- Email: [email protected]
Violaxanthin esters from Viola tricolor flowers
23X
Il.
Baltaev.
U.,
Abubakirov, 12.
Horn,
Gorowitz, N. K. (1978)
M.
B..
Rashkes.
Ja.
V.
and
Khrm. Prrr. Sordirt. 463.
D. H. S.. Wilkie, J. S., Sage, B. and O’Connors.
Hegnauer, Hiller.
T., Ogawa.
Phlrrnrtrzie 32. 365.
S.. Nishimoto,
N.. Arihara.
S. and
yu;ukugolruZtrs\lri 87. 1414.
E. D. and Wilson. 1. D. (19X0) Progress if1Ed.ww
Morgan,
17.
Resrurch
(Hoffmann.
J. A..
ed.)
pp.
29 43.
Elsevier,
.4msterdam.
Basel.
K.. Keipert.
K. and Voigt. G. (1977)
Bue. K. (1967)
R. (1964) Ch~~~l~~ru.~~~~l~)tn~~, tier PP~trrzrrr. Vol. 3. p.
409. Birkhauser, 14.
Hiller,
Takemoto,
J. D.
(1976) J. Inw(./. Phy.siol. 22, 901. 13.
15. 16.
M. and Linzer. B. (1966) Phi~rmtr-_ie 21.
713.
VIOLAXANTHIN
ESTERS
FROM
P. HANSMANN Institut fur Biologie II, Zellbtologre.
Key
Word Index-Ciolu
rricohr:
Universitat
Violaceae;
I/IO,!,/4TRZCOLOR FLOWERS H. KLEINIG
and
Freiburg.
vlolaxanthm
Schamlestr.
I, D-7X00
ester: /I-hydroxy
Frcihurg.
W’. Gcr-many
acids.
-The pattern of fatty acid esters of violaxanthin and the minor xanthophylls in the petals of 1 iol~ rricolor (yellow varieties) is unusually complex. This is due to the fact that P-hydroxy acids I 12:O. IA:& 16:O) take part in the esterification in addition to the usual acids (12:0, 14:0, 16:O. 18:O). Abstract
The xanthophylls in flower petals are usually esterified with saturated Cl2 C,, acids ([1~~3]; for review see [4]). The yellow varieties of T/i& tricolor contain as their main pigment violaxanthin (ca 75 ‘I, of total carotenoid) which occurs in an unusually complex ester pattern as has been shown by TLC [5]. In the present investigation the acid moieties of these violaxanthin esters have been analysed. The source of carotenoid esters were whole petals or isolated lipid globules (plastoglobuli) in which the esters are mainly localized [6]. Violaxanthin diesters were the main components while monoesters and free violaxanthin only occurred in trace amounts. The diesters were separated by Si gel TLC into three fractions in comparable concentrations. The correspondmg fatty acids upon saponification or transmethylation of these fractions were the usual saturated acids in the less polar fraction, saturated and. additionally, unknown more polar acids in the middle fraction in a molar ratio of (‘0 1: 1, and exclusively polar acids in the polar fraction. The saturated acids were 12:0, 14:O. 16:O. and 18:O acids as identified by CC. The polar acids were I(-hydroxy 12:O. 14:0, and 16:O acids as revealed by MS and ‘H NMR. This means that in the three ester fractions the two hydroxyl groups of violaxanthin were either both esterified with the usual acids, or one hydroxyl with a usual acid and one hydroxyl with a P-hydroxy acid, or both hydroxyls with fl-hydroxy acids. respectivrely. The /Ihydroxy group itself was not esterified as was shown by acetylation. The minor xanthophylls of t&~/a tricolor, lutein, lutein epoxide, and neoxanthin. apparently showed corresponding ester patterns. This is to our knowledge the first report of xanthophyli /I-hydroxy acid esters from flower petals.
I iolrrrrlcrdruL.
Flower petals or lipid globules -6 from were extracted
mu.xitm~
with acetone. Violaxanthin
separated by Si gel TLC
using petrol
free fatty acids with C‘HCI,
EtOAc
acid methyl esters with petrol performed
using ethanolic
pyrrdine using Ac:O.
using
CH,N?.
esters
2.4m
x 3.2mm.
(NH,j.
were
by
wI(s
done
methylatmn by
in
Gc‘
usrng
(column,
isothermal
at 11IO’,
1ID).
NH,;’
(46),241
from C‘ri
group
(100).169
:
-7: (34).
from
MeOH]’
from GIL (5).
‘1~ NMR
(300MH~.
C‘,&
-M d H -~ H,O
{ ’ from C‘,,, ‘- from C,’
[Yl -t-I:’
from GIL (291. 209 [\I (XI.
’
from C‘, ,) (2 I. 276
fromC‘,,(66).231
+ H ~ HLO_’
-~ MeOH]-
(3 l-1. \. COOMe).
+ Ii
from C‘rJ (JO). 73X [M i NH,
IM 4 H - H,O].
(AB-part
by the McLaffcrty
pr-obe) ?OOeV. 1)) r (rel. Int.): 30-1 [M $ NIH,:
Crz (IO). 713 [M - Hz0
(1001.7-I. formed
group
triggered by the ester carbonyl
(4). 159 [M -C H]
-
or
separated
methyl ester (7). 2X7 [M
from C,,,
t
wa\
(probe) 7OeV. jr) r (rel. 111~). 103. formed by r-cleavage
rearrangement
[M
and fatty
(7: I ). Saponification
packed with IO”,, FGSSX.
to the hydroxy CIMS
(IO:?:
Acctylatton
N,rOhle
carrrcr gas Nz at 3Oml mm EIMS
Et,0
KOH.
1I,
MclCO (40:6:3).
Fatty acid methyl esters were prepared by
transmethylation Methyl
Et10
HCO:H
subsp.
esters were
IXI
[M
from + H
t H ~ HZ0
CDC‘I,?): 0 3.008 (I H. h,_ \. H,,). 3.716
2.X23 (I H. II. J = 3 I-IL.C>Il), 2.514 and 1.410
of ABX-spectrum.
1 H each. (‘1~1: (x). .I ,,, = 16.2 Hf.
J \\ = 3 Hr. J,,, = 9 H/ (X =z H,j ). chemical shaft and large value of J ,II is indicative l’or the x-position to the carbonyl). (‘(I 1.53 (I H).ca
1.43 (2 H)andc’rr
I.258 (<‘ii 17 I-1. CHZ
tr, J z 6.X Hr. Me). t!pon OH-signal
disappeared
slightly sharper.
addition
CH,).O.XXO
(3 H.
of DLO to the solution the
and the multiplet
signal of H,, became
239 Acknowledgements-We are indebted to Dr. G. Englert and W. Meister, F. Hoffmann-La Roche, Basel, for recording and interpretation of theNMR and MS. This work was supported by Deutsche Forschungsgemeinschaft (SFB 46, Universitit Freiburg). REFERENCES
3. Kleinig, H. and Nitsche, H. (1968) Phytochemistry 7, 1171. 4. Goodwin, T. W. (1980) The Biochemistry of the Carotenoids Vol. 1. Chapman & Hall, London. 5. Nitsche, H. (1966) Staatsexamensarbeit UniversitLt, Heidelberg. P. (1980) Diplomarbeit Universitlt, Freiburg. 6. Hansmann, 7. Ryhage, R. and Stenhagen, E. (1960) Ark. Kemi 15, 545.
1. Egger, K. (1964) Ber. Dtsch. Ges. 77, 145. 2. Egger, K. (1968) Z. Naturforsch. 23b, 733.
Phytochemistry, Vol. 21, No. Printed in Great Britain.
1, pp. 239-240,
1982.
0031-9422j82/010239%02 C 1982 Pergamon
$03.00/O
Press Ltd.
A TRIGLUCOSIDE OF HECOGENIN FROM FRUITS OF AGAI/E CANTALA I. P.
VARSHNEY, D. C. JAIN and H. C. SRIVASTAVA*
Department of Chemistry, University of Garhwal, Srinagar (Garhwal) 246174, India; * Chemistry Division, Ahmedabad Textile Industry’s Research Association, Ahmedabad 380015, India (Received 3 March 1981)
Key Word Index--Agaue
cantala; Agavaceae; saponin; hecogenin triglucoside.
Abstract-The fruits of Agaue cantala contain a steroidal saponin which is a glycoside of hecogenin with three molecules of glucose. Its structure has been established as a-D-glucopyranosyl(1 + 4)cc-D-glucopyranosyl(1 + 4)a-Dglucopyranosyl( 1 + 3)hecogenin.
A review of the literature showed that while leaves of different Agaue species contain a number of steroidal saponins [l j no work has been reported on the fruits of these plants. The blackish fruits of Agaue cantala were therefore collected from Srinagar-Garhwal, and dried and powdered. The defatted powder was extracted with ethanol which was concentrated to give a cream-coloured syrupy mass, which was purified in the usual manner [2j. The light cream-coloured powder obtained on TLC showed the presence of one major component, which was finally purified by column chromatography. The purified substance (1) had mp 240-243”, [x]~ + 95.5” (c, 1% MeOH), and its IR spectra showed bands at 860, 900, 920 and 980cm-’ indicating that ring F is closed and there is no sugar attached to C-26. On acid hydrolysis (2N H2S04) it yielded a genin (44.8 ‘;/,), identified as hecogenin by mmp and co-chromatography with an authentic sample. The hydrolysate after neutralization was deionized with ion-exchange resins [Amberlite IRA 400 and IR 120 (H+)]. The solution thus obtained was concentrated to a syrupy mass which by PC, GLC (silyl derivative) [3] and HPLC showed only the presence of glucose. The molar ratio of aglycone:glucose was 1:3 based on genin weight. In order to determine the sequence and mode of linkage of sugars 1 was completely methylated and this was confirmed by IR spectra. The permethylated saponin was subjected to methanolysis and the resulting methyl glycosides of the methylated sugars thus obtained were identified by GLC as 2,3,4,6-tetra-O-methyl-cc-I,-glucose
(1 mol),
and 2,3,6-tri-O-methyl-cc-D-glucose (2 mol). On enzymic hydrolysis with p-glucosidase, no sugar was liberated indicating that the glucose molecules are LXlinked. On treatment with NaI04 no free sugar was liberated, suggesting that all three glucose molecules are linked in such a manner that they are all oxidized by periodate. The molecular rotation calculations of the saponins based on Klyne’s rule [4j also support the alinkage of the sugars. The observed (M), value, + 874.78, is in reasonable agreement with the calculated (M), value, + 882.38. Thus on the basis of above results 1 is identified as IX-Dglucopyranosyl( 1 -+ 4)a-D-glUCOpyranOSyl( 1 + 4)cr-Dglucopyranosyl( 1 + 3)hecogenin.
EXPERIMENTAL All specific rotations are equilibrium values. Whatman filter paper No. 1 was used for PC: for sugars the solvents were n-BuOH-pyridine-H,O (6:4:3) or EtOAc-pyridine-H,O (22: 10: 1) and sprayed with p-anisidine HCl or alkaline AgNO,. TLC of 1 was carried out on Si gel in n-BuOH-HOAc-H,O (4:1:5) or CHCl,-MeOHpHZO (13:7:2). The sprays were 5% H2S04 or cinnamaldehyde reagent. CC was carried out using 8% OS-138 on Chromosorb-W (NAW) column with N2 and FID. HPLC was carried out using column-p Bondapak/carbodydrate, solvent system acetonitrile-H,O (4: 1). Isolation and pur[fication of 1. Dried and powdered fruits of cantala collected from plants growing in Agave Srinagar-Garhwal were defatted (petrol a-60”), followed by
Il.
Baltaev.
U.,
Abubakirov, 12.
Horn,
Gorowitz, N. K. (1978)
M.
B..
Rashkes.
Ja.
V.
and
Khrm. Prrr. Sordirt. 463.
D. H. S.. Wilkie, J. S., Sage, B. and O’Connors.
Hegnauer, Hiller.
T., Ogawa.
Phlrrnrtrzie 32. 365.
S.. Nishimoto,
N.. Arihara.
S. and
yu;ukugolruZtrs\lri 87. 1414.
E. D. and Wilson. 1. D. (19X0) Progress if1Ed.ww
Morgan,
17.
Resrurch
(Hoffmann.
J. A..
ed.)
pp.
29 43.
Elsevier,
.4msterdam.
Basel.
K.. Keipert.
K. and Voigt. G. (1977)
Bue. K. (1967)
R. (1964) Ch~~~l~~ru.~~~~l~)tn~~, tier PP~trrzrrr. Vol. 3. p.
409. Birkhauser, 14.
Hiller,
Takemoto,
J. D.
(1976) J. Inw(./. Phy.siol. 22, 901. 13.
15. 16.
M. and Linzer. B. (1966) Phi~rmtr-_ie 21.
713.
VIOLAXANTHIN
ESTERS
FROM
P. HANSMANN Institut fur Biologie II, Zellbtologre.
Key
Word Index-Ciolu
rricohr:
Universitat
Violaceae;
I/IO,!,/4TRZCOLOR FLOWERS H. KLEINIG
and
Freiburg.
vlolaxanthm
Schamlestr.
I, D-7X00
ester: /I-hydroxy
Frcihurg.
W’. Gcr-many
acids.
-The pattern of fatty acid esters of violaxanthin and the minor xanthophylls in the petals of 1 iol~ rricolor (yellow varieties) is unusually complex. This is due to the fact that P-hydroxy acids I 12:O. IA:& 16:O) take part in the esterification in addition to the usual acids (12:0, 14:0, 16:O. 18:O). Abstract
The xanthophylls in flower petals are usually esterified with saturated Cl2 C,, acids ([1~~3]; for review see [4]). The yellow varieties of T/i& tricolor contain as their main pigment violaxanthin (ca 75 ‘I, of total carotenoid) which occurs in an unusually complex ester pattern as has been shown by TLC [5]. In the present investigation the acid moieties of these violaxanthin esters have been analysed. The source of carotenoid esters were whole petals or isolated lipid globules (plastoglobuli) in which the esters are mainly localized [6]. Violaxanthin diesters were the main components while monoesters and free violaxanthin only occurred in trace amounts. The diesters were separated by Si gel TLC into three fractions in comparable concentrations. The correspondmg fatty acids upon saponification or transmethylation of these fractions were the usual saturated acids in the less polar fraction, saturated and. additionally, unknown more polar acids in the middle fraction in a molar ratio of (‘0 1: 1, and exclusively polar acids in the polar fraction. The saturated acids were 12:0, 14:O. 16:O. and 18:O acids as identified by CC. The polar acids were I(-hydroxy 12:O. 14:0, and 16:O acids as revealed by MS and ‘H NMR. This means that in the three ester fractions the two hydroxyl groups of violaxanthin were either both esterified with the usual acids, or one hydroxyl with a usual acid and one hydroxyl with a P-hydroxy acid, or both hydroxyls with fl-hydroxy acids. respectivrely. The /Ihydroxy group itself was not esterified as was shown by acetylation. The minor xanthophylls of t&~/a tricolor, lutein, lutein epoxide, and neoxanthin. apparently showed corresponding ester patterns. This is to our knowledge the first report of xanthophyli /I-hydroxy acid esters from flower petals.
I iolrrrrlcrdruL.
Flower petals or lipid globules -6 from were extracted
mu.xitm~
with acetone. Violaxanthin
separated by Si gel TLC
using petrol
free fatty acids with C‘HCI,
EtOAc
acid methyl esters with petrol performed
using ethanolic
pyrrdine using Ac:O.
using
CH,N?.
esters
2.4m
x 3.2mm.
(NH,j.
were
by
wI(s
done
methylatmn by
in
Gc‘
usrng
(column,
isothermal
at 11IO’,
1ID).
NH,;’
(46),241
from C‘ri
group
(100).169
:
-7: (34).
from
MeOH]’
from GIL (5).
‘1~ NMR
(300MH~.
C‘,&
-M d H -~ H,O
{ ’ from C‘,,, ‘- from C,’
[Yl -t-I:’
from GIL (291. 209 [\I (XI.
’
from C‘, ,) (2 I. 276
fromC‘,,(66).231
+ H ~ HLO_’
-~ MeOH]-
(3 l-1. \. COOMe).
+ Ii
from C‘rJ (JO). 73X [M i NH,
IM 4 H - H,O].
(AB-part
by the McLaffcrty
pr-obe) ?OOeV. 1)) r (rel. Int.): 30-1 [M $ NIH,:
Crz (IO). 713 [M - Hz0
(1001.7-I. formed
group
triggered by the ester carbonyl
(4). 159 [M -C H]
-
or
separated
methyl ester (7). 2X7 [M
from C,,,
t
wa\
(probe) 7OeV. jr) r (rel. 111~). 103. formed by r-cleavage
rearrangement
[M
and fatty
(7: I ). Saponification
packed with IO”,, FGSSX.
to the hydroxy CIMS
(IO:?:
Acctylatton
N,rOhle
carrrcr gas Nz at 3Oml mm EIMS
Et,0
KOH.
1I,
MclCO (40:6:3).
Fatty acid methyl esters were prepared by
transmethylation Methyl
Et10
HCO:H
subsp.
esters were
IXI
[M
from + H
t H ~ HZ0
CDC‘I,?): 0 3.008 (I H. h,_ \. H,,). 3.716
2.X23 (I H. II. J = 3 I-IL.C>Il), 2.514 and 1.410
of ABX-spectrum.
1 H each. (‘1~1: (x). .I ,,, = 16.2 Hf.
J \\ = 3 Hr. J,,, = 9 H/ (X =z H,j ). chemical shaft and large value of J ,II is indicative l’or the x-position to the carbonyl). (‘(I 1.53 (I H).ca
1.43 (2 H)andc’rr
I.258 (<‘ii 17 I-1. CHZ
tr, J z 6.X Hr. Me). t!pon OH-signal
disappeared
slightly sharper.
addition
CH,).O.XXO
(3 H.
of DLO to the solution the
and the multiplet
signal of H,, became
239 Acknowledgements-We are indebted to Dr. G. Englert and W. Meister, F. Hoffmann-La Roche, Basel, for recording and interpretation of theNMR and MS. This work was supported by Deutsche Forschungsgemeinschaft (SFB 46, Universitit Freiburg). REFERENCES
3. Kleinig, H. and Nitsche, H. (1968) Phytochemistry 7, 1171. 4. Goodwin, T. W. (1980) The Biochemistry of the Carotenoids Vol. 1. Chapman & Hall, London. 5. Nitsche, H. (1966) Staatsexamensarbeit UniversitLt, Heidelberg. P. (1980) Diplomarbeit Universitlt, Freiburg. 6. Hansmann, 7. Ryhage, R. and Stenhagen, E. (1960) Ark. Kemi 15, 545.
1. Egger, K. (1964) Ber. Dtsch. Ges. 77, 145. 2. Egger, K. (1968) Z. Naturforsch. 23b, 733.
Phytochemistry, Vol. 21, No. Printed in Great Britain.
1, pp. 239-240,
1982.
0031-9422j82/010239%02 C 1982 Pergamon
$03.00/O
Press Ltd.
A TRIGLUCOSIDE OF HECOGENIN FROM FRUITS OF AGAI/E CANTALA I. P.
VARSHNEY, D. C. JAIN and H. C. SRIVASTAVA*
Department of Chemistry, University of Garhwal, Srinagar (Garhwal) 246174, India; * Chemistry Division, Ahmedabad Textile Industry’s Research Association, Ahmedabad 380015, India (Received 3 March 1981)
Key Word Index--Agaue
cantala; Agavaceae; saponin; hecogenin triglucoside.
Abstract-The fruits of Agaue cantala contain a steroidal saponin which is a glycoside of hecogenin with three molecules of glucose. Its structure has been established as a-D-glucopyranosyl(1 + 4)cc-D-glucopyranosyl(1 + 4)a-Dglucopyranosyl( 1 + 3)hecogenin.
A review of the literature showed that while leaves of different Agaue species contain a number of steroidal saponins [l j no work has been reported on the fruits of these plants. The blackish fruits of Agaue cantala were therefore collected from Srinagar-Garhwal, and dried and powdered. The defatted powder was extracted with ethanol which was concentrated to give a cream-coloured syrupy mass, which was purified in the usual manner [2j. The light cream-coloured powder obtained on TLC showed the presence of one major component, which was finally purified by column chromatography. The purified substance (1) had mp 240-243”, [x]~ + 95.5” (c, 1% MeOH), and its IR spectra showed bands at 860, 900, 920 and 980cm-’ indicating that ring F is closed and there is no sugar attached to C-26. On acid hydrolysis (2N H2S04) it yielded a genin (44.8 ‘;/,), identified as hecogenin by mmp and co-chromatography with an authentic sample. The hydrolysate after neutralization was deionized with ion-exchange resins [Amberlite IRA 400 and IR 120 (H+)]. The solution thus obtained was concentrated to a syrupy mass which by PC, GLC (silyl derivative) [3] and HPLC showed only the presence of glucose. The molar ratio of aglycone:glucose was 1:3 based on genin weight. In order to determine the sequence and mode of linkage of sugars 1 was completely methylated and this was confirmed by IR spectra. The permethylated saponin was subjected to methanolysis and the resulting methyl glycosides of the methylated sugars thus obtained were identified by GLC as 2,3,4,6-tetra-O-methyl-cc-I,-glucose
(1 mol),
and 2,3,6-tri-O-methyl-cc-D-glucose (2 mol). On enzymic hydrolysis with p-glucosidase, no sugar was liberated indicating that the glucose molecules are LXlinked. On treatment with NaI04 no free sugar was liberated, suggesting that all three glucose molecules are linked in such a manner that they are all oxidized by periodate. The molecular rotation calculations of the saponins based on Klyne’s rule [4j also support the alinkage of the sugars. The observed (M), value, + 874.78, is in reasonable agreement with the calculated (M), value, + 882.38. Thus on the basis of above results 1 is identified as IX-Dglucopyranosyl( 1 -+ 4)a-D-glUCOpyranOSyl( 1 + 4)cr-Dglucopyranosyl( 1 + 3)hecogenin.
EXPERIMENTAL All specific rotations are equilibrium values. Whatman filter paper No. 1 was used for PC: for sugars the solvents were n-BuOH-pyridine-H,O (6:4:3) or EtOAc-pyridine-H,O (22: 10: 1) and sprayed with p-anisidine HCl or alkaline AgNO,. TLC of 1 was carried out on Si gel in n-BuOH-HOAc-H,O (4:1:5) or CHCl,-MeOHpHZO (13:7:2). The sprays were 5% H2S04 or cinnamaldehyde reagent. CC was carried out using 8% OS-138 on Chromosorb-W (NAW) column with N2 and FID. HPLC was carried out using column-p Bondapak/carbodydrate, solvent system acetonitrile-H,O (4: 1). Isolation and pur[fication of 1. Dried and powdered fruits of cantala collected from plants growing in Agave Srinagar-Garhwal were defatted (petrol a-60”), followed by