Enzymes inhibitory constituents from Buddleja crispa

Article abstract of DOI:10.1515/znb-2005-0319

Steroidal galactoside 1 and aryl esters 2 and 3 have been isolated from Buddleja crispa, along with ginipin 4, gardiol 5, 1-heptacosanol 6, and methyl benzoate 7, isolated for the first time from this species. The structures of all of the compounds were determined by spectroscopic techniques and chemical studies. The steroidal galactoside 1 is an inhibitor of lipoxygenase. Compounds 1-3 displayed inhibitory activity against butyrylcholinesterse, while compounds 2 and 3 further showed inhibition against acetylcholinesterase.

Full text of DOI:10.1515/znb-2005-0319

Enzymes Inhibitory Constituents from Buddleja crispa
a
a
b
a
a
Ijaz Ahmad , Abdul Malik , Nighat Afza , Itrat Anis , Itrat Fatima ,
a
c
a
Sarfraz Ahmad Nawaz , Rasool Bukhsh Tareen , and M. Iqbal Choudhary
a
International Center for Chemical Sciences, HEJ Research Institute of Chemistry,
University of Karachi, Karachi-75270, Pakistan
Pharmaceutical Research Centre, PCSIR Labs. Complex, Karachi
Department of Botany, University of Baluchistan, Pakistan
b
c
Reprint requests to Prof. Dr. A. Malik. E-mail:abdul.malik@iccs.edu
Z. Naturforsch. 60b, 341 – 346 (2005); received July 9, 2004
Steroidal galactoside 1 and aryl esters 2 and 3 have been isolated from Buddleja crispa, along
with ginipin 4, gardiol 5, 1-heptacosanol 6, and methyl benzoate 7, isolated for the first time from
this species. The structures of all of the compounds were determined by spectroscopic techniques
and chemical studies. The steroidal galactoside 1 is an inhibitor of lipoxygenase. Compounds 1 – 3
displayed inhibitory activity against butyrylcholinesterse, while compounds 2 and 3 further showed
inhibition against acetylcholinesterase.
Key words: Buddleja crispa, Buddlejaceae, Steroid Galactoside, Aryl Esters, Enzyme Inhibition
Introduction
structure elucidation of (22R)-stigmasta-7,9(11)-dien-
2 α-ol-3 β-O-β-D-galactopyranoside 1, nonyl ben-
2
The genus Buddleja (family Buddlejaceae) is found zoate 2 and hexyl p-hydroxy-cinnamate 3 along with
in the temperate regions of America, Asia and South ginipin 4 [14], gardiol 5 [15], 1-heptacosanol 6 [16]
Africa. It is a genus of about 100 species represented and methyl benzoate 7, reported for the first time from
in Pakistan by four species [1]. The flowers, leaves this species.
and roots of various species of Buddleja are used in
Acetylcholinesterase (AChE; EC 3.1.1.7) and bu-
traditional medicine in several parts of the world [2]. tyrylcholinesterase (BChE; EC 3.1.1.8) are serine hy-
Several known pharmacological activities and folkoric drolases that share about 55% of amino acid sequence
uses are attributed to the genus. For example B. asiata, identity, and have similar, yet distinct catalytic proper-
which is indigenous to China, India, and Java, is known ties. The different specificity for substrates, irreversible
for its uses as an abortifacient and for treatment of skin inhibitors and reversible ligands is dictated by the dif-
diseases [3]. Flowers and buds of B. asiata produces ference in amino acid residues of the active sites of
a yellow coloured essential oil which is active against AChE and BChE [17].
several pathogenic fungi [4]. In central and southern
AChE is a key component of cholinergic brain
region of Chile the aqueous extract of B. globase is synapses and neuromuscular junctions. The major bi-
used for stomach ulcers, wounds and burns [5]. The ological role of the enzyme is the termination of im-
medicinal importance of the genus prompted us to pulse transmission by rapid hydrolysis of the cationic
carry out phytochemical studies on one of its species neurotransmitter acetylcholine [18]. According to the
namely B. crispa which is a densely tomentose shrub. cholinergic hypothesis, the memory impairment in
The wood of B. crispa is fairly hard and is used for the patients with senile dementia of Alzheimer’s type
fuel, found in southern part of Pakistan. No phyto- results from a deficiency in cholinergic function in
chemical investigation has so far been carried out on the brain [19]. Hence the most promising therapeu-
this species. A literature survey of the genus Buddleja tic strategy for activating central cholinergic functions
revealed the isolation of various natural products, in- has been the use of cholinomimetic agents. The aim
cluding sterols [3, 5], aryl esters [5], triterpenoid glyco- of AChE inhibitors is to boost the endogenous lev-
sides [6], phenylethanoids[7, 8], flavonoids [9], pheno- els of acetylcholine in the brains of Alzheimer’s dis-
lic fatty acid esters [10], diterpenes [11] and sesquiter- ease patients and thereby, to boost cholinergic neu-
penes [12, 13]. Herein we report the isolation and rotransmission. It has also been found that butyryl-
0
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I. Ahmad et al. · Enzymes Inhibitory Constituents from Buddleja crispa
cholinesterase inhibition may also be an effective tool 1H, H-7) and 5.56 (J = 6.1 Hz, 1H, H-11) were as-
for the treatment of AD and related dementias [20]. signed to the olefinic protons. The position of the dou-
BChE (E.C 3.1.1.8) is produced in the liver and en- ble bonds were further confirmed by COSY experi-
riched in the circulation. In addition, it is also present ments, which established the J connectivities between
in adipose tissue, intestine, smooth muscle cells, white H -6 and H-7 and between H -12 and H-11. A pair of
2
2
matter of the brain and many other tissues [21]. The doublet at δ = 0.81 (J = 6.9 Hz, H-26) and δ = 0.91
H
H
exact physiological function of BChE is still elusive. It (J = 6.9 Hz, H-27) and a multiplet at δ = 1.81 (H-25)
H
acts as a scavenger for anticholinesterase compounds.
confirmed the presence of an isopropyl group. The po-
Lipoxygenases (EC 1.13.11.12) constitute a fam- sition of the ethyl group at C-24 was confirmed by the
1
ily of non-haem iron containing dioxygenases that are presence of 3H triplet at δH = 0.89 in the H NMR
widely distributed in animals and plants. In mam- and also confirmed by the mass fragmentation which
malian cells these are key enzymes in the biosynthesis showed a fragment at m/z 85 due to the loss of C6H13.
1
of variety of bioregulatory compounds such as hydrox- In addition the H NMR also showed signals for the β-
yeicosatetraenoic acids (HETEs), leukotrienes, lipox- linked sugar moiety which appeared at δH = 4.65 (d,
ins and hepoxylines [22]. It has been found that these J = 7.8 Hz, H-1’), 3.27– 3.45 (m, H-2’, 3’, 5’), 3.31
(t, J = 1.6, H-4’), 3.63 (dd, J = 11.9, 5.5) and 3.84
lipoxygenase products play a role in a variety of disor-
ders such as bronchial asthma, inflammation [23] and (dd, J = 11.9, 2.3, H-6’). In COSY spectrum the more
tumor angiogenesis [24]. Lipoxygenases are therefore downfield oxymethine proton at δH = 3.66 showed
potential target for the rational drug design and discov- cross peaks with four other protons and it could con-
ery of mechanism-based inhibitors for the treatment sequently be assigned to the more usual C-3 position;
of bronchial asthma, inflammation, cancer and autoim- its downfield shift being due to glycoside formation.
mune diseases.
Its larger coupling constant allowed us to assign α and
axial configuration to H-3. The comparatively upfield
oxymethine proton at δH = 3.28 showed connectiv-
ity to three other protons including H-20 (δH = 1.73),
thereby confirming the presence of hydroxyl moiety at
C-22.
Results and Discussion
The methanolic extract of shade-dried whole plant
material (30 kg) of B. crispa was evaporated in vacuo,
the residue suspended in H O, and successively par-
titioned with n-hexane, EtOAc, and BuOH. Silica gel
column chromatography of the EtOAc extract afforded
compounds 1,4,5 while the n-hexane extract provided
compounds 2, 3, 6 and 7.
The 13C NMR spectrum (BB and DPET) showed 35
carbon signals, in which there are 6 methyl, 10 methy-
lene, 15 methine and 4 quaternary carbons. The low-
2
13
field region of C NMR spectrum showed four sig-
nals at δ = 145.7 (C-9), 133.2 (C-8), 123.1 (C-7),
C
Compound 1 was obtained as colorless crystalline 119.4 (C-11) which could be assigned to olefinic car-
solid which gave characteristic colour reactions of bons. One of the oxymethine resonated at δ = 72.5.
C
+
sterols. It showed an [M+H] peak in the HRFABMS The downfield shift of C-3 (δ = 79.3) confirmed
at m/z 591.4243 corresponding to molecular formula the linkage of the sugar moiety at this position. The
C
1
3
C H O (calcd. for C H O , 591.4245). The UV
C NMR showed signals for the sugar moiety at δ =
35
59
7
35 59
7
C
spectrum showed absorptions at λmax 235, 242 and 100.6 (C-1’), 73.2 (C-2’), 76.6 (C-3’), 70.2 (C-4’),
2
50 which are characteristic of ∆ 7,9(11)-sterols [25]. 76.7 (C-5’) and 61.2 (C-6’). The sugar could be identi-
1
13
The IR spectrum showed bands due to hydroxyl groups fied as galactose by comparing its H and C chem-
− −1
1
(
3490 cm ) and double bond (1650, 890 cm ). The ical shifts with those reported in literature and fur-
EIMS showed peaks at m/z 572 [M-H O] , 428 [M- ther confirmed through acid hydrolysis of 1 which
hexose] and 410 [M-hexose-H O] . The peak at m/z provided various products, among which the glycone
+
2
+
+
2
2
71 represented the loss of both the sugar and side could be separated and identified as galactose by com-
chain from the molecular ion peak.
paring the retention time of its trimethylsilyl (TMS)
1
The H NMR spectrum displayed signals for six ether with that of the standard in gas chromatogra-
methyl protons at δ = 0.81 (d, J = 6.9, 3H, H-26), phy (GC). The sign of optical rotation allowed us to
H
0
.87 (s, 3H, H-18), 0.89 (t, J = 7.2, 3H, H-29), 0.91 (d, assign D-configuration to the galactose moiety.
J = 6.9, 3H, H-27), 0.93 (d, J = 7.0, 3H, H-21), 1.02 (s,
The stereochemistry at C-22 could be established by
1
3
3
H, H-19). A pair of doublets at δ = 5.85 (J = 5.4 Hz, comparing C chemical shifts with literature. In 22-R
H
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I. Ahmad et al. · Enzymes Inhibitory Constituents from Buddleja crispa
343
Fig. 1. Important HMBC, NOESY and
COESY interaction of 1.
hydroxysterols the signal of C-22 was observed com- 129.5 and 128.3 were assigned to the aromatic ring.
paratively upfield and that of C-20 downfield relative to The signals between δ = 32.6 and δ = 22.6 (all
C
C
the corresponding S-epimer [26, 27]. In 1 the chemical methylenes in DEPT) were indicative of a long chain
shifts of both C-20 (δ = 42.8) and C-22 (δ = 72.5) hydrocarbon.
C
C
corresponded to those of 22-R epimer. The β config-
The mass spectrum showed fragment at m/z 122,
uration of both H-20 and H-22 could further be au- 105 and 77. On the basis of these evidences 2 could
thenticated by NOESY spectrum in which the pro- be identified as nonyl benzoate. Alkaline hydrolysis of
tons at C-18 showed correlations with both H-20 and 2 provided benzoic acid and nonanol, respectively, pro-
H-22. The configuration at C-24 could also be assigned viding conclusive evidence for the assigned structure.
‘
2
R’ based on similar chemical shift difference between This compound is reported as a new natural product
4R- and 24S-epimers [28]. Thus structure of 1 could following its earlier synthesis by Breusch et al. [29].
Compound 3 was also obtained as an oil. The
be assigned as (22R)-stigmasta-7,9(11)-dien-22α-ol-3
+
β-O-β-D-galactopyranoside. The HMBC correlations HREIMS showed an [M] peak at m/z 248.1405 cor-
were in complete agreement of the assigned structure responding the molecular formula C H O . It gave
1
5
20
3
(Fig. 1).
greenish-brown color with FeCl3 and the IR spec-
Compound 2 was isolated as an oil. The molecu- trum showed absorption bands at 3470 (free OH),
lar formula C H O was determined by HREIMS 1700 (C=O), 1670 (C=C), 1610, 1530, 1460 (aro-
1
6
24
2
and 13C NMR spectra. The IR spectrum showed ab- matic), 1365 (methyl) 1260, 1160 (C–O) and 720 cm
−1
−
1
sorption bands at 1720, 1610 and 1460 cm
sug- [-(CH2)X -]. The UV spectrum was typical of cinnamic
gesting the presence of ester and aromatic function- acid ester [30] showing λmax at 235 and 325, respec-
1
alities. The H NMR spectrum showed the presence tively.
1
of monosubstututed benzene [multiplets at δ = 7.36
The H NMR spectrum showed characteristic p-
H
(2H), 7.47 (1H) and 7.97 (2H), respectively] and a ter- hydroxycinnamoyl moiety [aromatic protons showing
minal methyl group at δ = 0.82 (3H, t, J = 7.0 Hz). AA’, BB’ pattern with δ = 7.39 (2H, d, J = 8.4 Hz)
H
H
The signal of oxymethylene protons was observed at and δH 6.87 (2H, d, J = 8.4 Hz); trans olefinic pro-
= 4.26 while another methylene group was ob- tons at δ = 7.64 (1H, d, J = 16 Hz) and δ = 6.29
δ
H
H
H
served at δ = 1.50. The unresolved multiplate at δ = (1H, d, J = 16 Hz)]. In addition it showed a three pro-
.22 integrating for twelve protons was due to 6 further tons triplet for the terminal methyl group at δ = 0.87
H
H
1
H
1
3
methylene groups of long chain. The C NMR spec- (6.34 Hz) and OCH CH R grouping [δ = 4.19, 2H,
2
2
H
trum (BB & DEPT) showed the signals at δ = 166.7 t, J = 6.69 Hz and δ = 1.65, 2H, m)]. The unresolved
C
H
which could be assigned to the ester carbonyl. The multiplet at δ = 1.29 integrating for 6 protons showed
H
signal at δ = 65.1 was assigned to the oxygenated the presence of 3 further methylene groups. The mass
C
methylene while the resonance at δ = 132.7, 130.5, spectrum showed peaks at m/z 164, 147 and 119. The
C
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I. Ahmad et al. · Enzymes Inhibitory Constituents from Buddleja crispa
Table 1. In vitro quantitative inhibition of AChE, BChE and Department of Botany, University of Baluchistan, Quetta,
LOX enzymes by compounds 1 – 3.
Pakistan.
Extraction and isolation: The shade-dried plant (30 kg)
a
Compounds
IC50 ± SEM [µM]
AChE
–
53.5± 1.2
32.2± 0.5
0.5± 0.01
–
BChE
LOX
were chopped and extracted thrice with MeOH (60 l) at rt
for 96 h. The methanolic extract was evaporated in vacuo
to give a dark greenish residue (800 g), which was parti-
tioned between n-hexane and water. The water fraction was
further extracted with EtOAc and n-BuOH. The EtOAc frac-
tion (40 g) was subjected to column chromatography elut-
1
2
3
46.7± 0.2
73.2± 1.2
22.5± 0.6
8.5± 0.001
–
6.1± 0.5
–
–
–
b
Galanthamine
c
Baicalein
22.7± 0.05
a
Standard mean error of five determinations; b positive control used
in AChE and BChE inhibiting assays;c positive control used in LOX ing with n-hexane-EtOAc in increasing order of polarity to
inhibiting assay.
give eight fractions. The fraction obtained from n-hexane-
EtOAc (3:7) was rechromatographed over flash silica using
compound 3 could, therefore, be identified as hexyl es- n-hexane-EtOAc (6:4-2:8) as solvent systems to give two
ter of p-hydroxy cinnamic acid. The structure was cor- successive fractions. The first fraction was further purified by
roborated by 13C NMR spectrum which included peaks column chromatography on silica gel using n-hexane-EtOAc
at δ = 168.6 for the ester carbonyl and further peaks (4:6) as eluent to afford compound (1) (16 mg). The frac-
C
tion obtained from n-hexane-EtOAc (6:4) was further pu-
rified by column chromatography over silica gel using n-
hexane-EtOAc (8:2-5:5) as eluents to afford compound (4)
at δ = 145.2 and δ = 114.8 for the olefinic carbons.
C
C
The alkaline hydrolysis of 3 provided p-hydroxy cin-
namic acid and hexanol, respectively.
(11 mg) and (5) (15 mg), respectively. The n-hexane fraction
Compound 1 showed moderate inhibitory poten-
tial against LOX with IC50 value (6.1 ± 0.5) µM
(30 g) was subjected to column chromatography eluting with
n-hexane and n-hexane-CHCl in increasing order of polarity
3
(
(
(
Table 1) and weak inhibitory activity against BChE
to give five fractions. The fraction obtained from n-hexane-
CHCl3 (8:2) was rechromatographed over flash silica us-
46.7± 0.2) µM whereas the standard inhibitor of LOX
baicalein) and BChE (galanthamine) have IC5 values
0
ing n-hexane- CHCl (9:1-6:4) as solvent systems to give
3
of (22.5± 0.2) µM and (8.5± 0.01) µM, respectively. three successive fractions. The second fraction was a mix-
Compounds 2 and 3 displayed weak inhibitory activ- ture of two compounds and afforded compounds 2 (40 mg)
ity against AChE with IC50 values of (53.5± 1.2) µM and 3 (35 mg) by silica gel column chromatography using
and (32.2± 0.5) µM, and also against BChE with IC
n-hexane, n-hexane-CHCl3 (9:1, 8:2) as eluents. The first
fraction which was also a mixture of two compounds, was
purified through column chromatogrphay over silica gel us-
5
0
values of (73.2 ± 1.2) µM and (22.5 ± 0.6) µM, re-
spectively. The positive control (galanthamine) used in
the assays showed IC50 values of (0.5± 0.001) µM and
ing n-hexane-CHCl (9:1) as eluent to afford compounds 6
3
(18 mg) and 7 (15 mg), respectively.
(8.5± 0.01) µM against AChE and BChE, respectively.
Acid hydrolysis of compound 1
Experimental Section
A solution of 1 (8 mg) in MeOH (5 ml) containing 1 N
General: Optical rotations were taken on a JASCO DIP- HCl (4 ml) was refluxed for 4 h, concentrated under reduced
3
60 digital polarimeter. IR spectral data were measured on pressure, and diluted with H O (8 ml). It was extracted with
2
a JASCO 302-A spectrophotometer in CHCl . UV spec- EtOAc and the residue recovered from the organic phase was
3
tra were obtained on a Hitachi UV-3200 spectrophotometer. found to be an inseperatable mixture of products. The aque-
NMR spectra were run on a Bruker instrument. Chemical ous phase was concentrated and D-galactose was identified
2
0
◦
shifts δ are shown in ppm relative to TMS as internal stan- by the sign of its optical rotation ([α] + 80.0 , c = 0.02,
D
dard and coupling constant J are described in Hz. EI-, FAB-, H O). It was also confirmed based on comparison of the re-
2
and HREI-MS were recorded on a JEOL JMS-HX-110 and tention time of its TMS ether (α-anomer 3.8 min, β-anomer
JMS-DA-500 mass spectrometers, m/z (relative. int). Silica 5.2 min) with a standard.
gel 60 – 200 mesh and 200 – 440 mesh (Merck) was used for
column chromatography, respectively. Silica gel plates (Si 60
F254, Merck) were used for TLC.
Alkaline hydrolysis of 2 and 3: The ester 2 (20 mg) in ben-
zene (25 ml) was refluxed with 5% methanolic potassium hy-
droxide (15 ml) for 4 h, concentrated in vacuo, diluted with
Plant material: The whole plant material was collected water (30 ml) and extracted with ether. The organic layer on
◦
◦
in March 2003 from Baluchistan and identified as Buddleja removal of solvent afforded a liquid (Fp −5 , Bp 214 ), char-
+
crispa Benth. by Prof. Rasool Bakhsh Tareen, Department acterized as nonanol (M peak at m/z 144, superimposable
of Botany, University of Baluchistan, Pakistan. A voucher IR). The aqueous layer was acidified with dil. hydrochlo-
specimen (BBU-101) is deposited in the herbarium of the ric acid and extracted with dichloromethane. The residue re-
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I. Ahmad et al. · Enzymes Inhibitory Constituents from Buddleja crispa
345
covered from the organic phase crystallized from H O, m. p.
Compound 3. – Oil. – IR (KBr) ν = 3470, 1700, 1670,
2
◦
−1
1
23 C: It was characterized as benzoic acid (m.m.p., IR and 1610, 1530, 1460, 1365, 1280, 1160, 720 cm . – UV
Co-TLC).
(MeOH) λmax = 235, 325. – HREIMS m/z 248, 164, 147,
1
Corresponding hydrolysis of 3 provided hexanol (char- 119. – H NMR (400 MHz, CDCl3) δ = 0.87 (3H, t,
acterized through B. P, IR and co-TLC) and p-hydroxy cin- J = 6.34 Hz, Me), 1.29 [6H, br m, (CH2)3], 1.65 (2H,
namic acid, colourless crystals from H O, m. p. 212 C (char- m, OCH2CH2R), 4.19 (2H, t, J = 6.69 Hz, OCH2CH2R),
◦
2
acterized through m.m.p., Co-TLC, IR).
6.29 (1H,d, J = 16.0 Hz, =CH), 6.87 (2H,d, J = 8.4 Hz
Compound 1: – Colourless crystalline. – M. p. 258 – H-3 and H-5), 7.39 (2H,d, J = 8.4 Hz H-2 and H-6),
◦
13
2
(
1
60 C. – [α]D −29.9 (c = 0.02, MeOH). – UV λmax 7.64 (1H,d, J = 16 Hz CH=). – C NMR (100 MHz,
MeOH) 235, 240, 250. – IR (KBr) ν = 3490 (OH group), CDCl3) δ = 168.7 (C-3’), 158.7 (C-4), 145.2 (C-1’),
−1
650, 890 cm
(double bonds). – Positive HRFABMS 130.0 (C-2 and C-6), 126.6 (C-1), 116.1 (C-3 and C-5),
+
m/z 591.4243 [M+H] (calcd. 591.4245 for C H O ). – 114.8 (C-2’), 65.1 (C-1”), 31.4 (C-2”), 28.6 (C-3”),
35
59
7
HREIMS m/z 572, 428, 410, 271. – 1H NMR (90 MHz, 25.6 (C-4”), 22.5 (C-5”), 14.0 (C-6”).
CD OD) δ = 0.81 (d, J = 6.9 Hz, 3H, H-26), 0.87 (s,
3
In vitro cholinesterase inhibition assay
3
6
H, H-18), 0.89 (t, J = 7.2 Hz, 3H, H-29), 0.91 (d, J =
.9, 3H, H-27), 0.93 (d, J = 7.0, 3H, H-21), 1.02 (s, 3H,
Electric-eel AChE (EC 3.1.1.7), horse-serum BChE (EC
H-19), 1.39 (m, 1H, H-4β), 1.59 (m, 1H, H-2β), 1.88 (m, 3.1.1.8), acetylthiocholine iodide, butyrylthiocholine chlo-
H, H-4α), 1.96 (m, 1H, H-2α), 3.28 (m, 1H, H-22), 3.66 ride, 5,5’-dithiobis [2-nitrobenzoic acid] (DTNB) and galan-
m, 1H, H-3), 5.56 (d, J = 6.1 Hz, 1H, H-11), 5.85 (d, thamine were purchased from Sigma (St. Louis, MO, USA).
J = 5.4 Hz, 1H, H-7), 4.65 (d, J = 7.8 Hz, H-1’), 3.27 – All other chemicals were of analytical grade. AChE and
.45 (m, H-2’,3’,5’), 3.31 (t, J = 1.6 Hz, H-4’), 3.63 dd BChE inhibiting activities were measured by the spectropho-
J = 11.9, 5.5 Hz) and 3.84 dd (J = 11.9, 2.3) (H-6’). – tometric method developed by Ellman et al. [31]. Assay
1
(
3
(
1
3
C NMR (100 MHz, CD OD) δ = 37.7 (C-1), 30.5 (C-2), conditions and protocol was the same as described previ-
3
7
1
4
2
4
4
2
7
9.3 (C-3), 35.5 (C-4), 40.6 (C-5), 31.0 (C-6), 123.1 (C-7), ously [32].
33.2 (C-8), 145.7 (C-9), 37.0 (C-10), 119.4 (C-11),
In vitro lipoxygenase inhibition assay
4.9 (C-12), 42.6 (C-13), 57.9 (C-14), 25.8 (C-15),
7.9 (C-16), 52.7 (C-17), 13.6 (C-18), 19.6 (C-19),
Lipoxygenase inhibiting activity was measured by slightly
2.8 (C-20), 12.6 (C-21), 72.5 (C-22), 30.0 (C-23), modifying the spectrometric method developed by A. L. Tap-
1.5 (C-24), 28.9 (C-25), 17.5 (C-26), 20.6 (C-27), pel [33]. Lipoxygenase (1.13.11.12) type I-B and linoleic
3.6 (C-28), 11.9 (C-29), 100.6 (C-1’), 73.2 (C-2’), acid were purchased from Sigma (St. Louis, MO, USA). All
6.6 (C-3’), 70.2 (C-4’), 76.7 (C-5’), 61.2 (C-6’).
other chemicals were of analytical grade. The assay condi-
Compound 2. – Oil. – IR (KBr) ν = 1720, 1610, 1460. tions and protocol was the same as described previously [34].
UV (MeOH) λmax = 227, 282. – HREIMS m/z 248, 122,
–
1
1
Determination of IC50 values
05, 77. – H NMR (400 MHz, CDCl ) δ = 0.82 (3H,
3
t, J = 7.0 Hz, Me), 1.22 [12H, br m, (CH ) ], 1.50 (2H,
The concentrations of the test compounds that inhibited
2
6
m, OCH CH R), 4.26 (2H, m, OCH CH R), 7.47 (IH, m), the hydrolysis of substrates (acetylthiocholine, butyrylthio-
2
2
2
2
13
7
.97 2H, m), 7.36 (2H,m). – C NMR (100 MHz, CDCl3) choline and linoleic acid) by 50% (IC50) were determined
δ = 166.7 (C-1’), 132.7 (C-4), 130.5 (C-1), 129.5 (C-2, by monitoring the effect of various concentrations of these
6
2
2
), 128.3 (C-3,5), 65.1 (C-1”), 32.62 (C-2”), 31.8 (C-7”), compounds in the assays on the inhibition values. The IC50
5.7 (C-4”), 29.6 (C-5”), 29.4 (C-6”), 26.8 (C-3”), values were then calculated using the EZ-Fit Enzyme Kinet-
2.6 (C-8”), 14.2 (C-9”).
ics program (Perrella Scientific Inc., Amherst, USA).
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