Acylglucosylsterols from two Aegiphila species

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Acylglucosylsterols from two Aegiphila species Article in Phytochemistry · May 1994 DOI: 10.1016/S0031-9422(00)97032-8

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Pergamoa

0031-9422(93)30134-Z

ACYLGLUCOSYLSTEROLS

Phyrockemmy. Vol 36, No. I, pp. 167-170. 1994 Copyright 0 1594 Fkvier Sckocc Ltd Pruned 10 Great Britain. All ri@s reserved 0031.9422/94 %00+0.00

FROM TWO AEGIPHILA

SPECIES

SUZANA G. LEITAO,*MARIAAUXILIAD~RA C. KAPLANand FRANCODELLEMoNACHEt Nhcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Cidade Universitiria, 21941-590 Rio de Janeiro, Brazil; tCentro Chimica dei Recettori de1 C.N.R., Universid Cattolica, Largo F. Vito 1, 00168, Roma, Italy (Received 29 July 1993)

Key Word Index--Aegiphila

Ihotzkyanu; A. obducta; Verbenaceae; acylglucosylsterols.

Abstract-From bark of Aegiphila lhotzkyana and A. obducta were isolated 3-O-[6’-0-n-acyl-fi-D-glucosyl]-22dehydroclerosterol; 3-O-[6’-O-n-acyl+D-glucosyl]-c~erosterol and 3-o-[6’-o-n-acyl-/?-D-ghXosyl]-stigmasterol derivatives. From leaves of A. obducta were isolated 3-0-[6’-O-n-acyl-~-~glucosyl]-22-dehydroclerosterol derivatives.

INTRODUCTION

In a previous study on the chloroform extracts of Aegiphila lhotzkyana and A. obducta we have reported the isolation and identification of sterol and sterol-glucosides from the barks and leaves [l]. Continued studies on these extracts led to the isolation, from the bark of both species, of a mixture of the acylglucosylsterols l-3. Fractionation of the chloroform extract from the leaves of A. obducta, however, led to the isolation of the acylglucosylsterol (AGS) mixture 1 (la-k), where 22-dehydroclerosterol was the sole steryl moiety. The identification of the components of the AGS mixture l-3 could be established directly within the crude mixtures by comparison with the ‘H and i3C NMR data of the corresponding glucosides (4-6), previously isolated from the same plants [l]. RESULTSAND DISCUSSION Fractionation of the chloroform extract from the leaves of A. obducta led to the isolation of the AGS mixture 1 (la-k). The ‘HNMR spectrum of 1 showed signals attributable to a steryl moiety, consistent with those of 22dehydroclerostero1, found in the leaves of the same plant [l]. Signals for a sugar moiety were also present. The signal of the anomeric proton at 64.97 (d, J =7.26 Hz) suggested the presence of glucose (@configuration) which was confirmed by “C NMR. Additionally, it showed characteristic signals for an unsaturated long chain fatty acid at 60.88 (brt, o-Me), 1.26 (brs, nCH,-), 2.91 (t, J =4.5 Hz, -CH,-C=O) and 5.49 (m, -CH =CH-). The above spectral data suggested that 1 might be an acylglucosyl sterol. In fact, 13CNMR signals at 6173.5 (CO,-), 34.5 (-CH,-CO-), 30.0 (n-CH,-), 14.3

*Author to whom correspondence

should be addressed.

(-CH,-Me), 127.5-128.6 and 130.5-132.1 (-CH=CH-) confirmed the presence of an unsaturated fatty acid ester moiety in the molecule. Comparison of 13CNMR data of 1 with those of 3-0-[/?-D-glucosyl]-22-dehydroclerosterol (4) Fable l] indicated the site of linkage of the n-acyl group as being C-6’ of glucose. The glucose resonance values of 1 remained practically unchanged, except from that of C-6’ which was shifted ca 1.90 ppm downfield due to acylation. These data indicated that 1 was 3-O-[6’-O-n-acyl-/?-Dglucosyl]-22dehydroclerosterol. Fractionation of the chloroform extract from the barks of both A. lhotzkyana and A. obducta led to the isolation of the AGS mixture l-3 as a chromatographically unitary fraction by TLC. Their ‘H and 13C NMR spectra suggested that the two mixtures consisted of 6’-0-n-acyl derivatives of 22dehydroclerosterol gtucoside (4); clerosterol glucoside (5); and stigmasterol glucoside (6); previously isolated from the same plant sources [l]. In the ‘H NMR spectrum of 1-3, aglycone proton signals of the known sterols were readily identified at 65.08 (dd, J= 15.2 and 8.4 HI H-23, 3); 5.23 (dd, .I= 15.2 and 8.8 Hz, H-22, 3); 4.83 and 4.87 (m, Hz-26, 2); 5.29 and 5.34 (m. H-23 and H22, 1); 4.89 (m, H,-26, 1) and 5.40 (m, H-6, l-3), by comparison with data from the literature [2] and with those of 4-6 [l]. Also present were signals assignable to an unsaturated n-acyl moiety at 6 1.25 (br s, n-CH,-), 2.34 (t, J=7.5 Hz, z-CH,-) and 5.50 (m, -CH=CH-). The anomeric proton appeared as a doublet at 65.02 (d, J =7.7 Hz) indicative of a /?-glucosidic linkage. Comparison of i3C NMR data of l-3 with those of 4-6 (Table l), confirmed the proposed structures. In the “CNMR spectrum of l-3, besides the glucosylsterol signals, there were signals for an unsaturated long chain fatty acid ester-linked moiety at 6 173.7; 130.4; 130.2; 128.4; 34.6; 29.7-30.0; 27.5; 25.4; 23.0 and 14.3. Additionally, 13C resonance values of l-3 remained practically unchanged when compared with those of 4-6, except for C-2 and C-6’ values. The downfield shift (1.90 ppm) experimented by 167

s. G. LEITAOet cd.

168

\ dP v OR

HO

s’

6’

I’

’

4

0

o$

1’

R = n-rcyl (Table 2)

. . .&

. . .&

. . .A

2

1

3 -?

HO

HO

3

P

I’

0

\ /dP

OH0 2

C-6’ for the acylglucosyl derivatives was due to acylation of this carbon in 1-3. Finally, the “C chemical shifts of 2 were in good agreement with those of 3-0-[6’-O-stearyl/3-D-glucosyt]-clerosterol and 3-O-[6’-O-patmitoyl-/l-Dglucosyl]-clerosterol, isolated from the green fruits of Momordica charantia [3]. Saponification of 1, isolated from the leaves of A. obducta, and of 1-3, isolated from the barks of both A. obducta and A. Ihotzkyana, afforded the glucosylsterol 4 (A. obducta, leaves) and the mixture of glucosylsterols 4-6 (A. obducta and A. Ihotzkyanu, barks), respectively. The identity of 4 and of 4-6 was confirmed by ‘H NMR and co-TLC with authentic samples of the glucosylsterols previously isolated from the same plant sources. The corresponding fatty acid methyl esters were analysed by GLC and the results are summarized in Table 2. Also, 13C NMR values of fatty acid methyl esters from 1 were assigned by comparison with reported data for linoleic acid [4]. It is not a surprise that the main fatty acids esterifying the glucosylsterols are palmitic and stearic acids, since they are commonly found in nature. It is noteworthy, however, that there were odd number carbon fatty acids e.g. C,,, C,, and C,, esterifying 22dehydroclerosterol glucosides (Id, Ig and li) in leaves and

barks of A. obducta. Acylglucosylsterols have been isolated from higher plants (Cucurbitaceae, Araceae, Liliaceae, Musaceae, Thymelaceae, Solanaceae, etc.) [3,5-93, as well as from snake [IO] and chicken epidermis [ 111. As far as we know, this is the first report of the occurrence of la-k, 24-i and 3a-k.

EXPERIMENTAL

General. ‘H and 13CNMR were recorded at 300 and 75 MHz with TMS as int. standard. GLC: DBWAX-50, column temp. 70-200” (4” mitt- ‘), injector temp. 220”, carrier gas: He, detector HP 5988A/MS, gas chromatograph HP 5890. Analytical TLC: silica gel developed in CHCI,-MeOH, 19: 1 and spots visualized with HzOH,SO, -formal (1: 2: 1) spray and heating. Plant collection and extraction. As described in ref. [ 11. A new extract was made with 2.2 kg of leaves of A. obducta, following the same method as ref. [l]. Isolation uf 1 from the leaves of A. obducta. Part (26.0 g) of the CHCI, extract from the leaves of A. obducta was chromatographed on silica gel (CHCI, with increasing amounts of MeOH). Frs containing 1 (2.4 g eluted

Acylglucosylsterols

169

from two Aegiphilo species

Table 1. 13C NMR data of acylglucosyisterols l-3 and of glucosylsterols c6 isolated the bark of A. lhotrkyana and A. obducta, and from the leaves of A. obducta

c 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 1’ 2 3’ 4 5 6

1 37.7 30.4 75.2 39.4 141.0 121.9 32.2 32.2 50.5 37.0 21.4 39.9 42.4 24.6 t 36.1 12.2 19.5 40.6 21.1 137.6 130.7 52.3 148.6 110.2 20.4 26.1 12.4 102.9 15.2 78.6 71.7 78.4 64.7

2 37.7 30.4 75.2 39.4 141.0 121.9 32.2+ 32.2* 50.5 37.0 21.4 39.9 42.5 57.0156.9 24.6 t 56.4 12.0 19.5 35.8 18.9 34.1 t 49.8 147.7 112.0 17.9 26.8 12.3 102.9 75.2 78.6 71.7 78.4 64.7

3

4

5

6

37.7 30.3 75.2 39.4 140.0 121.9 32.2 32.2 50.5 37.0 21.4 39.9 42.4

37.5 39.3 78.1. 39.3 140.9 121.9 32.2 32.1 50.4 37.0 21.3 39.9 42.4 56.9 24.5 29.1 56.0 12.2 19.4 40.5 21.0 137.6 130.3 52.3 148.6 110.2 20.3 26.1 12.4 102.6 75.3 78.6 71.7 78.4 62.8

37.5 30.3 78.1* 39.3 140.9 121.9 32.2 32.1 50.4 37.0 21.3 40.0 42.5 57.0 24.5 28.5 56.3 12.0 19.4 35.8 18.9 34.0 29.7 49.8 147.7 12.0 17.9 26.8 12.3 102.6 75.3 78.6 71.7 78.4 62.8

37.5 30.3 78.1. 39.3 140.9 121.9 32.2 32.1 50.4 37.0 21.3 39.9 42.4 56.9 24.6 29.3 56.1 12.2 19.4 40.8 21.5 138.8 129.5 51.4 32.1 19.2 21.3 25.7 12.6 102.6 75.3 78.6. 71.7 78.4’ 62.8

24.6 t 56.2 12.2 19.5 40.8 21.5 138.9 129.5 51.5 32.2 21.4 19.2 25.7 12.6 102.9 75.2 78.6 71.7 78.4 64.7

n-Acyl moiety n-acyl 1-31

n-acyl

173.7 130.4 130.2 128.4 34.6 28.7/30.0 27.5 25.4 23.0 14.3

173.5 132.1 130.5 128.6 128.4 128.1 127.5 34.5 29.9-30.0 27.5 25.4 22.9 14.3

*Interchangeable values. toverlapped signal. SAegiphila obducta and A. lhoirkyana §Aegiphila obducta leaves.

PHY-rO36:1-L

bark.

15

from

S. G. LEITAOet al.

170

Table 2. Acyl groups of fatty acid methyl esters from saponification of AGS from A. obducta and A. lhotzkyana (per cent) R

1’

a palmitoyl (16:O) b stearyl (18:O) c miristyl (14:O) d margaryl (17:O) e oleyl (18: I) f linoleyl (18:2) g pentadecanoyl (15:O) II arachicoyl (20: 0) i nonadecanoyl(19:O) j palmitoleyl (16: I) k lauril (I2 : 0)

53.46 31.09 3.26 3.08 I .66 0.68 0.62 0.72 0.33 0.73 0.55

l-3t

1-3:

71.12 17.56 I .02 4.77 0.17 0.15 0.54 0.88 0.55 -

83.39 14.40 0.17 1.92 -. -_

*Aegiphila obducta leaves. tAegiphifa obducta bark. :Aegiphila Ihotzkyuna bark.

with CHCI,-MeOH, 19:l) were filtered twice on Sephadex LH-20 (CHCI,-MeOH, 9:l) to separate the chlorophyll. Final purification was achieved by silica gel CC (C,H, -EtOAc, I : 1) affording 173 mg of 1 as a greasy material. Isolation of 1-3 from the barks of A. obducta and A. Ihotzkyana. The CHCI, extracts from the bark of A. obducta (5.0 g) and A. Ihotzkyana (6.0 g) were chromatographed separately by silica gel CC (CHCI, with increasing amounts of MeOH). Frs containing 1-3 (eluted with CHCI,--MeOH, l9:1, 670mg A. obducta and 1.13 g A. Ihotzkyana) were purified by repeated CC (CHCI, -MeOH, 19: I and further C,H,-EtOAc, 1: I), affording 90 and I04 mg of 1-3, respectively, as greasy materials. Chemical modifications. Saponification of AGS (I 10 mg: A. obducta leaves, 30 mg 4. obducta barks, and 18 mg A. lhotzkyana barks) was carried out separately by treatment with CHCI,-MeOHM NaOH (2:7: 1) for I hr with reflux as described in ref. [3], affording 17 mg (A. ohducta, leaves), I1 mg and 5 mg (A. obducta and A. lhotzkyana bark, respectively) of fatty acid mixts. The resulting fatty acids were methylated with CH,N, -Et,0 and analysed by GLC. 3-0-[6’-O-n-acyl-~-~-glucosyl]-22-dehydroclerosterol (1). n-Acyl groups

(per cent): Table 2. ‘H NMR (pyridine-

d,, TMS): 60.69 (s, Me-18); 0.88 (br t, w-Me, n-acyl); 0.90 (m, Me-29); 0.96 (s, Me-19);

1.07 (d, J=6.3 Hz, Me-21); 1.26 (hrs, n-CH,-, n-acyl); 1.73 (s, Me-27); 2.91 (t, J =4.5 Hz, a-CH,-): 3.92 (m, H-3); 4.87 and 4.85 (m, H,-26); 4.97 (d, J = 7.3 Hz, H-l’); 5.28 (m, H-23); 5.33 (m, H-22); 5.37 (m, H-6) and 5.49 (m, -CH=CH , n-acyl). ‘jC NMR: Table I.

3-o-[6’-o-n-aCy[-j%D-ghKosy&stero/ mixture (l-3). nAcyl groups (per cent): Table 2. ‘H NMR (pyridine-d,, TMS): 60.88 (br t, w-Me, n-acyl); 1.25(br s, n-CH,, n-acyl); 2.34 (t, J=7.5 Hz, z-CH,-, n-acyl); 4.28 (t, J=8.6 Hz, H2’ x 3); 4.83 and 4.87 (m, H,-26,2); 4.89 (m, H,-26, 1); 5.02 (d, J=7.7 Hz, H-l’x 3); 5.08 (dd, J= 15.2 and 8.4 Hz, H23.3); 5.23 (dd,J= 15.2 and 8.4 Hz’, H-22,3); 5.29(m, H-23, 1); 5.34 (m, H-22, 1); 5.40 (m, H-6 x 3) and 5.50 (m, -CH =CH-, n-acyl). 13CNMR: Table I. Methyl esters ohtained from saponification of 1. ‘H NMR (pyridine-d,, TMS): 60.88 (t, J= 7.1 Hz, *Me); 1.26 (brs, n-CH,-); 1.61 (m); 2.05 (m, allylic H); 2.30 (t. J =7.5 Hz, r-CH,-); 2.80 (m): 5.40 (m, -CH=CH-). 13C NMR (pyridine-d,, TMS): i554.l (o-Me); 22.7 (-CH,- Me); 25.0 (fi-CH, -); 25.6 (--CH=CH-CH,CH=CH-); 27.2 ( -CH=CH- CH, --); 29.2-29.7 (nCH,--); 31.9 (-CH,-CH,-Me); 34.1 (a-CH,-); 51.4 (OMe); 127.7, 127.1, 128.3, 128.3, 130.3, 132.0 (-CH =CH--); 174.3 (-CO,-Me). Acknowledgements-We are indebted to Dr Franc0 Chialva from Centro Studi Maria Branca, Milano, Italy, for GLC analysis and to Dr Amtlia C. Kassis and Mr Jorge Luis da Silva from Universidade Federal de Ouro Preto for helping plant collection and identification. One of us (S.G.L.) is indebted to Conselho National de Desenvolvimento Cientifico e Tecnol6gico (CNPq, Brazil) for a fellowship. REFERENCES

I. LeitBo, S. G., Kaplan. M. A. C., Delle Monache, Akihisa, T. and Tamura, T. (1992) Phytochemistry

F., 31,

2813.

2. Akihisa, T., Tamura, W. C. M. C., Ghosh. Chem. Sot. Perkin

T., Matsumoto, P. and Thakur,

T., Kokke, S. (1990) J.

Trans I 2213.

3. Guevara, A. P., Lim-Sylianco, C. Y., Dayrit, F. M. and Finch. P. (I 989) Phytochemistry 28, 172 I. 4. Bus, J., Sies, I. R. and Liekenjie, M. S. F. (1976) Chem. Phys. Lipids 17, 50 I. 5. Della Greta, M., Molinaro, A.. Monaco, P. and Previtera, L. (1991) Phytnchemistry 30, 2422. 6. Tandon, M., Shukla, Y. N. and Thakur, R. S. (1990) Phytochemistry

29, 2957.

7. Ghosal, S. (1985) Phyrochemistry 24, 1807. 8. Hashimoto, T., Tori, M. and Asakawa, Y. (1991) Phytochemistry

30, 2927.

9. Bhattacharya, S. K., Gael, R. K., K&r, R. and Ghosal, S. (1987) Phytothempy Res. 1, 32. 10. Abraham, W., Wertz. P. W., Burken, R. R. and Downing, D. T. (1987) J. Lipid. Res. 28, 446. 11. Wertz, P. W., Stover, P. M., Abraham, W. and Downing, D. T. (1986) J. Lipid. Res. 27, 427.