31,No. 5,pp. 17651768,1992 Printed in Great

0031~9422/92 $5.00+0.00

Q 1992PergamonPresspfc

Britain.

STEROLS OF CAJANUS CAJAN L~GUMINOSAE

AND THREE OTHER SEEDS

TOSHIHIRO AKIHISA, YUTAKA NISHIMURA, NAOMI NAKAMURA, KARABI ROY,* PARTHASARATHI GHOSH,* SWAPNADIP THAKUR* and TOSHITAKE TAMURA

College of Science and Technology, Nihon University, 1-8, Kanda Surugadai, Chiyoda-ku, Tokyo 101, Japan; *Department of Chemistry, The University of Burdwan, Burdwan 713 104, west Bengal, India (Received 23 July 1991) Key Word Index-Cajanus cajan; Cicer arietinum; Pisum sativum; Vigna mungo; Gguminosae; seeds; sterols; 14a-methyl-24-alkyl-A y(l lb-sterols; 24a-ethyl-Sa-cholestclerosterol; 24-epiclerosterol; 24-alkyl-A *(‘%terols; 9(1I)-en-3/G01.

occurrence of 24-epiclerosterol, three A8(*4)-sterols [24a- and 24~-methyl-Sa~holest-8~14)-en-3~-ols and 24a-ethyl-5ct-cholest-8(14)-en-3~-olJ and four 14a-methyl-A 9(1‘)-sterols [14a,24a- and 14a,24/&dimethyl-5c-cholest-9( 1l)-en-3gols, 14a-methyl-24a-ethyl&-cholest-9( 1l)-en-38-01 and 14cc-methyl-24B-ethyl&-cholesta-9( 11),25dien-3a-ol] has been demonstrated in the seeds of four Leguminosae species, Cajanus cajan, Cicer arietinum, Pisum sativum and Vigna mungo. These compounds were minor sterol constituents. In addition, 24c+ethyl-Scr-cholest9(11)-en-3/%ol and ~-methylenepollinastanol were isolated and identified from I/. mungo seeds. Sitosterol was the principal sterol in all of the four Leguminosae seeds investigated. Abstract-The

INTRODUCIlON

Most of the plants belonging to the family Leguminosae so far examined were reported to contain 24-alkyl-A’sterols, viz., campesterol (2b) (most probably accompanied by its 24/%epimer, 2c), sitosterol (2f) and stigmasterol (2g), as the dominant sterols [1], whereas some species contain two 24-ethyl-A7-sterols, schottenol (3f) and spinasterol (24~-ethylcholesta-7,22-dien-3~-ol, 3g), as the major sterols [l]. Our recent study of the sterols of the seeds of three Leguminosae, Dolichos biforus, Lathyrus sativus and Lens culinaris, has shown the occurrence of several uncommon sterols: 24,25-dimethylcholesta-5,24(28)-dienol (2n) and 24c+ethylcholest-9(11)-enol (5f) in D. bijlorus, 24-epiclerosterol (Za), in addition to its 24/3-epimer, clerosterol(21), in L. sativus, and sterol 2n in L. ~~i~ris [ 11. These compounds always constituted the minor ster01s whereas 2f was the dominant sterol. Detection of the above uncommon sterols in the Leguminosae seeds prompted us to investigate further species of this family. This paper describe.8 our study on the sterols of the seeds of the four Leguminosae species, Cajanus cajan, Cicer ffrietinum, Pi~m sutiuum and Vigna mu~go, which led to the isolation and identification of several uncommon sterols including 2k, three A8(14)-sterols (4b, 4c and 4f) and four 14a-methyl-Ag(11)-sterols (6b, 6c, 6f and 61). BESC’LTSAND DISCZ;SSION

Sterol fractions of the seeds of Cujcznus cajun, Cicer arietinum, Pisum sativum and Vigna mungo were obtained

by the extraction of the seeds, alkaline hydrolysis of the extracted lipids followedIby the silica gel chromatograPHMO31:5-v

phy of the unsaponifiable lipids. The sterol fractions were acetylated and the resulting steryl acetates were subjected to argentation TLC followed by reverse-phase HPLC which enabled the isolation of individual sterols (as the acetates) with the exception of the pairs of C-24 epimeric 24-alkylsterols. Table 1 lists the names of 30 sterols, their chromatographic (GC and HPLC) data and the compositions of the sterol fractions from the seeds of the four Leguminosae species. Identifi~tion of individual sterols was performed by comparison of the GC.and HPLC data with those of reference compounds. All of the 24-alkylsterols possessing a chiral centre at C-24 isolated from C. cajan seeds were, when available in sufficient amount, subjected to 400 MHz ‘H NMR spectroscopy in order to determine their stereochemistry at C-24. Some of the 24-alkylsterols from the other three Leguminosae seeds were also examined by ‘H NMR spectroscopy. All of the 30 sterols identified in the Leguminosae seeds in this study (Table 1) are known compounds in higher plants. However, several of them have so far been detected in only a few plants and are uncommon as the natural products. Thus, we have recently demonstrated the occurrence of 24-epiclerosterol @k), in addition to its 24/&epimer, clerosterol(21), in three K~~u~hoe species of Crassulaceae [2, 31 which constituted the first case for the detection of a 24-alkyl-Az5-sterol possessing a 24~ stereochemistry in plants. (Recently we have revised to fithe stereochemistry at C-24 for several 24-alkyl-Az5-ster01s isolated from a fern, Polypodium formosanum [4], which was wrongly assigned previously as 24a [5].) Ster01 2k was detected in the seeds of four Leguminosae species (see Table 1) in this study, in addition to Lathyrus sativus (Leguminosae) seeds [1], suggesting its widespread occurrence accompanying sterol 21 in this family. 1765

T. AKIHISA et al.

a

f (24Rlaf

k (24Rla)

b (24RRla)

g (22‘%24S/a)

c (24Sll3)

h

d (22E,24RIB)

e

i [24(28)zl

j 12WWl

m (2Z24W)

~~-ex~nation of the stereochemistry at C-24 of clerosterof (21) detected so far in several plants [2] might be by use of high-field ‘HNMR and/or necessary r3C NMR. This study constitutes the second case for the detection of the three A8(r4)-sterols, 4b, 4c and 4f, from higher plants. These sterols were detected first in Dioscorea batatas (Dioscoreaceae) [6]. The other Aso4)-sterols known to occur in higher plants are cholest-8(14)-en3/S01 (4a) [6], 24sr-ethylcholesta-8(14),22-dien-38-01 (49) [7], 24(-4g [8,9] and 245-ethylcholesta-8(14),25-dien3@01 (4k and/or 41) [lo]. Although A804)-sterols are known to be produced from A7- and A’-sterols by isomerization in acetic acid in the presence of platinum and hydrogen [ll], the A8(14)-stero1s detected in this study are considered to be natural products rather than artefacts produced from the corresponding A7- or A*sterols during extraction and separation procedures, because the isomerization could only be achieved under drastic chemical conditions. The Asfi r)-sterot 5f isolated from F’fgna ~~~~0 seeds in

this study has recently been detected in the seeds of the other Leguminosae plant, Dolichos bijlorus Cl]. The occurrence of 14a-methyl-A g’“‘-sterols, as identified in the four Leguminosae seeds in this study, has previously been reported only in Cucurbita~ae species [12-L 51 and some other plants [16-191. Although it could not be confirmed due to unavailability of the reference compound, the possibility for the occurrence of the 24aepimer (6k) of 14ff-methyl-2~thyl-Agt1 ‘), 25-sterol, as a minor companion of the 24/Gepimer (61),isolated from the four Leguminosae seeds in this study, cannot be excluded since the other A”-unsaturated sterol, 24-ethylA3.2s-sterol, was shown to occur as the C-24 epimeric mixture (2k and 21) with the 24/3-epimer predominant. As far as the seed sterols are concerned, the four Leguminosae plants investigated in this study can be classified among the most common “main line” plant group since they contain sitosterol (2f) as the dominant sterol component [ZO]. The function and biogenesis of the minor sterols, including the uncommon ones, detected in this study remain to be clarified in the future.

1767

Sterols of Leguminosae Table

1. Chromatographic

data of the reference steryl acetates,

and compositions

of sterol fractions

from four Leguminosae

Compositions Acetate,

GC

HPLC

Cajanus cajan

lb lc If lg 2s 2b 2c 2d 2e

1.2811 1.2811 1.55 1.38 1.00 1.27)) 1.2711 1.13

1.3211 1.3211 1.48 1.24 1.00 1.1411 1.141( 0.93

0.6$ 0.23 0.5$ 0.51 0.1 10.83 2.9$ td

1.34 1.54 1.37

0.81 1.27 1.09

0.6 53.83 19.81

1.86 1.72 1.63 1.56 1.56 1.44 1.61 1.81 2.01 1.84 1.3411 1.3411 1.61 1.46 1.471) 1.4711 1.76 1.79

1.01 1.02 0.98 0.97 0.95 0.78 0.96 1.27 1.01 0.94 1.0311 1.0311 1.19 1.13 0.97 II 0.97 II 1.16 0.85

1.61

0.81

*All sterols possess the C-3/l hydroxyl

group.

All sterols, with the exception

Argentation prep. TLC: silica gel-AgN03 (4: 1) developed x 3 with CC1&H,C12 (5: 1). HPLC: Altex Ultrasphere ODS 5 y column (Beckman Altex; 25 cm x 10 mm i.d.), MeOH as mobile phase (flow rate, 4 ml min-‘); GC: DB-17 fused silica capillary column (30 m x 0.3 mm id.), column temp. 275”. RR, on HPLC and GC expressed relative to cholesteryl (2a) acetate. ‘H NMR

(400 MHz) spectra were determined in CDCl, with TMS as int. standard. Acetylation: Ac,O-pyridine at room temp. overnight. The following sterols were used as the reference compounds as their acetyl derivatives: a mixture of lb and lc, lf, lg, 2a, a mixture of 2b and Zc, 2d, Zf, 2g, 21,Zm and 31 [Z]; 2e, 2k, 2i, 2j, tk, 2n and 3i [21]; 3fand 5f [l]; 4b, 4c and 4f [63; 6b and 6e [15]; 6f and 7e [13]; and 61 [lfl.

Cicer arietinum

Pisum sativum

Vigna mung0

0.S

0.45

0.35

4.15

2.35

0.1 17.39

0.2 9.45

4.34 1.q 0.8 0.85 tr§

tr§ 0.2 66.e 0.20

0.2 72.Q 6.55

1.9 29 1.1 0.53 3.0f

0.5 1.6 0.7 0.2f 1.71

3.4 0.4 tr% 0.3$

0.5

1.3

0.1

0.3

0.2

0.5

t3 trS 0.11

0.13 trS 0.13

0.9$ 0.3$ trS O.l$

O.l$

trS

0.3$ 0.13 0.43 0.2$

0.1% trS 0.11 trS trS trS trS 1.3%

0.4

0.3

1.0

tr# 1.1

6.6 0.5 trS O.l$ trS 0.3 tr8 0.1 0.34 0.23 O.l$ 0.3$

of A5-sterols,

t Data for the reference steryl acetates (those accompanied with a 1)are for the C-24 epimeric acetate (RR, = 1.00). SStereochemistry at C-24 determined by ‘H NMR spectroscopy. gstereochemistry at C-24 undetermined (not examined by ‘H NMR spectroscopy).

EXPERIMENTAL

(%)

RR,?

Code Sterol 24a-Methylcholestanol 24fi-Methylcholestanol 24a-Ethylcholestanol 24m-Ethylcholest-22-enol Cholest-5-enol (cholesterol) 24a-Methylcholest-5-enol (campesterol) 24b-Methylcholest-5-enol 24/l-Methylcholesta-5,22-dienol (brassicasterol) 24-Methylcholesta-5,24(28)-dienol (24-methylenecholesterol) 2f 24a-Ethylcholest-5-enol (sitosterol) 24n-Ethylcholesta-5,22-dienol (stigmasterol) zp 2h 24-Ethylcholesta-5,24-dienol(24_ethyldesmosterol) 2i 2CEthylcholesta-5,24(28)Z-dienol (isofucosterol) 24-Ethylcholesta-5,24(28)&dienol (fucosterol) 2i 2k 24cc-Ethylcholesta-5,25dienol (24-epiclerosterol) 21 24fi-Ethylcholesta-5,25-dienol (clerosterol) 2m 24b-Ethylcholesta-5.22,25-trienol 2n 24,25-Dimethylcholesta-5,24(28)-dienol 24cz-Ethylcholest-7-enol (schottenol) 3f 24-Ethylcholesta-7,24(28)Z-dienol (avenasterol) 3i 31 24/?-Ethylcholesta-7,25-dienol 4b 24u-Methylcholest-8(14)-enol 4c 248-Methylcholest-8(14)-enol 4f 24a-Ethylcholest-8(14)-enol 24a-Ethylcholest-9( 1 1)-enol Sf 14cr,24a-Dimethylcholest-9( 1 l)-enol 6b 6c 14a,24B-Dimethylcholest-9(1 l)-enol 14a-Methyl-24a-ethylcholest-9(11)-enol 6f 14a-Methyl-24B-ethylcholesta-9(11X25-dienol 61 7e 14a,24-Dimethyl-9~,19-cyclocholest-24(28)-enol (24-methylenepollinastanol) Others, unidentified

seeds’

trS 0.5$

5:2$ 31.6

are Sa-sterols. mixture).

Standard

cholesterol

(2a)

Isolation and identi&ation of sterols. The seeds of Cajanus cajan (L.) Huth (cajan) (A), Cicer arietinum L. (chick pea) (B), Pisum satiuum L. (garden pea) (C) and Vigna mungo L. (black gram) (D) were collected locally in West Bengal (India). Dried and ground seeds [2.5 kg each for (A), (B) and (C); 8.0 kg for (D)] were extracted with MeOH in a Soxhlet extractor for 36 hr. The extracted lipids [(A) 350 g, (B) 370 g, (C) 340 g, (D) 655 g] were refluxed with 5% KOH in EtOH for 3 hr and then extracted with Et,0 which gave unsaponiliable lipids [(A) 3.0 g, (B) 2.3 g, (C) 2.5 g, (D) 11.8 g]. The unsaponifiable lipids were subjected to CC over silica gel. They were eluted with htxane, hexane-Eta0 (9: l), hexane-Et20 (4: l), hexane_EtOAc (6: 1) and then with hexane-EtOAc (4: 1). The frs which eluted with hexane-EtOAc (6: 1) gave the sterol mixture [(A) 1.26 g, (B)

1768

T. AKIHISA et al.

1.15 g,(C) 0.88 g,(D) 3.79 g]. The sterol mixtures were acetylated and the acetates were then subjected to argentation TLC followed by HPLC, in the same way as described prcv~ously [I], which enabled the lsolatlon of all sterols (as the acetates) hsted in Table 1. This procedure did not allow separation between the diastereoisomers at C-24 of 24-alkylsterols (lb/lc. Zb/Zc, 2k/21. 4b/4c, 6b/6c). Identification of individual sterols was performed on the basis of GC and HPLC data. Confirmation of the identification and the examination of the C-24 epimerlc ratio of the diastereolsomerlc 24-alkylsterols Isolated from C. cujan seeds were undertaken by ‘H NMR when available m sufficient amount. Several sterois from the other three Leguminosae seeds were also subjected to ‘H NMR analysis. The sterols which were subJected to ‘H NMR analysis are indicated in Table 1 Compositions of the sterol fractions hsted m Table 1 were determined based on the argentation TLC, GC. HPLC and, in some cases, by ‘H NMR data. Refer to our previous papers [l-4,6,7, 12-15, 17, 211 for the ‘HNMR data.

REFERENCES

1. Akihisa, T., Nishimura, Y., Roy, K., Ghosh, P., Thakur, S. and Tamura, T. (1991) Phytochemistry 30, 4029. 2. Akihisa, T., Kokke, W. C. M. C., Tamura, T. and Matsumoto, T. (1991) Lipids 26, 660. 3. Akihisa, T., Gebreyesus, T., Hayashi, H. and Tamura, T. (1992) Phytochemistry 31, 163. 4. Akihisa, T., Takahashi, S., Sahashi, N.. Kokke, W. C. M. C. and Tamura, T. (1992) Phytochemistry 31 (in press). 5. Ageta, H. and Aral. Y. (1984) Phytochemlstry 23, 2875. 6. Akihisa, T., Tanaka, N., Yokota, T., Tanno, N. and Tamura, T. (1991) Phytochemlstry 30, 2369.

7. Akihisa. T., Ghosh. P., Rosenstem, F. U. and Matsumoto, T. (1986) J. Am. 011 Chem. Sot. 63, 653. 8. Zalkow. L. H., Cabat, ti. A. and Chetty, G. L. (1968) Tetrahedron Letters 5727. 9. Abramson, H. N. and Kim. C. S. (1973) Phytochemistry 12. 351. 10. Fiescr, L F. and Fieser, M (1959) in Srermds, pp. 352, 354. 11. Gupta. M. M. and Shukla, Y. N (1986) Phytochermstry 25, 1423 12. Akihlsa. T., Shimizu, N., Ghosh, P.. Thakur, S., Rosenstein, F. U., Tamura. T. and Matsumoto. T. (1987) Phytochemistry 26, 1693. 13. Akihisa. T., Shimizu. N., Tamura, T. and Matsumoto. T. (1986) Lipids 21, 491. 14. Akihlsa. T., Tamura. T. and Matsumoto. T. (1987) Phytochemistry 26, 2412. 15. Aklhisa, T.. Kanari. M., Tamura, T. and Matsumoto, T. (1989) Phytochemistry 28. 1271. 16. Yano, K., Aklhsa, T., Tamura, T. and Matsumoto, T. (1989) Abst. Papers 28th Annual Meet. Jpn. Ozl Chem. Sot., 97. 17. Akihisa, T.. Tamura, T.. Matsumoto, T., Kokke. W. C. M. C., Ghosh, P. and Thakur. S. (1990) J. Chem. Sot. Perkm Trans I 2213. 18. Farmes. M., Cocallemen, S. and Soulier. J. (1988) Lipids 23, 349. 19. Aklhisa, T.. Sugawara, T., Yokota, T. and Tamura, T. (1992) Phytochemistry (submitted). 20. Nes, W. R. and McKean. M. L. (1977) Biochemistry of Steroids and Other Isopentenoids. University Park Press, MD. 21. Akihisa, T., Matsubara. Y.. Ghosh, P.. Thakur, S., Tamura, T. and Matsumoto, T (1989) Steroids 53. 625.