Structure elucidation of four new triterpenoid oligoglycosides from anagallis arvensis

Tetrahedron Vol.47,No. 28.pp.5215-5230.1991 Printed inGreatBritain

STRUCTURE

oo4o-lo24w1 s3.00+.00 0 1991 PeqmonPressplc

OF FOUR WEW TRITERPENOID ARVBNSI S ANAGALLIS

ELUCIDATION

OLIGOGLYCOSIDES

FROM

*

Shashi

B. Mahato

, Niranjan

P. Sahu, Sucharita Sen

Subodh

K. Roy and

Indian Institute of Chemical Biology 4, Raja S. C. Mullick Road, Jadavpur, Calcutta - 700032, India.

(Received in UK 15March 1991)

my

WOR%:

Anaqallis

Primulaceaet Triterpenoid saponins

arvensist

Abstract

characterisation of desglucoanagalloside, - Besides oligoglycosianagallisins A,B,D and E, four new triterpenoid des isolated from the aerial part of Anagallis arvensis were respectively defined to be anagalligenin B 3-c-{B-D-xylopyra(1-4)-[fl-D-glucopyranosylnosy1 (l-2)-fl-D-glucopyranosyl (l), (l-=4)-B-D-glucopyranosyl (1--2)]-a-L-arabinopyranoside) anagalligenone 3-0-{P-D-xylopyranosyl (ld2)-B-D-glucopyranosyl (l-4)-[p-D=glucopyranosyl (1-4)-p-D-glucopyranosyl anagalligenone 3-c-(@ (l-2)]-a-L-arabinopyranoside} (21, D-xylopyranosyl (l-+2)+-D-glucopyranosyl (l--+4)-[/3-D-glucopyranosyl-(1-2)]-a-L-arabinopyranoside) (4) and anagalligenin B-3-O-(~-D-glucopyranosyl(l~2)-[~-D-glucopyranosyl(l~4)]-a-Larab%opyranoside) (Z).The structural features were elucidated by a combination of fast - atom - bofbardmeqs mass spectrometry, C NMR spectroscopy. H and strategic chemical degradation, and

Anagallis of

India

arvensis

particularly

in vitro 2-4 polio virus

in

the

their

saponin chemical

of the

herb

fraction tion

of

. The

the

of

five

The

HPLC E

Bu"OH

separation

(l-5)

saponins

soluble

yielded

according gave

to

positive

. The

activity of

have

the

reported target

occurs plant

the

few

been

simplex plant

reported5.

The

and

turned

out to be new glycosides.

paper triterpenoid oligoglycosides

of

pure

decreasing

the

located and

occurrence

of the saponin elucida-

isolation

of

I

potential

and biolo-

AND DISCUSSION

MeOH

purification five

been

structure

reports

to

type

saponins wide

activities

isolation

parts

reported virus

has

This

fraction

in many

triterpenoid

biological for

wild has

Herpes of

a

been

ideal

four of which

chromatographic

which 1

against

Isolation

RESULTS

repeated

plain

antiviral

saponins.

the

interest

with

plant

herb

activity

characterization

characterization gical

small

in Gangetic

fraction.

coupled

made

a

antiviral

possess and

is

on

extract

of

silica

gel

glycosides order

of

Liebermann-Burchard

5215

the

designated

their test

polarity. for

whole

plant

on

followed

by

anagallisins

A-

column

the

five

triterpenoids

All

and

S. B. MAHATOet al.

Molisch an

test

for

aglycone

its

sugars.

identified

physical

and

Anagallisin

spectral

data

monosaccharide

constituents

L-arabinose

descending

by

authentic was

samples.

confirmed

tion.

The

acid did

by

aglycone,

hydrolysis not

were

show

made

to

technique the

of

use

yielded could

of

any

for

the

on

acid

the

40 h afforded

toan

by

by

the of

of The

D-xylose

and

comparison

with

L-arabinose

its

unit

specific

rota-

in

a

such,attempts

trace

yields,

apparently

- Na metal

aglycone

As

of the newer 6 solution . Although

having

isolable

technique

carbon.

application metal

solution

metal

the

of

olefinic

aglycone

of Bu"OH

the

due

water7

aglycone

to

its

at water-bath

(12) as the major

of

(12)

formation

temperature

product.

The mpr,

found to be identical with those 1 H and l3 C NMR data were compatible reported for anagalligenin 13 C values were made by the appliwith its structure. Assignments of the 9 13 with the C data of shift rules , comparison cation of known chemical values

[aI,

of

and

androsacenol", ment of

mass the

sugar was of

portion at -m/z moiety

further KC113

negative

- two

glycosylated

showed

FAB-MS

two

and

was

457

The

corresponding

from

to

[M-H]-

sequences

to

the

ion

losses

1263 which of

were

- hexose hexose

S = anagalligenin and pentose

moiety

- pentose

- S

I

- hexose

B, hexose

= arabinose

are

= glucose

or xylose

to

shown

pentose,

observed

a pentasaccharide

of

FAB-MS

assigned

the

and the

ion

[M+H saponin

by addition [M+K]+.

The

in Table

1.

hexose,

hexose-

three

hexoses

and which

in

displayed

[M+H]+

weight

hexoses-pentose

:

Pentose

determination units

FAB-MS

of the positive

at -m/z ion peaks

three

the

[M+Na]+,

molecular

ion

various

for

positive

ascribed

intense

exhibited

employed

Its

by determination an

Fast-atom-bombard-

B5.

of the monosaccharide

(1).

respectively.

A (1) contains

monosaccharide

A

hexoses-pentose,

pentoses

anagallisin

1225

H20]+

confirmed

which

anagalligenin

(FAB-Ms)~~~~~

anagallisin

1247, -

(12) were B 5rS . The

weight and sequence of

Fragmentations pentose,

the

spectrometry

molecular

glycone peaks

compound

D-glucose,

and GLC

liberated

comparison 4 sample .

authentic

determination

as

this

use

as

by

to be an artefact formed during 13 C NMR spectrum of the saponin the

(5,7-11)

by

an

alcoholic-alkali

using

isolated

of

(6)

appeared

genuine

prosapogenins

hydrolysis

A

configuration and

assignable the

those

identified

saponin

signal

in traces. However, (95OC)

however,

isolate

be

(1)

chromatography

absolute

alcoholic-alkali

the not

were

isolation

hydrolysis

of

with

paper

The its

A

as 23-hydroxyprimulagenin

indicated

possessing

that

the following

5217

Triterpenoid oligoglycosides

!

(7)

RI=\~

I

L -----------------

(14) -----_---

Rl=\H

/OH

, R=H

,.OMe

,R=Me -------

(I)

R,=
(2)

Rt=O

(13)

RI=\~

R=H

, R = H ,,OMe

,R

=Me

5218

s. B.

h’hI+ATOetd.

‘\

&

d@

0

RO RO-’

(12)

(20)

R =Ac;Rl

determine

(12) the

of

be

detected moiety

the

the

The

hydrolysis

two

with

pentoses

sodium

only

(HMPA)

yielded

the

no

linkages

anagallisin

linked

diol

revealed

the

with

NaH

-

MeI

permethylate

aglycone

worked

up

for

L-arabinose

suggesting

vicinal

were

the

and

that

and

is

the

linked

by permethylation,

of the partially A

to

hydrolysate.

constituent

contains

identification

of

in

sugar

saponin

are

R = Ac,Rl=O

metaperiodate

present

intersugar

and

triamide

hydrolysis

treated

as of

permethylation

phoric

the

monosaccharide

aglycone.

partial Thus

was

any

= H;R,=O

(25)

of

_-OH

R = H ;Rl=\H

(21)R

which

saponin

arabinose to the

(6)

/OAc =\H

R =Ac;Rl=O

detection could

RO RO-’

R = H ; R, = 0

(23)

CH20R RI

RI

,.OH R = H; RI =\H

(22)

To

\

methylated in

sugars.

hexamethylphos-

(13),

which

on

acid

yielded

2,3,4,6-tetra-O-methyl-D-glucose, 3,4,6-tri-g-methyl2,3,6-tri-O-methyl-D-glucose, 2,3,4-tri-0-methyl-D-xylose and 3-0-methyl-L-arablnose. Moreover, partial hydrolysis of the saponin with -

D-glucose,

alcoholic

-

alkali

sapogenins

Acid

(14-19). methylated using

(Table

p-Configuration

as

mentioned

permethylation

of

the

2)

identified

above

by

afforded

yielded

permethylates

conformation)

(4Cl

yielded

GLC

of

their

inferred 1

H NMR

from

the

spectrum

of

for

the

six

six

pro-

permethylates

various

partially

alditol

glucopyranosyl

(lC, conformation)

and a-configuration

were

in the

on

acetates

samples.

the xylopyranosyl, pyranoside

solution

which

hydrolysis

sugars

authentic

protons

metal

(5,7-11)

units

and

for the arabino-

of the respective anomeric -J values the permethylate (13). The attachment

(12) was revealed carbohydrate moiety at the C-3 of the aglycone 13 C NMR chemical shifts of anagallisin A and Its aglycone (Table from the 14,15 shift values . The the glycosylation 3) taking into consideration

of

the

carbon and of

0.7 the

shifts

atoms

C-2,

p.p.m. aglycone of

the

c-3

and

C-4

respectively (12). U-carbon

The

of in

saponin comparison

intersugar

atoms

as well

(1) were to

linkages as

those

the were of

shifted

by

- 1.3,

corresponding also

indicated

adlacent

carbon

6.5

signals by the atoms

5219

Triterpenoid oligoglycosides

Table

1.

Characteristic FAB

spectra

mass of

spectral

anagallisin

sin C (3), anagallisin Saponin

Significant

1

ions A

in

(11,

the

D (4) and anagallisin

ions

(relative

intensity)

and

B

(2),

negative anagalli-

E (5).

and assignment

m/za 1247 (100) [M+Na]+, 1225(37)[M+H]+, -455(50), 439(75) and 421(30).

457(55)[M+H-S-H20]+,

109(16.2)[m-H-x]-, m/z b 1223(lOO)[M-HI-, -767(20)[M-H-2g-xl-, 929(8.7)[M-H-g-x]-,

1061(26.9)[M-H-g]-,

719(2.5),

627(5.0),

571(6.2), 455(4.1),

and 219

441(8.4),

585(3.7),

439(5.6),

365(6.2),

249(12.4),

(19.4)

m/xc 1263(100)[m+K]+, -1026(lO)[M+K-g-H20]+, [M+K-x-29]+,

587(2.5),

473(5.6)[M-H-3g-x-ar]-,

527(7.5),

569(4.4),

747(3.4),

749(2.8),

605(7.5)[M-H-39-x]-,

453(7.2),

221(35.6)

1131(18)[M+K-xl+,

1083(16)[M+K-g-H,O]+,

951[M+K-x-g-H20]+,

645(14)[M+K-x-39]+,

880(5),

611(14),

, 407(22),

439(13)[M+K-S-2H,O]+

368(75),

1223(87)[M+H]+, m/x a 1245(100)[M+Na]+, -1083(12)[M+Na-g]+, 767(62)[M+H-2g-xl+,

2

positive

anagallisin

807(11)

457(12) [M+K-S-H201+,, 311(28)

and 191(86)

1113(25)[M+Na-xl+, 605(20)[M+H-3g-xl+,

and 455(58)[M+H-S-H,O]+

473(50)[M+H-3g-x-ar]+

m/za 1085(10)[M+Na]+, 1063(28)[M+H]+, 769(1O)[M+H-x-g]+,589(17) -457[M+H-S-H20]+ and 439[M+H-S-2H20]+ [M+H-genin]+,

3

m/xb 1061(100)[M-HI-, 929(34.2)[M-H-x]-, 901(8), 767t22.4) -585(5.8)[M-H-genin-2H]-,567 [M-H-x-g]-, 605(6)[M-H-x-2g]-, (5.8),

455(3.9)[M-H-x-2g-ar-H20]-,453

473(4.2)[M-H-x-2g-ar]-,

(4.51, 233(15),

441(7.1), 221t15.3)

439(5.3),

407(6.6),

365(12.6),

249(14.5),

and 219(15)

C

967(8) [M+K-XI+, 1099(lOO)[M+K]+, 1061(12)[~+~]+, m/z -643(5)[M+K-x-29]+ and 455 937(7)[M+K-g]+, 805(6)[M+K-x-g]+,

4

(2O)[M+K-S-H20]+ 767(18)[M-H-g]-, m/x b 929(lOO)[M-HI-, -(5.4) [M-H-S]- and 455(15)[~-~-~-~~01-

5

apositive

FAB,

g = glucose,

b

negative

x = xylose,

FAB,

l/3-shift) (Table

31, Moreover, acetates

disclosed

the points

added

ar = arabinose,

thylated

alditol

'KC1

to

of linkages.

from

Thus

the

S = sugar

identification

obtained

the

605(7)[M-H-2g]-,

sample

in positive

473

FAB,

moiety.

of the various

partially

me-

permethylates(l&19)(Table

the structure

of anagallisin

A is

2)

5220

S. B. IVLuwmet al.

2 : Partially

Table

methylated

permethylated alditol

sugars

compound

(13-19,241

by

and

acid

hydrolysis

identified

Partially

by GLC

methylated

sugars

(Rt)

2,3,4,6-tetra-O-methyl-D-glucose 3,4,6-tri-c-methyl-D-glucose

14

15

*17,18

2,3,6-tri-g-methyl-D-glucose

2.30

2,3,4-tri-0-methyl-D-xylose 3-0-methyl-L-arabinose -

0.54 1.42 1

2,3,4,6-tetra-O-methyl-D-glucose 2,3,6-tri-O-methyl-D-glucose -

2.34

3-0-methyl-L-arabinose -

1.45 1 1.36 2.30 1

2,3,4,6-tetra-O-methyl-D-glucose 3-g-methyl-L-arabinose

1.45 1

2,3,4,6-tetra-O-methyl-D-glucose

1.35

3,4-di-0-methyl-L-arabinose -

1

2,3,4,6-tetra-g-methyl-D-glucose

18

of their

1

3,4-di-0-methyl-L-arabinose 2,3,6-tri-c-methyl-D-glucose

17

2,3-di-0-methyl-L-arabinose -

1.03

19

2,3,4-tri-0-methyl-L-arabinose

0.55

24

2,3,4,6-tetra-c-methyl-D-glucose

* Retention

the

1.90

2,3,4,6-tetra-c-methyl-D-glucose

16

of

acetates.

Compound

13

obtained

1 1.86

3,4,6-tri-O-methyl-D-glucose 2,3,4-tri-g-methyl-D-xylose

0.55

3-g-methyl-L-arabinose

1.47

times

relative

to

Di-0-acetyl-2,3,4,6-tetra-O-methyl-D-

1,5,

glucitol. proposed

to beanagalligenin

a-L-arabinopyranoside The 1223 other

B3-g-P-D-xylopyranosyl

FAB-MS

ascribed fragment

(1 -221-

(1).

of to

(l-2)-8-D-glucopyra-

(1 -4)-p-D-glucopyranosyl

(1 -4)-[p-D-glucopyranosyl

nosy1

anagallisin [M+Na]+

ions

when

and

B

(2) displayed

[M+H]+

compared

with

ion

respectively. those

of

peaks These

at m/z -values

anagallisin

A

1245 and

(1)

and its

(Table

5221

Triterpenoid oligoglycosides

Table

3. Chemical sin

A

shifts

(l),

[bC

(20), anagallisin

Carbon

No.

( f O-l)]

anagallisin

C

(3),

of anagalligenin anagallisin

B (2) and anagallisin

E

(12),

B

(5),

anagalli-

anagalligenone

D (4)

t12ja

wb

(3)b

(5 )b

(20)a

wb

(4)b

1

38.8

39.3

39.1

39.1

38.7

39.7

39.1

2

27.2

25.9

26.0

25.8

27.0

26.0

25.9

82.7

76.2

82.0

82.3

3

76.2

82.7

82.7

4

42.8

43.5

43.5

43.2

42.7

43.6

43.6

5

49.5

48.0

48.0

48.3

49.6

48.5

47.8

19.1

18.6

18.9

18.8

19.4

6

18.5

19.5

7

33.3c

33.1=

33.1=

33.2

33.4

33.5

33.7

8

42.0

42.5

42.5

42.5

42.0

42.9

43.0

9

50.1

50.7

50.7

50.6

50.0

50.5

50.4

10

36.8

36.9

36.9

36.7

36.6

36.6

36.7

11

18.5

19.5

19.6

19.4

17.8

17.8

17.5

12

32.8'

32.9=

32.9=

31.7

31.6

31.9

31.9

13

86.4

86.5

86.5

86.3

86.2

86.1

86.3

14

44.4

44.6

44.6

44.4

49.7

49.8

50.0

15

34.4

34.3

34.3

34.3

45.4

45.5

45.8

16

77.4

77.4

77.4

77.4

213.3

212.1

212.0

17

44.7

44.6

44.6

44.8

56.1

56.1

56.2

18

51.4

51.5

51.6

51.5

54.5

55.5

54.6

19

39.4

39.3

39.4

39.4

40.0

40.1

40.2

20

31.7

31.7

31.7

31.7

31.6

31.7

31.7

21

36.8

36.9

36.9

36.8

35.2

35.2

35.7

22

29.8

30.0

30.2

29.9

24.8

24.7

25.0

23

69.2

65.1

65.1

65.1

71.6

64.7

64.9

24

13.1

13.0

13.1

13.0

11.3

13.2

13.1

25

16.9

16.9

17.0

16.9

16.3

16.9

16.8

26

17.6

17.7

17.8

17.7

17.8

17.5

17.6

27

18.6

18.5

18.5

18.5

21.7

21.7

21.7

28

77.8

77.7

77.5

77.4

75.1

77.3

77.5

29

24.6

24.7

24.8

24.8

23.6

24.9

23.5

30

33.5

33.6

33.7

33.6

33.3

33.2

33.4

A-l

103.4

103.6

103.9

103.6

103.6

A-2

80.1

80.3

80.5

80.5

80.3

A-3

73.0

73.2

73.5

72.8

73.1

A-4

75.7d

75.5d

75.5d

75.gd

75.6d

5222

s. B. hdAHAT0et al.

Carbon

No.

(12)a

A-5

64.0

G-l

104.2=

G-2

83.3

G-3

77.9f

(2)b

(4)b

64.2

64.0

64.2

104.0=

104.8f

103.af

84.6

74.5

84.0

84.7

77.ae

78.0

70.1=

77.ae

103.8

71.4

71.4

71.5

71.4

71.2

78.0f

77.ge

78.1

78.2e

78.0

G-6

62.6

62.6

62.6

62.7

62.8

G'-1

104.5=

104.2=

104.9f

104.8f

G'-2

75.4a

75.8'

75.2'

76.0'

75.1d

G'-3

76.0

76.2

77.7

76.1'

77.a=

G'-4

78.1f

71.4

71.5

78.3e

71.5

G'-5

75.5a

77.ge

78.1

76.2'

78.0

62.6

62.4

62.4

62.0

62.4

104.7

X-l

106.5

106.9

106.9

107.0

x-2

75.4

75.sa

74.9

75.9

x-3

77.9

77.8

77.3

77.4e

x-4

70.4

70.5

70.6

70.5

x-5

67.1

67.2

67.3

67.2

G"-1

104.5e

104.7f

G"-2

74.5

74.8

G"-3

76.9

77.3e

G"-4

71.4

71.5

G"-5

77.gf

78.2e

G"z6

62.6

62.5

b. in C5D5N,

CDC13,

f may be reversed 1)

revealed

weight

ages

the

in the FAB-MS

in

the

i.e.

(20)

mass

units

two

glycone (Table

from Bu"OH

B

1) and

13C NMR

(2) yielded

with

Na

472.

the

1H NMR metal

anagaligenone 13 MS, 'H and C NMR

less

other

= glucose

(Table

The

that

c,d,e

for

40

molecular ions

molecular

aglycone and

the

acid

(21) which

ion (see

furnished was of

link-

same

by

hydrolysis was

(25), treatment h

(12).

their

to be

3). Although

aglycone

characterization

fragment

of

appeared

its acetate 95'C

possesses

constituents

data

rearranged

Its

(2)

than

saponins

at

data.

G,G',G"

saponin

the

of

(20).

The

of

carbohydrate

of both

its MS and

at -m/z

the

part

aglycone,

displayed

of

X = xylose,

column.

aglycone

composition

of anagallisin cterised

A = arabinose,

on each vertical

that 472

of

However,

its

64.3

(20)a

G-4

ain

(2)

(5)b

G-5

G'-6

the

(3)b

Wb

chara-

of saponin the

genuine

accomplished

compound

Experimental)

by

(20)

was

and

the

5223

Triteqenoid oligoglycoskks

13C

NMR

nin

B

is

data

are

which

compatible

a genuine

spectrum

of

attributed It may

saponin

structure an

that

of

showed

this

source.

(l-

besides

On

B

the

has

anagalligenone

report

of

of

defined

the

foregoing

(1 --2)]-CC-L-arabinopyranoside in

its

FAB-MS

the on

acid

with

ascribed

to

studies its

D

structure

(12)

[M+H]+ the

(5)

containing

saccharide

moiety

NMR

chemical

was

confirmed

by

partial

by

Consequently, pyranosyl

turned

constituents evidences

suggested

and

as

its

well

identity

of

the

to

and

evident

from

saponin.

its direct

hydrolysis

of

anagallisin

E

FAB-MS

ion peaks

at -m/z 1099 and 1061 Hydrolysis and permethylation led

to

the

establishment

of

3-0-{H-D-xylopyranosyl (l-2)-P-D-gluco(1 -2)]-CL-L-arabinopyranoside) (4).

out

L-arabinose was

shifts

These (3)

respectively. 13 C NMR data

as anagalligenone

E

compound

in its

(l-4)-[S-D-glucopyranosyl

Anagallisin B

and

in con3unctionwith

pyranosyl

of

aglycone.

it affor-

B5.

(4) showed

[M+K]+

the

shifts

desglucoanagalloside Anagallisin

as

ions

[M+H]+

hydrolysis

at -m/z 1085 and 1063 respectively. Moreover, D-glucose, D-xylose and L-arabinose as carbohydrate (6)

3-0-

(2).

[M+Naj+and

ded

23-hydroxyprimulagenin A 13 as the C NMR chemical

evidence

(1 -4)-[fl-D-glucopyrano-

2)-fl-D-glucopyranosyl

showed

(20).

of anaga-

anagalligenone

(l-4)-P-D-glucopyranosyl (3)

NMR

at 212.1

compound

isolation

as

(20)

13C

the

signal

of

Anagallisin

C

by

carbonyl

to those

basis

been

of anagallige-

suggested

the

first

those

That

was

comparable

is the

with

(20).

artifact

signals

anagallisin

-fl-D-xylopyranosyl syl

the

a natural

from

comparison

not

other

be mentioned

by

and

(2) which

C-16,

structure

assigned

with

sapogenol

to

lligenone the

were

be

a

trisaccharide

D-glucose.

The

permethylation Finally

comparison

with

the

is defined

(l-c2)-[P-D-glucopyranosyl

A

as

anagalligenin

structure studies

the

anagallisin

of

of

structure

of

prosapogenin as

tri-

as

the

l3C

saponin

(5) obtained

described

anagalligenin

the

as well

previously.

B 3-g-/3-D-gluco-

(1 --4]-a-L-arabinopyranoside

(5).

BXPERIXENTAL The

plant

material

and a voucher All apparatus the

and

Whatman saturated

are

were

No.1

solution

GLC

was

performed

the

columns(i)

at

on

ECNSS-M,

solvent

aniline a

Gas

-

a

carried Paper

system

oxalate

Hewlett 3% on

on

was

(60:30:5).

with of

TLC

Indian

Botanic

at the herbarium

measured

uncorrected.

CHC13-MeOH-H20

paper

identified

has been deposited

points

melting

solvent

was

specimen

in

Packard Chrome

Garden,

capillary out

on

melting

silica

chromatography Bu"OH-C5H5N-H2Q

water model

was

used

5730A

Q at 190°C

for

Howrah

of IICB. point

gel was

G with done

on

(6:4:3);

a

for

staining.

instrument alditol

using

acetates

5224

S. B. MAHAToet al.

and

(ii) OV-225

acetates.

polarimeter. on

rotations

High

a spectra-

sorb

on Gas Chrome

Optical

performance

physics

S-lo-ODS

Q at 195'C were

liquid

model

for partially

measured

on

a

JASCO

chromatography

SP 8000B

instrument

methylated

alditol

DPI

digital

-

(HPLC)

with

360

was

performed

a column

of

Spheri-

a Micromeritics 771 refractive index detector in MeOH1 H20 (75:25) as mobile phase. H NMR spectra were recorded on a JEOL FX 13 -100 (99.6 MHz) instrument in CDC13. C NMR spectra were recorded either on a JEOL

and

FX-100

trometer

(300

spectrometer

MHz)

with

tetramethylsilane

ment

mass

spectra

equipped

with

Samples FAB

probe

applied

A

the

a saddle-field

an energy

dissolved sample

of

in

8

to

salt

to which

for

MS-g/50

by

mass

were

spectra

Isolation

the

were

dryness

A

total

to HPLC

gun

using

a

was Kg) chloroform and

The

under

under

Bu"OH

reduced

a

122

similar

The

separation

on

fast

air

atom at

the

the

of

copper

a

probe

and thoroughly

of the mass

the

mA

were

that

layer

added

beam

a

such

obtained

to

copper of

spectro-

on

a

Kratos

probe

xenon

potential

of

tip

produced 9

kV.

The

Electron-impact

recorder.

dried

a

The

on TLC

to

and

give of

various

(each were

on a reversed

washed

was

250

a

dark

silica

gel

ratios ml)

combined. phase

with

of

aerial

with

methanolic

pressure

was

column

powdered

extracted

methanol.

extract

fractions

spots

to

was

produced l-l.5

samples

added thin

were

galvanometer

reduced

chloroform

with of

-

pressure on

A

salt

loaded

successively

solvent

of

applied

operating UV

current

technique,

a

was

thoroughly

a tube

was

on

at 70 eV.

Saponins

chromatographed

performed

with

atom

recorded

the

water-Bu"OH.

giving

bombarded NP

(2

(60-800C),

was

11

recorded

of

arvensis

of

were

samples

mixed

was

1:3.

FAB-MS

of 8 kV.

deposited

thioglycerol

and

into the sourcce

Negative

voltage and

(xenon)

KC1

was

spectrometer

atom

addition

containing

introduced

The

at

t o which

(5O:SO)

mass

or

samples

primary

about

solution

spectrometer.

Jon-Tech

spectra

mixture

acquisition.

glycerol, an

salt

ul-')

glycerol

the

The

ul-l)

was

ratio

ug

spec-

respectively

Fast-atom-bombard-

an accelerating

operating

the

pg

was then

data

TC

source For

sample

The probe

mixed. meter

with

the

containing source.

(2-5

glycerol-thioglycerol tip

tip

CDC13

Positive

(2-10

either

the

kV.

DMSO-d6

of

or on a Bruker

and

on a VG-ZAB-SE

at

DMSO

MHz

in C5D5N

standard.

operating

layer

ion

at 25.05

MHz

obtained

L2H61

thin

into

75

were

in

probe

insertioon

using at

trip.

at

internal

source

dissolved

to

before

as

(FAB-MS)

a FAB

were

operating

operating

part

extract

on

A. ether

removal between

partitioned

water

and

brown

mass

(900

of

petroleum

evaporated (42 g).

g),

elution

chloroform

to This

being

- methanol.

were

collected

and

fractions

These

fractions

were

subJected

Spherisorb

S-lo-ODS

column

with

mo-

5225

Triterpenoidoligoglycosidcs

bile B

phase

MeOH

-

H20

C

E (25 mg) were

obtained.

anagallisin A

(1) - It

mp

ZM-HCl

followed

in

by

aq.

MeOH

mp

[aID

+

filtrate

The

and

pressure

a

and

authentic

conflrmed

tested

for

Three

by

its

The

other

up

of

in

acetate

and arabinitol

Three

Eydrolysis

in

of

sodium

(20

at

95'C

ml)

microneedles, 56.1;

H,

7.9:

was

hydrolysed

5 h. Usual gel

work

column

MeOH

as

gave

up 23-

needles

(45

acetate

free

phic

purification.

The

MeOH

to yield

250

CH20H-H20)

reduced

by

chromatography

paper to

was

the

preparative

with

that

was

manner

and

and was

L-arabinose.

reduced with

subjected

to

glucitol

using

chromatography

authentic was

acetylated

to

D-xylose

paper

of

filtrate

grade g)

BunOH

with

dissolved

Water

was

alkali,

major

dried

product

in

then

GLC

analysis

B

specimens.

was

the

subjected

to

obtained

was

(1) (200

treated

and

(12), mp

on

xylitol

acetate,

spectroscopic

added

and

thus

anagalligenin

ml)

NaBH4

Ac20-pyridine

authentic

(18

using

L-enantiomer

D-glucose,

arabinose

were detected

h.

from

of

Ag2C031

with

a

grade

BunOH

BunOH

layer

chromatogra-

crystallized

from

+ 7.4"

[al

248-250°C,

9 m/z 474 (39%), 456 (M+-H20) (lo), 455 (M -H20-H) -(16)r 407(M+-2H20-CH20H) (M+-H~O-CH~OH) (13), 424 (M+-H20-CH30H)

in

(27),425

needles

by

residue

(1.5

40

with under

(1) with Bun00 - Na Metal - Compound

A

metal for

the

neutralised concentrated

was

corresponding

peaks

washed

separated,

The

spectroscopic

solution

MeOH)

(a)

MS,

(65).

The

(~~~1,)60.84 (3Hls)

232

(43)r

Ac20-pyridlne

s) I 1.16

give

for

from

was

concentrated

usual

of Anagallisin

dissolved

'H NMR

anagallisin

: C,

a silica

corresponding

rotation

the

the

(1).

with

bath

on

filtrate

isolation

usual.

up

column

(12)r

anagallisin

and

(c - 0.3 in MeOH) (lit. l6 mp 246-255°C MS data were comparable to those of an

That

specific

as

worked

the

spots

actual

of

portion

worked

0.2

to

(Found

crystallized

carbohydrates

obtained.

comparison

(2

a water

hydrolysate

of

were

and

mg)

and

the

from portion

samples.

L-arabinose

(1:l) I

on

which

NMR

lH

mg),

(1) (150 mg)

(1) - Compound

+ 40.8"

blD

Its

MeOH

MeOH)

purification

(6)

(150

(250 mg)

sample.

filtered,

and

A

240-244'C, 47.6').

authentic

A

from

in

A

D

H, 7.94%).

(46 ml)

chromatographic

hydrooxyprimulagenin mg),

(5 0.5

C, 56.02:

of Anagallisin

Hydrolysis with

crystallized

5.81"

H20 requires

'5aH96'27'

anagallisin

anagallisin

(340 mg),

was

-

[q)

244-246"C,

Thus

(75:25).

anagallisin

(40 mg),

(2 - H20)

acetate

in the

(22)

usual

(3~~s) 0.96

(6xMe)

2.04

(70)r of

way

was

(~H,s),

(3H,s),

219

(a-CH~OH)

compound

2.08

(12)

obtained

0.98

as an

(3H,s)r

(3H,s),

2.10

(100)

prepared

1.00

and by

amorphous (3H,s),

(3H,s)

201

(a-

treatment powder, 1.12

(3 x OAc)

(3H,

S.B.MAHA-rOeral.

3.24

(lH, d, J: 8H2,

ABq,

J

12H2,

28-H),

and 5.08

(lH, d-like,

Periodate

Oxidation

To a solution wise

was

and

worked

and

the

of

of

at

in the

aqueous

chromatography

(1)

(d each,

usual

(1)

for

3 h,

way.

The

J -

3.70

5,

Only

and

and

lOHz,

one

Hydrolysis

in 90% EtOH (35 mg) kept

at

3.90

(2H,

3 - axial

peak

in water

was

for

was

of

the

(4 ml) was

room

residue

examined

was

GLC.

A

(30 mq)

metaperiodate

15°C

phase

and

4.86

Anagallisin

sodium

stirred up

(lH, d, 2 8Hz,28-H),

and

H)

J 5Hz).

of compound

a solution

mixture

3.64

4.76

-CH20Ac),

(3 ml)

temperature hydrolysed

drop-

and

the

overnight with

2M-HCl

by

paper to L-

carbohydrates

obtained

Product

added

corresponding

arabinose. of

Permethylation a solution and

Me1

(7

mixture phic

of

ml)

was

and

purification

4.30

glucose

d,

J -

unit),

The under was

(CDC13)

(lH,

reflux

with

yield

pyridine (ii).

unit),

2M-HCl

The

after

usual at

peaks

detected

hydrolysis and

with two

sodium drops

mixture

compound

(1)

water

was

(1)

8

was

six

to

preparative

benzene-acetone

A

TLC

was to

d,

and

left

for

in a

the

same

resinous

on

(3:l) to give

silica

being

reaction

48

heated mixture

with

Ag2C03

with

to

analysis

be

NaBH4 Ac20-

on

those

of

column alditol

2. Alcoholic

(10

formation the

unit).

on

acetylated

Alkali

followed

ethanol

ethanol

unit),

J 7Hz,l-H of 4.56 (lH, d, J

with

mixture

in absolute

(24 mg)

reduced

GLC

with

Prosapogenin

indicating

give

(13)

unit),

in Table

(1)

prosapogenins,

to

(lH,

then

identified

absolute

methylated

experiment

reaction

chromatogra-

was neutralsed

which

as shown

the

added

spots

The

h. and

5 h. The

and

subJected

and

(95 mg) in

(1 g)

containing

preceeding

solvent

of

revealed

mixture

subJected

metal

and

sugars

of

methylation

filtrate

residue

1 h

Anagallisin

of

- Compound

separation

a

were

Partial solution

up

3 up

hydrolysed

for

concentrated

for

of the methylated

(6 ml)

aqueous

was

was

(10 ml)

ml).

To

metal

by

was

this

their

treated solution

h.

TLC

examination

of

six

prosapogenins.

aglycone manner

as

substance gel

permethylated

To

(270 mg)

1-H of arabinose

4.44

-

NaH

permethylate

of glucose

(12 mg)

MeOH

90°C

acetates

1-H

Product

added

(lH, d, J 6H2, 1-H of xylose

in aq.

work

5.5H2,

(13)

and the

work

(IH, d, J

unit),

the

was for

Usual the

7H2,

4.65

filtrate

(1:l)

Five

J

of

(6 ml)

temperature

ether.

glucose

product

up as usual

filtered.

d,

Hydrolysis

furnished

gel

of

and

in HMPA

room

64.06

1-H

permethylated

worked

and

at

7H2, 4.56

(1)

diethyl

silica

, 1-H of glucose

7Hz

A

(50 mg)

with

on

'H NMR

(lH,

(1)

stirred

extracted

as powder:

to

Anagallisin

compound

the The

(12)

as

well

for

(1)

in

the

which

was

(108

plates

of

mg),

using

prosapogenins

as

developing (5)(4.5

Triterpenoid

mg) I (7) (6.4 mg), (6.0 mg). Pennethylate

(5) - 'H NMR

unit),

4.36

H

glucose

of

methylated alditol

sugars

as

unit),

4.38

(lH,

1-H

glucose

of

hydrolysis

Permethylate H

glucose

methylated

(lH, d, J

of glucose

acid

unit),

hydrolysis

in Table-2

d,

(5.2

5Hz,

mg)

1-H

4.41

it

by

GLO

(11)

and

of

arabinose

(lH, d, J

yielded

identified

the

7Hz,

l-

partially

analysis

of

the

(11)

-

B

H, 7.90: of

J

d,

,,O,,

C, 76.22:

NMR

3

7Hz,

This

'H

NMR

from

(11)

on

hydrolysis

acid

yielded

2.

(CDC13)

441

(lH, d, 3

6 4.19

(CDC13) 6 4.22

on

acid

in Table

5Hz,

(lH,

1-H

d,

J

of arabinose

5.5Hz,

of

1-H

of

Hz,

3.44, 23-H2),

(M+-CH20H)

H, 10.24%).

to

give

of arabinose

partially

methy-

(hydroxyl), 1.00

(2H, each

5.44

(lH,

(Found

for

the

1705

cm-l

: C,

was

hydrolyused

usual

work-up

(21)

which

[(iI,+ 19.9"

(ketone):

'H

(c NMR

(3~1,s)~ 1.22 (3H,s)

28-H2),

12-H): H,

to yield

(Found

ketone

218-220°C1

d, J 12Hzr

76.20:

MeOH

4 h. The

(3~1,s)~ 1.04

t-like,

: c,

(90 mg)

yielded mp

from MeOH)

H, 7.84%).

(2)

reflux

needles,

(~H,s),

3.62

1-H

the

in

C, 55.31;

purification

3420-3300 0.90

under

0.5

(c

(2) - Compound

(25 ml)

acetone

yielded

crystallized

18.2"

2H20 requires

MeOH

IR

(2) was

[aID-

B

(lH, d, 2 5Hz,

hydrolysis

2.

compound

256-26O"C,

(~H,s),

12

on

(lH, d, J 5.5Hz, 1-H of arabinose unit), 4.40 (lH, d, J 7Hz, l-

(CDC13) 6 4.21

- 'H NMR

aq.

6 x Me),

(M+-

4.19

in Table

Anagallisin

CHC13);

each

d,

of glucose

permethylate

chromatographic

(~~~1~)60.88 (togethr

1-H

This

C58Hg4027.

in

crystallized in

(CDC13)6

7Hz,

(2) - The mp

by

(lH,

(lH, d, J 7Hz, 1-H of glucose unit). On acid hydrothe methylated sugars as shown in Table 2.

shown

microneedles,

2M-HCl

4.41

4.41

compound

arabinose

followed

unit),

4.43

d, J 6.5Hz, 1-H of glucose unit). Acid hydrolysis furnished methylated sugars as shown in Table 2.

Permethylate

Hydrolysis

glucose

arabinose

(lH,

it furnished

55.39:

of

of

(lH, d, J 7Hz, 1-H of glucose unit). the methylated sugars shown in Table 2.

(10)

Anagallisin

1-H

1-H

5H2,

- 'H

unit),

The

6.5Hz,

(lH, d, J

(9)

Permethylate arabinose

(CDC13) 6 4.20

as shown

this permethylate

unit).

J -

d, J unit).

sugars

4.40

unit),

'H NMR

unit),

(lH,

Permethylate

0.3

1-H

shown

(8) - 'H NMR

4.35

of

-

liberated

unit),

with

(CDC13) 6 4.18

(10)

acetates. (7)

lated

(9) (3.6 mg),

mg),

(lH, d, J 7Hz, unit). On

Permethylate

lysis

(8) (7.2

5227

oligoglycosides

MS,

10.28;

3.70,

3.98

(2H,

m/z 472 (M+), 454 -C30H4805 requires

5228

S. B. Iikuwmeral.

compound

(21)

(1 ml)

at

water

acetate

(25),

(M+-AcOH),

525

2.0

Me),

(2H, each

4.73

I

(1 g) which

1.20

Ac20

Usual

cm-l

(M+-CH20Ac

(3H,s),

furnished

(2 ml) work

and

up

pyridine

afforded

the

(CO): MS

m/z 598 (M+), 538 -'H NMR (cDc13)60.84

- ACOH);

(3H,s),

2.04

(3H,s)

1.24

(3H,s)

(together

3

(together

x

OAc),

6

3.72,

(2)

from

436

(120 mg)

anagallisin acetone

A

in

MS, -m/z

(M+- 2H20,

25),

with

Bu"OH

(10 ml)

(1) furnished

microneedles,

mp

472 (M+, 12%),

454

248

and

Na

anagalligenone

(ion a, la),

264-266"C, (M+- H20,

217

E),

(a - - CH20H,

(2, 55).

'H

of

compound

acetate

(23)

(3H,s),

3.46

(2H, ABq,

as an

(lH,

d,

Ac20-pyridine

amorphous

(3H,s),

1.26 (3H,s)

J 12H2, -

with

(20)

(CDC13)60.84

NMR

1.24

xOAC),

B

for

in CHC13):

44),

the

CHC13);

anagallisin

(c 0.24

Acetylation

2

2.02

crystallised

100) and 223

3.90

465 (3H,s),

desscribed

(M+- CH20,

(3H,s)r

1.06

of

as

[al, - 9.16" 442

1705

(OAc),

h.

(lH,

Hydrolysis

(20)

1240

with

1

dr J 12H2, 28-H2), 3.94, 4.49 (2H, each d, J 12H2, 23-H2), each d, J 5, lOHz), 5.44 (lH, t-like, 12-H) (Found : H, 9.05; C36Hs407 requires C, 72.21, H, 9.09%).

4.84

C, 72.24:

metal

acetylated for

(M+-CH20Ac),

(3H,s),

3.85

was

temperature

IR 1735,

0_86(6H,s),

(3H,s), x

(20 mg)

bath

J EHz, CH20Ac),

solid,

0.88

(together

in

[a]D

(3H,s), 6

x

the

- 6.6"

0.94

Me),

usual

way

(c 0.6

(3H,;),

2.04,

in

1.04

2.08 (each

s,

28-H), 3.80 (lH, d, J EHz, 28-H), 3.74, 4.72, 4.84 (d each, J 5, 9Hz, 3 - axial

-H). Anagallisin

C

microneedles

(3) - The mp

not

available

and

l3 C

NMR)

compound

from

found

desglucoanagalloside

B5.

Anagallisin

(4))

(4)

with

llisin Na-metal none

D

D-xylose (4)

and

from MeOH

to yield

(4)

58.85:

HI

(15 ml)

aq.

Bu"OH

Bu"OH

in MeOH)

7.98%). MeOH

sugar

and

by

(12 ml)

(10 ml)

with

crystallized

6.9' (c 0.4

L-arabinose

in

agreement

those

from (Found

Hydrolysis

yielded

PC

and

on

at 95°C

GLC.

for

as

: C,

58.79:

of

microH,

anagallisin

-12-ene-3,

23,

indentified

Hydrolysis

treatment

for

MeOH

olean

constituents

reported

with

a

36 h furnished

of

as anaga-

solution

of

anagallige-

(20) (8 mg). Permethylation

mg)

C,

(21)

(60 mg)

(1 g) with

-

ZM-HCl

28-trihydroxy-16-one

in good

compound

[a],

requires

(80 mg)

D-glucose,

to be

- The

256-26O"C,

C52H84022

8.0: D

D

mp

crystallised

(c - 0.5 MeOH). The mp and [a], were the spectroscopic data (FAB-MS However,

literature.

were

prisms,

(13) was

[aID - 3.2"

236-238'C,

and

powder

MeI (22

(7 ml) mg)

'H

of in NMR

compound the

(4)

usual

(CDC13)6

(50 mg) way

4.32

using

yielded (lH,

d,

a J

HMPA

(7 ml),

permethylate 5H2,

1-H

NaH (24)

of

(320 as

arabinose

a

5229

Triterpemoid oligoglycosides

4.37

unit),

(lH,

unit)

1-H of glucose Hydrolysis (5 ml)

on

acetates

water

bath

E

: c, 60.6:

(5)

under

of

Compound the

4.22

(lH,

unit),

4

(55

Bloomfield,

of

the

Research

h yielded

-

(60

sugars

from

(5 0.4

Institute for

as

as

ZM-HCl

in aq.

of

this

'H

powder,

(35

Dr.

(18

mg)

and

previously

to

(12

positive

negative NMR

as

Schering

FAB-MS,

the

(CDCl,)a

7Hz,

1-H

described

FAB-MS,

spectrum for

NMR

J

of

earlier

2.

Pramanik,

B.

the

Centre,

mg)

in Table

Institute

Instrumentation

elemental

(Found

MeOH

A

described

white

(16)

as shown

for 13 C

the

as

constituents.

mg)

for

MeOH

in MeOH)

23-hydroxyprimulagenin

thank

U.S.A.

alditol

H, 8.44%).

with

Permethylated

was

up

2.

6.8O

(65 mg)

as the sugar

We

Dastidar

Sophisticated

(5)

in aq. MeOH

work

crystallised

C, 60.62;

permethylkate

Jersey,

University,

Ghosh

E

(16)

-

New

Food

Calcutta

[ 0 1,

requires

mg)

the methylated

Acknowledgements

and some

224-226OC,

(5)

2M-HCl

usual

in Table

7H2,

unit).

1-H of xylose

after

compound

(lH, d, J

4.24 (lH, d, -J 5Hz, 1-H of arabinose), 4.38 (lH, d, J 6.5Hz, 1-H of glucose unit).

Hydrolysis furnished

shown

mp

4.42

(15 mg) with

yielded

The

permethylate d,

h

-

for

(5)

(24)

sugars

and L-arabinose

yield

3

anagallisin

reflux

D-glucose

P.P.

for

(90 mg)

unit),

(lH, d, 3 6H2,

H, 8.4: C47H78018

Hydrolysis

AFRC

4.70

of the methylated

microneedles

glucose

and

of glucose

1-H

6.5Hz,

of the permethylate

Anagallisin

ml)

d, J

Prof.

of

Research, K.R.

A.

Price,

Banerjee,

anagalligenone,

NMR

Lucknow,

Dr.

spectra

for

a

few

and

Mr.

Regional

mass

spectra

analysis.

REFERENCES

2.

Amoros,

M.; Fauconnier,

B.; Girre, B.; Girre,

~01.1,

ed

Wealth

Manjunath, Delhi,

B.L.:

"The

1.

P-75

3.

Amoros,

M.;

4.

Amoros,

M.; Girre,

5.

Glombitza,

6.

Chen,

7.

Ogihara,

8.

Heitz,

9.

Y.: Nose,

Stothers, New York

10. Pal,

J.B.

H. Planta

M.: Ogihara,

S.; Billet,

L.

Raww

materials"

M. J-

"Carbon-13

1987 -26, 787 1987 548

Pharm.

SOC. Chem.

D.; Raulais, NMR

Pharm.

medica

Y. Chem.

Chem.

Ann.

D. Bull.

Bull.

Commun.

1987 354, 1653 1986

Sot. Chim.

Spectroscopy",

1417

Fr. 1971 2320

Academic

Press,

(1972)

B.C.: Roy,

CSIR,

Fr. 1977 -35, 371 L. Pl. Med. Phytoh. 1979 15, 122

R.L. Phytochemistry

Kurth,

Y.; Nose,

India,

(1948)

Fauconnier,

K.W.:

of

G.: Mahato,

S.B. Phytochemistry

1984 -23, 1475

New

S. B. h&UIATOetd.

11.

Williams,

12.

Fenselau,

13.

Mahato,

L.C.E.

Perkin 14.

Seo,

D-H.;

Bradley,

J. Am. Chem.

Sot. 1981

C. J. Nat. Prod. S.B.;

Sahu, N.P.:

Trans.

I 1989 2065

S.; Tomita,

Bojesen,

C.;

S.:

Taylor,

103, 5700

1984 47, Roy,

Y.; Tori,

Santikarn,

G.;

215

S.K.; Pramanik,

K.; Yoshimura,

B.N. J. Chem.

Y. J. Am.

Chem.

Sot.

Sot.

1978

100, 3331 15.

Kasai,

R. :

Okihara,

0.;

Asakawa,

1979 -35, 1427 T.: Kitatani, H.: Hinoh,

J.;

Mizutani,

K.:

Tanaka,

0.

Tetrahedron 16.

Kubota,

17.

Jensson,

P-E.:

Kenne,

J. Chem.

Commun.

Univ.

18.

Lonngren,

J.; Pilotti,

L.:

H.: Tetrahedron

Liedgren,

Stockholm, A. Acta

H.:

1976 No.8,

Chem.

Stand.

Lett.

Lindberg,

1969 771 B.:

23 1971 -25,1144.

Lonngren,