Vol.31,No. 6, pp. 2079-2086,1992 Printedin Great Britain.

Phytochemistry,

TRITERPENOID

003l-9422/92$5.00+ 0.00 6 1992PergamonPressLtd

SAPONINS FROM ARGANIA

SPINOSA

Z. CHARROUF,J. M. WIERUSZESKI,*S. FKIH-TETOUANI, Y. LEROY,* M. CHARROUF,B. FOURNET* de Chimie des Plantes, FacuIt6 des Sciences, B.P. 1014 Rabat RP, Morocco; *Laboratoire de Chimie Biologique et Unit& Mixte de Recherche du CNRS N”111, Universit6 des Sciences et Techniques de Lille Flandres-Artois, 59655 VilIeneuve d’Ascq-cedex,France

Laboratoire

(Receivedin revised form 29 July 1991) Key Word Index--Argania spitwsa; Sapotaceae; argan, kernel, triterpenoid saponin; oleanane saponin; arganine A, B, C, D, E, F, and mi-saponin A.

Abstract-Five new oleanane saponins named arganine A, B, D, E and F and two known saponins: arganine C and mi-saponin A were isolated from the kernel of Argania spinosu. The structures of these saponins were elucidated by using ‘H NMR, ‘H-‘H COSY NMR, ’ jC NMR, FAB mass spectrometry and chemical evidence.

INTRODUCTION Argania spinosa is an endemic tree of Morocco. It grows on the west side of the high Atlas mountains. The fruit of A. spinosa has an oleaginous kernel from which a well known edible oil [l-4], is used in folk medicine and cosmetics. In this paper, we describe the analyses of five new triterpenoid saponins and two other known compounds.

RESULTSANDDISCUSSION The crude saponins were isolated from the ethanolic extract of defatted A. spinosa kernet HPLC on an ODS column of the crude saponins results in seven pure compounds 1 to 7 (Fig. 1). The yields, and the carbohydrate compositions of these compounds are given in Table 1. All the saponins yielded neutral monosaccharides identified by GC after methanolysis; their absolute configurations were determined by GC of their trimethylsilylated (-)-Zbutylglycoside. D-ghCOSe, L-rhamnose, D-xylose and L-arabinose were the only sugars detected in the saponins 1,3,4 and 6. In saponins 2,5 and 7 we identified an additional D-apiOSe residue by GC-EI-mass spectrometry and GC-CI-mass spectrometry of the pertrimethylsilylated methylglycoside form (characteristic ions in the EI mass spectrum at m/z 275 and 103 and CI mass spectrum at m/z-398[M+ 18]+; 381 [M+H]+; 366 [M -32+18]+; 349 [M-32+I-I-J+). The alkaline hydrolysis under reductive conditions of crude saponins allowed the isolation of oligosaccharidicalditol fractions containing arabitol. We can conclude that in all the saponins the arabinose residue was linked by an ester linkage to the aglycone. The FAB-mass spectrum of arganine A (1) showed an [M +Na]+ ion at m/z 1423 and an [M-H]ion at m/z 1399 indicating the M, to be 1400. The FAB-mass spectrum in the negative mode showed two major peaks at m/z 843 and 483. These two peaks indicated the presence of a tetrasaccharide attached by an ester glycosidic linkage to the aglycone [S]. The first one corresponded to the [prosaponin -H] - ion and the second to 2 x 6deoxyhexose (d) + 2 x pentose (p) - 90 - H [2d + 2p - 90

bH CIC

6H bH

t)H 6H

API

Rha

Name

ArganineA Arganinc B Arganine C Arganine D Arganinc.E MisaponineA Arganine F

R3 Rha Api Rha Rha Api Rha APi

-HI- ion [S]. Significant peaks at m/z 1253,1237, 1121, 975 corresponded, respectively, to the loss of d, hexose (h), d+p, 2d+p (Table 2). This result, together with the

2019

2080

et al.

2. CHARROUF

1

r----, ,' 3' /' I' I'

___________~

r__________s~ ,' 1' , ,' l' 8' , ,'

I”

Y”

LY

-?”

Time (mh-d

Fig. 1. Analysis of crude saponins by reversed phase chromatography

on a Zorbax ODS column.

Table 1. Carbohydrate compositions and weights of fractions obtained by reversed phase chromatography of crude saponins (500 p(p)

Fraction

Name

Glc

Ara

1 2 3 4 5 6 7

Arganine A Arganine B Arganine C Arganine D Arganine E Mi-saponin A Arganine F

2.2 2.1 1.0 2.1 2.0 1.0 1.1

1.0 1.0 0.8 1.0 1.1 0.8 0.9

Molar ratio* Xyl Rha 2.2 1.1 1.6 2.2 1.2 1.7 1.1

1.0 1.0 1.0 1.0 1.0 1.0 1.0

Apit

Weight (pg)

0.4 0.4 0.4

195 40 16 21 17 19 16

*The molar ratio of xylose is taken as 1. tThe molar response is taken as 1. Table 2. FARMS ions profile of fractions 1 to 7 Ions [M-H][M-h-H][M - 2h - HI[M-d-H][M-p-H][M-2p-HI[M-(d+p)-H][M-(2d+2p)-HI[M - (2h + d) - H] CM - (2p + 2d + h) - HI[M-(2p+2d+H,O)-HICM-(d+3p)-HI[Aglycone - HI[Aglycone - H,O - HI[2d + 2p - HI[3p + d - 90 - H-J[2d+2p-90-H][2d + p - HICM-(2d+p)-HI[M-(3p+h+d)-HI-

1

2

3

4

5

6

7

1385 1237 1383 1369 1221 1207 1221 1207 1059 1045 1223 1059 1045 -~ ~1061 1075 1091 1237 -~ 1237 1075 1253 -1105 943 1121 943 959 1105 --~ 827 665 681 913 665 -647 665 827 843 503 503 503 519 519 503 ~-501 555 555 469 469 469 483 483 483 483 423 423 -_ _ _ .__ 975 __ _ 681-

1399 1237 ~ 1253 1121 843 681 519

Triterpenoid saponins from Argania spinosa

molar carbohydrate composition (Table l), indicated the presence of six neutral monosaccharides and gave information about the sequence of the tetrasaccharide linked by an ester linkage to the aglycone. Because D-XylOSe and L-arabinose are pentoses, their sequence cannot be determined by FAB mass spectrometry. Thus compound 1 was subjected to alkaline hydrolysis with NaOH-KBH, to yield the tetrasaccharidealditol. The carbohydrate composition of this was established as rhamnose, xylose and arabitol in the molar ratio 2: 1: 1. Its methylation analysis showed a 2-linked arabitol, a 3-linked xylopyranose, a 4-linked rhamnopyranose and a terminal rhamnopyranose. The prosaponin of arganine A was obtained by K&O, hydrolysis and subjected to methanolysis and methylation analysis to give only terminal glucopyranose and 6-linked glucopyranose. Permethylation of 1 confirmed the monosaccharide linkage. The ‘H NMR spectrum (Fig. 2 and Table 3), revealed six tertiary methyl signals at 60.88, 0.98, 1.06, 1.30, 1.34 and 1.60, two secondary methyls at 6 1.24 [d, J = 6.2 Hz, H-6 Rha terminal position (t)], 1.30 [d, J=6.2 Hz, H-6 Rha inner position (i)]; four hydroxymethine protons at 63.56 (d, J=3.7 Hz, H-3), 4.33 (ddd, J=3.3, 3.5; 3.7 Hz, H-2) and 4.47 (m, H-6 and H-16); one allylic proton for H-18 (a typical shift in the oleanane series) at 63.09 (dd, J = 14.5, 4.0 Hz) [6]; one olefinic proton signal at 65.42 (like t) and six anomeric proton signals at 65.61 (d, J =3.9Hz,H-lAra), 5.14(d,J=1.6 Hz,H-1 Rhat), 507(d, J= 1.0 Hz, H-l Rha i), 4.55 (d, J=7.7 Hz, H-l Xyl), 4.45 (d, J = 7.6 Hz, H-l Glc t) and 4.31 (d, J = 7.7 Hz, H-l Glc i). The ‘H-‘H NMR spectrum was useful to differentiate the hydroxymethine protons of sugars and those of triter.

2081

penoids. The 13CNMR spectrum (Table 4) showed one ester carbon signal at 6 177.1; two trisubstituted olefinic carbons at fi 124.2 (C-12) and 144.0 (C-13); six anomeric carbons at 6 106.5 (C-l Xyl), 104.5 (C-l-Glc i), 104.3 (C-l Rha t), 101.4 (C-l Rha i) and 94.2 (C-l Ara); five CH,O groups at 665.6,69.9,62.8,64.1 and 76.3 were revealed by 13CDEPT NMR. These spectral data suggested that arganin A was a bisdesmosidic saponin of an oleanane triterpene carboxylic acid type. By comparison of 13CNMR data of this type of triterpenoid glycoside reported in the literature [7-91, we allocated the hydroxyl groups to positions C-2, C-6, C-16 and C-23, the disaccharide Glc-Glc to C-3 by O-linkage, and the tetrasaccharide to C-28 by an ester linkage. The configurations of the C-2, C-3, C-6 and C-16 hydroxyl groups were determined to be fl,fl,b and W,respectively, by the J values of the protons. The a or /I configuration of the sugars were determined by JH1_H values of the anomeric protons. The last problem to ;be solved on the structure of arganine A was the configuration of the ester linkage of the arabinopyranosyl residue. The JHI_H2 value (3.9 Hz), of this group suggests at first that the arabinopyranosyl group in the 4C,-conformation was linked in the flconfiguration, but several cases have been reported on the occurrence of the predominant ‘C,-conformation for glycosylated a-L-arabinopyranosyl residue which is linked to the sterically hindered carboxylic acid group of a triterpene carboxylic acid [lo]. Therefore, the esterlinked arabinopyranosyl residue in this case was in the %,-conformation and the J value supports the a-config uration. On the basis of ‘H, ‘H-‘H and “CNMR spectroscopy, FAB-mass spectrometry, chemical composition and methylation analysis, the structure of arganine A was

5.10

I.‘~,”

I.20

s.00

1.w

4.20

I.00

‘%”

=.#a

1.40

1.10

i.00

2.20

2.00

Fig. 2. Comparison of ‘HNMR spectra of saponins 1 to 7. PHYTO

31:6-Q -

L.10

I&

1.40

L.10

L.00

1

Rhai

1

1

XYl

Api

19 Me

6 12 16 18

Aglycone 2 3

1

5 Me

1

Ara

Rhat

1

Glc t

5 Me

1

H

4.33 3.56 d, J = 3.7 4.47 m 5.42 4.47 3.09 dd, J = 14.5; 4.0 2.28 0.88; 0.98; 1.06; 1.30; 1.34; 1.60

4.31 d, J = 1.1 4.45 d, J = 1.6 5.07 d,J=l.O 3.16 1.30 d, J = 6.1 5.14 d, J = 1.6 3.99 m 1.24 d, J = 6.2 5.61 d, J = 3.9 4.55 d, J = 1.1

1

5.61 d, J = 3.9 4.55 d, J = 1.1 5.25 d. J = 2.9 4.33 3.56 d, J = 3.1 4.41 m 5.42 4.41 3.09 dd, J = 14.5; 4.0 2.28 0.88; 0.98; 1.06; 1.30; 1.34; 1.m;

4.31 d, J = 1.1 4.45 d, J = 1.6 5.07 d, J = 1.0 3.16 m 1.30 d, J = 6.1

2

= 7.8

= 3.9

= 6.2

= 1.7 m

= 6.2

= 1.4

= 1.6

4.34 m 3.58 d, J = 3.1 4.48 m 5.42 4.48 m 3.09 dd, J = 14.5; 4.0 2.28 0.88; 0.97; 1.05; 1.31; 1.34; 1.63

4.44 d, J 5.01 d, J 3.76 1.30 d, J 5.14 d, J 3.99 1.24 d, J 5.61 d, J 4.54 d, J

3

and oleanane tetramethylsilane

monosaccharides

relative to internal

3. ‘H NMR chemical shifts of constituent

Glci

Table

aglycones

= 1.1

= 3.9

= 6.2

= 1.6 m

= 6.2

= 1.0 m

= 1.1

= 1.1

2.94 dd, J = 14.3; 3.2 1.73 0.91; 0.95; 1.06; 1.14; 1.30; 1.59

4.33 m 3.56 d, J = 3.1 4.46 m 5.35 _

4.32 d, J 4.44 d, J 5.09 d, J 3.74 1.28 d, J 5.14 d, J 3.99 1.24 d, J 5.65 d, J 4.52 d, J -

4

= 3.9 m

= 2.9 m

= 1.5

= 3.9

= 6.2

= 1.1 m

= 1.6

= 1.1

2.94 dd, J = 14.0; 3.0 1.73 0.91; 0.95; 1.06, 1.14; 1.30; 1.59

5.65 d, J 4.53 d, J 5.25 d, J 4.33 3.56 d, J 4.46 5.35 -

4.32 d, J 4.44 d, J 5.05 d, J 3.76 1.28 d, J

5

= 7.5

= 3.8

= 6.2

= 1.7

= 6.2

= 1.0 m

= 7.6

2.94 dd, J = 14.0; 3.0 1.73 0.91; 0.95; 1.06; 1.14; 1.31; 1.62

4.3? m 3.57 d, J = 3.9 4.46 m 5.35

4.44 d, J 5.10 d, J 3.76 1.28 d, J 5.14 d, J 3.99 1.24 d, J 5.64 d, J 4.52 d, J

6

isolated from argama

at 25”)

for saponins

(6 = 0.0 ppm in C2H,02H

triterpenoid

= 4.0 m

= 3.0 m

= 7.5

= 4.0

= 6.2

= 1.2 m

= 7.6

shifts are

2.94 dd, J = 13.0; 3.0 1.73 0.9 1; 0.95; 1.06; 1.14; 1.31; 1.62

5.64 d, J 4.53 d, J 5.25 d, J 4.32 3.57 d, J 4.41 5.35 -.

4.43 d, J 5.11 d, J 3.16 1.28 d, J

7

fruit; chemical

Triterpenoid saponins from Argania spinosa Table 4. “CNMR chemical shifts of the aglycone moiety of compounds 1 and 2 (a in ppm) C

1

2

C

1

2

1 2 3 4 5 6 I 8 9 10 11 12 13 14 15

41.1 69.9 83.9 43.5 49.5 71.6 41.4 40.0 49.1 37.2 24.7 124.2 144.0 44.0 36.4

41.1 69.9 83.9 43.5 49.1 71.6 41.4 40.0 49.7 31.2 24.7 124.2 144.0 44.0 36.4

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

14.1 50.5 42.3 46.5 31.4 36.5 31.9 65.5 16.4 18.1 19.2 25.2 111.1 33.4 25.2

14.1 50.5 42.3 46.5 31.4 36.4 31.8 65.5 16.4 18.1 19.2 25.2 177.1 33.4 25.2

established as 3-0-fl-D-glucopyranosyl-(1+6)-fl-D-glucopyranosyl-2B,3B,68,16a,23-pentahydroxyolean-12-en-28oic acid 28-0-a-L-rhamnopyranosyl-(1+3)-j-D-XylOpyranosyl-( 1-r4)-a-L-rhamnopyranosyl-( l-+2)-a+arabinopyranoside. The FAB-mass spectrum of arganine B (2) was helpful in the determination of its M,. The positive FAB-mass spectrum showed a pseudomolecular ion at m/z 1409 assigned to [M + 23]+ and a peak at m/z 867 corresponding to [prosaponin+23]+. In the negative FAB-mass spectrum (Fig. 3) three intense peaks appeared at m/z 1385, 843 and 469 assigned to [M-H]-, [M-(3p+d) -HI-, [3p+d-90-H-[S] and significant peaks at 1253,1223,1121,1061,681,559,519 ascribed respectively to [M-p-I-I-J-, [M-h-H]-, [M-2p-HI-, [M -2h-HI-, [M-(3p+d+h)-HIand [aglycone -H] -. Compound 2 was subjected to alkaline hydrolysis with NaOH-KBH, to yield the tetrasaccharide-aldito1 which gave the same results as arganine A except for a terminal L-rhamnopyranose which was replaced by Dapiofuranose. The ‘HNMR spectrum of 2, (Fig. 2 and Table 3), exhibited signals assignable to six anomeric protons at 65.61 (d, .I= 3.9 Hz, H-l Ara), 5.25 (d, J=2.9 Hz, H-1 Api), 5.07 (d, J= 1.2 HZ, H-l Rha), 4.55 (d, J = 7.6 Hz, H-l Xyl), 4.45 (d, J = 7.6 Hz, H-l Glc t) and 4.31 (d, J = 7.7 Hz, H-l Glc i). There were also six tertiary methyls at 80.88, 0.98, 1.05, 1.30, 1.34 and 1.60, one secondary methyl at 6 1.30 (d, J= 6.2 Hz, H-6 Rha), one olefinic proton at 6 5.42 (t-like, H-12) and one allylic proton at 63.09 (dd, J = 14.5; 4 Hz, H-18). The proton signals due to the sugar moiety displayed characteristic doublets of apiose at 65.27 (d, J=2.9 Hz, H-l Api), 3.99 (d, J= 2.9 Hz, H-2 Api), 4.12 (d, J=9.6Hz, H-5a Api), 3.78 (d, J=9.6Hz, H-5b Api) and 3.60 (2H, d, J= 1.1 Hz, H-4 Api). The 13C NMR spectrum (Table 4) showed signals corresponding to one ester carbon at 6 177.1, two trisubstituted olefinic carbons at S 144.0 (C-13) and 124.2 (C-12), six anomeric carbons at 694.2 (C-l Ara), 106.5 (C-l Xyl), 111.3 (C-l Api), 101.4 (C-l Rha), 104.5 (C-l Glc i) and 104.3 (C-l Glc t). Seven CH,O groups exhibited signals at 662.8; 64.1; 65.2; 65.5; 66.9; 69.9; 75.1.

2083

On the basis of ‘H, ‘H-‘H and 13CNMR spectroscopy, FAB-mass spectrometry, chemical composition and methylation analyses, the structure of arganine B was established as 3-O-~-D-~U~pyraUOSyl-(1+6)-/?D-glucopyranosyl-28,38,68,16a,23-pentahydroxyolean12-en-28-oic acid 28-O-P-D-apiofuranosyl( 1+3)-/?-D-xylopyranosyl-( 1+4)-a-L-rhamnopyranosyl-( l-+2)-a-L-arabinopyranoside. The molar carbohydrate composition of arganine C (3) (Table 1) and the negative ion FAB-mass spectrum (Table 2) with the major ions at m/z 1237 [M-H]-, 681 [M-(2p+2d)-HIand 483 [2d+2p-90-H]indicated that 3 is a bisdesmosidic saponin with a tetrasaccharide constituted by two rhamnoses, one arabinose and one xylose linked by ester linkage and a glucosyl moiety attached by O-linkage. The sequence of the tetrasaccharide was determined by peaks at m/z 1091 [M-d -HI-, 959 [M-(d+p)-HIin the negative ion FABmass spectrum and the identification of arabitol by reductive alkaline hydrolysis. Methylation analysis yields 2,3,4,6-tetra-O-methylglucopyranose, 2,3,4-tri-o-methylrhamnopyranose, 2,3-d&O-methylrhamnopyranose, 2,4di-0-methylxylopyranose in the same molar ratio confirming the presence of glucopyranose linked to the aglycone and a linear tetrasaccharide moiety with a rhamnopyranose at the non-reducing terminal position. The ‘H NMR spectrum of 3, (Fig, 2), showed signals of six tertiary methyl groups at 6 0.88,0.97, 1.05, 1.31, 1.34 and 1.63 and two secondary methyl groups at 6 1.30 and 1.24. It also showed five anomeric proton signals at 64.44 (d, 3=7.6 Hz, H-l Glc), 4.54 (d, 3=7.8 Hz, H-l Xyl), 5.07 (d, J= 1.4 Hz, H-l Rha i), 5.14 (d, J= 1.7 Hz, H-l Rha t) and 5.61 (d, J = 3.9 Hz, H-l Ara). Detailed comparison of the ‘H (Fig. 2) and ‘H-‘H COSY NMR spectrum of 3 with those of 1 showed that 3 lacked the signals due to the inner glucosyl residue and confirmed the presence of the same aglycone. On the basis of ‘HNMR, FAB-mass spectrometry and chemical composition, the structure of arganine C was established as 3-O-/?-D-glucopyranosyl-2fi,3/?,68,16a, 23-pentahydroxyolean-12-en-28-oic acid 28-O-a-L-rhamnopyranosyl-(l 4 3)-fi-D-xylopyranosyl-(l+4)-a+rhamnopyranosyl-(l+2)-a-L-arabinopyranoside. This saponin has been isolated previously from Crossopteryx jih+fiqa [ 111. Arganine D (4) comprised an aglycone and six monosaccharides as confirmed by GC molar carbohydrate composition (Table 1) methylation analysis (2,3,4,6-tetra0-methylglucose, 2,3,4-tri-O-methylrhamnose 2,3-di-Omethylrhamnose, 2,4-di-0-methylxylose and 3,4-di-Omethylarabinose in the same molar ratio) and negative ion FAB-mass spectrometry with the ion at m/z 1383 [M -HIand 503 [aglycone -HI-. The ions at m/z 827 [M -(2p+2d)-HIand483 [2d+2p-90-H]indicated that arganine D contained a tetrasaccharide consisting of two deoxyhexoses and two pentoses. This tetrasaccharide was linked to the aglycone by an ester linkage as indicated by the identification of arabitol after alkaline hydrolysis under reducing conditions. The sequence of this tetrasaccharide was deduced from the negative ion FAB-mass spectrum with the ions at m/z 1237 [M-d -HI-, 1105 [M-(d+p)-HIand methylation analysis as Rha (l-3) Xyl (1+4) Rha (1+2) Ara. The methylation data showed an additional 2,3,4-tri-o-methylglucose in comparison with the methylation analysis of 3. This result indicated that 4 is a bisdesmosidic saponin

2. CHARROUF et al.

2084

.-

1223

Glc

/’ --c

rd

i3q ; 1253 -.-.....e.*

1043

i 975 .. . . . . . ..m....

127

1385 [

0

; 1121 I_..._......F

1I

100

50

519

M-HI’

_

IZS3

1443 is%0

1685 I '. 1586

, . .

,

.

*

'

1

l600

Fig. 3. Negative ion FAB-mass spectrum of arganine B (2). with the same tetrasaccharide as 3 and a disaccharide Glc (l--+6) Glc linked by O-linkage to the aglycone. The ‘H NMR spectrum (Table 3) showed signals of six tertiary methyl groups at 60.91,0.95, 1.06, 1.14, 1.30 and 1.59 and two secondary methyl groups at 6 1.28 and 1.24. It also showed six anomeric protons at 64.32 (d, J =7.7 Hz, H-l Glc i), 4.44 (d, J=7.7 Hz, H-l Glc t), 4.52 (d, J= 7.7 Hz, H-l Xyl), 5.09 (d, J= 1.0 Hz, H-l Rha i), 5.14 (d, J= 1.6 Hz, H-l, Rha t) and 5.65 (d, J = 3.9 Hz, H1, Ara). Signals at 65.35 (t-like) and 2.94 (dd, J= 14.3, 3.2 Hz) corresponding, respectively, to vinylic and allylic protons are characteristic of oleanic acid derivatives [6]. In the ‘H-‘H COSY NMR spectrum, the signal at 64.46 co~esponding to one hydroxymethine proton was correlated to three signals. The same signal was correlated to five signals in the case of 1.This result confirmed that the aglycone of 1 contained one more oxygen atom than the aglycone of 4 and indicated that the hydroxyl group of 1

at C-16 was absent in the case of 4. Thus the aglycone of arganine D is the protobassic acid. On the basis of these results the structure of arganine D was established as 3~-~-D-giUCOpyranOSyl-(1 -&)-B-D-glucopyranosyl-protobassic acid 28-0-cr-t-rhamnopyranosylj 1-+3)+-D-xylopyranosyl-( l-+4)-sr-L-rhamnopyranosyl-(1+2)-a-L-arabinopyranoside. Carbohydrate analysis of arganine E (5) (Table 1) indicated the presence of glucose, arabinose, xylose, rhamnose and an additional D-apiofuranose residue identified by GC-mass spectrometry of the permethylsilylated methylglycoside in a molar ratio 2.0; 1.1; 1.0; 1.2; 0.4, respectively. This result was confirmed by methylation analysis with the identification of 2,3,4,6-tetra-Omethylglucose, 2,3,4-tri-0-methylglucose, 2,3,5-tri-Omethy!apiose, 2,4-di-O-methylxylose. 2,3-di-o-methylrhamnose and 3,4-di-0-methylarabinose in the same molar ratio. The negative ion FAB-mass spectrum also

Triterpenoid saponins from Argania spinosa

indicated that the only difference between arganine D and E is the presence of D-apiofuranose in 5 instead of rhamnopyranose in 4 at the non-reducing terminal position in the tetrasaccharide moiety: pseudomolecular ion at m/z 1369 [M - H J - for 5 instead of 1383 for 4, [M -h -HIat m/z 1207 for 5 instead of 1121 for 4, [M-2h -H]at m/z 1045 for 5 instead of 1059 for 4 and the same ion at m/z 1237 corresponding to the elimination of the non-reducing monosaccharide from the total arganine D and E. In the same way, ions corresponding to the tetrasaccharide linked by an ester bound to the aglycone showed the same mass difference value of 14 (4: [2d + 2p -90-H]=483; 5: [3p+d-90-H]=469).The combination of molar carbohydrate composition, methylation data, FAB-mass spectrometry and the identification of arabitol after alkaline hydrolysis under reducing conditions estabhshed the sequence of the tetrasaccharidie moiety as Api (l-+3) Xyl (1+4) Rba (142) Ara. The ‘H NMR spectrum of 5 showed the same chemical shifts for the protons of the aglycone in comparison with the aglycone of 4. However, the proton signals due to the sugar moiety displayed a characteristic doublet signal of apiose at 6 5.25 (d, J =2.9 Hz, H-l Api), 3.99 (d, J = 2.9 Hz, H-2 Api), 4.12 (d, J = 9.6 Hz, H-5a Api), 3.78 (d, J = 9.6 Hz, H-5b Api) and 3.60 (2H, d, J = 1.1 Hz, H-4 Api). On the basis of these results the structure of arganine E was established as 3-O-#?-D-glucopyranosyl(1+6)-/3-D-glucopyranosyl protobassic acid 28-0-fl-Dapiofuranosyl-( I--) 3)8-D-xylopyranosy1-((1+4)-a-L-rhamnopyranosyl( l-+2)&-L-arabinopyranoside. Mi-saponin A (6) possessed the same carbohydrate composition as arganine C (Table I). The methylation analysis gave the same methyl-ethers as arganine C, 2,3,4,6-tetra-0-methylglucose,2,3,4-triand 2,3-di-Omethylrhamnoses, 3,4-di-0-methylarabinose and 2,4,-di0-methylxylose. The 1M, and the monosaccharide sequence were deduced from the negative ion FAB-mass spectrum which exhibited a pseudo-molecular ion [M -H] - at m/z 1221 and an ion [M-h -H]- at m/z 1059 indicating the presence of a glucosyl residue linked to the aglycone. The tetrasaccharidic sequence Rha-Xyl-RhaAra was deduced from peaks at m/z 1075 [M-d-H]-, 943 [M-(d+p)-HI-, 665 [M-(2d+2p)-HIand 423 [2d + p - HI-. The presence of arabinose linked to the aglywne by an ester bond was also deduced by the identification, after alkaline hydrolysis under reducing conditions, of one arabitol residue. Comparison of the NMR data (Table 3) with those of arganine D showed the lack of signals due to one anomeric proton of glucose. On the basis of carbohydrate composition, methylation data, comparison of HPLC mobility with an authentic sample, FAB-mass spectrometry and NMR spectroscopy, the structure of 6 was established as 3-O-~-D-glUCOpyranOSyl protobassic acid 28-O-ar-L-rhamnopyranosyl-(i +3)$-Dxylopyranosyl-(l+4)-a-L-rhamnopyranosyl-(1-,2)-~~arabinopyranoside which was previously isolated by Kitagawa et al. [12] from the seeds kernels of lWadhuca Zongij”2ia (L.) Macbride (Sapotaceae) and named misaponin A: Arganine F (7) had a negative ion FAB-mass spectrum (Table 2) which showed three intense peaks at m/z 1207 665 [M-(d+3p)-H]and 469 [3p [M-HI-, + d -9O- H] -. This result, together with the molar carbohydrate composition (Table 1) indicated that 7 is a bisdesmosidic saponin with a tetrasaccharide constituted by one methylpentose (rhamnose) and three pentoses

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(arabinose, xylose and apiose) and a glucosyl moiety. Peaks at m/z 1075 [M-p-H]-, 943 CM-2p-HI-, 469 [3p + d - 90 - H] -, and the identification of arabitol after alkaline hydrolysis under reducing conditions, established the sequence of the tetrasaccharide as Api-XylRha-Ara. Methylation analysis with the identification of 2,3,4,6-tetra-0-methylglucose, 2,3,5-tri-0-methylapiose, 2,4-di-0-methylxylose,2,3-di-0-methyhhamnose and 3,4di-0-methylarabinose indicated that 7 had the same tetrasaccharide as 5. The ‘HNMR spectral data of compound 7 (Table 3 and Fig. 2) gave the same chemical shifts for the protons of the aglycone in comparison with those of compounds 4, 5 and 6 establishing the nature of the aglywne as protobassic acid. The proton signals due to the sugar moiety in comparison with those of 5 lacked the signals due to the glucosyl moiety and exhibited the same characteristic doublets signals due to an apiosyl residue. On the basis of these results, the structure of 7 was established as 3-0-/3-D-glucopyranosyl protobassic acid 28-0~-D-apiofuranosyl-(l-+3)-~-D-xylopyranosy1-(l-+4)-a-Lrhamnopyranosyl (1 -+ 2)-ol-L-arabinopyranoside.

EXPERIMENTAL Material. Argania fruit was from Tamanar (Essaouira, Morocco). IsoIation and puri,fication of saponins. Argania kernels (1 kg) were ground and defatted by hexane and then extracted with EtOH-H,O (4: 1). The ethanolic extract was coned by rotary evapn giving a brown residue (120 g). This residue was suspended in Hz0 and extracted with Et,0 and n-BuOH, successively. The n-BuOH extract was dissolved in MeOH and precipitated by Et,O. Dissolution in MeOH and precipitation with Et,0 was repeated x 3 to yield the crude saponins fr. (5 g). Semi-prep. HPLC was performed on a liquid chromatograph equipped with an LDC variable-wavelength detector connected to a computing integrator. The crude saponins fr. (0.5 mg) dissolved in 300 ~1 of MeOH was injected on an ODS Zorbax column (25 x 0.94 cm I.D.). The column was equilibrated with H20. After injection, a linear gradient to MeCN-H,O (7: 18) was applied for 10 ruin, isocratic elution during 10 min, linear gradient to MeCN-H,O (7:13) for 10 min, isocratic efution during 10 min, linear gradient to MeCN-H,O (1: 1) for 5 min and then isocratic elution during 5 min. MeCN in each fr. was evapd in a rotary evaporator and dry saponin was obtained after freeze-drying. MoIa carbohydrate composibion and D,L configurations. Monosaccharides were analysed by GC as their trimethylobtained after methanolysis silylated methylglycosides (0.5 M HCI in MeOH, 24 hr, 80”) and trimethylsilyIation according to Kamerhng et al. [13] modified by Montreuil et al. [14]. The configuration of the glycosides was established by capillary CC of their trimethylsilylated (-)-2-butylglycosides [15]. AEkaEinehydrolysis with F&O,. A soln of saponin in MeOH was treated with K&O, (12 hr, 8OO”). The reaction mixture was then neutralized with Dowex 50 x 8 HC and filtered. The filtrate was extracted by n-BuOH to yield the prosaponin, Alkaline hydrolysis with NaOH-KBH,. The saponin in MeOH was treated with 0.1 M NaOH-1 M KBH, and heated at 40” for I2 hr. The reaction mixture was then neutralized with Dowex 50 x 8 H’ and boric acid was eliminated as methylborate by co-distillation with MeOH ( x 3). The residue was partitioned into n-BuOH-H,O (1: 1) and the aq. phase containing the tetrasaccharide-alditol was freeze-dried.

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CHARROUF et al.

Methylation analysis. The saponin was methylated with dimethylsulphoxide-lithium methylsuphinylcarbanion-methyl iodide according to the procedure described by Paz-Parente et al. [16]. The tetrasaccharide-alditol obtained after alkaline hydrolysis was methylated with DMSO-NaOH-Me1 [17]. The methyl ethers were obtained either (a) after hydrolysis (4M TFAA, 100’) and analysed as partially polyol-acetates by GCMS [18] or (b) after methanolysis 0.5 M HCI in MeOH, 24 hr, 80”) and analysed as partially methylated methyl glycosides by GC-MS [19]. GC and GC-MS analysis. The gas chromatograph was fitted with a FID. A capillary column (0.3 mm x 25 mm) OV 101 was used. GC-MS analysis was carried out with a Riber-Mag R lo10 mass spectrometer using an electron energy of 70 eV and an ionizing current of 0.2 mA. Fast-Atom-Bombardment mass spectrometry (FAB-MS). A Kratos concept II HH (Kratos Manchester, U.K.) High resolution mass spectrometer equipped with the DS 90 (DGDG/30) data system was used in this study. The mass spectrometer was operated at 8 KeV accelerating potential. An ion Tech Model B 11 NF saddle field fast atom source energy with the B-50 current regulated power supply was used with Xenon employed as the bombarding atom (operating conditions: 7 kV, 1.2 mA). The mass range 2000-120 was scanned at 10 set/decade. The native saponins (100 pg) were dissolved in MeOH (10 pl) and 1 pl was loaded on the copper tip with 2 ~1 of glycerol-water (1: 9) as matrix for analysis in negative ion mode and glycerol-NaCl as matrix for analysis in positive ion mode. NMR spectroscopy analysis. ‘H and 13CNMR spectra were recorded at 25” with a Bruker AM-400 WB F.t. spectrometer (Centre Commun de Mesures, UniversitC de Lille FlandresArtois). All the programs used were from the Bruker library. Proton NMR spectra of the different samples, dissolved in 0.4 ml MeOH-d, were analysed with a spectral width of 4 kHz for 32 K frequency-domain and time domain data point. The 13CNMR spectra at 100 MHz were obtained with a spectral width of 25 kHz for 32 K frequency and time domain data point, using the standard program POWGATE (1H broad band with composite pulse decoupling, Dl +Ac9=2s, PW 90”=4.8 ps, Sl =S2 = 1 Watt). The chemical shifts (a) for ‘H and ‘“C are expressed in ppm relative to internal tetramethylsilane (6 =O.O ppm in the two cases).

Acknowledgements-This work was supported by the Centre National de la Recherche Scientilique (Uniti Mixte de Recherche du CNRS no 111, Directeur Professeur And& Verbert) and by the Umversitt des Sciences et Techniques de Lille Flandres-Artois. The authors are grateful to the Conseil

RCgional du Nord-Pas de Calais, the CNRS, the Minis&e de la Recherche et de I’Enseignement Sup&ieur, the Association pour la Recherche sur le cancer for their contribution in the acquisition of the 400 MHz NMR and the concept II HH mass spectrometer. The authors thank Guy Ricart for help in MS analysis.

REFERENCES 1. Huyghebaert, A. and Hendrick, H. (1974) Oleagineuse 29,29. 2. Farines, M., Soulier, J., Charrouf, M. and Soulier, R. (1984) Revue Francaise des Corps Gras, 11,443. 3. Farines, H., Soulier, J., Charrouf, M. and Soulier, R. (1984) Revue Francaise des Corps Gras 7-8, 283. 4. Farines, M., Charrouf, M. and Soulier, J. (1981) 106”me Congris Nat. des Sot. Savantes Fast II, 327. 5. Fraisse, D., Tabet, J. C., Becchi, M. and Raynaud, J. (1986) Biomed. Environ. Mass Spectrometry 13, 1. 6. Furuya, T., Orihara, Y. and Hayachi, C. (1987) Phytochemistry 26, 715. 7. Ischii, H., Tori, K., Tozyo, T. and Yoshimura, Y. (1984) J. Chem. Sot. Perkin Trans I 661. 8. Niranjan, P., Suboth, S.. Roy, K. and Hamato, S. B. (1989) Phydochemistry 28, 52. 9. Nagao, T.. Okabe, H. and Yamanchi, T. (1990) Chem. Pharm Bull. 38, 783.

10. Ishii, H., Kitagawa, I., Matsushita, K., Shirakawa, K., Tori, K., Tozyo, T., Yoshikawa, M. and Yoshimura, Y. (1981) Tetrahedron Letters 22, 1529.

11. Gariboldi, P., Verotta, L. and Gabetta, B. (1990) Phytochemistry 29, 2629. 12. Kitagawa, I., Inada, A. and Yusioka, I. (1975) Chem. Pharm. Bull. 23, 2268. 13. Kamerling, J. P., Gerwig, G. J., Vliegenthart, J. F. G. and Clamp, J. R. (1975) J. B&hem. 151, 491.

14. Montreuti, J., Bouquelet, S., Debray, H., Fournet, B., Spik, G. and Strecker, G. (1986) in Carbohydrate Analysis a Practical Approach (Chaplin, M. F. and Kennedy, J. F., eds), p. 143. IRL Press, Oxford. 15. Gerwig, G. J., Kamerling, J. P. and Vliegenthart, J. F. G. (1978) Carbohydr. Res. 62, 349. 16. Paz-Parente, J., Cardon, P., Leroy, Y., Montreuil, J., Fournet, B. and Ricart, G. (1984) Carbohydr. Res. 141, 41. 17. Ciucanu, I. and Kerek, F. (1984) Carbohydr. Res. 131, 209. 18. Fournet, B., Dhalluin, J. M., Leroy, Y. and Montreuil, J. (1981) J. Chrotnotogr. 153, 91. 19. Fournet, B., Strecker, G., Leroy. Y. and Montreuil, J. (1981) Anal. Biochem. 116,498.

Triterpenoid saponins from Argania spinosa.

Five new oleanane saponins named arganine A, B, D, E and F and two known saponins: arganine C and mi-saponin A were isolated from the kernel of Argani...
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