\
PERGAMON
Phytochemistry 49 "0888# 0254Ð0257
Two picrotoxin derivatives from Anamirta cocculus
Santosh K[ Agarwal\ Sudhir S[ Singh\ Sushma Verma\ Sushil Kumar
Central Institute of Medicinal and Aromatic Plants\ P[O[ * CIMAP\ Lucknow 115 904\ India
Received in revised form 0 October 0887
Abstract
The berries of the plant Anamirta cocculus a}orded picrotin\ picrotoxinin\ methyl picrotoxate and two new sesquiterpene g!
lactones\ dihydroxypicrotoxinin and picrotoxic acid\ whose structures were determined by spectroscopic methods[ Þ 0888 Elsevier
Science Ltd[ All rights reserved[
Keywords] Anamirta cocculus^ Menispermaceae^ Sesquiterpene lactones^ Dihydroxypicrotoxinin^ Picrotoxic acid^ Picrotoxinin^ Methyl picrotoxate^
Picrotin
0[ Introduction
1[ Results and discussion
Anamirta cocculus "Linn[# Wight et Arn[\ syn[ Anamirta
paniculata and commonly known as Jahre!mahi is a liana
occurring in several regions of SouthÐeast Asia "Bentley
+ Trimen\ 0879#[ In India\ it is mainly found in Orissa\
Eastern Bengal\ Khasia hills\ Deccan "Cuddapah\
Malabar + Mysore#\ etc[ The berries have been used in
India to stupefy _sh and to poison crows and very small
quantities are su.cient for this purpose "Wealth of India\
0837^ Drury\ 0862#[ They have also been employed as
cattle poison "Hooper + Field\ 0826^ Venkatachalan\
0834#[ The use of berries in small quantities is described
in a number of pharmacopoeias "Osol\ Pratt\ + Altschule\
0859^ Morton\ 0866#\ though these are highly toxic and
fatal if consumed by humans[ The symptoms produced
are colic pains\ nausea\ vomiting\ tetanic convulsions and
occasionally delirium "Wealth of India\ 0837#[ The active
poisonous principle of the berries is picrotoxin\ a sesqui!
terpene mixture "Hegnauer\ 0858^ Taylor + Battersby\
0858^ Boullay\ 0879#[ Other reported compounds belong!
ing to this class are picrotoxinin "Corey + Pearce\ 0867#\
picrotin "Corey + Pearce\ 0879# and methyl picrotoxate
"Porter\ 0856^ Sarma\ Rambabu\ Anjaneyulu\ + Rao\
0876^ Pradhan\ Mamdapur\ + Sipahimalani\ 0889#[ The
present paper describes the isolation and structure of
two new minor sesquiterpene lactones of the picrotoxane
group from A[ cocculus seeds\ in addition to those pre!
viously reported[
Three known sesquiterpene lactones\ picrotoxinin "0#\
methyl picrotoxate "1# and picrotin "2#\ as well as two
new sesquiterpene lactones\ dihydroxypicrotoxinin "3#
and picrotoxic acid "4#\ were isolated and characterised
from the seeds of A[ cocculus compounds 0 and 2 were
the major constituents[
Dihydroxypicrotoxinin "3# was assigned the molecular
formula C04H07O7 "ðMŁ¦m:z 215 and elemental analy!
sis#[ Its IR spectrum showed the presence of two g!lactone
moieties at 0689 and 0659 cm−0 and primary and tertiary
hydroxyl groups at 2239 "broad# cm−0[
The 0H NMR spectrum exhibited two methyl singlets
at d 0[25 "C8ÐCH2# and 0[13 "C6ÐCH2# and the doublets
of a ÐCH1OH group at d 2[34 and 2[59[ Other signals are
listed in Table 0 and indicated that 3 belonged to the
picrotoxane group with two hydroxyls at C!7 and C!09
instead of the double bond present in picrotoxinin "0#[
The 02C NMR spectrum of 3 showed the presence of
04 carbons "Table 1# two of which were lactone carbonyl
carbons at d 065[7 and 062[4 and seven were oxygen!
bearing carbons[ 3 formed an acetonide "5# when reacted
with dry acetone and anhydrous copper sulphate indi!
cating the vicinal nature of the two hydroxyls[ Thus\ the
structure of 3 was established for dihydroxypicrotoxinin[
Picrotoxic acid "4# was assigned the molecular formula
C04H07O6 "ðMŁ¦m:z 209 and elemental analysis#[ The
presence of a g!lactone moiety\ a tertiary hydroxyl and a
free carbonyl group were revealed by IR bands at 0675\
2335 and 0605 cm−0 respectively[ The 02C NMR spec!
trum of 4 showed the presence of 04 carbons "Table 1# of
which two were carbonyl carbons at d 065[9 and 069[4[
Corresponding author[
9920!8311:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[
PII] S 9 9 2 0 ! 8 3 1 1 " 8 7 # 9 9 5 8 1 ! X
S[ K[ Agarwal et al[ : Phytochemistry 49 "0888# 0254Ð0257
0255
One of them was assigned to a g!lactone and the other to
a carboxyl group carbon[
In the 0H NMR spectrum\ singlets at d 0[10 and 0[61
were attributed to the usual tertiary methyl on C!0 and
the vinylic methyl on C!7\ in addition two singlets at d
3[77 and 3[74 represented the C!09 methylene and one
oxymethine proton at d 3[91 "Table 0#[
Acetylation of 4 with Ac1O and C4H4N yielded a
monoacetate "6# as indicated by the appearance of new
singlet at d 1[99 and the down_eld shift of the C!2 methine
signal[
Picrotoxinin "0# was treated with dil[ NaOH to give
picrotoxic acid as described in an earlier paper "Hathway\
0846#[ Synthetically prepared 4 was found to be identical
Table 0
0
H NMR data of compounds 0Ð4 and 6
H
0
1
2
3
4
6
1
2
3
4
5
6
8
09
3[83 d"2#
4[90 dd"4\2#
2[31 bs "W0:13#
1[82 d"4#
0[59 s
0[12 s
0[89 s
3[88 s
3[66 s
1[76 d"04#
1[91 dd"04\2#
2[69 d"2#
Ð
Ð
3[11 d"1#
2[71 d"1#
1[87 d"01#
1[79 d"01#
0[32 s
0[10 s
0[65 s
3[76 s
3[72 s
2[98 d"04#
1[08 dd"04\2#
3[06 d"2#
2[58 s
Ð
Ð
Ð
Ð
3[73 d"3#
0[43 s
0[19 s
0[39 s
0[39 s
3[49 d"2#
4[95 dd"4\2#
2[33 d"2#
1[77 d"3#
0[78 s
0[13 s
0[25 s
2[34 d"01#
2[59 d"01#
1[88 d"01#
1[96 dd"01\2#
3[19 d"2#
Ð
Ð
3[21 d"2#
3[91 dd"2\5#
2[39 bs "W0:13#
1[50 d"01#
0[37 s
0[10 s
0[61 s
3[77 s
3[74 s
1[77 d"04#
1[97 dd"04\2#
2[57 d"2#
Ð
Ð
3[89 d"2#
3[59 dd"2\5#
2[34 m
1[84 d"09#
0[38 s
0[12 s
0[60 s
3[78 s
3[74 s
1[84 d"04#
1[04 dd"01\2#
2[79 d"2#
Ð
1[99 s
00
01
05
06
1[75 d"04#
1[01 dd"04\2#
2[64 d"2#
Ð
Ð
S[ K[ Agarwal et al[ : Phytochemistry 49 "0888# 0254Ð0257
Table 1
C NMR data of compounds 0Ð4
02
C
0
1
2
3
4
0s
1d
2d
3d
4d
5s
6q
7s
8q
09 t
00 t
01 d
02 s
03 s
04 s
05 q
55[19
70[39
71[49
34[09
52[69
63[49
09[79
030[79
12[19
001[19
31[89
41[49
49[09
065[69
064[19
Ð
47[19
70[74
68[69
36[19
36[79
74[49
09[24
030[69
11[09
001[79
31[89
61[79
79[29
065[09
062[29
41[29
58[49
68[19
71[19
41[39
52[09
63[49
09[49
75[09
17[49
18[89
33[99
42[79
41[69
065[29
064[09
Ð
69[49
71[79
68[59
40[89
52[39
63[89
09[39
75[29
12[89
69[09
33[49
38[79
49[69
065[79
062[49
Ð
59[99
72[99
70[49
36[49
34[99
76[99
00[49
034[99
11[49
003[99
32[49
41[99
37[49
065[99
069[49
Ð
Carbon multiplicity by DEPT for compounds 0 and 1[
with naturally occurring picrotoxic acid by co!TLC\
mmp\ 0H NMR\ EIMS and IR[ Thus picrotoxic acid was
assigned as 4[
1[ Experimental
1[0[ General
Mps] uncorr[^ 0H and 02C NMR] Varian FT!79A "79
MHz#\ Bruker "199 MHz and 399 MHz#\ chemical shifts
in d "ppm# with TMS as int[ standard[ EIMS] 69 eV^ CC]
Silica gel "Ranbaxy\ 59Ð019 mesh#^ TLC] Silica gel G
"Ranbaxy#^ detection of spots by exposure to I1 vapour
and:or by spraying with 4) vanillinÐsulphuric acid soln[
followed by heating at 094> for 4 min[
1[1[ Plant material
The seeds of the A[ cocculus were purchased from
local market and identi_ed in our Botany Department\
where a voucher specimen has been maintained[
1[1[0[ Extraction and isolation
The air dried and powdered seeds "4[9 kg# were
extracted with MeOH "4×09[9 l#\ in a perculator at room
temp[ After _ltration\ the combined dark brown soln[
was evapd[ to dryness under red[ pres[ below 59>[ The
methanolic extract was obtained as a dark brown gummy
mass "179 g#[ A part of the MeOH extract "099 g# was
chromatographed on a silica gel column and eluted suc!
cessively with hexane\ hexaneÐEtOAc "1]0#\ "0]0#\ "0]1#\
"0]2# mixtures and EtOAc[ The eluates were monitored
by TLC and grouped into 8 frs[
Fr 1 eluted with hexaneÐEtOAc "0]0# was rechro!
0256
matographed on a silica gel column using hexaneÐ
EtOAc "2]0#\ "1]0#\ "0]0#\ "0]1# and "0]2# mixtures as eluate\
yielding compound 0 "mp 109Ð01>\ 299 mg#\ 1 "mp 063>\
59 mg# and 2 "mp 155Ð56>\ 0[2 g#\ with identical mp\ IR
values\ ðaŁD\ and 0H NMR to those of picrotoxinin\
methyl picrotoxate and picrotin\ respectively[
Fr 5 eluted with hexaneÐEtOAc "0]3# was rech!
romatographed on a silica gel column using hexaneÐ
EtOAc "0]0#\ "0]1#\ "0]2#\ EtOAc and EtOAcÐMeOH
"4)# mixtures as eluate yielding compounds 3 "39 mg#
and 4 "14 mg#[
1[1[1[ Dihydroxypicrotoxinin "3#
Colourless needles\ mp 159Ð51>^ ðaŁD06 −54> "c0[24 in
−0
] 2239 broad "ÐOH#\ 0689 and 0659
EtOH#^ IR nKBr
max cm
"for two g!lactones#\ 0017^ 0H NMR "199 MHz\ CD2OD#]
Table 0^ 02C NMR "49 MHz\ CD2OD#] Table 1^ EIMS
m:z "rel[ int[#] 215 ðMŁ¦"09#\ 183"00#\ 165"5#\ 149"19#\
121"21#\ 195"7#\ 074"03#\ 049"05#\ 024"34#\ 013"05#\
009"27#\ 86"16#\ 72"03#\ 64"84#\ 56"01#\ 44"57#\ 32"099#^
"Found] C\ 44[35^ H\ 4[75[ C04H07O7 requires] C\ 44[10^
H\ 4[41)#[
1[1[2[ Acetonide "5# of 3
3 "09 mg# in dry Me1CO "0[4 ml# and anhydrous CuSO3
"1×49 mg# were stirred together for 5 h[ The reaction
mixture was _ltered and the _ltrate was evaporated\ again
dissolved in CHCl2 and _ltered[ The _ltrate was con!
centrated and crystallised to give colourless needles "6
mg#\ mp 109Ð01>^ 0H NMR "79 MHz\ CDCl2#] d 0[15\
0[18 "1×CH2\ acetonide# and other proton signals^ EIMS
m:z "rel[ int[#] 223 ðMŁ¦ "14#[
1[1[3[ Picrotoxic acid "4#
−0
]
White crystalline needles\ mp 197Ð09>^ IR nKBr
max cm
2335 "t!OH#\ 0675 "for one g!lactone#\ 0605 "ÐCOOH#^
0
H NMR "399 MHz\ DMSO!d5#] Table 0^ 02C NMR "19
MHz\ DMSO!d5#] Table 1^ EIMS m:z "rel[ int[#] 209 ðMŁ¦
"01#\ 181"30#\ 162"13#\ 153"03#\ 136"05#\ 107"11#\ 075"16#\
058"18#\ 041"38#\ 027"52#\ 014"41#\ 000"099#\ 84"57#\
68"18#\ 56"48#\ 44"41#\ 33"54#^ "Found] C\ 47[23^ H\ 4[65[
C04H07O6 requires] C\ 47[95^ H\ 4[70)#[
0 "09 mg# was treated with 9[0 N NaOH "4 ml# in
MeOH "4 ml# at room temp for 14 h[ After removal of
excess NaOH with 9[90 N HCl\ the reaction product was
treated with Et1O and the organic layer seperated and
evaporated to yield picrotoxic acid "4\ 5 mg#\ identical
with that described above "by co!TLC\ mmp\ 0H NMR\
EIMS#[
1[1[4[ Mono!acetate "6# of 4
Compound 4 "4 mg# was dissolved in Ac1O "9[4 ml#!
C4H4N "9[4 ml# and left to stand at room temperature[
Usual workup yielded a colourless amorphous com!
−0
] 2395 "t!OH#\ 0685 "one g!
pound "6#^ IR nKBr
max cm
lactone#\ 0626 "ester C1O#\ 0608 "ÐCOOH#^ 0H NMR "79
0257
S[ K[ Agarwal et al[ : Phytochemistry 49 "0888# 0254Ð0257
MHz\ CDCl2#] Table 0^ EIMS m:z "rel[ int[#] 241 ðMŁ¦
"00#[
Acknowledgements
The authors are grateful to the Head\ RSIC at
CDRI\ Lucknow\ for spectral data[
References
Bentley\ R[\ + Trimen\ H[ "0879#[ Medicinal plants!I "Chap[ 03#[
Boullay\ P[ F[ G[ "0879#[ Ann[ Chim[ Phys[ "Paris#\ 198\ 0700[
Corey\ E[ J[\ Pearce\ H[ L[ "0867#[ J[ Am[ Chem[ Soc[ 090\ 4730[
Corey\ E[ J[\ Pearce\ H[ L[ "0879#[ Tetrahedron Lett[ 10\ 0712[
Drury\ H[ "0862#[ The useful plants of India "p[ 401#[ London] William
H[ Allen + Co[
Hathway\ D[ E[ "0846#[ J[ Chem[ Soc[ 3842[
Hegnauer\ R[ "0858#[ Chemotaxonomie der P~anzen "Vol[ 4\ p[ 77#[
Basel\ Stuttgart] Birkhaauser Verlay[
Hooper\ D[\ Field\ H[ "0826#[ Useful plants and drugs of Iran and Iraq[
Bot[ Ser\ Field Mus[ Nat[ Hist[ Chicago\ 8\ 130 Pub[ 276[
Morton\ J[ F[ "0866#[ Major medicinal plants "p[ 82#[ USA] Charles C[
Thomas Publ[
Osol\ A[\ Pratt\ R[\ + Altschule\ M[ D[ "0859#[ The US Dispensatory
and Physicians Pharmacology\ 0166[
Porter\ I[ A[ "0856#[ Chem[ Rews[\ 56\ 330[
Pradhan\ P[\ Mamdapur\ V[ R[\ Sipahimalani "0889#[ Indian J[ Chem[\
18B\ 565[
Sarma\ N[ S[\ Rambabu\ M[\ Anjaneyulu\ A[ S[ R[\ Rao\ C[ B[ S[ "0876#[
Indian J[ Chem[\ 15B\ 078[
Taylor\ W[ I[\ + Battersby\ A[ R[ "0858#[ In C[ J[ Coscia "Ed[#\ Cyclo
pentenoid terpene derivatives "p[ 036#[ New York] Marcel Dekker[
Venkatachalan "0834#[ Mon[ Bull[ Banglore Cattle Soc[\ 3\ 36[
Wealth of India "0837#[ PID\ CSIR "New Delhi#\ 0\ 64[