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Phytochemical studies on the seedextract of Piper nigrum Linn.Bina S. Siddiqui a , Tahsin Gulzar a , Azhar Mahmood b , SabiraBegum a , Bushra Khan b , Munawwer Rasheed a , Farhana Afshan a

& Rajput M. Tariq ca H. E. J. Research institute of Chemistry, International Centre forChemical Sciences, University of Karachi, Karachi-75270, Pakistanb Department of Chemistry, University of Karachi, Karachi-75270,Pakistanc Department of Zoology, University of Karachi, Karachi-75270,PakistanVersion of record first published: 20 Aug 2006.

To cite this article: Bina S. Siddiqui , Tahsin Gulzar , Azhar Mahmood , Sabira Begum , Bushra Khan ,Munawwer Rasheed , Farhana Afshan & Rajput M. Tariq (2005): Phytochemical studies on the seedextract of Piper nigrum Linn., Natural Product Research: Formerly Natural Product Letters, 19:7,703-712

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Natural Product Research, Vol. 19, No. 7, October 2005, 703–712

Phytochemical studies on the seed extract of

Piper nigrum Linn.

BINA S. SIDDIQUIy*, TAHSIN GULZARy, AZHAR MAHMOODz,SABIRA BEGUMy, BUSHRA KHANz, MUNAWWER RASHEEDy,

FARHANA AFSHANy and RAJPUT M. TARIQx

yH. E. J. Research institute of Chemistry, International Centre for Chemical Sciences,University of Karachi, Karachi-75270, Pakistan

zDepartment of Chemistry, University of Karachi, Karachi-75270, PakistanxDepartment of Zoology, University of Karachi, Karachi-75270, Pakistan

(Received 9 April 2004 )

The petroleum ether extract of dried ground seeds of Piper nigrum Linn. and some column frac-tions of this extract were subjected to GC and GC-MS analysis, resulting in the identification offourteen compounds (1–14) by using NIST Mass spectral search program 1998 and the Kovat’sretention indices. Ten of the compounds (1, 2, 4–12) are reported for the first time from thisplant. All the fractions showed insecticidal activity against the fourth instar larvae of Aedesaegypti and against the fourth instar larvae of Anopheles stephensi Liston, determined by theWHO method.

Keywords: Piper nigrum Linn.; Piperaceae; GC and GC-EI-MS analysis; Aedes aegypti;Anopheles stephensi

1. Introduction

Piper nigrum L., (black pepper) belongs to the family Piperaceae. This species bearsa great commercial, economical and medicinal potential, due to the attributed physio-logical activities [1–5]. In search of plant-based potent insecticides [6–10], currentinvestigations on the petroleum ether extract of seeds of P. nigrum have resulted inthe identification of fourteen compounds. The petroleum-ether extract of the seeds ofP. nigrum and its fraction obtained through column chromatography were subjectedto GC and GC-EI-MS analysis (table 1). The components of these fractions wereidentified mainly by the NIST Mass spectral search program 1998 [11] and byKovat’s retention indices [12,13]. Literature survey revealed that out of these fourteencompounds, ten compounds (1, 2, 4–12) are reported for the first time from this genusand three compounds, 3, 13 and 14, have already been reported from this species[5,10,14–16]. Two compounds, 1 and 3, were previously reported from another species

*Corresponding author. E-mail: bina@khi.comsats.net.pk

Natural Product Research

ISSN 1478-6419 print: ISSN 1029-2349 online � 2005 Taylor & Francis Group Ltd

http://www.tandf.co.uk/journals

DOI: 10.1080/14786410512331330657

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P. marginatum through the GC and GC-EI-MS analyses [15]. In continuation of ourstudies [17–20] on indigenous medicinal plants possessing pesticidal activity againstAe. aegypti and An. stephensi, the present work has been undertaken on the seedsof P. nigrum. The fractions showing pesticidal activity against the fourth instarlarvae of Ae. aegypti (table 2a) and against the fourth instar larvae of An. stephensiListon (table 2b) by the WHO method [21] were analysed by GC and GC-EI-MS.The components of these fractions were identified, mainly by NIST (NationalInstitute of Standards and Technology).

1.1. GC and GC-EI-MS

Fourteen compounds were identified: six of them are hydrocarbons, five are esters andthe other two are acids, along with piperine. It is interesting to note that all the hydro-carbons characterized until now were aliphatic, and that no aromatic hydrocarbonhad been identified as yet from the above-mentioned selected fractions. These aliphatic

Table 1a. LC50 of fractions of the seeds of P. nigrum L.

LC50(ppm)

Fraction Ae. aegypti An. stephensi

PEP 8.0 16.0Fr-1 50.0 100.0Fr-2 6.0 12.0Fr-3 31.0 70.0Fr-5 5.0 10.0Fr-10 20.0 40.0Fr-11 11.0 22.0Fr-12 40.0 80.0Fr-13 25.0 50.0Fr-14 45.0 90.0Fr-15-100 20.0 48.0

Table 1b. GC-analysis of fractions of the seeds of P. nigrum L.

Fraction Compounds identified % RI

PEP 2 2.13 15704 1.87 165010 9.88 231611 23.69 2416

Fr-1 9 7.96 2349Fr-2 5 2.27 1683Fr-3 1 1.83 1537Fr-5 3 14.18 1570

16504 1.87

Fr-10 6 6.23 **Fr-11 7 2.10 2137

8 1.98 2206Fr-12 8 1.98 2206Fr-13 11 23.69 2416Fr-14 12 7.10 2247

13 6.20 2477Fr-15-100 14 5.04 **

**Identified by co-injection.

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hydrocarbons include open chain, mono-, bi- and tri-cyclic compounds; saturated andunsaturated hydrocarbons namely 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)-[1S-(1�,2�,4�)]-cyclohexane (1), tricyclo[6.2.1.0(4,11)]undec-5-ene,1,5,9,9-tetramethyl-(iso-caryophyllene-II) (2), 4,11,11-trimethyl-8-methylene-,[1R-(1R*,4E,9S*)]-bicyclo [7.2.0]undec-4-ene (3), decahydro-4a-methyl-1-methylene-7-(1-methylethenyl)-,[4aR-(4a�,7�,8a�)]-naphthalene (4), 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methylethenyl)-,[1S-(1�,7�,8a�)]-naphthalene (5), nonacosane (6). Esters obtained include methylhexadecanoate (7), ethyl hexadecanoate (8), methyl 14-methyl heptadecanoate (9),methyl-trans-8-octadecanote (10), ethyl-cis-9-octadecanaote (11). The two acids wereidentified as normal saturated acids, hexadecanoic acid (12) and octadecanoicacid (13), along with a known compound 1-[5-(1,3-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]-,(E,E)-piperidine (14).

1.2. Characterization of constituents through GC/GC-EI-MS

1.2.1. 1-Ethenyl-1-methyl-2,4-bis(1-methylethenyl)-[1S-(1�,2�,4�)]-cyclohexane,(1). (common name: �-Elemene) GC-EIMS: m/z (rel. int.)¼ 204 (C15H24, M

þ; 4), 189

Table 2a. Biological activities of fractions from fruits of P. nigrum L. showing their final dose in ppm andtheir mean mortalities with S.D. values against fourth instar larvae of Ae. aegypti L. (O. T. wild-strain).

Entry Dose (ppm) MþS.D. Dose (ppm) MþS.D. Dose (ppm) Mþ S.D.

PEP Fr-1 Fr-20 ctl 04� 4 ctl 02� 4 ctl 04� 41 4.0 14� 5 12.5 08� 4 2.0 08� 42 6.0 28� 4 25.0 14� 5 4.0 24� 53 8.0 50� 7 37.5 24� 5 6.0 50� 74 10.0 72� 4 50.0 50� 7 8.0 77� 45 12.0 94� 5 62.5 76� 5 10.0 94� 5

Fr-3 Fr-4 Fr-50 ctl 02� 4 ctl 00� 0 ctl 00� 01 11.0 10� 7 2.0 04� 4 1.0 18� 42 21.0 28� 4 4.0 10� 7 3.0 34� 53 31.0 50� 7 6.0 20� 7 5.0 50� 74 41.0 76� 5 8.0 36� 5 7.0 76� 55 51.0 82� 8 10.0 50� 7 9.0 88� 4

Fr-6 & Fr-7 Fr-8 Fr-90 ctl 00� 0 ctl 02� 4 Ctl 00� 01 5.0 08� 4 5.0 06� 5 15.0 20� 72 10.0 20� 7 10.0 14� 5 30.0 34� 53 15.0 34� 5 15.0 22� 4 45.0 50� 74 20.0 50� 7 20.0 36� 5 60.0 72� 45 25.0 77� 4 25.0 50� 7 75.0 90� 7

Fr-10 Fr-11 Fr-120 ctl 00� 0 ctl 02� 4 ctl 00� 01 10.0 22� 4 7.0 20� 7 10.0 10� 72 15.0 34� 5 9.0 34� 5 20.0 22� 43 20.0 50� 7 11.0 50� 7 30.0 36� 54 25.0 66� 5 13.0 77� 4 40.0 50� 75 30.0 84� 5 15.0 94� 5 50.0 66� 5

Fr-13 Fr-14 Fr-15-100

0 ctl 04� 4 ctl 02� 4 ctl 00� 01 15.0 22� 4 15.0 22� 4 10.0 20� 72 20.0 34� 5 30.0 36� 5 15.0 36� 53 25.0 50� 7 45.0 50� 7 20.0 50� 74 30.0 74� 5 60.0 76� 5 25.0 72� 45 35.0 96� 4 75.0 94� 5 30.0 84� 5

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(26), 175 (11), 161 (33), 147 (38), 133 (29), 121 (55), 107 (62), 93 (100), 81 (92), 67 (62),53 (36).

Table 2b. Biological activities of fractions from fruits of P. nigrum L. showing their final dose in ppm andtheir mean mortalities with S.D. values against fourth instar larvae of An. stephensi Liston. (O. T. wild-strain).

Entry Dose (ppm) Mþ S.D. Dose (ppm) Mþ S.D. Dose (ppm) Mþ S.D.

PEP Fr-1 Fr-20 ctl 00� 0 ctl 02� 4 ctl 00� 01 8.0 20� 7 25.0 14� 5 4.0 22� 42 12.0 34� 5 50.0 30� 7 8.0 36� 53 16.0 50� 7 100.0 50� 7 12.0 50� 74 20.0 74� 5 150.0 76� 5 16.0 74� 55 24.0 88� 4 200.0 94� 5 20.0 90� 7

Fr-3 Fr-4 Fr-50 ctl 02� 4 ctl 00� 0 ctl 00� 01 10.0 08� 4 5.0 06� 5 5.0 28� 42 30.0 20� 7 10.0 14� 5 10.0 50� 73 50.0 34� 5 15.0 28� 4 15.0 76� 54 70.0 50� 7 20.0 50� 7 20.0 88� 45 90.0 78� 4 25.0 82� 8 25.0 98� 4

Fr-6 & Fr-7 Fr-8 Fr-90 ctl 00� 0 ctl 02� 4 ctl 04� 41 10.0 04� 4 10.0 08� 4 30.0 14� 52 20.0 12� 4 30.0 36� 5 60.0 34� 53 30.0 30� 7 50.0 50� 7 90.0 50� 74 40.0 50� 7 70.0 78� 4 120.0 68� 45 50.0 74� 5 90.0 96� 4 150.0 94� 5

Fr-10 Fr-11 Fr-120 ctl 02� 4 ctl 00� 0 ctl 00� 01 20.0 30� 7 11.0 34� 5 20.0 08� 42 40.0 50� 7 22.0 50� 7 40.0 20� 73 60.0 76� 5 33.0 66� 5 60.0 36� 54 80.0 84� 5 44.0 84� 5 80.0 50� 75 100.0 98� 4 55.0 96� 4 100.0 72� 4

Fr-13 Fr-14 Fr-15-100

0 ctl 00� 0 ctl 00� 0 ctl 00� 01 25.0 28� 4 30.0 12� 4 12.0 08� 42 50.0 50� 7 60.0 32� 4 24.0 14� 43 75.0 72� 4 90.0 50� 7 36.0 30� 74 100.0 84� 5 120.0 78� 4 48.0 50� 75 125.0 98� 4 150.0 96� 4 60.0 74� 5

Check for all fractions¼ 06� 5, ctl¼ control, S.D.¼ standard deviation and M¼Mean mortalities in %.

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1.2.2. Tricyclo[6.2.1.0(4,11)]undec-5-ene,1,5,9,9-tetramethyl-(isocaryophyllene-II)

(2). GC-EI-MS: m/z (rel. int.)¼ 204 [C15H24, Mþ] (20), 189 (28), 175 (12), 161 (34),

148 (39), 133 (100), 119 (48), 105 (64), 93 (79), 81 (38), 69 (53), 55 (24).

H

H

1.2.3. 4,11,11-Trimethyl-8-methylene-,[1R-(1R*,4E,9S*)]-bicyclo[7.2.0]undec-4-ene

(3). Common name: �-caryophyllene) GC-EI-MS: m/z (rel. int.)¼ 204 (C15H24, Mþ;

16), 189 (18), 175 (10), 161 (31), 148 (32), 133 (92), 120 (42), 105 (50), 93 (100), 79(55), 69 (73), 55 (30), 41 (55).

3

12

7

5

9

1011

1.2.4. Decahydro-4a-methyl-1-methylene-7-(1-methylethenyl)-,[4aR-(4a�,7�,8a�)]-naphthalene (4). (common name: (þ)-�-Selinene) GC-EI-MS m/z (rel. int.)¼204 [C15H24, Mþ] (100), 189 (55), 175 (14), 161 (56), 147 (36), 133 (45), 121 (56),105 (77), 93 (75), 81 (35), 67 (37), 55 (32).

1.2.5. 1,2,3,5,6,7,8,8a-Octahydro-1,8a-dimethyl-(1-methyl-ethenyl)-,[1S-(1�, 7�, 8a�)]-naphthalene (5). (common name: Eremophilene) GC-EI-MS m/z (rel. int.)¼ 204[C15H24, M

þ] (85), 189 (64), 175 (28), 161 (67), 147 (53), 133 (54), 119 (47), 107 (100),93 (95), 79 (80), 67 (56), 55 (39).

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1.2.6. Nonacosane (6). GC-EI-MS:m/z (rel. int.)¼ 408 [C29H60, Mþ] (13), 379 (5), 351

(5), 337 (9), 309 (5), 281 (9), 253 (7), 225 (8), 211 (15), 197 (12), 169 (15), 155 (17),141 (23), 127 (30), 113 (25), 99 (30), 85 (76), 71 (83), 57 (100), 43 (39).

14

29

1

1.2.7. Methyl hexadecanoate (7). GC-EI-MS: m/z (rel. int.)¼ 270 [C17H34O2, Mþ](28), 239 (12), 227 (13), 213 (13), 199 (6), 185 (5), 171 (4), 157 (3), 143 (20), 129 (7),115 (4), 101 (9), 87 (69), 74 (100), 69 (14), 55 (26), 43 (29).

O

O

10116

1.2.8. Ethyl hexadecanoate (8). GC-EI-MS: m/z (rel. int.)¼ 284 [C18H36O2, Mþ] (25),

255 (3), 239 (11), 213 (3), 199 (4), 157 (12), 143 (6), 115 (7), 101 (63), 88 (100), 83 (10),73 (13), 55 (21), 43 (27).

O

O

10116

1.2.9. Methyl 14-methyl heptadecanoate (9). GC-EI-MS m/z (rel. int.)¼298 [C19H38O2, M

þ] (60), 267 (13), 255 (30), 241 (5), 212 (5), 199 (16), 185 (5), 171 (3),157 (5), 143 (24), 129 (10), 111 (6), 97 (9), 87 (74), 74 (100), 55(18), 43 (17).

O

O

1451

17

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1.2.10. Methyl trans-8-octadecanote (10). GC-EI-MS m/z (rel. int.)¼ 296 [C19H36O2,Mþ] (15), 264 (70), 222 (16), 207 (6), 180 (10), 166 (5), 152 (7), 137 (10), 123 (17),110 (19), 97 (45), 83 (65), 69 (66), 55 (100), 42 (60).

O

O

38

61

18

4

1.2.11. Ethyl cis-9-octadecanaote (11). GC-EI-MS m/z (rel. int.)¼ 310 [C20H38O2,Mþ] (6), 281 (5), 264 (37), 236 (3), 222 (14), 194 (3), 180 (7), 166 (8), 155 (6), 137 (8),123 (15), 111 (19), 97 (39), 83 (49), 69 (54), 55 (100), 41 (43).

O

O

3

9

61 3

18

1.2.12 Hexadecanoic acid (12). GC-EI-MS: m/z (rel. int.)¼ 256 [C16H32O2, Mþ] (82),

227 (6), 213 (37), 199 (8), 185 (26), 171 (15), 157 (14), 143 (8), 129 (43), 115 (20), 97 (26),87 (20), 83 (32), 73 (100), 55 (67).

HO

O

10116

1.2.13. Octadecanoic acid (13). GC-EI-MS: m/z (rel. int.)¼ 284 (C18H36O2, Mþ; 100),

255 (5), 241 (24), 227 (5), 213 (4), 199 (6), 185 (29), 171 (7), 157 (5), 143 (6), 129 (34),115 (10), 97 (18), 83 (28), 73 (70), 69 (33), 60 (56), 55 (71), 43 (74).

HO

O

12118

1.2.14. 1-[5-(1,3-Benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]-,(E,E)-piperidine (14). (com-mon name: Piperine) GC-EI-MS: m/z (rel. int.)¼ 285 (C17H19NO3, M

þ; 93), 201 (100),173 (41), 143 (20), 135 (33), 112 (59), 121 (65), 84 (42).

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O

O

N

O

2. Experimental

2.1. General

Column chromatography (CC): silica gel 9385 (Merck, 0.040–0.063mm). GC:Shimadzu-GC-9A gas chromatograph, FID at 260�, N2 at 1.0ml/min, SPB-5� capillarycolumn (30m� 0.53mm ID; 0.3 m df); split ratio 1:30 injector temp. was 240�; thecolumn temperature was maintained at 50� for the first 5min and then raised to 240�

(3�/min) followed by 5min at 240�. GC-EI-MS: Hewlett-Packard 5890 gas chromato-graph, combined with a Jeol, JMS-HX 110 mass spectrometer with source at 270�

at 70 eV. Injector was set at 270o with splitting ratio 1:30. The analyses were performedon the aforementioned program on an equivalent column HP-5� (25m� 0.22mm and0.25 mmdf). A mass spectral survey was performed using the NIST Mass SpectralSearch Program 1998.

2.2. Extraction and purification

The seeds of P. nigrum were purchased from the local market in Karachi. Air-dried,ground seeds of P. nigrum (5 kg) were extracted with petroleum ether (3� 10 l) atroom temperature for 72 h. Evaporation of the solvent from combined extracts invacuo afforded a dark brown viscous residue (102.1 g). A portion (91.8 g) of this residuewas partitioned between petroleum ether and 90% MeOH. The petroleum-ether phasewas dried over anhydrous Na2SO4 and the solvent evaporated in vacuo to give a brown-ish syrupy concentrate (PEP, 63 g). A portion (30mg) of this concentrate was subjectedto GC and GC-EI-MS analyses (vide Experimental) to obtain compounds 2, 4, 10and 11. A part (PEP, 21.0 g) was subjected to a silica gel CC which was eluted withpetroleum ether>cyclohexane>cyclohexane/CHCl3>CHCl3>CHCl3/EtOAc>EtOAc> MeOH in order of increasing polarity to obtain 25 fractions. Compounds9 and 5 were identified through the GC and GC-EI-MS analyses from Fr-1 (petroleumether–cyclohexane, 9.9:0.1 eluate; 28.3mg), and Fr-2 (petroleum ether–cyclohexane,9.8:0.2 eluate; 49.7mg), respectively, while 1 from Fr-3 (petroleum ether–cyclohexane,9.5:0.5 eluate; 86.8mg), 3, and 4 from Fr-5 (petroleum ether–cyclohexane, 9.0:1.0eluate; 93.2mg) and 6 from fr-10 (cyclohexane–CHCl3, 6:4! 1:1 eluate; 39.5mg)were identified by GC and GC-EI-MS analyses. In Fr-11 (cyclohexane–CHCl3,1:1! 3:7 eluate; 55.6mg), compounds 7 and 8 were identified through the GC andGC-EI-MS analyses. Compound 8 was identified when a portion of Fr-12 (cyclo-hexane–CHCl3, 2:8 eluate; 3.1mg) was subjected to GC and GC-EI-MS analysis.Compound 11 was identified from Fr-13 (cyclohexane–CHCl3, 1:9 eluate; 2.7mg)and 12, 13 were identified from Fr-14 (CHCl3–EtOAc, 100%-7:3 eluate; 79.6mg)through GC and GC-EI-MS analysis. Fr-15 (CHCl3–EtOAc, 1:1 eluate; 9.4 g) was

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subjected to silica gel CC and eluted with petroleum ether>petroleum ether/EtOAc >EtOAc>CHCl3>CHCl3/MeOH> MeOH); to obtain 15 fractions.4.1mg of fr-100 (petroleum ether–EtOAc, 6.0:4.0 eluate) was subjected to theGC and GC-EI-MS analyses, and only 14 could be identified.

3. Pesticidal activity

3.1. Rearing technique

The fourth instar larvae of Anopheles stephensi Liston (Orangi Town wild strain), amalaria vector mosquito and fourth instar larvae of Ae. aegypti L. (O.T. wild strain),a yellow fever mosquito, were collected from a semi-natural pond, especially con-structed for this research work. The size of this pond was 8� 4 ft with a depth of 2 ft.The egg strips of the identified mosquitoes i.e. An. stephensi and Ae. aegypti weredipped into the pond. The larvae in the pond were fed by prawns, dried and groundinto a powder. The pond was covered with a net having small holes so that the inter-mixing of the other mosquito species may be avoided and the release of mosquitoesfrom the pond into the environment may be checked. The pupae from the pond werecollected every day and kept in mosquito cages for adult emergence. These adultswere fed with Albino rats twice a week and the filter paper strips were kept in bowlsof 6 in. diameter. The egg strips were dried for a day and then dipped into the pondfor larvae hatching.

3.2. Biological test (screening procedure)

Ten young fourth instar larvae of Ae. aegypti and An. stephensi were collected ina 250ml beaker having 5ml of tap water separately and the beaker was filled up tothe level of 200ml. The fractions were tested at 28� 1�C at five final concentrations(dose in ppm; table 2a and 2b selected from the preliminary tests of each fraction).The control and the check were also set. The observation was recorded after 24 h.

3.3. Accurate tests

The WHO method was followed for the application. A batch of 10 insects (4th instarmosquito larvae) was released in a 250ml beaker, containing 200ml filtered tapwater. The concentrations finally selected in the preliminary tests of each fractionwere applied. A group of 7 beakers was set up, five for different concentrations andone each for the control and check, separately for Ae. aegypti and An. stephensi.Each experiment was repeated five times. The experiment was discarded, if the mortal-ity was found to be more than 10% in control. The mortality was recorded after 24 hand the readings were subjected to Abbot’s formula [22].

3.4. Calculations of LC50

The lethal concentrations (LC50) were calculated using PROBIT analysis for Ae. aegyptiand An. stephensi [23].

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