New norterpenoids and a sphingolipid from Carissa opaca

Article abstract of DOI:10.1080/10286020.2015.1064904

Chemical investigations on the aerial parts of Carissa opaca resulted in the isolation and characterization of two new nor-triterpenoids (compounds 1 and 2) and a new sphingolipid (compound 3) together with six known compounds. The structures of all the isolates were established using spectral data. All the isolated compounds showed DPPH radical scavenging and enzyme inhibitory activities against enzymes acetylcholinesterase, butyrylcholinesterase, and lipoxygenase.

Full text of DOI:10.1080/10286020.2015.1064904

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
New norterpenoids and a sphingolipid from
Carissa opaca
Shehla Parveen, Muhammad Saleem, Naheed Riaz, Muhammad Ashraf,
Qurat-ul-Ain, Muhammad Farrukh Nisar & Abdul Jabbar
To cite this article: Shehla Parveen, Muhammad Saleem, Naheed Riaz, Muhammad Ashraf,
Qurat-ul-Ain, Muhammad Farrukh Nisar & Abdul Jabbar (2016) New norterpenoids and a
sphingolipid from Carissa opaca, Journal of Asian Natural Products Research, 18:3, 222-231,
DOI: 10.1080/10286020.2015.1064904
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Date: 26 March 2016, At: 23:27

Journal of Asian Natural Products Research, 2016
Vol. 18, No. 3, 222–231, http://dx.doi.org/10.1080/10286020.2015.1064904
New norterpenoids and a sphingolipid from Carissa opaca
a
Shehla Parveen , Muhammad Saleem *, Naheed Riaz *, Muhammad Ashraf ,
a
a
ab
b
c
a
Qurat-ul-Ain , Muhammad Farrukh Nisar and Abdul Jabbar
Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur,
a
b
Bahawalpur 63100, Pakistan; Department of Biochemistry and Biotechnology, Baghdad-ul-Jadeed
c
Campus, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan; Department of
Botany, University of Gujrat, Gujrat 50700, Pakistan
(Received 19 February 2015; final version received 17 June 2015)
Chemical investigations on the aerial parts of Carissa opaca resulted in the isolation
and characterization of two new nor-triterpenoids (compounds 1 and 2) and a new
sphingolipid (compound 3) together with six known compounds. The structures of all
the isolates were established using spectral data. All the isolated compounds showed
DPPH radical scavenging and enzyme inhibitory activities against enzymes
acetylcholinesterase, butyrylcholinesterase, and lipoxygenase.
Keywords: Carissa opaca; nor-triterpenoid; sphingolipid; acetylcholinesterase
inhibition; butyrylcholinesterase inhibition; lipoxygenase inhibition
1
.
Introduction
phytochemical investigations on this plant.
Herein, we report the isolation and
characterization of two new nor-triter-
penoids 30-nor-2a,3b-dihydoxyurs-12-
ene (1), 30-nor-2a,3b,23-trihydroxyurs-
Carissa opaca, an important member of the
genus Carissa, is an evergreen thorny shrub
commonly found in the semi-arid coastal
areas of the tropical regions. In Pakistan, it
is wildly growing in Punjab, Himalayas,
and up to the height of 6000 ft in Murree [1].
C. opaca is used in folk medicine with
hepatoprotective, aphrodisiac, purgative
1
2-ene (2), a new sphingolipid (3) (Figure
) together with 3b,27-dihydroxylup-12-
1
ene,
lupeol-b-hydroxyoctadecanoate,
pinoresinol, (2)-carinol, (2)-carissanol,
and arjunolic acid ).
[2], and wound-healing properties [3].
Fruits are effective to cough, fever, eye
disorder [4], diarrhea, snakebite [5], and
asthma [6]. In Pakistan, its leaves are used
as a substitute in cardiac disorders [6,7].
The root’s paste is used for the treatment of
horn injuries and maggot wounds in animals
2. Results and discussion
Compound 1 was isolated as a colorless
amorphous solid. Its HR-EI-MS showed
þ
the molecular ion peak [M] at m/z
428.3660 consistent to the molecular
formula C H O with six double bond
[3]. The bark is effective for chicken pox
2
9 48 2
and skin diseases [8]. In Thailand, its stem is
used as healing agent and as a stimulant [9].
The decoction of fresh leaves of C. opaca is
effective in the treatment of jaundice,
various neuromuscular diseases, and hepa-
titis [1]. These remarkable properties of
C. opaca motivated us to carry out the
equivalences (DBE). The Infrared (IR)
spectrum showed the absorption bands due
2
to hydroxyl groups (3415 cm ) and
1
2
olefinic system (1630 cm ). The
1
1
H
NMR spectrum of compound 1 (Table 1)
showed signals for six tertiary methyl
groups at d 1.11, 1.01, 1.01, 0.96, 0.84, and
*Corresponding authors. Email: drsaleem_kr@yahoo.com; nrch322@yahoo.com
q 2015 Taylor & Francis

2
Journal of Asian Natural Products Research
223
2
9
1
9
21
22
1
8
1
1
13
2
5
1
26
H
2
8
14
16
HO
HO
1
0
8
H
2
7
3
5
6
H
R
24
2
3
1
2
R = CH3
R = CH2OH
O
OH
3'
1
'
22'
HN
CH3
2
'
OH
2
0
1
6
HO
CH3
2
3
4
1
5
15
OH
3
Figure 1. Compounds 1–3 isolated from Carissa opaca.
0.80 (3H each, s) corresponding to the
signals at d 24.0, 17.2, 17.5, 17.8, 17.6, and
(d 30.7), C-19 (d 40.4), and C-18 (d 54.3).
1
3
Twenty-nine carbon signals in the
C
1
3
29.3, respectively, in C NMR spectrum
and a secondary methyl group at d 0.89
NMR spectrum of compound 1 (Table 1)
revealed presence of seven methyl, nine
methylene, seven methine, and six qua-
ternary carbon atoms. All the assignments
were accomplished through COSY,
HSQC, and HMBC information and
comparing the data with salvins A and B
[10]. The stereochemistry both at C-2 and
C-3 was determined by NOESY corre-
lations (Figure 3), molecular modeling,
and coupling constants. The larger coup-
ling constant of H-3 (J ¼ 9.8 Hz) and
absence of NOESY correlation with H-25
confirmed its axial and a orientation,
whereas the multiplicity of H-2 (ddd,
J ¼ 9.8, 9.5, 3.5 Hz) and the NOESY
interaction between H-2 with H-25 con-
firmed it b and axial orientation. The
above discussion confirmed compound 1
to be 30-nor-2a,3b-dihydoxyurs-12-ene,
which is a new natural product and is
named carisursane A.
(
3H, d, J ¼ 6.5 Hz) corresponding to the
carbon signal at d 21.5. The signal
resonating at d 5.23 (1H, dd, J ¼ 4.0,
3.5 Hz) was attested for an olefinic proton.
The H NMR spectrum showed the
presence of two oxymethines at d 2.91
1
(
1H, d, J ¼ 9.8 Hz) and 3.61 (1H, ddd,
J ¼ 9.8, 9.5, 3.5 Hz), which showed
COSY correlations to each other,
suggesting their adjacent positions.
A doublet methine at d 2.21 (1H,
d, J ¼ 11.5 Hz, H-18) suggested that
compound 1 could be the ursane-type
triterpenoids and the absence of C-30 was
confirmed through COSY correlation in
which H-29 (d 0.89) showed correlation
with H-19 (d 1.36–1.34) with further
extension to H-18 (d 2.21) and CH -20
2
(
d 1.64–1.61, 1.49–1.46). This infor-
mation was further supported by HMBC
correlations (Figures 2) in which H-29
showed correlations with the carbons C-20
Compound 2 was obtained as colorless
amorphous powder whose HR-EI-MS

2
24
S. Parveen et al.
3
d
d
d
d

4
Journal of Asian Natural Products Research
225
23-trihydroxyurs-12-ene, which is a new
natural product and named carisursane B.
Compound 3 was obtained as a color-
less amorphous solid. The molecular
formula C H NO was determined by
H
4
2
83
5
H
H
H
the data of HR-EI-MS showing the
molecular ion peak at m/z 681.6280 with
two DBE. The IR absorption bands at
HO
HO
H
-1
3335, 1644, and 1623 cm indicated the
presence of hydroxyl, amide, and olefinic
1
groups. The H NMR spectrum of
H
R
compound
3 displayed signals for
Figure 2. Important HMBC correlations of
compounds 1 and 2.
a secondary amide at d 7.40 (1H,
d, J ¼ 8.5 Hz), two oxygenated methylene
groups at d 3.71 (1H, dd, J ¼ 12.0, 4.5 Hz)
and 3.63 (1H, dd, J ¼ 12.0, 2.5 Hz), and
three oxymethine groups at d 3.94 (1H, dd,
J ¼ 8.0, 3.5 Hz), 3.42 (1H, dt, J ¼ 6.3,
4.2 Hz), and 3.39 (1H, dd, J ¼ 4.5, 3.9 Hz).
A signal resonating at d 3.99–3.96 (1H, m)
þ
showed the molecular ion peak [M] at m/z
444.3610 corresponding to the molecular
formula C H O with six DBE. The IR
2
9 48 3
and NMR spectra showed close resem-
blance to those for compound 1 except for
the absence of a tertiary methyl group and
the presence of an oxymethylene group at d
1
3
corresponded to a carbon atom in
C
NMR spectrum at d 51.4, which was
assigned to the azomethine group. These
data indicated compound 3 to be a
sphingolipid [11]. The signal appeared at
d 5.32 (1H, dt, J ¼ 16.5, 5.0 Hz) and 5.28
(1H, dt, J ¼ 16.5, 5.5 Hz), indicating the
presence of double bond in hydrocarbon
chain. The presence of aliphatic chain was
due the signals at d 1.35–1.22 and
terminal methyl groups at d 0.77 (6H,
3
.53 (1H, d, J ¼ 11.0 Hz) and 3.39 (1H, d,
J ¼ 11.0 Hz) corresponding to the carbon
at d 71.3. That indicated that one of the
tertiary methyl groups has been oxidized to
secondary alcohol in compound 2 and its
position was confirmed at C-23 by HMBC
correlation (Figure 2) in which H-24 (d
0
.77) correlated with C-23 (d 71.3), H-23
d 3.53, 3.39) with C-24 (d 17.5), and H-3
d 3.59) with C-23 (d 71.3), confirming the
1
3
(
t, J ¼ 7.0 Hz). The C NMR spectra (BB
and DEPT) of compound 3 displayed the
signal for amide carbonyl group at d 175.6,
and d 75.5, 72.1, 71.8, 60.9 and 51.4 were
(
oxidation of C-23. The above discussion
confirmed compound 2 as 30-nor-2a,3b,
CH3
CH3
H
R
CH3
H
CH3
CH3
HO
HO
CH3
H
H
H
Figure 3. Important COSY and NOESY correlations of compounds 1 and 2.

2
26
S. Parveen et al.
5
O
OH
OH
HN
CH3
HO
CH3
OH
Figure 4. Important HMBC and COSY correlations of compound 3.
attributed to three oxymethine groups, an
oxymethylene group, and an azomethine
group, respectively. The presence of
double bond was confirmed due to the
presence of carbon atoms at d 130.6,
and 2-acetamino-1,3,4-triacetoxyeicosene
at m/z 512. The position of the double bond
was fixed between C-15 and C-16 by
permanganate/periodate oxidative cleavage
[16] of 2-acetamino-1,3,4-triacetoxyeicosene
yielding a mixture of carboxylic acids, which
on methylation and GC–MS analysis
provided peaks for 2-acetamino-1,3,4-triace-
toxypentadecanoic acid at m/z 473 and
pentanoic acid at m/z 102. The configuration
at the stereogenic centers could be deter-
mined by the optical rotation of compound 3
1
29.6. The coupling constants and
chemical shifts C-14 and C-17 (d 32.4)
next to the double bond indicated its
trans geometry [12,13]. The entire
sequence of the molecule was fixed by
HSQC, COSY, and long-range HMBC
correlations (Figure 4). The length of the
fatty acid chain was fixed by the
characteristic fragments at m/z 339 and
([a] þ 33.1) and its methanolysis products
D
([a ] 2 7.2 and þ17.2), which were
D
2
double bond at m/z 308 [M 2 2H O] and
80 and the amide chain containing a
þ
comparable to those of the sphingolipids
with (2S,3S,4R,2 R) configurations [17,18].
0
2
2
25 (Figure 5). Methanolysis [14] of
Based on the above evidence, the structure
of compound 3 could be assigned as
(2S,3S,4R,15E)-2-{[(2R)-2-hydroxydocosa-
noyl]amino}eicos-15-ene-1,3,4-triol and
named carisnin.
compound 3 with methanolic HCl pro-
vided aliphatic oxygenated amine and
methyl ester of the fatty acid. These
fragments were acetylated [15] and ana-
lyzed by GC–MS and were identified as
methyl-2-acetoxydocosanoate at m/z 412
These compounds (1–9) were evalu-
ated for their DPPH radical scavenging
3
39
2
80
-
H
3
3
70
98
O
OH
2
-H O
HN
-
2H
2
O
225
OH
HO
2
-2H O
2
-2H O
3
08
OH
2
-3H O
-
CH
3
OH
2
-H O
3
83
9
7
43
6
48
408 439 453
2
65
Figure 5. EI-MS mass fragmentations of compound 3.

6
Journal of Asian Natural Products Research
227
and enzyme inhibitory activities against
enzymes acetylcholinesterase (AChE),
butyrylcholinesterase (BChE), and lipox-
ygenase (LOX) and results are shown in
Table 2.
3.3 Extraction and isolation
The shade-dried plant material of Carissa
opaca (10 kg) was ground and extracted
thrice with methanol (35 L). The methanolic
extract was concentrated under reduced
pressure to get a dark greenish mass
(455 g), which was subjected to silica gel
column chromatography eluting with n-
hexane, n-hexane:dichloromethane (DCM),
DCM, DCM:methanol, and methanol in
increasing order of polarity to get seven
fractions (1–7). Fraction 1 (2 g) obtained
from n-hexane:DCM (5.5:4.5) on silica gel
column chromatography using isocratic
3
.
Experimental
3
.1 General experimental procedures
IR spectra were recorded on Shimadzu 460
spectrometer (Duisburg, Germany).
1
13
H (400, 500 MHz), C NMR (100,
25 MHz), and 2D NMR (HMQC, HMBC,
1
and COSY; 400, 500 MHz) spectra were
recorded on Bruker spectrometer (Zurich,
Switzerland). The chemical shift values (d)
are reported in ppm and the coupling
constants (J) are in Hz. EI-MS, HR-EI-MS,
FAB-MS, and HR-FAB-MS were recorded
on Finnigan (Varian MAT, Waldbronn,
Germany) JMS H £ 110 with a data system
and JMSA 500 mass spectrometers, respect-
ively. The gas chromatography (GC) was
performed on a Shimadzu gas chromatograph
(50% n-hexane/DCM) mobile phase yielded
3b,27-dihydroxylup-12-ene (25 mg). Frac-
tion 2 (2.5 g) obtained from the main column
using n-hexane:DCM (0.5:9.5) on elution
with pure DCM yielded lupeol-b-hydro-
xyoctadecanoate (27 mg). Fraction 3 (3 g)
subjected to silica gel eluting with DCM
further yielded two sub-fractions. Sub-
fraction 1 (1.9 g) was washed out with ethyl
acetate to obtain arjunolic acid (17 mg),
whereas sub-fraction 2 (3.8 g) when sub-
jected to column chromatography eluted
with DCM:MeOH: (9.8:0.2) yielded pinor-
esinol (26 mg) from the head fractions and
(GC-9A, Noisiel, France) (3% OV-1 silanized
chromosorb W, column temperature 180 C,
8
injection port and detector temperature 275–
3
8
00 C, flow rate 35 ml/min, flame-ionization
detector). Chromatographic separations were
carried out using aluminum sheets pre-coated
with silica gel 60 F254 (20 cm £ 20 cm,
(2)-carinol (37 mg) from the tail fractions.
Fraction 4 (4.8 g) obtained from DCM:
MeOH: (9.7:0.3) was subjected to silica gel
column chromatography eluted with DCM:
MeOH: (9.6:0.4) and divided into two sub-
fractions. Sub-fraction 1 (1.7 g) on purifi-
cation using silica gel eluting with the same
mobile phase yielded (2)-carissanol
0
.2 mm thick; E. Merck; Darmstadt,
Germany) for thin-layer chromatography
TLC) and silica gel (230–400 mesh,
(
Darmstadt, Germany) for column chroma-
tography. TLC plates were visualized under
UV at 254 and 366 nm and by spraying with
ceric sulfate reagent solution (by heating).
(19 mg), whereas sub-fraction 2 (2.9 g) on
repeated silica gel column chromatography
eluting with DCM:MeOH (9.6:0.4)
yielded 30-nor-2a,3b-dihydoxyurs-12-
ene (1, 21 mg) from the head fractions
and 30-nor-2a,3b,23-trihydroxyurs-12-
ene (2, 23 mg). Fraction 5 (2.5 g) obtained
at 6% MeOH in DCM was subjected to
repeated silica gel column chromatog-
raphy using the same solvent system to
obtain carisnin (3, 25 mg). The remaining
fractions contained salts and sugars.
3
.2 Plant material
The whole plants of Carissa opaca
Stapf ex Haines were collected from
Quaid-i-Azam University, Islamabad,
Pakistan, in June 2008 and were identified
by Mr. Farrukh Nisar, Plant Taxonomist,
Department of Botany, University of
Gujrat, Gujrat, Pakistan, where a voucher
specimen is deposited (CO-46/08-BUG).

2
28
S. Parveen et al.
7

8
Journal of Asian Natural Products Research
229
3
.3.1 Carisursane A
30-nor-2a,3b-dihydroxyurs-12-ene; 1)
3.4 Methanolysis
(
Compound 3 (12 mg) was refluxed with
6 ml of 1N HCl and 25 ml of MeOH for
15 h. The reaction mixture was then
extracted with n-hexane to obtain the
corresponding fatty acid methyl ester,
which was analyzed by GC–MS after
2
4
Colorless amorphous solid; [a ]D þ 75.2
-
cm :
1
(
c ¼ 0.0026, MeOH); IR (KBr) n
max
13
415, 2935, 1630, 830; H and C NMR
1
3
spectral data; see Table 1; HR-EI-MS: m/z
þ
4
4
28.3660 [M] (calcd for C H O ,
2
9
48
2
28.3654).
acetylation with Ac O-Py. The aqueous
2
layer was evaporated, and the residue was
acetylated. Purification over Sephadex
LH-20 and elution with CH Cl /MeOH
3.3.2 Carisursane B (30-nor-2a,3b,23-
trihydroxyurs-12-ene; 2)
2
2
1:1 gave the corresponding acetylated
2
4
sphingosine, which was analyzed by
GC–MS.
Colorless amorphous solid; [a ]D þ 58.5
2
1
(
c ¼ 0.002, MeOH); IR (KBr) n
cm
520, 2935, 1655, 830; H and C NMR
:
max
13
1
3
spectral data; see Table 1; HR-EI-MS: m/z
3.4.1 Methyl ester derived from
compound 3
þ
4
4
44.3610 [M] (calcd for C H O ,
48
2
9
3
44.3603).
1
a] 2 7.2; H NMR (CDCl , 300 MHz):
[
D
3
0
d 4.09 (1H, t, J ¼ 6.3, H-2 ), 3.51 (3H,
s, MeO), 1.99 (3H, s, MeCO), 1.32–1.19
(
3
2
.3.3 Carisnin (2S,3S,4R,15E)-2-{[(2R)-
-hydroxydocosanoyl]amino}eicos-15-
0
0
38H, br s, CH -3 –21 ), 0.84 (3H,
2
0
t, J ¼ 6.6, CH –22 ); GC–MS: m/z 412.
3
ene-1,3,4-triol; 3)
2
4
White amorphous solid; [a ]D þ 33.1
3.4.2 Acetylsphingamine derived from
compound 3
(
c ¼ 0.0021, MeOH); IR (KBr): n
max
3
1
335, 3215, 2920, 1644, 1623,
2
1
1
602 cm ; H NMR (CDCl , 400 MHz):
1
3
[a ] þ 17.2;
D
H
NMR
(CDCl3,
d 5.32 (2H, dt, J ¼ 16.5, 5.0 Hz, H-15),
.28 (2H, dt, J ¼ 16.5, 5.5 Hz, H-16), 7.40
300 MHz): d 7.51 (1H, d, J ¼ 7.6, NH),
5.31 (2H, dt, J ¼ 16.2, 5.1, H-15,16),
4.68–4.65 (1H, m, H-4), 4.22–4.20 (1H,
m, H-2), 4.36 (1H, dd, J ¼ 11.3, 4.5, H-1),
4.21 (1H, dd, J ¼ 5.6, 3.1, H-3), 2.00
(12H, 4 £ MeCO), 1.26–1.15 (26H, br s,
CH -5–14,17–19), 0.85 (t, J ¼ 6.5, CH –
5
(
1H, d, J ¼ 8.5 Hz, NH), 3.99–3.96 (1H,
m, H-2), 3.94 (1H, dd, J ¼ 8.0, 3.5 Hz,
0
H-2 ), 3.71 (1H, dd, J ¼ 12.0, 4.5Hz, H-1a),
3
.63 (1H, dd, J ¼ 12.0, 2.5 Hz, H-1b), 3.42
(
1H, dt, J ¼ 6.3, 4.2 Hz, H-4), 3.39 (1H,
2
3
dd, J ¼ 4.5, 3.9 Hz, H-3), 1.94–1.89 (4H,
20); GC–MS: m/z 511.
0
m, H-14,17), 1.70–1.67 (1H, m, H-3 a),
1
m, H-3 b), 1.31–1.28 (1H, m, H-5b),
.61–1.58 (1H, m, H-5a), 1.49–1.46 (1H,
0
3.5 Oxidative cleavage of the double
bond in compound 3
0
1
.33–1.22 (56H, br s, H-6–13,18–19,4 –
0
0
2
1 ), 0.77 (6H, t, J ¼ 7.0 Hz, H-20,22 );
To the solution of methyl ester of
compound 3 (4 mg) in acetone, added
1 ml of 0.04 M solution of K CO , 6 ml of
1
3
C NMR (CDCl , 100 MHz): d 175.6
3
0
(
(
C-1 ), 130.6 (C-16), 129.6 (C-15), 75.5
0
2
3
C-3), 72.1 (C-4), 71.8 (C-2 ), 60.9 (C-1),
an aqueous solution 0.025 M KMnO and
4
0
5
3
2
6
6
1.4 (C-2), 34.2 (C-3 ), 32.4 (C-14,17),
0
0.09 M NaIO in a 100 ml round-bottom
4
1.8 (C-5), 22.5–31.7 (6–13,18–19,4 -
0
flask. The reaction was allowed to proceed
8
at 37 C for 18 h. After acidification with
0
1 ), 13.8 (C-20,22 ); HR-EI-MS: m/z
þ
81.6280 [M] (calcd for C H NO ,
4
5 N H SO , the solution was decolorized
2
2
83
5
4
81.6271).
with a 1M solution of oxalic acid and

2
30
S. Parveen et al.
9
extracted with Et O (3–10 ml). The
2
compounds were calculated using the
EZ-Fit enzyme kinetics software (Perella
Scientific Inc., Amherst, USA).
combined organic extract was dried over
Na SO , filtered, and concentrated. The
2
4
resulting carboxylic acids were methylated
with ethereal solution of diazomethane and
analyzed by GC–MS.
3
.6.4 Lipoxygenase assay
LOX activity was assayed according to the
reported method [21] but with slight
modifications. The percentage inhibition
was calculated by the formula given
below:
3
.6 Biological activity assays
3
.6.1 DPPH radical scavenging activity
The DPPH radical scavenging activity was
examined by comparison with that of a
known antioxidant, quercetin, using the
method developed by Lee and Shibamoto
Control2Test
Control
Inhibitionð%Þ ¼
£ 100
[19] with slight modification of decolora-
Disclosure statement
tion of the samples and was calculated
according to the formula:
No potential conflict of interest was reported by
the authors.
Acontrol 2 Asample
Antiradicalactivity ¼
Acontrol
Funding
We are thankful to Higher Education Commis-
sion (HEC) of Pakistan for providing financial
aid to the indigenous Ph.D. scholars of our
laboratory.
£
100:
Each sample was assayed in triplicate
and the mean values were calculated.
References
3
.6.2 Acetylcholinesterase assay
[
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(
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