Isotamarixen - A New Antioxidant and Prolyl Endopeptidase-Inhibiting Triterpenoid from Tamarix hispida

Article abstract of DOI:10.1055/s-2004-815458

A new pentacyclic triterpenoid, 3α-(3″,4″-dihydroxy- trans-cinnamoyloxy)-D-friedoolean-14-en-28-oic acid (1) has been isolated along with two known compounds, rhamnocitrin (2) and isorhamnetin (3) from the aerial parts of Tamarix hispida Willd. Compound 1 was found to be a potent antioxidant. In addition, compounds 1 - 3 showed significant inhibitory activity against prolylendopeptidase (PEP).

Full text of DOI:10.1055/s-2004-815458

carbons ꢀC-2¢, C-3¢, C-1¢¢, C-2¢¢, C-3¢¢ to C-6¢¢), an ester carbonyl
C-1¢), a carboxyl carbon ꢀC-28) and one double bond carbon ꢀC-
14 and C-15) resonated at d = 115.5, 146.5, 127.4, 115.0, 146.4,
49.2, 117.7, 122.7, 168.0, 182.2, 161.3 and 116.2, respectively. The
Isotamarixen ± A New Antioxidant
and Prolyl Endopeptidase-Inhibiting
Triterpenoid from Tamarix hispida
ꢀ
1
position of the COOH-moiety at C-17 was deduced by a quatern-
2
1
1
2
ary carbon signal at d = 52.1 which was assigned to C-17. The
Nurgul Sultanova , Talat Makhmoor , Amsha Yasin , Z. A. Abilov ,
1
13
2
1
1
complete H- and C-NMR chemical shifts and multiplicities of
each protonated carbon are listed in Table 1. The HMBC long-
range coupling, data in which H-18 exhibited an interaction
with C-28 ꢀd = 182.2), further confirmed the location of COOH
at C-17. The H-3 ꢀd = 4.69) showed a long-range correlation
V. B. Omurkamzinova , Atta-ur-Rahman , M. Iqbal Choudhary
Abstract
A new pentacyclic triterpenoid, 3a-ꢀ3¢¢,4¢¢-dihydroxy-trans-cin- with the C-1¢ ꢀd = 168.0), indicating that the 3¢¢,4¢¢-dihydroxy-
namoyloxy)- -friedoolean-14-en-28-oic acid ꢀ1) has been isolated cinnamoyl group was located at C-3. The complete carbon-hy-
D
along with two known compounds, rhamnocitrin ꢀ2) and iso- drogen connectivities are presented in Table 1. To confirm the po-
rhamnetin ꢀ3) from the aerial parts of Tamarix hispida Willd.
Compound 1 was found to be a potent antioxidant. In addition,
compounds 1 ± 3 showed significant inhibitory activity against
prolylendopeptidase ꢀPEP).
Table 1 1H- and 13C-NMRdata of 1 *CD OD, CDCl )
3
3
C/H No
Compound 1
HMQC connectivity
dC
dH
The leaves of the plant Tamarix hispida Willd ꢀTamaricaceae) are
used in traditional medicine in the treatment of dysentery, rheu-
matism, and ulcers [1]. Our phytochemical investigation on T.
hispida led to isolation of a new triterpenoid with antioxidant
and enzyme inhibiting potential. It is the first report of isolation
of a triterpene from this species.
1
2
36.4
2H m 1.30
2H m 1.95
4.69 t *2.5)
±
H-25
c
23.5
79.4
38.3
51.7
19.4
42.0
40.0
50.2
37.1
18.2
34.1
37.6
161.3
116.2
32.8
52.1
42.7
34.6
30.4
34.3
32.1
28.4
22.2
15.7
26.6
22.8
182.2
32.5
29.3
168.0
115.5
146.5
127.4
115.0
146.4
149.2
117.7
122.7
*3)
3
H-23, H-24
4
H-3, H-23, H-24
5
1H 1.35
H-23, H-24, H-25
6
2H m 1.40; 1.60
2H m 1.40; 1.95
±
*5,7)c
H-26
H-26
H-26
H-25
*12)c
H-27
H-27
H-26, H-27
*16)c
*15)c
±
7
Compound 1 was isolated as a colorless amorphous solid with IR
absorptions at 1259 ꢀa,b-unsaturated ester) and 3411 ꢀhydroxy)
8
±
1
9
1H 1.55
±
cm . The UV spectrum showed absorptions at 216, 244, 255,
05 and 372 nm. The 1H-NMR signals of seven 3H singlets at
10
3
11
12
13
14
15
16
17
18
19
20
21
2H m 1.55; 1.65
2H m 1.6; 1.76
±
d = 0.87 ± 0.98 represented the seven tertiary methyls. An olefi-
nic proton appeared as a double doublet at d 5. = 5.57 ꢀ1H, dd,
J = 8.0 Hz, J = 3.5 Hz, H-15). The chemical shift, multiplicity,
coupling constants of the olefinic proton and chemical shifts of
the tertiary methyl signals were in agreement with those of tar-
axer-14-en skeleton [2]. The H-3 ꢀd = 4.69) was deduced to be in
a b conformation from the coupling constant ꢀJ = 2.5 Hz) of its
signal. The coupling relationship between aromatic proton sig-
nals, appearing at d = 7.03 ꢀ1H, d, J = 2.0 Hz, H-2¢¢), 6.76 ꢀ1H, d,
J = 8.0 Hz, H-5¢¢) and 6.92 ꢀ1H, dd, J = 8.0 Hz, J = 2.0 Hz, H-6¢¢),
indicated a 1,3,4-trisubstituted aromatic ring. Two 1H doublets
at d = 6.26 and 7.51 ꢀJ = 16.0 Hz) represented the trans-substi-
tuted olefinic H-2¢ and H-3¢, which indicated the presence of O-
6
5
±
5.57 dd *3.5;8)
2H m 1.96; 2.37
±
1H m 2.37
2H m 1.1; 1.24
±
H-27
H-29, H-30
H-29, H-30
H-29, H-30
*21)c
H-24
H-23
±
2H m 1.05; 1.6
2H m 1.5; 1.7
3H s 0.87
3H s 0.92
3H s 0.97
3H s 0.98
3H s 0.94
±
22
23
24
25
26
27
3
¢¢,4¢¢-dihydroxycinnamoyl group in 1. The a-position of dihy-
3
±
droxycinnamoyl group at C-3 was deduced by the small JH,H
2.5 Hz) of the equatorial H-3 signal. The 13C-NMR spectrum of
showed seven methyls, ten methylenes, ten methine and
±
ꢀ
1
2
2
3
8
9
0
±
3H s 0.96
3H s 0.93
±
H-30
H-29
H-3
twelve quaternary carbon signals. The signals for eight olefinic
1
¢
Affiliation: 1 HEJ Research Institute of Chemistry, International Center for Che-
2¢
3¢
6.26 d *16)
7.51 d *16)
±
*3')
c
2
*2')c
mical Sciences, University of Karachi, Karachi, Pakistan ´ Department of
Chemistry, Al-Farabi Kazakh State National University, Almaty, Kazakhstan
1
¢¢
H-2, H-6
H-1
Correspondence: Prof. M. Iqbal Choudhary ´ HEJ Research Institute of Chemis-
try ´ University of Karachi ´ Karachi-75270 ´ Pakistan ´ Fax: +9221 9243190,
2¢¢
3¢¢
7.03 d *2)
±
H-2
9
243191 ´ E-mail: hej@cyber.net.pk
4
¢¢
5¢¢
¢¢
±
H-2, H-5, H-6
*H-6)c
*H-5)c
Received: January 31, 2003 ´ Accepted: June 28, 2002
6.76 d *8)
6.92 dd *2,8)
6
Bibliography: Planta Med 2004; 70: 65±67 ´ ꢁ Georg Thieme Verlag Stuttgart ´
New York ´ ISSN 0032-0943 ´ DOI 10.1055/s-2004-815458
C
Proton cross-peaks in 1H- H COSY-45 spectrum.
1
8
Letter¼ Planta Med 2004; 70: 65±67

sition of the cinnamoyl moiety, basic hydrolysis of compound 1 on a Gallenkamp apparatus. FAB-MS was measured on a Jeol HX
was carried out. The HREI of 1 showed the highest peak at m/ 110 mass spectrometer. EI-MS and HREI-MS were recorded on a
1
z = 438.3507 corresponding to the formula C H O ꢀcalc. Varian MAT 311A mass spectrometer. The H-NMR spectra were
4
3
0
46
2
13
38.3497). The FAB-MS ꢀ-ve) showed the pseudomolecular ion recorded on Bruker AM 500 MHz spectrometer, while C-NMR
at m/z = 617.432 corresponding to the molecular formula spectra were recorded at 125 MHz on the same instrument.
C H O . The mass difference between HREI- and FAB-MS data
3
9
53
6
was due to the loss of a 3¢¢,4¢¢-dihydroxycinnamoyl group The aerial parts ꢀ3 kg) of T. hispida Willd. were collected from the
C H O ). The characteristic fragments at m/z = 248, 203, 189 southern parts of Kazakhstan in September 2000. The plant was
ꢀ
9
7
4
and 133 resulting from the retro-Diels-Alder cleavage suggested identified by Dr. Lapshina and a voucher specimen ꢀ# 5239) was
a traxer-14-ene skeleton with a carboxylic group. On the basis of deposited at Department of Botany, Al-Farabi Kazakh National
all spectral observations, compound 1 was deduced to be 3a- University, Almaty.
ꢀ
3¢¢,4¢¢-dihydroxy-trans-cinnamoyloxy)-D-friedoolean-14-en-28-
oic acid ꢀ1).
Dried and crushed plant material of T. hispida ꢀ2 kg) was macer-
ated in 50% acetone-H O ꢀ2”5 L) at room temperature. The ex-
2
The compound 3a-hydroxytaraxer-14-en-28-oic acid ꢀ1a) is the tract was filtered and concentrated. The concentrated extract
+
hydrolyzed product of 1. The EI-MS of 1a ꢀm/z [M] = 456) corre- ꢀ50 g) was dissolved in water ꢀ1 L) and defatted with petroleum
1
sponds to the molecular formula C H O . In the H-NMR spec- ether, followed by chloroform and ethyl acetate extractions. The
3
0
48
3
trum of 1a, H-3 appeared as a broad singlet at d = 4.41 ꢀW1/2
.56 Hz) which indicated that H-3 was equatorially oriented. A the DPPH free radical assay, was chromatographed over silica gel
triterpene, 3a-acetyltaraxer-14-en-28-oic acid, previously isolat- ꢀ900 g) with gradient eluents ꢀCH Cl , 2.5 L ; 0.5 ± 100% MeOH in
=
ethyl acetate extract ꢀ10 g) which was found to be most active in
2
2
2
ed from Phytolacca acinosa is structurally related to 1 except that CH Cl , each 2.5 L) to afford frs. 1 ± 4. Fr. 2 ꢀ3.0 g) obtained from
2
2
the 3a-acetyl group is present at C-3 [3]. Compounds 2 and 3 the 5% MeOH was chromatographed over silica gel ꢀ240 g) using
were identified as rhamnocitrin and isorhamnetin, respectively, hexane/EtOAc ꢀ0 ± 100% EtOAc, each 100 mL) as eluents to yield
by comparison of their spectral data with the literature values four fractions ꢀfrs. 2.1 ± 2.4). Fr. 2.2 ꢀ500 mg) obtained from the
[4], [5], [6], [7].
20% EtOAc was rechromatographed over silica gel ꢀ40 g) and
eluted with hexane/EtOAc ꢀ9:1 ® 8:2 ® 7:3, each 10 mL) afford-
Compound 1 having a caffeoyl moiety exhibited significant anti- ed compound 2 ꢀ10 mg) and fr. 2.2a on elution with hexane/
oxidant activity by scavenging the DPPH and superoxide radicals EtOAC ꢀ9:1). The fr. 2.2a ꢀ50 mg) was chromatographed over si-
ꢀ
Table 1). The IC50 value of compound 1 is almost equal to both lica gel ꢀ4.5 g) and eluted with hexane-EtOH-BuOH ꢀ8:1.5:0.5, 5
the standards indicating its effectiveness as a free radical scaven- mL) to afford a partially purified compound, which was purified
ger. On the other hand, compound 1a which does not have a caf- by preparative TLC using pre-coated silica gel plates with solvent
feoyl moiety was found to be inactive. It has been reported that system CH Cl /MeOH ꢀ9.8:0.2) to furnish compound 1 ꢀ20 mg).
2
2
the phenolic groups contribute to the free radical scavenging Fr. 2.4 ꢀ250 mg) obtained from the hexane/EtOAc ꢀ6:4) was chro-
ability of chemical compounds, the radical scavenging potential matographed over a flash silica gel ꢀ15 gm) column and eluted
of 1 may therefore very well be due to the presence of the o-dihy- with hexane/EtOAc ꢀ8:2 ® 7:3 ® 6:4, each 10 mL) to afford
droxy groups present at the caffeoyl moiety. Compounds 1 ± 3 three frs. 2.4a ± 2.4c. Fr. 2.4b ꢀ20 mg) obtained from the hexane/
demonstrated significant inhibitory activities against PEP as EtOAc ꢀ7:3) was purified by preparative TLC using pre-coated si-
6
6
compared to the positive control bacitracin [8] ꢀTable 1). This is lica gel plates with solvent system MeOH/CHCl ꢀ3:7) to afford
3
the first report of a triterpene that showed potent inhibitory ac- compound 3 ꢀ12 mg).
tivity against PEP ꢀEC 3.4.21.26). This enzyme has recently gained
pharmaceutical interest since its specific inhibitors showed anti- 3a-ꢀ3¢¢,4¢¢-Dihydroxy-trans-cinnamoyloxy)-
D-friedoolean-14-en-
amnesic effects [9], [10], [11]. The hydroxy function at C-7 in 28-oic acid ꢀ1), C H O ; amorphous solid; Rf ꢀCH Cl :MeOH,
3
9
54
6
2
2
compounds 2 and 3 seems to be responsible for the inhibitory ac- 9.7:0.3): 0.3, ꢀEtOAc:hexane, 4:6): 0.5; m.p. 196 ± 198 8C; UV
2
7
tivity. Cinnamic acid, methyl caffeate and pyrocatechol were also ꢀMeOH): lmax= 216, 244, 255, 305, 372 nm; [a] : ±228 ꢀc 0.04,
D
screened and only methyl caffeate showed inhibition with MeOH); IR ꢀKBr): nmax = 3411 ꢀOH),1259 ꢀa,b-unsaturated ester),
±
1
IC50 = 341.37 mM. These results indicate that the activity of com- 1691 ꢀCOOH), 1600, 812 ꢀC = C) cm ; FAB-MS ꢀ±ve): m/z =
±
1
pound 1 is probably due to the triterpenoid moiety itself or the 617.432 [M ± 1] . EI-MS: m/z = 438, 248, 203, 189, 133; H-NMR
13
ester linkage between caffeic acid and the triterpenoidal moiety. ꢀ500 MHz, mixture of CD OD and CD Cl ) and C-NMR ꢀ125
3
3
3
MHz, mixture of CD OD and CDCl ), see Table 1.
3
3
Material and Methods
The compound 1 ꢀ6 mg) was refluxed in a solution of 3% KOH in
MeOH ꢀ5 mL) for 2 hours at 608C. Work-up in the usual way af-
Thin layer chromatography was carried out on precoated silica forded a product that was chromatographed over a silica-gel col-
gel sheets ꢀE. Merck, 60 F254). Column chromatography was also umn using CHCl as eluent to afford 3a-hydroxytaraxer-14-en-
3
performed using silica gel ꢀ230 ± 400 mesh) and spots were de- 28-oic acid ꢀ1a) ꢀ3 mg) which was identified on the basis of IR,
1
tected at 254 and 366 nm and also by using ceric sulphate spray- EI-MS and H-NMR data [3]. EI-MS: m/z = 456.36 [C H O ],
3
0
48
3
+
ing reagent. UV spectra were recorded on a Hitachi UV 3200 [M ± H O] , 438, 248, 203, 189, 133; IR ꢀKBr): n
spectrophotometer, while IR spectra were recorded on a Jasco 1690 ꢀCOOH), 1460, 1386, 1061, 993 ꢀC = C) cm ; [a] : ±13.58
= 3448 ꢀOH);
2
max
±
1
27
D
3
02-A spectrophotometer. Optical rotation was measured on a ꢀc 0.04, CHCl ); m.p. 270 ± 2728C.
3
Schmidt + Haensch Polartronic D. Melting points were measured
Letter¼ Planta Med 2004; 70: 65±67

Table 2 In vitro antioxidant and PEP inhibitory activities of compounds 1 ± 3
Compound
IC50 ꢀmM)*
DPPH Scavenging activity
Superoxide scavenging activity
PEP Enzyme inhibition
1
29  0.5
306  1.4
0.250  0.021
±
1
2
3
a
”
±
±
”
±
±
32.64  0.84
18.94  0.25
Bacitracinꢀꢀ for PEP
PGꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
129.26  3.28
6
7
30  0.27
44  2.00
106  1.7
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
BHAꢀꢀ
ꢀ
”
IC50 values are the mean  standard mean *SEM) error of three assays. ꢀꢀ Used as standard. ꢀꢀꢀ Not used as standard in these assays.
Not active. - Not determined. PG = propyl gallate. BHA = 3-t -butyl-2-hydroxyanisole.
7
Antioxidant activities were determined by using the DPPH ꢀ1,1-
diphenyl-2-picrylhydrazyl radical) scavenging and NADH/PMS
Noor AK, Kamil M, Ilias M. Constituents of Tamarix dioica Roxb. ꢀTa-
mariscineae). Indian J Chem 1979; 18B: 390
Kimura KI, Kanou F, Yamashita Y, Yoshimoto T. Prolyl endopeptidase
inhibitors derived from Actinomycetes. Bioscience Biotechnology Bio-
chemistry 1997; 61: 1754±6
Fan W, Tezuka Y, Komatsu K, Namba T, Kadota S. Prolyl endopeptidase
inhibitors from the underground part of Rhodiola sacra S. H. Fu. Biolo-
gical & Pharmaceutical Bull 1999; 22: 157±61
Toid K, Shinoda M, Miyazaki A. A novel prolyl endopeptidase inhibitor,
JTP-4819, its behavioral and neurochemical properties for the treat-
ment of Alzheimer's disease. Neuroscience 1998; 9: 17±29
Lee HJ, Song KS. Kynapcin-12, A new p-terphenyl derivative from
Polyozellus multiplex, inhibits prolyl endopeptidase. The Journal of An-
tibiotics ꢀTokyo) 2000; 53: 714±9
8
9
ꢀb-nicotinamide adenine dinucleotide/phenazine methosul-
phate) superoxide anions scavenging assays [12], [13] while the
PEP inhibitory activities were measured by the method of Yoshi-
moto et al. [14].
10
References
11
1
Sokolov LD. Rastitelnye Resursy USSR. USSR: L. Academ. Nauk, 1986:
1
00 ± 05
12
2
3
4
5
6
Lee SK, Zakaria H, Chung H, Luyengi L, Gamez EJC, Mehta RJ, Kinghorn
D, Pezzuto JM. Evaluation of the antioxidant potential of natural pro-
ducts. Combinatorial Chemistry and High Throughput Screening
Corbett RE, Cumming SD. Linchens and fungi, part X, 14 a-taraxerane.
Journal of Chemical Society Perkin Transactions I, 1972: 2827±9
Razdan TK, Harkar S. Phytolaccanol and epiacetylaleuritoric acid, two tri-
terpenoids from Phytolacca acinosa. Phytochemistry 1982; 21: 2339±42
Sharma SK, Parmar VS. Novel constituents of Tamarix species. Journal
of Scientific and Industrial Research 1998; 57: 873±90
Nawwar MAM, Sherbeiny AEA, Ansari MAE. Plant constituents of
Tamarix aphylla flowers ꢀTamaricaceae). Experientia 1975; 31: 1118
Sissi HIE, Nawwar MAM, Saleh NAM. Plant constituents of Tamarix
nilotica leaves ꢀTamaricaceae). Experientia 1973; 29: 1064
1998; 1: 35±46
13
Soares JR, Dinis TCP, Cunha AP, Almeida LM. Antioxidant activities of
some extracts of Thymus zygis. Free Rad Res 1997; 26: 469±78
Yoshimoto T, Walter R, Tsuru D. Proline-specific endopeptidase from
Flavobacterium. Journal of Biological Chemistry 1980; 255: 4786±92
14
Letter¼ Planta Med 2004; 70: 65±67