New constituents from the dried fruit of Piper nigrum Linn., and their larvicidal potential against the Dengue vector mosquito Aedes aegypti

Article abstract of DOI:10.1016/j.phytol.2013.01.006

Six bioactive compounds were isolated from the seeds extract of Piper nigrum Linn. following a larvicidal activity guided isolation against 4th instar larvae of Aedes aegypti L., a Dengue vector mosquito and a carrier of yellow fever. Their structures were elucidated using spectroscopic methods including HR-EI-MS, FAB-MS, ¹H and ¹³C NMR (Broad Bond Decoupled, & DEPT), and 2D-NMR techniques (¹H-¹H COSY, NOESY, HMQC, HMBC, & 2D-J-resolved). These include three new constituents namely pipilyasine (1), pipzubedine (2) and pipyaqubine (3), and three known constituents pellitorine (4), pipericine (5) and piperine (6). The larvicidal activity was determined by WHO method.

Full text of DOI:10.1016/j.phytol.2013.01.006

Phytochemistry Letters 6 (2013) 219–223
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
New constituents from the dried fruit of Piper nigrum Linn., and their larvicidal
potential against the Dengue vector mosquito Aedes aegypti
a
b,c
b,
d
b
Tahsin Gulzar , Nizam Uddin , Bina Shaheen Siddiqui *, Syed N.H. Naqvi , Sabira Begum ,
e
Rajput Muhammed Tariq
a
Department of Applied Chemistry, G. C. University, Allama Iqbal Road, Faisalabad, Pakistan
b
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
c
Center of Excellence in Marine Biology, University of Karachi, Karachi-75270, Pakistan
d
Department of Pharmacology, Baqai Medical University, Karachi, Karachi-74600, Pakistan
e
MAHQ, Biological Research Center, University of Karachi, Pakistan
A R T I C L E I N F O
A B S T R A C T
Article history:
Six bioactive compounds were isolated from the seeds extract of Piper nigrum Linn. following a larvicidal
activity guided isolation against 4th instar larvae of Aedes aegypti L., a Dengue vector mosquito and a
carrier of yellow fever. Their structures were elucidated using spectroscopic methods including HR-EI-
Received 8 November 2012
Received in revised form 9 January 2013
Accepted 26 January 2013
Available online 21 February 2013
1
13
1
1
MS, FAB-MS, H and C NMR (Broad Bond Decoupled, & DEPT), and 2D-NMR techniques ( H– H COSY,
NOESY, HMQC, HMBC, & 2D-J-resolved). These include three new constituents namely pipilyasine (1),
pipzubedine (2) and pipyaqubine (3), and three known constituents pellitorine (4), pipericine (5) and
piperine (6). The larvicidal activity was determined by WHO method.
Keywords:
Piper nigrum Linn
Larvicidal activity
Aedes aegypti L.
Pipilyasine
ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Pipzubedine
Pipyaqubine
1
. Introduction
Piper (family Piperaceae) is a very large genus consisting of
serious disease vectoring insect pest. Its control primarily depends
on continued applications of synthetic pesticides which are the
most effective larvicides (Rozendaal, 1997) but result in the
development of resistance and undesirable effects on non-target
organisms, and cause environmental hazards (Brown, 1983; Hayes
and Laws, 1991; Rozendaal, 1997).
about 2000 species. Several species of Piper have been used as spice
and in traditional medicine and bear an immense commercial,
economical and medicinal importance (Dymock et al., 1891;
Nadkarni and revised by Nadkarni, 1996; Siddiqui et al., 1997).
Piper nigrum (black pepper) is an acrid, hot, pungent, and a
climbing perpetual shrub (Irvine, 1961; Krishnamurthi, 1969). Its
different parts have been used as medicinal agents for the
treatment of bronchitis, gastrointestinal diseases, and rheumatism
These problems call for the discovery of new easily available
low cost agents which may control mosquito larvae without
producing any cross-resistance. Considering that plants constitute
a rich source of bioactive chemicals our group has been engaged in
search of botanical pesticides for several years and has reported
several new insecticidal compounds including amides and amide
dimers from the fruits of P. nigrum Linn (Siddiqui et al., 1997,
2004a,b,c, 2005; Wei et al., 2004). The present study is a
continuation of these efforts to obtain new constituents from P.
nigrum L. and to investigate their potential against fourth instar
larvae of A. aegypti strains. These findings have led to the isolation
of six larvicidal compounds including three new constituents,
pipilyasine (1), pipzubedine (2) and pipyaqubine (3) together with
three known compounds pellitorine (4) (Kiuchi et al., 1988;
Siddiqui et al., 2004c), pipericine (5) (Siddiqui et al., 1997), and
piperine (6) (Siddiqui et al., 1997) (Fig. 1).
(
Parmar et al., 1997). Phytochemical investigations on this species
undertaken by various groups have led to the isolation of a number
of compounds including alkaloids (Siddiqui et al., 1997, 2004a,b,c,
2
005; Wei et al., 2004), propenylphenols, lignans, neolignans,
terpenes, steroids, pyrones, piperolides, chalcones, flavones and
flavanones (Parmar et al., 1997; Jagella and Grosch, 1999). The
yellow fever mosquitoe, Aedes aegypti (L.) is a widespread and
*
Corresponding author. Tel.: +92 21 99261716; fax: +92 21 99261713 14.
E-mail address: siddiqui_bina@yahoo.com (B.S. Siddiqui).
1
874-3900/$ – see front matter ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.phytol.2013.01.006

2
20
T. Gulzar et al. / Phytochemistry Letters 6 (2013) 219–223
Fig. 1. Structures of compounds isolated from the seeds extract of Piper nigrum Linn.
1
3
2
. Results and discussion
observed in the C NMR spectrum at
d
C
28.6–29.9 and
d
C
23.2
0
which were assigned to CH -7-11, CH
2
2
-2 and CH
2
-12 respec-
Compounds 1–6 were isolated from the ethanol extract of the
dried seeds of P. nigrum L., through solvent fractionation and
chromatographic techniques.
tively (Table 1) on the basis of comparison with the data of
similar partial structures (Breitmaier et al., 1979; Moriyama
et al., 1986). A methylene group adjacent to a double bond in the
Pipilyasine (1) was obtained as a white amorphous powder.
chain was evident from the signals at
33.1, CH -6 in HMQC spectrum. The MS fragments at m/z
86.0979 (C
H
d 2.28 (2H, q, J = 6.7 Hz),
Its molecular formula, C18
H
33NO, was determined from its exact
d
C
2
+
mass at m/z 279.2571 (calcd 279.2562) in the HR-EI-MS which
revealed three unsaturations in the molecule. The IR spectrum of
showed absorptions at 3289 for NH of amide, 1659 for amide
5
H12N) indicated the presence of an N-isopentyla-
+
mide moiety and the fragments at m/z 208.1707 (C13
H
22NO) ,
+
+
1
193.1585 (C13
H
21O) , 165.1654 (C12
H
21
)
and the base peak at
0
ꢀ1
+
RC 55 ONHR , and 1635 cm for the aliphatic C 55 C. Characteristic
absorption band at 256.5 ( = 19,200) in the UV spectrum
m/z 114.0925 (C
6
H
12NO) showed that the N-isopentylamide
e
moiety is bonded to the acyl carbon (Fig. 2). These data match
well with the values reported for compounds with similar
partial structures (Kikuzaki et al., 1993; Kiuchi et al., 1988).
Acid hydrolysis of 1 with conc: HCl and work up (Manosroi
et al., 1999) yielded 3-methylbutyl amine, as identified by GC (co-
injection of authentic samples of similarly branched amines; vide
experimental). In the light of above discussion, the structure of 1
was elucidated as 1-[(2E, 4E)-tridecadienoyl]-N-isopentylamide.
Pipzubedine (2) was obtained as a white amorphous powder
suggested the presence of a conjugated amide moiety (Kikuzaki
et al., 1993). The H NMR spectrum confirmed the presence of a
1
trans-
a,b,g,d-double bonds conjugated with a carboxyl group
by the presence of a doublet at
d
H
5.75 (1H, d, J = 15.8 Hz),
21.3, CH-2, two double doublets at
.9 Hz), 142.7, CH-3, and 6.16 (1H,dd, J = 16.2, 9.9 Hz),
28.2, CH-4, and a double triplet at
.7 Hz), 141.1, CH-5 (Table 1). The conjugation of trans-
-double bond moiety with the carboxyl was confirmed
by HMBC connectivities of H-2 and H-3 with C-1 (Fig. 2). Typical
d
C
1
9
1
6
d
H
7.16 (1H, dd, J = 15.8,
d
C
d
H
d
C
H
d 6.05 (1H, dt, J = 16.2,
d
C
a
,
b
,
g
,
d
and its molecular formula, C22H41NO, was obtained from the exact
mass at m/z 335.3193 (calcd. 335.3188) in the HR-EI-MS. Analysis
of the UV, IR, NMR, DEPT, HSQC and HMBC data (Table 2 and Fig. 3)
and their comparison with those of 1 revealed that the two
compounds are similar, except that the alkenoyl group consists of
seventeen carbons instead of thirteen carbons in 1. Hence, the
structure of 2 was elucidated as 1-[(2E, 4E) – heptadecadienoyl]-N-
isopentylamide.
1
0
H NMR signals at
d
H
3.14 (2H, q, J = 6.6 Hz),
d
C
46.7, CH
2
-1 ,
d
H
0
1
1
d
.81 (1H, m),
d
C
28.1, CH
2
-3 and
d
H
0.93 (6H, d, J = 6.6 Hz),
d
C
0
0
0
9.7, CH
3
-4 and CH
3
-5 , and HMBC correlation between CH-3 at
1
3
H
1.81 and
2
C Signal for CH at 28.6–29.9 displayed an
isopentyl bonded to the nitrogen atom (Kiuchi et al., 1988). A
1
13
terminal methyl group was also indicated by the H and
NMR signals at 0.87 (3H, t, J = 6.7 Hz) 14.5, CH
Further, a broad singlet of fourteen protons was present in the
C
d
H
d
C
3
-13.
Pipyaqubine (3) was obtained as a white amorphous powder
and its molecular formula was obtained as C22H39NO by HR-EI-MS,
region
d
H
1.29–1.41 (14 H, br s) and the correlated carbons were
which gave the exact mass at m/z 333.3034 (calcd 333.3031). The
Table 1
a
NMR spectroscopic data (500 MHz, CDCl
3
) of pipilyasine (1).
Position
d
H
(Multiplicity, J (Hz))
d
C
HSQC
1
2
3
4
5
6
–
165.1
121.3
142.7
128.2
141.1
33.1
C
5.75, d (15.8)
7.16, dd (15.8, 9.9)
6.16, dd (16.2, 9.9)
6.05, dt (16.2, 6.7)
2.28, q (6.7)
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
–
2
2
2
3
2
2
7
1
1
1
2
3
4
5
–11
2
1.29–1.41, br. s
1.29–1.41, br. s
0.87, t (6.7)
28.6–29.9
23.2
3
14.5
0
3.14, q (6.6)
46.7
0
0
0
0
1.29–1.41, br. s
1.81, m
28.6–29.9
28.1
0.93, d (6.6)
19.7
3
3
0.93, d (6.6)
19.7
NH
5.45, br. s
–
a
Chemical shift values are in ppm and assignments are based on DEPT, HSQC,
COSY, and HMBC experiments.
Fig. 2. (a) Mass fragmentation pattern and (b) important HMBC (1) correlations
observed for pipilyasine (1).

T. Gulzar et al. / Phytochemistry Letters 6 (2013) 219–223
221
Table 2
NMR spectroscopic data (500 MHz, CDCl
Table 3
a
a
3
) of pipzubedine (2).
NMR spectroscopic data (500 MHz, CDCl
3
) of pipyaqubine (3).
Position
d
H
(Multiplicity, J (Hz))
d
C
HSQC
Position
d
H
(Multiplicity, J (Hz))
d
C
HSQC
1
2
3
4
5
6
–
165.1
121.2
142.6
127.9
141.2
32.9
C
1
–
163.3
C
5.76, d (15.9)
7.16, dd (15.9, 10.1)
6.13, dd (16.1, 10.1)
6.04, dt (16.1, 6.6)
2.29, q (6.6)
CH
CH
CH
CH
2
5.94, d (16.3)
7.25, dd (16.3, 10.2)
6.11, dd (11.8, 10.2)
6.04, m
119.7
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
3
141.1
4
125.0
5
140.3
CH
CH
CH
CH
CH
CH
CH
CH
CH
–
2
2
2
3
2
2
6
2.30, q (6.8)
27.6
2
2
2
3
2
2
7
1
1
1
2
3
4
5
–13
1.29–1.38, br. s
1.24–1.27, br. s
0.87, t (6.8)
29.1–31.4
22.8–24.6
14.7
7–16
17
18
1.25–1.34, br. s
1.35, m
28.3–31.7
24.1
4–16
7
0.89, t (6.7)
14.4
0
0
0
0
0
3.10, q (6.4)
47.0
1 , 4
3.41–3.52, m
1.80–1.99, m
44.6–46.1
24.9–26.2
0
0
0
0
1.29–1.38, br. s
1.77, m
29.1–31.4
28.3
2 , 3
a
Chemical shift values are in ppm and assignments are based on DEPT, HSQC,
COSY, and HMBC experiments.
0.92, d (6.5)
19.6
3
3
0.92, d (6.5)
19.6
NH
5.43, br. s
–
a
Table 4
Chemical shift values are in ppm and assignments are based on DEPT, HSQC,
COSY, and HMBC experiments.
LC50 of extract, fractions and pure compounds (1–6) against 4th instar larvae of A.
aegypti, a dengue vector mosquito and a carrier of yellow fever.
Crude extract/fractions and pure compounds
LC50 (ppm)
ꢀ
1
IR spectrum exhibited bands at 1655.8 and 1615.2 cm for amide
EtOH extract
35.0
29.5
20.0
28.0
22.0
31.0
20.0
25.0
10.0
50.0
0
RC 55 ONHR and C 55 C. The UV spectrum displayed an absorption
=22,600). The H NMR spectrum (Table 3)
of 3 revealed the presence of a pyrrolidine ring by two multiplets at
PE soluble fraction
PE insoluble fraction
Pipilyasine (1)
Pipzubedine (2)
Pipyaqubine (3)
Pellitorine (4)
Pipericine (5)
1
maximum at 262.3 nm (
e
0
0
d
H
3.41–3.52 (4H, m),
d
C
44.6–46.1, CH
2
-1 and CH
2
-4 , and the
0
0
other at 1.80-1.99 (4H, m)
d
H
d
C
24.9–26.2, CH
2
-2 and CH
2
-3 . It
further showed signals related to four olefinic protons, one with a
trans and the other with a cis geometry. Thus these protons
Piperine (6)
Azadirachtin
displayed signals at
7.25 (1H, dd, J = 16.3, 10.2 Hz),
and protons located on the trans double bond conjugated with
the carboxyl group and at 6.11 (1H, dd, J = 11.8, 10.2 Hz),
25.0, CH-4, and 6.04 (1H, m), 140.3, CH-5 due to the protons
dH 5.94 (1H, d, J = 16.3 Hz),
C
d 119.7, CH-2, and
d
H
d
C
141.1, CH-3 demonstrating
a
b
+
+
+
d
H
d
C
150.0912 (C
263.2381 (C18
discussed above led to elucidate the structure of pipyaqubine as N-
9
H
12NO) , 183.2112 (C13
H
27) , 235.2436 (C17
H31) and
+
1
d
H
d
C
H31O) in the HR-EI-MS (Fig. 4). The spectral data
of the second double bond with a cis geometry and conjugated with
the first double bond. H-2 and H-3 also showed HMBC
connectivities with C-1. These data indicated the presence of a
1
3
[(2E,4Z)-octadecadienoyl]-pyrrolidine (3). The
C NMR data
(Table 3) are consistent with the assigned structure and match
well with the values reported for compounds with similar partial
structures (Kikuzaki et al., 1993; Kiuchi et al., 1988).
trans-
group (Table 3). A methylene protons signal adjacent to the double
bond was observed at 2.30 (2H, q, J = 6.8 Hz, H-6), 27.6, CH
. The up field C NMR shifts of C-6 due to cis shielding confirmed
a,b- and cis-g,d-double bond conjugated with the carboxyl
d
H
d
C
2
-
The toxicity of the ethanolic extract, fractions and pure
constituents 1–6, was determined against fourth instar larvae of
A. aegypti L. by WHO method (WHO, 1970). The results (Table 4)
showed that the extract, both the hexane soluble and insoluble
fractions obtained from the extract (vide experimental) were active
against yellow fever and Dengue fever vector mosquito at the
larval stage. The pure compounds 1 and 2 obtained from the
hexane soluble fraction and 3–6 obtained from the hexane
insoluble fraction were also toxic against the mentioned larvae.
13
6
the geometry of the double bond at C-4 as Z (Crombie, 1955). The
H NMR further showed that remaining part of the chain was same
as of 1 and 2. The assignments of these protons were established by
the analysis of H, H-COSY, HMBC, HMQC and J-resolved
1
1
1
spectrum. Additional support was obtained by the fragment ions
+
+
4 8 5 8
in the HR-EI-MS at m/z 70.0655 (C H N) , 98.0602 (C H NO) ,
Fig. 3. (a) Mass fragmentation pattern and (b) important HMBC (1) correlations
observed for pipzubedine (2).
Fig. 4. (a) Mass fragmentation pattern and (b) important HMBC (1) correlations
observed for Pipyaqubine (3).

2
22
T. Gulzar et al. / Phytochemistry Letters 6 (2013) 219–223
An important observation is that compounds 1–5 with a straight
chain alkenoyl have almost similar toxicities (LC50 20.0–31.0 ppm)
while 6 which has a methylenedioxy benzene ring at the end of the
pentadienoyl group is significantly more toxic (LC50 10.0 ppm).
Moreover, all these constituents exhibited a higher larvicidal
activity against fourth instar larvae of A. aegypti than azadirachtin
hexane–ethyl acetate (8:2) gave two major bands. These were
further purified in the same solvent system furnishing two
compounds pipilyasine (1; 12.8 mg), and pipzubedine (2;
18.5 mg) in order of polarity. A portion of the hexane insoluble
fraction (29.3 g) referred to above was subjected to flash CC using
hexane, hexane–ethyl acetate, and ethyl acetate–methanol (in order
of increasing polarity). On mixing various eluates on the basis of TLC,
9 fractions were obtained (Fr-I–Fr-IX). Fr-I (0.13 g) which was eluted
with hexane and hexane–ethyl acetate (100:0–90:10) was purified
through PTLC in solvent system (hexane–ethyl acetate, 8.0:2.0) to
furnish pipyaqubine (3; 11.8 mg; hexane–ethyl acetate, 8.3:1.7). Fr-
III (hexane–ethyl acetate, 7.0:3.0 eluate; 3.9 g) was subjected to
(LC50 50.0 ppm; (Siddiqui et al., 2009).) which is known as a most
active natural insect growth regulator reported from Azadirachta
indica (Vietmeyer, 1992)
3
. Experimental
0
0
3.1. General methods
column chromatography to get 27 fractions (1 –27 ) using hexane,
hexane–ethyl acetate, ethyl acetate, dichloromethane, and dichlor-
0
UV Spectra: Hitachi-U-3200 spectrophotometer;
l
max in nm. IR
omethane–methanol. Fr-7 (hexane–ethyl acetate, 8.0:2.0 eluate;
ꢀ
1
Spectra: Jasco-A-302 spectrophotometer;
Finnigan-Mat-311A mass spectrometer; source at 2508 and
0 eV; m/z (rel.%). HR-EI-MS: Jeol-JMS-HX-110 mass spectrometer;
n
in cm . EI-MS:
149 mg) on purification over PTLC in solvent system hexane–ethyl
0
acetate (9.0:1.0) afforded pellitorine (4; 6.2 mg) while Fr-11
7
(hexane–ethyl acetate, 7.0:3.0 eluate; 934 mg) furnished pure
pipericine (5; 3.5 mg) on PTLC using the solvent system hexane–
ethyl acetate (8.0:2.0). Fr-IV on PTLC with solvent system
dichloromethane–methanol (19:1) yielded 6 (20.2 mg).
EI, source at 2508 and 70 eV; m/z (rel.%). Vacuum liquid
chromatography (VLC): silica gel 60PF254 (Merck). Flash column
chromatography (FCC): Aldrich flash column chromatograph;
silica gel 9385 (Merck, 0.040–0.063 mm). Prep. TLC (PTLC): silica
1
gel 60PF254 (Merck). H NMR, COSY, NOESY, and J-resolved: Bruker
3.2.1. Pipilyasine (1)
1
spectrometers operating at 400 and 500 MHz for H NMR and at
White amorphous powder; IR (KBr) 3289, 2950, 1635, 1609, 1561,
13
ꢀ1
1
1
00 and 125 MHz for C NMR; chemical shifts (
d
) in ppm, coupling
1240, 1137 cm ; UV (MeOH)
l
max 256.5 nm (
e = 19,200); for H
13
consants (J) in Hz. The chemical shifts were referenced to the
(CDCl , 500 MHz) and C (CDCl , 125 MHz) NMR spectroscopic data,
3 3
1
13
+
residual solvent signals. The assignments of H and C nuclei are
see Table 1. EI-MS m/z 279 (M ; 39), 208 (25), 194 (9), 193 (39), 180
1
13
based on H-, COSY-45, J-resolved, C- (broad band and DEPT),
HMQC and HMBC spectra.
(15), 166 (22), 165 (36), 140 (7), 139 (5), 114 (100), 113 (22), 99 (16),
+
86 (31), 85 (14), 71 (51); HR-EI-MS m/z 279.2571 (M , C18
H
33NO:
+
+
GC analysis – Shimadzu-GC-9A gas chromatograph with FID at
calcd. 279.2562), 208.1707 (C13
H
22NO) , 194.1549 (C12
H
H
20NO) ,
+
+
+
2
60 8C was used. N
2
as carrier gas was passed at a flow rate of 1.0 ml/
193.1585 (C13
165.1654 (C12
114.0925 (C
H
21O) , 180.1395 (C11
H
18NO) , 166.1239 (C10
16NO) ,
1
+
+
+
min using SPB-5 capillary column (30 m ꢁ 0.53 mm ID; 0.3
m
df).
H
21) , 140.1074 (C
8
H
14NO) , 139.1486 (C10
H
19) ,
+
+
+
The split ratio was 1:30 and injector temperature was 240 8C. The
column temperature was maintained at 50 8C for the first 5 min and
then raised to 240 8C at a rate of 3 8C/min with 5 min final time. GC-
EI-MS: Hewlett-Packard 5890 gas chromatograph, combined with a
Jeol, JMS-HX-110 mass spectrometer with source at 2708 at 70 eV.
Injector was set at 2708 with splitting ratio 1:30. Analysis was
6
H
12NO) , 113.1339 (C
8
H
7
) , 99.1162 (C
7
H15) , 86.0979
+
+
+
5 6 5
(C H12N) , 85.1015 (C H13) , 71.0865 (C H11) .
3.2.2. Pipzubedine (2)
White amorphous powder; IR (KBr) 3275, 2950, 1651, 1627,
ꢀ
1
1563, 1231, 1139 cm ; UV (MeOH)
l
C
max 261.4 nm (
e = 18,600);
1
13
performedontheaforementionedprogramonanequivalentcolumn
for
H
(CDCl
3
,
500 MHz) and
(CDCl 125 MHz) NMR
3
,
1
+
HP-5 (25 m ꢁ 0.22 mm and 0.25
m
m df). Mass spectral survey was
spectroscopic data, see Table 2. EI-MS m/z 335 (M ; 32), 278
(15), 264 (27), 249 (38), 221 (46), 195 (10), 180 (13), 169 (18), 166
(22), 155 (16), 140 (9), 114 (100), 86 (51), 71 (26); HR-EI-MS m/z
performed using NIST Mass Spectral Search Program 1998. Hexane
used was of the boiling range 60–80 8C.
+
+
3
35.3193 (M , C22
H
41NO: calcd. 335.3188), 278.2491 (C18
H
32NO) ,
H
+
+
+
3.2. Extraction, isolation and purification
264.2321 (C17 30NO) , 249.2220 (C17
H
H
H
29NO) , 221.2279 (C16
29) ,
+
+
+
195.2119 (C14
27) , 180.1381 (C11
H
18 NO) , 169.1959 (C12
H
25) ,
+
+
+
The dried seeds of P. nigrum (10 kg) were purchased from the
166.1238 (C10
H
16NO) , 155.1789 (C11
H
23) , 140.1071 (C
8
H
14NO) ,
+
+
+
local market in Karachi, crushed and extracted (ꢁ5) with ethanol at
room temperature. After concentration of the combined extract in
vacuo, a syrupy residue was obtained (2 kg). It was left overnight at
room temperature when a semi-crystalline residue separated out
which was filtered and re-crystallized from methanol to yield
whitish crystalline needles of piperine (750 g). The mother liquor
was partitioned between ethyl acetate and water. The ethyl acetate
6 5 5
114.0911 (C H12NO) , 86.0961 (C H12N) , 71.0868 (C H11) .
3.2.3. Pipyaqubine (3)
White amorphous powder; IR (KBr) 2948, 1655, 1615, 1603,
ꢀ
1
1045, 955, 817 cm ; UV (MeOH)
l
max 262.3 nm (
e = 22,600); for
1
13
H (CDCl , 500 MHz) and C (CDCl , 125 MHz) NMR spectroscopic
3 3
+
data, see Table 3. EI-MS m/z 333 (M ; 26.5), 263 (14.1), 262 (13.5),
phase (378 g) was treated with a 4% aqueous solution of Na
remove acidic components. The residue (315 g) obtained on
washing (water), drying (Na SO ), decolorizing (activated charcoal
2
CO
3
to
235 (12.6), 209 (3.9), 206 (11.9), 183 (23.4), 150 (24.6), 127 (15.4),
124 (2.8), 98 (75.8), 85 (42), 71 (30.2), 70 (100), 57 (97.0); HR-EI-
+
2
4
MS m/z 333.3034 (M , C22
H
39NO: calcd. 333.3031), 263.2381
+
+
+
bed) and removal of the solvent from the ethyl acetate layer was
divided into hexane soluble (50.0 g) and hexane insoluble (205.0 g)
fractions. A part (25.0 g) of hexane soluble fraction was subjected
to VLC using hexane, hexane–ethyl acetate, ethyl acetate,
dichloromethane, and dichloromethane–methanol (in order of
increasing polarity). The fractions were combined on the basis of
their TLC to afford 13 fractions (Fr-1–Fr-13). Fr-9 (hexane–ethyl
acetate, 7.8:2.3–7.5–2.5 eluate; 5.3 g) on purification over PTLC in
solvent system hexane–ethyl acetate (8:2–7:3) produced several
bands, which on repeated purification in solvent system
(C18
(C15
H
H
31O) , 262.2177 (C17
H
18NO) , 235.2436 (C17
H
31) , 209.2278
+
+
+
29) , 206.1544 (C13
H20NO) , 183.2112 (C13
H27) , 150.0912
+
+
+
(C
(C
9
H
H
9 7
12NO) , 127.1486 (C H19) , 124.0768 (C H10NO) , 98.0602
+
+
+
+
5
8
5 4 8 4 9
NO) , 71.0861 (C H11) , 70.0655 (C H N) , 57.0702 (C H ) .
3.3. Pesticidal activity
3.3.1. Rearing technique
The 4th instar larvae of A. aegypti L. (O.T. wild strain), a yellow
fever and dengue fever vector mosquito were collected from

T. Gulzar et al. / Phytochemistry Letters 6 (2013) 219–223
223
Brown, A.W.A., 1983. Insecticide resistance as a factor in the integrated control of
Culicidae. In: Laird, M., Miles, J.W. (Eds.), Integrated Mosquito Control Meth-
odologies. Academic, New York, pp. 161–235.
Crombie, L., 1955. Amides of vegetable origin. Part IV. The nature of pellitorine and
anacyclin. J. Chem. Soc. 999–1006.
Dymock, W., Warden, C.J.H., Hooper, D., 1891. Pharmacographia Indica. The Insti-
tute of Health and Tibbi Research, Karachi (Republished under the auspices of
Hamdard National Foundation of Pakistan, p. 398).
Hayes, W.J., Laws, E.R., 1991. Handbook of Pesticide Toxicology. Academic, San
Diego, CA.
semi-natural pond, especially established for this research work.
The size of this pond was 8 ꢁ 4 feet with a depth of 2 feet. The egg
strips of identified mosquito i.e. A. aegypti was dipped into the
pond. The larvae in the pond were fed by dried and grinded prawns
as a powder. The pond was covered with a mosquito net, so that the
intermixing of other mosquito species may be avoided and the
release of mosquitoes from pond into the environment may be
checked. The pupae from the pond were collected daily and kept in
mosquito cages for adult emergence. These adults were fed by
Albino rats twice a week and the filter paper strips were kept in
bowls of 6 in. diameter. The egg strips were dried for one day and
then dipped into the pond for larvae hatching. These larvae at the
stage of 4th instar were used for this research work.
Irvine, F.R., 1961. Woody Plants of Ghana, Woody plants of Ghana. Oxford Univer-
sity Press, London p. 40.
Jagella, T., Grosch, W., 1999. Flavour and off-flavour compounds of black and white
pepper (Piper nigrum L.) I. Evaluation of potent odorants of black pepper by
dilution and concentration techniques. Eur. Food Res. Technol. 209, 16–21.
Kikuzaki, H., Kawabata, M., Ishida, E., Akazawa, Y., Takei, Y., Nakatani, N., 1993. LC-
MS analysis and structural determination of new amides from Javanese long
pepper (Piper retrofractum). Biosci. Biotechnol. Biochem. 57, 1329–1333.
Kiuchi, F., Nakamura, N., Tsuda, Y., Kondo, K., Yoshimura, H., 1988. Studies on crude
drugs effective on visceral larva migrans. IV. Isolation and identification of
larvicidal principles in pepper. Chem. Pharm. Bull. 36, 2452–2465.
Krishnamurthi, A., 1969. The Wealth of India. Council of Scientific and Industrial
Research, New Delhi p. 99.
Manosroi, A., Abe, M., Manosroi, J., 1999. Comparison of antioxidant activity of
extract from seeds of white pepper (Piper nigrum, Linn.) to commercial anti-
oxidants in 2% hydroquinone cream. J. Cosmet. Sci. 50, 221–230.
Moriyama, T., Mandai, T., Kawada, M., Otera, J., Trost, B.M., 1986. Synthesis of
trichonine via double elimination reaction and its structural reinvestigation. J.
Org. Chem. 51, 3896–3897.
3.3.2. Biological test (screening procedure)
Ten young 4th instar larvae of A. aegypti were collected in
2
50 ml beaker having 5 ml of tap water separately and the beaker
was filled up to the level of 200 ml. The extracts, fractions and
compounds were tested at 28 ꢂ 2 8C at five final ppm doses. The
control and check were also set. The observation was recorded after
24 h.
3
.3.3. Accurate tests
Nadkarni, K.M., 1996. Indian Materia Medica. Popular Prakashan, Bombay (revised
by Nadkarni A.K.).
Parmar, V.S., Jain, S.C., Bisht, K.S., Jain, R., Taneja, P., Jha, A., Tyagi, O.D., Prasad, A.K.,
Wengel, J., Olsen, C.E., 1997. Phytochemistry of the genus Piper. Phytochemistry
46, 597–673.
Raymond, M., Prato, G., Ratsira, D., 1993. PROBIT analysis of mortality assays
displaying quantal response. Version 33 License 193019.
Rozendaal, J.A., 1997. Mosquitos and Other Biting Diptera, Vector Control: Methods
for Use by Individuals and Communities. World Health Organization, Geneva,
Switzerland pp. 5–177.
The WHO method (1970) was followed for the application. A
group of 7 beakers was set up, five for different concentrations and
one each for control and check, separately for A. aegypti. Each
experiment was repeated five times. The experiment was
discarded if the mortality was found more than 10% in control
or in check, but in the present experiments the mortality was zero
in control and check. The mortality was recorded after 24 h and
readings were subjected to Abbot’s formula (Abbott, 1925).
Siddiqui, B.S., Ali, S.K., Ali, S.T., Naqvi, S.N.H., Tariq, R.M., 2009. Variation of major
limonoids in Azadirachta indica fruits at different ripening stages and toxicity
against Aedes aegypti. Nat. Prod. Commun. 4, 473–476.
Siddiqui, B.S., Begum, S., Gulzar, T., Noor, F., 1997. An amide from fruits of Piper
nigrum. Phytochemistry 45, 1617–1619.
Siddiqui, B.S., Gulzar, T., Begum, S., Afshan, F., 2004a. Piptigrine, a new insecticidal
amide from Piper Nigrum Linn. Nat. Prod. Res. 18, 473–477.
3
.3.4. Calculation of LC50
The lethal concentrations (LC50) were calculated using PROBIT
analysis for A. aegypti (Raymond et al., 1993).
Siddiqui, B.S., Gulzar, T., Begum, S., Afshan, F., Sattar, F.A., 2004b. Two new
insecticidal amide dimers from fruits of Piper nigrum Linn. Helv. Chim. Acta
Acknowledgements
87, 660–666.
Siddiqui, B.S., Gulzar, T., Mahmood, A., Begum, S., Khan, B., Afshan, F., 2004c. New
insecticidal amides from petroleum ether extract of dried Piper nigrum L. whole
fruits. Chem. Pharm. Bull. 52, 1349–1352.
Siddiqui, B.S., Gulzar, T., Begum, S., Afshan, F., Sattar, F.A., 2005. Insecticidal amides
from fruits of Piper nigrum Linn. Nat. Prod. Res. 19, 143–150.
This research was supported by Indigenous 5000 Ph.D.
fellowship program of the Higher Education Commission of
Pakistan.
Vietmeyer, N.D., 1992. National Research Council, Report of an ad hoc panel of the
Board on Science and Technology for International Development, Neem: a tree
for solving global problems, Washington, DC, USA.
References
Wei, K., Li, W., Koike, K., Pei, Y., Chen, Y., Nikaido, T., 2004. New amide alkaloids from
the roots of Piper nigrum. J. Nat. Prod. 67, 1005–1009.
WHO, 1970. Insecticide resistance and vector control. Seventeenth Report of the
WHO Expert Committee on Insecticides, World Health Organization Technical
Report Series, 443, pp. 1–279.
Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J.
Econ. Entomol. 18, 265–267.
Breitmaier, E., Haas, G., Voelter, W., 1979. Atlas of Carbon-13 NMR Data. Heyden &
Son Ltd, London.