Cabalion, Pierre

Lussignol, M.

Paphassarang, S.

Raynaud, J.

Horizon / Pleins textes

163 
A NEW OLEANOLIC GLYCOSIDE FROM POLYSCZAS SCUTELLARIA 
S. PAPHASSARANG, J. RAMAUD,. M. LUSSIGNOL, 
D f ~ ” t t  & Botmiqw, Bìologit nllw!uin tt P b ” g n a r i r ,  Puvlri k P b m n d r  k Lp, 
U n i w i r f  CL#& Brrnard, 8 A m u r  Rorkdtllu, 69008 Lyoa, Frank 
and P. CABAWON 
UR4G (Substawnr N&rdfu Glnrlrtr Biologiqut) DIpr Sanrf, 213, L l j ä y r r t ,  72010 Pm‘f, Frana 
A a m c r . - A  new triterpenoid uponin, named polysciurponin P, [8], h u  been i- 
lared from Polp-i,umrefl.m’u l a v a  and identified u 3-O-[ß-~glucopynnosyI( 1+4>.ß-~-glu- 
copynnosyl( 1~2>ß-~-glucumnopynnosyl] olanolic acid 28-0-ß-m Q lucopynnoside. The 
rtructuriwu atlblished by chemicd urd spectroscopic mans (hbms, ’ C nmr, gc-ms). , 
In previous work, we reported the iso- 
lation and stiucture determination of 
polysciasaponins P, [21, P6 131, P4 E51 
(l), P, 141, and P, VI (2) from the leaves 
of Polyscicrs scutellaria (Burm.f.) Posh. 
(Araliaceae) used in the islands of the 
Western Pacific Ocean, especially in 
Vanuatu, ’ as an anti-inflammatory. In 
che present paper we report the isolation 
and structure elucidation of polysciasa- 
ponins P3 161 and P, 181; Pl is a new 
oleanolic glycoside. 
An EtOH extract of dried leaves was 
fractionated. The n-BuOH fraction 
yielded a crude mixture of saponins. 
Separation of the n-BuOH extract by CC 
and preparative tlc on Si gel afforded 
pure saponins P, and P3. 
On acid hydrolysis, polysciasaponins 
P, and P, afforded oleanolic acid El] as 
the aglycone, identified by comparison 
with P standard sample (tlc, ms, and gc- 
ms). The sugars obtained from the hy- 
drolysates were identified by tlc and gc 
OR, 
Glc=ß-bglucopyranosyl. ’ . 
i 
164 
Olanolic acid 
c-3 . . . . . . . . . . .  
c-12. . . . . . . . . . .  
c-28 . . . . . . . . . . .  
C-13.  . . . . . . . . . .  
Journal ofNatUral Products [Vol. 53, No. 1 
as glucuronic acid and glucose for Pl  
(1:3), glucuronic acid and glucose for P, 
(1:2). 
The mol wt and the sugar sequence of 
Pl and P3 were established by fabms in 
negative ion mode. The following sig- 
nals were observed: mlz 11 17 for P I ,  mlz 
9SS for P, corresponding to depronated 
molcculat ion IM - HI-; m/z 9SS for Pl 
and d z  793 fbr P3 resulting from the loss 
of a glucosyl moiety [M - H - 1621'. 
On miifd acid hydrolysis, P l  initially 
gave P,, P,, P,, the,n P,; polysciasapo- 
nin P3 initially gave P, and P,. 
Basic hydrolysis of polysciasaponin P, 
yielded P,, 3-O-[ß-~-glucopyranosyt( IC, 
4)-ß-~-gI ucopyranosyl( IC+2)-ß-D-glu- 
curonopyranosyll oleanolic acid; basic 
hydrolysis of. P, afforded P,, 3-O-Iß-D- 
glucopyranosyl( 1+%2)-ß-D-glucurono- 
pyranosyl]; oleanolic acid. These results 
indicated that polysciasaponins Pl and 
P, are ester glycosides of P, and P,, re- 
spectitely. The structure of polyscia- 
saponin P, was established ps 3-0-[ß-b 
ghcopyranosyl( I+%4)-ß-D-glucurono- 
pyranosyl] oleanolic acid (I), and poly- 
sciasaponin P, as 3 - O - [ ß - ~ g l u m p y m ~  
lic acid (4). Upon comparison ofthe I3C- 
nmr spectrum of P, with that of P, 
(Table I), all of the carbon signals of the 
syl( l w 2 > ß D - g l u c u r o ~ l l  oleano- 
Grbon Polyxiurponin P 
c- I" 
c-2" 
c-3" 
C-4" 
c-5" 
C-6" 
Gluccnc 
c- I" 
c-2" 
c-3" 
c-4" 
c-S" 
C-6" 
Glvcov 
C- 1- 
c-2- 
c-3n 
C-4" 
c-s- 
C-6" 
. .  
* .  
. .  
. .  
. .  
. .  
. .  
. .  . .  
. .  
* .  
. .  
89.52 
122.10 
144.12 
176.28 
(-3.6 ppm) 
104.40 
83.26 
76.43' 
71.85' 
76.46' 
174.3s 
104.71' 
73.9Ib 
78. IOd 
80.97 
77.63¿ 
62.40' 
104.67' 
74.7Ib 
77.63' 
7 1.34' 
77.18' 
62.31' 
95.54 
73.74b 
78.73.J 
7 1 .gor 
78.47¿ 
62.97' 
I 
%gnals wirh rhe same superscript arc intcrchang 
Polyxiruponin P, 
89 
122.2 
144.52 
179.9 
104.6' 
82.9 
76.4h 
71.15 
76.1' 
174.4 
104.4' 
74.5 
77.20' 
80.5 
78.07' 
62.2 
104.Y 
74.5 
77.25' 
71.16 
77.8?' 
61.9' 
.. 
Jen-"Pcb 19901 Paphamrang et al,: 
iglycone ind glucosyl moieties appeared 
a t  dmost. the same positions, demomtnt- 
ing that PI is rn meer glycoside of P2. 
The chemicnl shift of C-28 of the nglycone 
moiety of Pl  w u  displaced downfield by 
3.6 (176.3 ppm for PI; 179.9 ppm for 
P2), indicating that the carboxy group is 
esterified with glucosyl moiety. 
The second molec'tlle of glucose in 
polysciaseponin P, might be linked 
either with COOH-28 of oleanolic acid 
or COOH-6' of glucuronic ncid. Methyl- 
ited derivative of P3 obtained with 
ß-glucosidase and ß-glucuronidase. Ole- 
snolic acid, ind not i n  aglycone mechyl- 
ester, was obtained, c!early indicating that 
one ß-D-glucase is linked to the C-28 at 
the carboxylic acid of olcanolic acid.' 
In conclusion, the structure of poly- 
xiasaponin PI was cstablishd u 34-[ß- 
fanosy1 ( Iw2)-ß-D-glucuronopyranosyl] 
olcPnolic acid-28d-ß-~-gIucopyranoside 
(81 and the structure of polyxiasaponin 
P, 1s 3-O-[ß-~-gIucopyranosyl( 1-2)- 
ß- glucuronopymnosyl] olennolic acid 
saponin P, is identical wirh chikusetsu- ~ 
saponin V, isolated from Panax species 
(3-9). 
EXPERIMENTAL 
CH2N2 WPS SuCCeUiVely hydrolyzed with 
D-glucopymnosy~ ( 1*4)-ß-D-gluCOpy- 
28 %- ß-D-glucopyranosidc (61. Polyxia- 
GENERAL EXPBIIHEMAL PROCEDURES,- 
G-mR wu perfòrmed on a SES4 cared quin t  
mpillsry colnmn with a Hcvlctr-Pukard in elec- 
tronic impcr mode. Pibms vere obnined on i 
VG Micromus ZAB-HP spmromercr in the 
ncptive ion mode; umplcs vere surpendcd in 
plyethylene glycol; "C-nmr spectn were re- 
corded on i Bmcker WP 200 in C,D,N. T h e  
chcmicd shifts are given in ppm. TMS w u  wed 
u inremil scindird. TIC vu arried out on S i  gel 
60 (Merck). Ihc rlc systems employed were: for 
u p n i n s  r-BuOH-HOAc-HIO (4: 1: 1) (system 
1). ErOAc-McOH-H,O (20:J:2) (system 2). ind 
CHCI,-McOH-H,O (30: 1S:2) (system 3); and 
for iglymncs CHCI,-MeOH (97:3) (system 4) 
rod &H,-EtOAc (3:l) (iysrcm S). 
PLANT MATERIAL.-~%C leaves of P. iwd- 
Lri. vére collmed from Vinuitu by P. Gbdion  
(ORSTOM). A voucher specimen i s  d c p i r e d  in 
the Hcbririm of Boriny and Phirmicognosy, . 
Piculrt de Phirmocie. Lyon. 
l 
i 
i 
! 
Oleanolic Glycoside 16s 
EXTRACTION A M )  ISOLATION O F M E  SAPO. 
E a H .  The EtOH rrt& VIS concenrrared, di- . 
move lipid mrtcrid. The H,O solution w u  a- 
trutcd wirh EtOAc, then m-BuOH. T h e  m- 
BuOH layer vu ~ p o r a t c d  ro dryness to give i 
crude uponin fncrion (21 8). The nepanrion vu 
cirried out on a column packed with,Si gel 60 
(Merk) usin8 i mlvenr g d i e n t  of CHCI,- 
MeOH-H,O (70:2:2 to 70:40 IO). Six  fnrrioru 
were collected. Prution 4 grw polysciurponin 
P, (30 mg); fnrtion 6 grw plysciwponin PI .  
Siponins PI  rnd P, were funher nepnted on rlc 
(Si pl) with r-BuOH-HOAc-H,O (4: 1: I) (de- 
tection virh indine) to give pure PI  and P,. 
M e T H v u n o N  BY CH,N,.-To UI h20 soolu- 
rion ofwpnin  (IS mg), fmhly prrp.ted CH,N, 
in Et,O w added in M~IS. The m i m r e  vu 
kept i t  37' for 1 h. I& mcrion pradUCf obtained 
on workup formed a yellow product. 
N I N S . - ~ W  OJO 1) mrc cxrrured with hoc 
luted With HZO, id "ed Wich CHCI, to IV- 
ACID HYDROLYSIS.--'(~~ uponin ( 5  mg in 
MeOH-HzO (2: 1) (I ml)] vu refluxed in O. 1 M 
HCI (I ml) for 6 h. The aglycay w exrnrted 
with CHCI, and identified with an ruthmtic 
umple by tlc an Si gel ind gc-ms &er dylrtion. 
The qucocu layer w u  ncutnlivd with N,N- 
ocryl-methylamine md evaporated ro dryneu. 
The sugin wem identifid by gc iftcr silylirim 
rnd by tlc on Si gel using m-BuOHiPIOH-H,O 
(S:3:1), or on cellulole ruing r-BuOH-C,H,N- 
HCI (5:3:2); compounds vere viswlited by 
spnying virh iniline hydrogen phthilite and 
then tKlringJI 10'. 10 min). 
P A R ~ A L  ACID HYDROLYSIS.-?)~C uponin 
(5, mg) in M q H  (0.2 ml) and 1 M HCI (0.2 ml) 
w u  kept i t  70'; after 80 min the m i m r e  vu 
rcpcnted by tlc on Si gel with m-BuOH-HOAc- 
H 2 0  (4: 1: 1) to &min the prtiilly hydrolyzed 
. 
FradUCtS. 
ENZYMATIC HYDROL&IS.-T~C enzymatic 
hydrolysis wu carried out vith I ml of saponin 
dur ion  (0.5 ml, pH 6) and 5 mg of ß- 
gluccnidiu (Extruynrhhe EC 3.2.1.21 300 U/ 
mg) or 0 .3  ml of ß-glucuronidi.u (Merck An. 
4 1  14, 12 Ulml, from Htf ixpwwia)  during 24 h 
i r  37'. After extraction with CHCI, (srpogenin), 
glucose or glucuronic acid wu identified in che 
rqueocis p h u c  ind olcmolic acid in rhe organic 
Isycr. 
ALKALINE HYDROLYSls.-Alhline hydroly- 
sis w u  performed i r  80"for 3 h wirh 5 mg o f s i p  
nin in 1 M NoOH ( 5  ml). After ucidificarion by 1 
M HCI (pH S), monodesmmide w u  ortncred 
POLYSCIASAPONIN Pl  (8l.-White povdcr: 
mp 263'; rlc R,0.22 (system l ) ,  0.03 (system 2). 
0.02 (system 3); fibms n/ r  11 17 tM - HI', 95s 
' 
with W-RUOH. 
166 Journal ofNatuml Products V O I .  53, NOI. 1
[M - H - 162]-; I3C nmr (C,D,M 8 89.5 (c- 
3), 122.10 (C-12). 144.1 (C-13). 176.3 G28)v 
104.4 (C-1'). 104.7 (C-I"), 104.6(C-l"), 95.5 
(C- 1.3. 
POLYSC~WAPONIN P, [6].-Amorphous 
powder: mp 260'; tlc Rf0.38 (system l), 0.09 
(system 2), 0.06 (system 3); hbms dz 9SS 
[M - HI', 793 [M- H - 1621'. Enzymatic hy- 
drolysis of the methylate derivative of P, afforded 
olenolic wid, identified by comprison with an 
authentic sample. 
A C ~ D  HynRoLvsis.-Polyscipaponins P I  and 
P, gare olnmlic acid identified by tlc [R,0.71 
(system 4). 0.56 (system 5)) and by gc-ms after 
silylarion: ms m/z IM]+ 600, [M - COOS¡ Me,]' 
483, [M-280]+ 320, [M-3211+ 279, 
[hf - 3971+ 203, [M - 4 11]+ 189. The sugars 
were identified by tlc and &y gc after silylrtion. 
The sugar components were Glc-GlcA (3: 1) for 
saponin PI,  Glc-GlcA (21) for uponin P,. 
P,. P, and P,; P, yielded P, followed by P,. 
gave P,. 
PARTMI. ACID HYDROLYSK-Pl gave P2, 
ALKAUNE HYDROI,YSIS.-P~ affotded Pz; P, 
ACKNOPVLEDCvMENlS 
The authors are gnteful to Prof. K. Hor- 
tettman (University de Luanne)  for cdm. 
duloside E sample. 
LITERATURE CITED 
1. S. Paphusrmng,J. Raynrud, M. Luuignol, 
and M. Becchi, P b y t œ b m i i ~ ,  28, 1539 
( 1989). 
S. Plphusarang, J. b y m u d ,  M. Luuignol, 
and M. Becchi, J .  Ndt. Pd., 52, 239 
( 1989). 
N, Kondo, Y. Muumoto, and J. Shoji, 
C h .  Pbanrr. Bull., 19 ,  1103 (1971). 
N. Kondo, J. Shoji, and O. Ta&, C h .  
P h .  Bull., 21, 270s (1973). 
T.M. Ln and A.D. Mardcr0sian.J. P h .  
Sci., 70,89(1981). 
H.L. Li, 'Flom of Taiwan: A Check List of 
the Vucular Plants of Tuukn.- Ed. 
Epoch, Taiwan, 1979, Vol. VI, p. 6 6 5 .  
T. Morin, R. i t m i ,  O. Tuuh, J. Zhou, 
T. Yang, and J. Shoji, C k .  Pbmn.  Bull., 
30,434 1 (1982). 
S. &nada m d  J. Shoji, Shy&g#ku Zutbi, 
32.96 (1978). 
T. Yang, 2. Jiang. M. Wu, J. Zhou, mdO. 
Tanalu, A## P h .  Sin.. 19,232 (1984). 
2. 
3. 
4. 
5 .  
6. 
7. 
8. 
9. 
R u r i d  24 April 1989 
,". 


A new oleanolic glycoside from Polyscias scutellaria

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