\

PERGAMON Phytochemistry 49 "0888# 0300Ð0304

Alkaloids and other constituents from Tribulus terrestris

Tian!Shung Wua\\ Li!Shian Shib\ Shang!Chu Kuob\c
a
Department of Chemistry\ National Chen` Kun` University\ Tainan\ Taiwan\ R[O[C[
b
Graduate Institute of Pharmaceutical Chemistry\ China Medical Colle`e\ Taichun`\ Taiwan\ R[O[C[
c
National Research Institute of Chinese Medicine\ Taipei\ Taiwan\ R[O[C[

Received in revised form 19 October 0886

Abstract

Three new compounds\ terrestribisamide\ 14R!spirost!3!en!2\01!dione and tribulusterine\ together with 09 known compounds\
N!p!coumaroyltyramine\ terrestriamide\ hecogenin\ aurantiamide acetate\ xanthosine\ fatty acid ester\ ferulic acid\ vanillin\ p!
hydroxybenzoic acid and b!sitosterol\ were isolated and characterized from dried fruits of Tribulus terrestris[ Structures of these
compounds were determined by spectral analysis[ Þ 0888 Elsevier Science[ All rights reserved

Keywords] Tribulus terrestris ^ Zygophyllaceae ^ fruits ^ amide ^ steroid ^ alkaloid[

0[ Introduction immunosuppressive activities[ This led us to reinvestigate

{{Jili||\ the fruits of Tribulus terrestris\ is a famous tra!
ditional Chinese medicine[ In the Shern!Nong Pharma!
copoeia "the oldest known pharmacological work in
China#\ it is described as a highly valuable drug used to
restore the depressed liver for the treatment of fullness in
the chest and mastitis and also used to dispel the wind and
clear the eyes for the treatment of acute conjunctivitis\
headache and vertigo "Xie + Huang\ 0877#[ Tribulus ter!
restris is also reported to have antimicrobial\ anti!
hypertension\ diuretic\ antiacetylcholine and haemolytic
activity and to stimulate spermatogenesis and libido "Jit
+ Nag\ 0875^ Bose\ Sai_\ Vijayvargia\ + Bhatnager\ 0853^
Tomova\ 0877^ Sharma\ Norula\ + Varadarajan\ 0866#[
Alkaloids\ steroids\ ~avonoids and carbohydrates have
been isolated from this plant "Duan + Zhou\ 0882^
Chiang\ 0866^ Mahato\ Nianjan\ Ganguly\ Miyaharo\ +
Kawasaki\ 0870^ Saleh + El!Hadidi\ 0871^ Saleh\ Ahmed\
+ Abdalla\ 0871^ Mahato\ Shau\ Pal\ Chakravarti\ Chak!
ravaryi\ + Ghosh\ 0867^ Perepelitsa + Kintya\ 0864^
Tomova\ Gyulemetova\ Zarkova\ Peeva\ Pangarova\ +
Simova\ 0870^ Zafar\ Lalwani\ + Siddiqui\ 0878^ Chiu +
Chang\ 0875^ Nag\ Mathur\ + Goyal\ 0868^ Mahato\
Niranjan\ Ganguly\ Miyaharo + Kawasaki\ 0870^
Mathur\ Nag\ + Goyal\ 0866^ Vasi + Kalintha\ 0871#[
During our continuing search for novel bioactive natu!
ral products\ a crude extract of the dried fruits of T[
terrestris showed signi_cant anti!in~ammatory and
 Corresponding author[
its constituents[ We report here the isolation and struc!
tural elucidation of three new compounds and ten known
compounds from methanolic extracts of the dried fruits[
1[ Results and discussion
Terrestribisamide "0# was obtained as colourless
powder\ and its HRFAB mass spectrum indicated a
pseudomolecular formula C13H18N1O5[ The UV absorp!
tions at 208\ 183\ 123 and 119 nm were similar to ter!
restriamide "4#\ being characteristic of 2\3!dioxygenated
cinnamic acid derivatives "Ren\ Chen\ Yang\ + Zhu\
0883#[ The presence of hydroxyl and amide groups was
inferred by IR absorption bands at 0541 and 2389 cm−0
correlated with two D1O!exchangeable signals at d 6[88
"1H\ t\ J  4[1 Hz# and 8[3 "1H\ s# in 0H NMR spectrum[
The 0H NMR spectrum of 0 showed the presence of a
sharp singlet signal at d 2[67 "5H#\ due to the methoxyl
group and methylene groups at d 0[34 "3H\ m# and d 2[03
"3H\ m#[ The presence of disubstituted cinnamate groups
was con_rmed by ABX!type signals at d 5[65 "1H\ d\
J  7[9 Hz#\ 5[85 "1H\ dd\ J  7[9\ 0[4 Hz# and 6[98 "1H\
d\ J  0[4 Hz#\ together with ole_nic protons at d 5[39
"1H\ d\ J  04[5 Hz# and 6[18"1H\ d\ J  04[5 Hz# attribu!
table to H!6\ 6?\ 4\ 4?\ 5\ 5?\ 1\ 1? and 7\ 7?\ respectively[
The NOESY spectrum "Fig[ 0# of 0 showed a cross!peak
between the methoxyl group at d 2[67 and H!1\ 1? at d
6[98\ indicating that the methoxyl group was substituted
at C!2\ 2?[ These data were in agreement with those for

9920!8311:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[
PII] S 9 9 2 0 Ð 8 3 1 1 " 8 6 # 9 0 9 7 5 Ð 7
0301 Tian!Shung Wu : Phytochemistry 49 "0888# 0300Ð0304

Fig[ 0[ NOESY correlations for compounds 0 and 2[

 Tian!Shung Wu : Phytochemistry 49 "0888# 0300Ð0304 0302

the FAB mass spectrum which showed a parent peak at of 14S!spirost!3!en!2\01!dione "Domingaez\ Waston\
m:z 330 and a fragment ion at m:z 110[ Based on these Garcia\ + Martinez Davila\ 0874#\ except for the chemical
results\ we assigned structure 0 to terrestribisamide[ shift of the chiral centre at C!14[ The chemical shift of C!
14R!Spirost!3!en!2\01!dione "1# was isolated as an 14 and C!16 in the 02C NMR spectrum of 14S!spiroketal
optically active colourless powder[ The molecular for! saponins are d 16 and 05\ respectively\ but those of 14R!
mula C16H27O3 was determined by high resolution mass spiroketal saponins are d 29 and 06\ respectively "Agra!
spectrometry[ A positive reaction with the Leibermann! wal\ Jain\ Gupta\ + Thakur\ 0874#[ The relative ster!
Burchard reagent\ together with its molecular formula\ eochemistry of 1 was R as deduced from the chemical
indicated that 1 was a steroid[ The UV spectrum with an shift at d 29[0 and 06[0 of C!14 and 16[ These results
absorption maximum at 127 nm was characteristic of an suggested structure 1 for 14R!spirost!3!en!2\01!dione[
a\ b!unsaturated ketone system in the molecule[ The IR Tribulusterine "2# was obtained as yellowish powder\
spectrum exhibited absorption bands at 0695 and 0563 which gave a positive reaction with Dragendro} |s
cm−0 due to carbonyl and a\ b!unsaturated ketone\ reagent\ indicating it to be an alkaloid[ The
respectively[ Compound 1 has four hydrogen atoms less pseudomolecular formula was established as C05H02N1O3
than hecogenin "5# by comparison of their mass spectra[ by FAB mass spectrometry[ The UV spectrum showed a
The 0H and 02C NMR spectra of 1 were similar to those close resemblance to that of perlolyrine "Je}roys\ 0869#[
of 5\ except for the A!ring moiety[ The H!2 and H!4 Its IR spectrum showed bands at 2209\ 0511 and 0418
signals in 5 were absent in 1\ whereas that for H!3 in 5 cm−0\ for hydroxyl\ amine and aromatic groups\ respec!
was shifted to down_eld to d 4[66 "0H\ s# and that for H! tively[ The 0H NMR spectrum exhibited a signal at d
08 at d 9[89 in 5 down_eld shifted to d 0[16 in 1[ The 02C 8[25 "0H\ br\ s\ exchangeable with D1O# for NH[ Four
NMR spectrum showed down_eld shifts of H!2\ H!3 and mutually coupled aromatic protons at d 7[00 "0H\ d\
H!4 from d 69[7\ 26[7 and 33[5 in 5 to d 087[5\ 057[4\ J  7[9 Hz#\ 6[59"0H\ dd\ J  6[3\ 0[5 Hz#\ 6[46"0H\ td\
013[5 in 1\ forming an a\ b!unsaturated ketone moiety[ J  6[3\ 0[5 Hz#\ 6[18"0H\ ddd\ J  7[9\ 6[3\ 0[5 Hz#\
The 02C NMR spectrum of 1 "Table 0# was similar to that together with ortho!coupled signals at d 7[33 and 6[75
"J  4[5 Hz#\ indicated the presence of a 0!substituted!b!
carboline nucleous in the molecule[ The signals at d 6[10
and d 5[41 "each 0H\ d\ J  2[1 Hz# were attributed to H!
Table 0 4? and H!3? of the furan ring[ An aryl hydroxyl methyl
02
C NMR spectral data of 14R!spiro!3!en!2\ 01!dione "1#\ Hecogenin
proton appeared at d 3[73[ The NOESY spectrum "Fig[
"5# and 14S!spiro!3!en!2\01!dione "03#
0# showed a cross!peak of H!3? "d 5[41# with the hydroxyl
C 1 5 03 methyl signal "d 3[73# and H!4? "d 6[10#\ indicating the
connection between C!0 of b!carboline and C!1? of the
0 24[1 25[3 24[2 furan ring[ On the basis of the above results\ structure 2
1 21[2 20[9 21[8
was assigned to tribulusterine[
2 087[5 69[7 087[5
3 013[5 26[7 013[6 The known compounds\ N!p!coumaroyltyramine
4 057[4 33[5 057[4 "Okuyma\ Shibata\ Kawada\ Osoda\ + Noguchi\ 0875#\
5 22[4 17[1 22[6 terrestriamide "Ren et al[\ 0883#\ hecogenin "David\
6 20[2 20[2 20[0 Harold\ Christal\ + Karen\ 0889#\ aurantiamide acetate
7 23[1 23[2 23[3
"Banerji + Das\ 0864#\ xanthosine "Britmaier + Voelter\
8 43[3 44[3 43[5
09 27[5 25[9 27[6 0867#\ fatty acid ester\ ferulic acid "Wu\ Yeh\ + Wu\
00 26[9 26[7 26[0 0884#\ vanillin "Wu et al[\ 0884#\ p!hydroxybenzoic acid
01 100[8 102[5 100[8 "Wu et al[\ 0884# and b!sitosterol "Wu et al[\ 0884# were
02 43[6 44[9 43[7 isolated and characterized by comparison of their spectral
03 43[6 44[6 43[7 data with corresponding literature values[
04 20[9 20[4 20[0
05 67[8 68[0 68[0
2[ Experimental
06 42[3 42[3 42[2
07 04[7 04[8 04[8
08 05[6 00[8 05[8 Mps ] uncorr[ 0H NMR "199 and 399 MHz# and 02C
19 31[0 31[0 31[6 NMR "49 MHz# were recorded in CDCl2\ except where
10 02[0 02[1 02[9 noted[ Chemical shift values are given d with TMS as int[
11 098[1 098[1 098[6
standard[ MS were recorded using a direct inlet system[
12 20[9 20[0 14[7
13 17[5 17[6 15[9 UV were determined in MeOH and IR recorded in KBr[
14 29[9 29[0 16[9
2[0[ Plant material
15 55[7 55[7 54[1
16 06[9 06[0 05[9
Tribulus terrestris was bought from a market and ident!
 Data from "Domingaez et al[\ 0874#[ i_ed by Prof[ C[ S[ Kouh[ A voucher specimen is
0303 Tian!Shung Wu : Phytochemistry 49 "0888# 0300Ð0304
deposited in the Department of Chemistry[ National
Cheng Kung University\ Tainan\ Taiwan\ R[O[C[
2[1[ Extraction and isolation
A MeOH extract of dried fruits "09 kg# was treated
with CHCl2\ n!BuOH and H1O\ respectively[ The CHCl2
layer was concd to dryness and subjected to silica gel CC
eluting with CHCl2 containing increasing proportions of
Me1CO\ to give 7 frs[ Fr[ 2 was rechromatographed on
silica gel CC and eluted with EtOAcÐnÐhexane "0 ] 8# to
yield 5 "024[1 mg#\ 1 "65[3 mg#\ 6 "38[6 mg#\ 8 "6[1 mg#
and 02 "213[5 mg#\ respectively[ Fr[ 4 was rechro!
matographed on silica gel CC eluted with MeOHÐCHCl2
"0 ] 14# to a}ord 3 "6[7 mg#\ 4 "61[4 mg#\ 09 "4[5 mg# and
00 "2[1 mg#\ successively[ The n!BuOH layer was concd
and subjected to Diaion HP!19 CC\ eluting with a gradi!
ent of H1OÐMeOH to a}ord 5 frs[ Fr[ 1 was rech!
romatographed on silica gel CC using MeOHÐCHCl2
"0 ] 8#\ to yield 7 "262 mg# and unknown A "10 mg#[ Fr[ 3
was rechromatographed on silica gel CC and eluted with
MeOHÐCHCl2 "0 ] 19#\ to a}ord 3 "16[4 mg#\ 4 "019[2
mg#\ 0 "3[2 mg#\ 2 "9[3 mg#\ 01 "2[3 mg# and unknown B
"11[1 mg#\ successively[ The H1O layer was concd and
recrystallized from MeOH to yield 7 "2[68 g#[
2[2[ Terrestribisamide "0#
Colourless powder "MeOH#\ mp 032 ½ 024>C[
HRFABMS ] Calcd[ for C13H18N1O5\ 330[1915 ðM¦0Ł¦\
found 330[1901[ UV lmax nm ] 208\ 183\ 123\ 119[ IR
nmax cm−0 ] 2499\ 1816\ 0541\ 0405\ 0163[ FABMS m:z ]
330ðM¦0Ł¦\ 110\ 196\ 082\ 066\ 43\ 036\ 027\ 026\ 025\
019\ 096\ 80\ 89[ 0H NMR "DMSO!d5# ] d 0[34 "3H\ m\
H!1\2#\ 2[03 "3H\ m\ H!0\3#\ 2[67"5H\ s\ 1×OCH2#\ 5[39
"1H\ d\ J  04[5 Hz\ H!6?\ 6ý#\ 5[65 "1H\ d\ J  7[9 Hz\
H!4?\ 4ý#\ 5[85 "1H\ dd\ J  7[9\ 0[4 Hz\ H!5?\ 5ý#\ 6[98
"1H\ d\ J  0[4 Hz\ H!1?\ 1ý#\ 6[18 "1H\ d\ J  04[5 Hz\
H!7?\ 7ý#\ 6[88 "1H\ t\ J  4[1 Hz\ NH#\ 8[30 "1H\ br s\
OH#[
2[3[ 14R!Spirost!3!en!2\01!dione "1#
Colourless powder "CHCl2#\ mp 139Ð130>[ HREIMS ]
calcd[ for C16H27O3 315[1669 ðMŁ¦\ found 315[1656[
ðaŁD−23[7> "c 9[917\ CHCl2#[ UV lmax nm ] 127[ IR nmax
cm−0 ] 1816\ 0695\ 0563[ EIMS m:z ] 315ðMŁ¦\ 287\ 256\
243\ 201\ 158\ 028\ 015\ 58\ 44[ 0H NMR ] d 4[66 "0H\ s\
H!3#\ 3[23 "0H\ m\ H!05#\ 2[38 "0H\ dd\ J  09[7\ 1[6 Hz\
H!15#\ 2[22 "0H\ t\ J  09[7 Hz\ H!15#\ 1[45Ð0[94 "11H\
m#\ 0[16 "2H\ s\ H!07#\ 0[09 "2H\ s\ H!08#\ 0[95 "2H\ d\
J  6[9 Hz\ H!10#\ 9[67 "2H\ d\ J  5[07 Hz\ H!16#[ 02C
NMR ] Table 0[
2[4[ Tribulusterine "2#
Yellowish powder "CHCl2#\ mp 073Ð074>[ UV lmax
nm ] 267\ 256\ 180\ 173\ 161\ 144\ 126[ IR nmax cm−0 ] 2209\
1811\ 1741\ 0418\ 0266\ 0249\ 625[ FABMS "rel[ int[# m:z ]
154ðM¦0Ł¦ "09#\ 150 "00#\ 038 "00#\ 021 "099#\ 094 "31#[
0
H NMR ] d8[25 "0H\ br s\ NH#\ 7[33 "0H\ d\ J  4[5 Hz\
H!2#\ 7[00"0H\ d\ J  7[9 Hz\ H!4#\ 6[75 "0H\ d\ J  4[5
Hz\ H!3#\ 6[59 "0H\ dd\ J  6[3\ 0[5 Hz\ H!7#\ 6[46 "0H\
td\ J  6[3\ 0[5 Hz\ H!6#\ 6[18 "0H\ ddd\ J  7[9\ 6[3\ 0[5
Hz\ H!5#\ 6[10 "0H\ d\ J  2[1 Hz\ H!4?#\ 5[41 "0H\ d\
J  2[1 Hz\ H!3?#\ 3[73 "0H\ s\ CH1#[
Acknowledgements
The author is grateful for _nancial support from the
National Council of R[C[O[ "NSC 76!1003!M!995!901#[
This work was supported in part by grants from the
National Research Institute of Chinese Medicine[
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