Phytochemistry Letters 6 (2013) 219–223

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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
Tahsin Gulzar a, Nizam Uddin b,c, Bina Shaheen Siddiqui b,*, Syed N.H. Naqvi d, Sabira Begum b,
Rajput Muhammed Tariq e
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
Received 8 November 2012 activity guided isolation against 4th instar larvae of Aedes aegypti L., a Dengue vector mosquito and a
Received in revised form 9 January 2013 carrier of yellow fever. Their structures were elucidated using spectroscopic methods including HR-EI-
Accepted 26 January 2013
MS, FAB-MS, 1H and 13C NMR (Broad Bond Decoupled, & DEPT), and 2D-NMR techniques (1H–1H COSY,
Available online 21 February 2013
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
Keywords:
piperine (6). The larvicidal activity was determined by WHO method.
Piper nigrum Linn
Larvicidal activity
ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Aedes aegypti L.
Pipilyasine
Pipzubedine
Pipyaqubine

1. Introduction
Piper (family Piperaceae) is a very large genus consisting of
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
(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,
2005; 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).
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).
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).

1874-3900/$ – 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
220 T. Gulzar et al. / Phytochemistry Letters 6 (2013) 219–223

Fig. 1. Structures of compounds isolated from the seeds extract of Piper nigrum Linn.

2. Results and discussion
Compounds 1–6 were isolated from the ethanol extract of the
dried seeds of P. nigrum L., through solvent fractionation and
chromatographic techniques.
Pipilyasine (1) was obtained as a white amorphous powder.
Its molecular formula, C18H33NO, was determined from its exact
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
1 showed absorptions at 3289 for NH of amide, 1659 for amide
RC5 5ONHR0 , and 1635 cm1 for the aliphatic C5 5C. Characteristic
absorption band at 256.5 (e = 19,200) in the UV spectrum
suggested the presence of a conjugated amide moiety (Kikuzaki
et al., 1993). The 1H NMR spectrum confirmed the presence of a
trans-a,b,g,d-double bonds conjugated with a carboxyl group
by the presence of a doublet at dH 5.75 (1H, d, J = 15.8 Hz), dC
121.3, CH-2, two double doublets at dH 7.16 (1H, dd, J = 15.8,
9.9 Hz), dC 142.7, CH-3, and dH 6.16 (1H,dd, J = 16.2, 9.9 Hz), dC
128.2, CH-4, and a double triplet at dH 6.05 (1H, dt, J = 16.2,
6.7 Hz), dC 141.1, CH-5 (Table 1). The conjugation of trans-
a,b,g,d-double bond moiety with the carboxyl was confirmed
by HMBC connectivities of H-2 and H-3 with C-1 (Fig. 2). Typical
1
H NMR signals at dH 3.14 (2H, q, J = 6.6 Hz), dC 46.7, CH2-10 , dH
1.81 (1H, m), dC 28.1, CH2-30 and dH 0.93 (6H, d, J = 6.6 Hz), dC
19.7, CH3-40 and CH3-50 , and HMBC correlation between CH-30 at
dH 1.81 and 13C Signal for CH2 at 28.6–29.9 displayed an
isopentyl bonded to the nitrogen atom (Kiuchi et al., 1988). A
terminal methyl group was also indicated by the 1H and 13C
NMR signals at dH 0.87 (3H, t, J = 6.7 Hz) dC 14.5, CH3-13.
Further, a broad singlet of fourteen protons was present in the
region dH 1.29–1.41 (14 H, br s) and the correlated carbons were
observed in the 13C NMR spectrum at dC 28.6–29.9 and dC 23.2
which were assigned to CH2-7-11, CH2-20 and CH2-12 respec-
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
chain was evident from the signals at dH 2.28 (2H, q, J = 6.7 Hz),
dC 33.1, CH2-6 in HMQC spectrum. The MS fragments at m/z
86.0979 (C5H12N)+ indicated the presence of an N-isopentyla-
mide moiety and the fragments at m/z 208.1707 (C13H22NO)+,
193.1585 (C13H21O)+, 165.1654 (C12H21)+ and the base peak at
m/z 114.0925 (C6H12NO)+ showed that the N-isopentylamide
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
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.
Pipyaqubine (3) was obtained as a white amorphous powder
and its molecular formula was obtained as C22H39NO by HR-EI-MS,
which gave the exact mass at m/z 333.3034 (calcd 333.3031). The

Table 1
NMR spectroscopic dataa (500 MHz, CDCl3) of pipilyasine (1).

Position dH (Multiplicity, J (Hz)) dC HSQC

1 – 165.1 C
2 5.75, d (15.8) 121.3 CH
3 7.16, dd (15.8, 9.9) 142.7 CH
4 6.16, dd (16.2, 9.9) 128.2 CH
5 6.05, dt (16.2, 6.7) 141.1 CH
6 2.28, q (6.7) 33.1 CH2
7–11 1.29–1.41, br. s 28.6–29.9 CH2
12 1.29–1.41, br. s 23.2 CH2
13 0.87, t (6.7) 14.5 CH3
10 3.14, q (6.6) 46.7 CH2
20 1.29–1.41, br. s 28.6–29.9 CH2
30 1.81, m 28.1 CH
40 0.93, d (6.6) 19.7 CH3
50 0.93, d (6.6) 19.7 CH3
NH 5.45, br. s – –
a Fig. 2. (a) Mass fragmentation pattern and (b) important HMBC (1) correlations
Chemical shift values are in ppm and assignments are based on DEPT, HSQC,
COSY, and HMBC experiments. observed for pipilyasine (1).
 T. Gulzar et al. / Phytochemistry Letters 6 (2013) 219–223 221

Table 2 Table 3
NMR spectroscopic dataa (500 MHz, CDCl3) of pipzubedine (2). NMR spectroscopic dataa (500 MHz, CDCl3) of pipyaqubine (3).

Position dH (Multiplicity, J (Hz)) dC HSQC Position dH (Multiplicity, J (Hz)) dC HSQC

1 – 165.1 C 1 – 163.3 C
2 5.76, d (15.9) 121.2 CH 2 5.94, d (16.3) 119.7 CH
3 7.16, dd (15.9, 10.1) 142.6 CH 3 7.25, dd (16.3, 10.2) 141.1 CH
4 6.13, dd (16.1, 10.1) 127.9 CH 4 6.11, dd (11.8, 10.2) 125.0 CH
5 6.04, dt (16.1, 6.6) 141.2 CH 5 6.04, m 140.3 CH
6 2.29, q (6.6) 32.9 CH2 6 2.30, q (6.8) 27.6 CH2
7–13 1.29–1.38, br. s 29.1–31.4 CH2 7–16 1.25–1.34, br. s 28.3–31.7 CH2
14–16 1.24–1.27, br. s 22.8–24.6 CH2 17 1.35, m 24.1 CH2
17 0.87, t (6.8) 14.7 CH3 18 0.89, t (6.7) 14.4 CH3
10 3.10, q (6.4) 47.0 CH2 10 , 40 3.41–3.52, m 44.6–46.1 CH2
20 1.29–1.38, br. s 29.1–31.4 CH2 20 , 30 1.80–1.99, m 24.9–26.2 CH2
30 1.77, m 28.3 CH a
Chemical shift values are in ppm and assignments are based on DEPT, HSQC,
40 0.92, d (6.5) 19.6 CH3
COSY, and HMBC experiments.
50 0.92, d (6.5) 19.6 CH3
NH 5.43, br. s – –
a
Chemical shift values are in ppm and assignments are based on DEPT, HSQC, Table 4
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)

IR spectrum exhibited bands at 1655.8 and 1615.2 cm1 for amide EtOH extract 35.0
RC5 5ONHR0 and C5 5C. The UV spectrum displayed an absorption PE soluble fraction 29.5
maximum at 262.3 nm (e=22,600). The 1H NMR spectrum (Table 3) PE insoluble fraction 20.0
Pipilyasine (1) 28.0
of 3 revealed the presence of a pyrrolidine ring by two multiplets at
Pipzubedine (2) 22.0
dH3.41–3.52 (4H, m), dC 44.6–46.1, CH2-10 and CH2-40 , and the Pipyaqubine (3) 31.0
other at dH1.80-1.99 (4H, m) dC 24.9–26.2, CH2-20 and CH2-30 . It Pellitorine (4) 20.0
further showed signals related to four olefinic protons, one with a Pipericine (5) 25.0
Piperine (6) 10.0
trans and the other with a cis geometry. Thus these protons
Azadirachtin 50.0
displayed signals at dH 5.94 (1H, d, J = 16.3 Hz), dC 119.7, CH-2, and
dH 7.25 (1H, dd, J = 16.3, 10.2 Hz), dC 141.1, CH-3 demonstrating a
and b protons located on the trans double bond conjugated with
the carboxyl group and at dH6.11 (1H, dd, J = 11.8, 10.2 Hz), dC 150.0912 (C9H12NO)+, 183.2112 (C13H27)+, 235.2436 (C17H31)+ and
125.0, CH-4, and dH6.04 (1H, m), dC 140.3, CH-5 due to the protons 263.2381 (C18H31O)+ in the HR-EI-MS (Fig. 4). The spectral data
of the second double bond with a cis geometry and conjugated with discussed above led to elucidate the structure of pipyaqubine as N-
the first double bond. H-2 and H-3 also showed HMBC [(2E,4Z)-octadecadienoyl]-pyrrolidine (3). The 13C NMR data
connectivities with C-1. These data indicated the presence of a (Table 3) are consistent with the assigned structure and match
trans-a,b- and cis-g,d-double bond conjugated with the carboxyl well with the values reported for compounds with similar partial
group (Table 3). A methylene protons signal adjacent to the double structures (Kikuzaki et al., 1993; Kiuchi et al., 1988).
bond was observed at dH 2.30 (2H, q, J = 6.8 Hz, H-6), dC 27.6, CH2- The toxicity of the ethanolic extract, fractions and pure
6. The up field 13C NMR shifts of C-6 due to cis shielding confirmed constituents 1–6, was determined against fourth instar larvae of
the geometry of the double bond at C-4 as Z (Crombie, 1955). The A. aegypti L. by WHO method (WHO, 1970). The results (Table 4)
1
H NMR further showed that remaining part of the chain was same showed that the extract, both the hexane soluble and insoluble
as of 1 and 2. The assignments of these protons were established by fractions obtained from the extract (vide experimental) were active
the analysis of 1H, 1H-COSY, HMBC, HMQC and J-resolved against yellow fever and Dengue fever vector mosquito at the
spectrum. Additional support was obtained by the fragment ions larval stage. The pure compounds 1 and 2 obtained from the
in the HR-EI-MS at m/z 70.0655 (C4H8N)+, 98.0602 (C5H8NO)+, hexane soluble fraction and 3–6 obtained from the hexane
insoluble fraction were also toxic against the mentioned larvae.

Fig. 3. (a) Mass fragmentation pattern and (b) important HMBC (1) correlations Fig. 4. (a) Mass fragmentation pattern and (b) important HMBC (1) correlations
observed for pipzubedine (2). observed for Pipyaqubine (3).
222 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
(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
3.1. General methods
UV Spectra: Hitachi-U-3200 spectrophotometer; lmax in nm. IR
Spectra: Jasco-A-302 spectrophotometer; n in cm1. EI-MS:
Finnigan-Mat-311A mass spectrometer; source at 2508 and
70 eV; m/z (rel.%). HR-EI-MS: Jeol-JMS-HX-110 mass spectrometer;
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
gel 60PF254 (Merck). 1H NMR, COSY, NOESY, and J-resolved: Bruker
spectrometers operating at 400 and 500 MHz for 1H NMR and at
100 and 125 MHz for 13C NMR; chemical shifts (d) in ppm, coupling
consants (J) in Hz. The chemical shifts were referenced to the
residual solvent signals. The assignments of 1H and 13C nuclei are
based on 1H-, COSY-45, J-resolved, 13C- (broad band and DEPT),
HMQC and HMBC spectra.
GC analysis – Shimadzu-GC-9A gas chromatograph with FID at
260 8C was used. N2 as carrier gas was passed at a flow rate of 1.0 ml/
min using SPB-51 capillary column (30 m  0.53 mm ID; 0.3 m df).
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
performed on the aforementioned program on an equivalent column
HP-51 (25 m  0.22 mm and 0.25 mm df). Mass spectral survey was
performed using NIST Mass Spectral Search Program 1998. Hexane
used was of the boiling range 60–80 8C.
3.2. Extraction, isolation and purification
The dried seeds of P. nigrum (10 kg) were purchased from the
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
phase (378 g) was treated with a 4% aqueous solution of Na2CO3 to
remove acidic components. The residue (315 g) obtained on
washing (water), drying (Na2SO4), decolorizing (activated charcoal
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
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
column chromatography to get 27 fractions (10 –270 ) using hexane,
hexane–ethyl acetate, ethyl acetate, dichloromethane, and dichlor-
omethane–methanol. Fr-70 (hexane–ethyl acetate, 8.0:2.0 eluate;
149 mg) on purification over PTLC in solvent system hexane–ethyl
acetate (9.0:1.0) afforded pellitorine (4; 6.2 mg) while Fr-110
(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).
3.2.1. Pipilyasine (1)
White amorphous powder; IR (KBr) 3289, 2950, 1635, 1609, 1561,
1240, 1137 cm1; UV (MeOH) lmax 256.5 nm (e = 19,200); for 1H
(CDCl3, 500 MHz) and 13C (CDCl3, 125 MHz) NMR spectroscopic data,
see Table 1. EI-MS m/z 279 (M+; 39), 208 (25), 194 (9), 193 (39), 180
(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+, C18H33NO:
calcd. 279.2562), 208.1707 (C13H22NO)+, 194.1549 (C12H20NO)+,
193.1585 (C13H21O)+, 180.1395 (C11H18NO)+, 166.1239 (C10H16NO)+,
165.1654 (C12H21)+, 140.1074 (C8H14NO)+, 139.1486 (C10H19)+,
114.0925 (C6H12NO)+, 113.1339 (C8H7)+, 99.1162 (C7H15)+, 86.0979
(C5H12N)+, 85.1015 (C6H13)+, 71.0865 (C5H11)+.
3.2.2. Pipzubedine (2)
White amorphous powder; IR (KBr) 3275, 2950, 1651, 1627,
1563, 1231, 1139 cm1; UV (MeOH) lmax 261.4 nm (e = 18,600);
for 1H (CDCl3, 500 MHz) and 13C (CDCl3, 125 MHz) NMR
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
335.3193 (M+, C22H41NO: calcd. 335.3188), 278.2491 (C18H32NO)+,
264.2321 (C17H30NO)+, 249.2220 (C17H29NO)+, 221.2279 (C16H29)+,
195.2119 (C14H27)+, 180.1381 (C11H18 NO)+, 169.1959 (C12H25)+,
166.1238 (C10H16NO)+, 155.1789 (C11H23)+, 140.1071 (C8H14NO)+,
114.0911 (C6H12NO)+, 86.0961 (C5H12N)+, 71.0868 (C5H11)+.
3.2.3. Pipyaqubine (3)
White amorphous powder; IR (KBr) 2948, 1655, 1615, 1603,
1045, 955, 817 cm1; UV (MeOH) lmax 262.3 nm (e = 22,600); for
1
H (CDCl3, 500 MHz) and 13C (CDCl3, 125 MHz) NMR spectroscopic
data, see Table 3. EI-MS m/z 333 (M+; 26.5), 263 (14.1), 262 (13.5),
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-
MS m/z 333.3034 (M+, C22H39NO: calcd. 333.3031), 263.2381
(C18H31O)+, 262.2177 (C17H18NO)+, 235.2436 (C17H31)+, 209.2278
(C15H29)+, 206.1544 (C13H20NO)+, 183.2112 (C13H27)+, 150.0912
(C9H12NO)+, 127.1486 (C9H19)+, 124.0768 (C7H10NO)+, 98.0602
(C5H8NO)+, 71.0861 (C5H11)+, 70.0655 (C4H8N)+, 57.0702 (C4H9)+.
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
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.
3.3.2. Biological test (screening procedure)
Ten young 4th instar larvae of A. aegypti were collected in
250 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  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
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).
3.3.4. Calculation of LC50
The lethal concentrations (LC50) were calculated using PROBIT
analysis for A. aegypti (Raymond et al., 1993).
Acknowledgements
This research was supported by Indigenous 5000 Ph.D.
fellowship program of the Higher Education Commission of
Pakistan.
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