Lupene-type triterpenes from Periploca aphylla

Article abstract of DOI:10.1021/np990426v

Two new lupane derivatives, 3β,6α-dihydroxylup-20(29)-ene (1) and 6α- hydroxylup-20(29)-en-3β-octadecanoate (2), have been isolated from the stems of Periploca aphylla, in addition to β-sitosterol and lupeol. The structures of 1 and 2 were determined by spectral and chemical methods. Compound 1 showed strong inhibition of α-glucosidase type VI and a moderate antibacterial activity.

Full text of DOI:10.1021/np990426v

J . Nat. Prod. 2000, 63, 881-883
881
Lu p en e-Typ e Tr iter p en es fr om P er iploca a ph ylla
Ghulam Mustafa,† Erum Anis,† Saeed Ahmed,† Itrat Anis,† Habib Ahmed,‡ Abdul Malik,*,†
Syed Shahzad-ul-Hassan,† and M. Iqbal Choudhary†
International Center for Chemical Sciences, HEJ Research Institute of Chemistry, University of Karachi,
Karachi-75270, Pakistan, and Department of Botany, Government Degree College Matta, Swat, Pakistan
Received August 30, 1999
Two new lupane derivatives, 3â,6R-dihydroxylup-20(29)-ene (1) and 6R-hydroxylup-20(29)-en-3â-octade-
canoate (2), have been isolated from the stems of Periploca aphylla, in addition to â-sitosterol and lupeol.
The structures of 1 and 2 were determined by spectral and chemical methods. Compound 1 showed strong
inhibition of R-glucosidase type VI and a moderate antibacterial activity.
The genus Periploca (Asclepiadaceae) consists of about
12 species, which are erect leafless shrubs, distributed in
Europe, Asia, and tropical Africa. Phytochemical studies
on various species of genus Periploca including P. sepium,
P. leavigatis, and P. gracea have resulted in the isolation
of various triterpenes, lignans, and flavonoids.1-3 The plant
P. aphylla Decne is widely distributed in the northern part
of Pakistan and finds various medicinal uses in the
indigenous system of medicine.4 The milky juice is used
as an external application to tumors and swellings and is
said to be useful in cerebral fever and as a stomachic.5
Earlier work on this plant by Mitsuhashi et al. has resulted
in the isolation of several known steroids and triterpenes.6
In the present investigation a methanolic extract of the
stems of P. aphylla showed positive activity in the brine
shrimp lethality test.7 Further biological screening of the
methanolic extract revealed strong antibacterial activity8
against Gram-positive (Bacillus cerus, Streptococcus pyo-
genes) and Gram-negative (Escherichia coli, Proteus mira-
bilis, Salmonella typhi) bacteria. This prompted us to carry
out bioassay-directed isolation studies on this plant. Herein,
we report the isolation and structure elucidation of two new
triterpenes of the lupane type, namely, 3â,6R-dihydroxylup-
20(29)-ene (1) and 6R-hydroxylup-20(29)-en-3â-octade-
canoate (2), along with the two known compounds â-sito-
sterol and lupeol.
Compound 1 was assigned the molecular formula
C30H50O2 by HRMS. It gave positive Liebermann-Burchard
and CeSO4 tests for a triterpene. The IR spectrum sug-
gested that it contained hydroxyl groups (3400 cm-1) and
a terminal double bond (3045, 1650, and 888 cm-1). The
1H NMR spectrum revealed six methyl groups [δH 0.75,
0.80, 0.96, 0.97, 1.02, 1.21 (3H, s, each)], an isopropenyl
group [δH 1.68 (3H, s), 4.57 and 4.67 (1H, d, J ) 2.3 Hz)],
and two oxymethine protons in proximity to hydroxyl
groups [δH 3.16 (1H, dd, J ) 10.8 and 5.46 Hz) and 3.81
(1H, dt, J ) 10.5 and 4.8 Hz)]. The hydroxyl groups were
shown to be secondary by their easy acetylation to the
corresponding diacetate 1a in which the signals at δH 3.16
and 3.81 were respectively shifted downfield to δH 4.45 and
5.51. The EIMS of 1 exhibited important peaks at m/z 442
[M+], 427 [M - CH3], 424 [M - H2O], 409 [M - H2O -
CH3], 406 [M - 2 H2O], 236, 218, 205, 203, and 189. This
fragmentation pattern supported the lup-20(29)-ene skel-
eton having both the hydroxyl groups in rings A and B.9
The broad-band and DEPT 13C NMR spectra showed the
presence of seven methyl, 10 methylene, seven methine,
and six quaternary carbon atoms, which provided further
evidence for a lup-20(29)-ene skeleton for 1. The positions
of the two hydroxyl groups were determined through an
HMBC experiment in which the protons of the geminal
2
methyl groups (δH 1.02, 0.75) at C-4 showed J correlations
with C-4 (δC 38.8) and 3J interactions with C-3 (δC 77.9)
and C-5 (δC 55.4), confirming the position of one hydroxyl
group at C-3. The position of the second hydroxyl was
confirmed at C-6 since 2J correlations of the oxymethine
proton signal (δH 3.81) to C-5 (δC 55.4) and C-7 (δC 44.0)
and 3J interactions with C-4 (δC 38.8) and C-10 (δC 32.5)
were observed in the HMBC spectrum. The magnitude
of the coupling constants of the proton at C-6 allowed
us to assign an R and equatorial configuration to this
hydroxyl group, since when a hydroxyl group is â and axial,
a W1/2 value of J ) 7.5 Hz would be expected.10 Conclu-
sive evidence for the structure was provided by oxida-
tion of 1 to the diketo derivative 1b, whose physical and
spectral data were in complete agreement with those of lup-
20(29)-ene-3,6-dione reported in the literature.11 Compound
1 was therefore assigned the structure 3â,6R-dihydroxylup-
20(29)-ene.
Compound 2 was assigned the molecular formula
C
48H84O3 by HREIMS. It also gave positive Liebermann-
* To whom correspondence should be addressed. Tel.: (92-21) 4968733.
Fax: (92-21) 4963373,4963124. E-mail: gm4u@cubexs.net.pk.
† HEJ Research Institute of Chemistry.
Burchard and CeSO4 tests for a triterpene. Analysis of the
IR spectrum suggested the presence of a hydroxyl group
(3385 cm-1), a terminal double bond (3050, 1630, 888 cm-1),
‡ Government Degree College Matta.
10.1021/np990426v CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/11/2000

882 J ournal of Natural Products, 2000, Vol. 63, No. 6
Notes
and an ester group (1735 cm-1). The 1H NMR spectrum
exhibited signals for six methyl groups [δH 0.76, 0.81, 0.84,
0.89, 1.0, 1.30 (3H, s, each)], an isopropenyl group [δH 1.65
(3H, s), 4.56, 4.68 (1H, d, J ) 2.3 Hz)], an oxymethine
proton in proximity to an ester moiety [δH 4.48 (1H, dd,
J ) 5.2, 10.1 Hz)], and another oxymethine proton in
proximity to a hydroxyl group [δH 3.95 (1H, dt, J ) 5.0,
specimen is deposited in the herbarium (accession no. 1997-
1294) of the Department of Botany, Government Degree
College Matta, Swat, Pakistan.
Extr a ction a n d Isola tion . The dried plant material (28
kg) was extracted three times with MeOH at room tempera-
ture. The methanolic extract was evaporated in vacuo, yielding
a brownish residue, which was suspended in H2O and then
extracted with n-hexane. The aqueous layer was further
extracted with chloroform. The fractionation was controlled
through a brine shrimp lethality test. The chloroform fraction,
which showed potent cytotoxicity, was chromatographed over
Si gel using mixtures of n-hexane and EtOAc of increasing
polarity as eluants. The fraction obtained from n-hexane and
EtOAc (19:1) showed strong cytotoxicity, and its further
chromatography over Si gel using mixtures of n-hexane and
EtOAc (9:1) as eluent yielded 6R-hydroxylup-20(29)-en-3â-
octadecanoate (2, 65 mg) and lupeol (80 mg) from less polar
fractions and 3â,6R-dihydroxylup-20(29)-ene (1, 55 mg) and
â-sitosterol (65 mg) from polar fractions. The physical and
spectral data of â-sitosterol and lupeol showed complete
agreement with those reported in the literature.16,17 Strong
brine shrimp toxicity was observed for compound 1 compared
to very weak activity of 2.
10.7 Hz)]. The ester function was also confirmed by the 13
C
NMR spectrum of 2, which showed an ester carbonyl
carbon signal at δC 170.2. The 13C NMR spectrum (broad-
band and DEPT) of 2 corroborated the presence of eight
methyl, 26 methylene, seven methine, and six quaternary
carbons. These were assigned on the basis of comparison
with 1 as well as from HMQC and HMBC experiments.
The mass spectrum of 2 exhibited peaks at m/z 708 [M+],
409 [M - C18H36O2 - CH3], 391 [M - C18H36O2 - CH3
-
H2O], 218, 205, 203, and 189. This fragmentation pattern
suggested that compound 2 was also of the lup-20(29)-ene
type, having an ester moiety and a hydroxyl group in rings
A and B.
The alkaline hydrolysis of 2 in methanolic KOH yielded
2a and methyl octadecanoate, with the latter confirmed by
comparison with an authentic sample from its GC retention
time. The spectral and physical data of 2a were in complete
conformity to those of compound 1. The attachment of the
octadecanoate moiety in 2 was determined through a
comparison of the 1H NMR spectrum of 2 with that of 1
and also through an HMBC experiment in which the
protons of the geminal methyl groups (δH 1.02, 0.76) at C-4
3â,6r-Dih yd r oxylu p -20(29)-en e (1): gummy solid; [R]20
D
+14° (c 0.4, CHCl3); IR (CHCl3) νmax 3400, 3045, 1650, 888
cm-1; 1H NMR (CDCl3, 400 MHz) δ 3.16 (1H, dd, J ) 5.5, 10.8
Hz, H-3), 3.81 (1H, dt, J ) 4.8, 10.5 Hz, H-6), 1.02 (3H, s,
H-23), 0.75 (3H, s, H-24), 0.96 (3H, s, H-25), 1.21 (3H, s, H-26),
0.97 (3H, s, H-27), 0.80 (3H, s, H-28), 4.57 (1H, d, J ) 2.2 Hz,
H-29), 4.67 (1H, d, J ) 2.3 Hz, H-29), 1.68 (3H, s, H-30); 13C
NMR (CDCl3, 75 MHz) δ 38.6 (t, C-1), 27.5 (t, C-2), 77.9 (d,
C-3), 38.8 (s, C-4), 55.4 (d, C-5), 67.8 (d, C-6), 44.0 (t, C-7),
38.2 (s, C-8), 50.5 (d, C-9), 32.5 (s, C-10), 22.1 (t, C-11), 25.4
(t, C-12), 37.9 (d, C-13), 41.8 (s, C-14), 27.4 (t, C-15), 35.7 (t,
C-16), 43.2 (s, C-17), 48.5 (d, C-18), 48.2 (d, C-19), 150.9 (s,
C-20), 30.0 (t, C-21), 40.2 (t, C-22), 28.1 (q, C-23), 18.5 (q, C-24),
16.7 (q, C-25) 16.0 (q, C-26), 14.6 (q, C-27), 18.0 (q, C-28), 109.3
(t, C-29), 19.3 (q, C-30); EIMS (70 eV) m/z 442 [M]+, 424 (21),
409 (3), 406 (18), 236 (5), 218 (18), 205 (35), 203 (10), 189 (55);
HREIMS m/z 442.6404 (calcd for C30H50O2, 442.64019).
Acetyla tion of 1. Compound 1 (20 mg) was dissolved in
pyridine (1 mL) with Ac2O (3 mL), and the mixture left
overnight at room temperature. Ice was added to the reaction
mixture, and it was extracted with EtOAc. The EtOAc layer
was evaporated to yield the diacetate 1a . Gummy solid: IR
(CHCl3) νmax 1740, 1665, 1250, 880 cm-1; 1H NMR (CDCl3, 300
MHz) δ 4.45 (1H, m, H-3), 5.51 (1H, m, H-6), 0.90 (6H, s, H-23,
27), 1.32, 1.24, 1.02 (3H each, s, H-20, 25, 24), 1.78 (3H, s,
H-28), 2.16 (6H, s, COCH3-3,6); EIMS (70 eV) m/z 526 [M]+
(9), 466 (8), 406 (6), 258 (15), 218 (100), 189 (15); HREIMS
m/z 526.4827 (calcd for C34H54O4 526.4821).
2
3
showed J correlations to C-4 (δc 38.5) and J interactions
with C-3 (δC 81.4) and C-5 (δC 56.0), confirming the position
of the octadecanoate moiety at C-3. The oxymethine proton
2
(δH 4.48) at C-3 also showed J correlations to C-2 (δC 23.6)
and C-4 (δC 38.5) and a 3J correlation to the carbonyl
carbon (δC 170.2) of the octadecanoate moiety in the HMBC
spectrum. The hydroxyl group at C-6 was assigned an R
and equatorial configuration for the same reasons as
described earlier for compound 1. On the basis of the above
accumulated evidence the structure of 2 was established
as 6R-hydroxylup-20(29)-en-3â-octadecanoate.
Enzyme inhibitory activity of 1 against R-glucosidase
type VI12-14 showed that it was a potent inhibitor, giving
an IC50 value of 42.5 µM. The in vitro antibacterial activity
of 1 was tested against Gram-positive (B. cereus, Staphy-
lococcus pyogenes, and Streptococcus pyogenes) and Gram-
negative (E. coli, P. mirabilis, and S. typhi) bacteria using
an agar well diffusion method.15 Amoxcillin and ampillicin
were used as standard drugs for comparison. Compound 1
showed moderate activity against S. pyogenes (zone of
inhibition 18 mm compared to 35 mm for the standard
drugs at the same concentration level). Compound 2 was
inactive in the assays used in this investigation.
Oxid a tion of 1. Chromium trioxide-pyridine complex (5
mL) and 1 (45 mg) were stirred at room temperature for 12 h.
Dilution of the reaction mixture with cold water, extraction
with diethyl ether, and successive washing with dilute hydro-
chloric acid (2 M) and water afforded white needles of lup-
20(29)-ene-3,6-dione (1b) from chloroform-hexane: mp 170
°C; [R]20 -33° (c 1.5, CHCl3). The physical and spectral data
D
of 1b showed complete agreement with the literature values.11
6r-Hyd r oxylu p -20(29)-en -3â-octa d eca n oa te (2): gummy
Exp er im en ta l Section
solid; [R]20 +26° (c 0.4, CHCl3); IR (CHCl3) νmax 3385, 3050,
D
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined in glass capillary tubes using a Buchi 535 melting
point apparatus, and specific rotations were measured on a
Schimdt and Haensch Polartronic D polarimeter. IR spectra
1735, 1630, 1380, 1230, 888 cm-1; 1H NMR (CDCl3, 400 MHz)
δ 4.48 (1H, dd, J ) 5.2, 10.1 Hz, H-3), 3.95 (1H, dt, J ) 5.0,
10.7 Hz, H-6), 1.02 (3H, s, H-23), 0.76 (3H, s, H-24), 0.84 (3H,
s, H-25), 1.30 (3H, s, H-26), 0.89 (3H, s, H-27), 0.81 (3H, s,
H-28), 4.56 (1H, d, J ) 2.2 Hz, H-29a), 4.68 (1H, d, J ) 2.3
Hz, H-29b), 1.65 (3H, s, H-30), 2.35 (2H, t, J ) 5.7 Hz, H-2′),
1.21-1.29 (30H, br s, H-3′-H-17′), 0.74 (3H, t, J ) 3.5 Hz,
H-18′); 13C NMR (CDCl3, 75 MHz) δ 38.5 (t, C-1), 23.6 (t, C-2),
81.4 (d, C-3), 38.5 (s, C-4), 56.0 (d, C-5), 68.2 (d, C-6), 43.9 (t,
C-7), 38.1 (s, C-8), 50.3 (d, C-9), 31.9 (s, C-10), 21.9 (t, C-11),
25.2 (t, C-12), 36.9 (d, C-13), 41.7 (s, C-14), 27.4 (t, C-15), 35.6
(t, C-16), 43.0 (s, C-17), 48.3 (d, C-18), 48.0 (d, C-19), 150.7 (s,
C-20), 29.9 (t, C-21), 40.0 (t, C-22), 28.1 (q, C-23), 18.2 (q, C-24),
16.6 (q, C-25), 16.0 (q, C-26), 14.5 (q, C-27), 18.2 (q, C-28), 109.3
1
were recorded on a J ASCO A-302 spectrophotometer. H and
13C NMR spectra were performed on a Bruker AM-400 NMR
spectrometer in CDCl3 solution with tetramethylsilane (TMS)
as internal standard. EIMS and HRMS were taken on Finni-
gan MAT-112 and Finnigan MAT-312 instruments, respec-
tively. Si gel (Merck 254, 70-230 mesh) was used for column
chromatography.
P la n t Ma ter ia l. The dried plant material was collected
from Swat (North West Frontier Province, Pakistan) and
identified as Periploca aphylla by Mr. Habib Ahmed. A voucher

Notes
J ournal of Natural Products, 2000, Vol. 63, No. 6 883
(t, C-29), 19.3 (q, C-30), 170.2 (s, C-1′), 34.6 (t, C-2′), 27.1 (t,
C-3′), 25.5 (t, C-4′), 29.4-29.9 (t, C-5′-C-16′), 22.7 (t, C-17′),
14.1 (q, C-18′); EIMS (70 eV) m/z 708 [M]+ (20), 409 (80), 391
(15), 218 (18), 205 (30), 203 (100), 189 (55); HREIMS m/z
708.6410 (calcd for C30H50O2, 708.6419).
Hyd r olysis of 2. Compound 2 (25 mg) was refluxed with
5% methanolic KOH for 3 h. The reaction mixture was
extracted with CHCl3. The CHCl3 solution, after washing with
H2O and drying over anhydrous Na2SO4, was evaporated and
passed over a Si gel column using n-hexanes-EtOAc (9:1) as
eluent to afford 2b (13 mg) and methyl octadecanoate (8 mg).
The latter was identified by comparison with an authentic
sample of methyl octadecanoate (Aldrich Chemical Co., St
Louis, MO), by its GC retention time, while the spectral data
of 2b were completely comparable with those of 1.
En zym e In h ibitor y Activity. Enzyme inhibitory activity
against R-glucosidase type VI (Sigma G6136) was observed
spectrophotometrically at pH 6.8 and at 37 °C using 0.7 mM
p-nitrophenyl R-D-glucopyranoside (PNP-G) as a substrate and
0.017 units/mL enzyme, in 50 mM sodium phosphate buffer
containing 100 mM NaCl, with 0.3 mM 1-deoxynojirimycin
used as positive control (IC50 ) 0.3 mM).13 The absorption
increase at 400 nm due to the hydrolysis of PNP-G by
R-glucosidase was studied continuously with a spectropho-
tometer (Molecular Devices, Sunnyvale, CA).14
An tiba cter ia l Activity. Antibacterial activity of 1 and 2
was determined by the disk diffusion method15 using Nutrient
Agar medium. For this purpose, sterile disks 6 mm in diameter
of filter paper were prepared. From a stock solution of 10 mg/
mL, 10 µL, i.e., 100 µg of the sample was applied to each disk.
The disks were air-dried and then placed on a lawn of the test
organism. The plates were incubated at 37 °C for 24 h, and
then zones of inhibition were observed around each disk,
measured, and recorded.
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NP990426V