Available online on www.ijppr.

com
International Journal of Pharmacognosy and Phytochemical Research 2016; 8(6); 977-996

ISSN: 0975-4873
Research Article

Antibacterial and Phytochemical Analysis of Piper nigrum using Gas

Chromatography – Mass Spectrum and Fourier-Transform Infrared
Spectroscopy
Ghaidaa Jihadi Mohammed1, Aseel Mohammad Omran2, Haider Mashkoor Hussein1
1
College of Science, Al-Qadisiya University, Iraq
2
College of Pharmacy, Babylon University, Iraq

Available Online: 9th June, 2016

ABSTRACT
The aims of the study were to investigate the presence of phytochemical compounds from the fruits of Piper nigrum, using
methanolic extraction and report the main functional components by using fourier-transform infrared spectroscopy. The
phytochemical compound screened by GC-MS method. A total of 55 bioactive phytochemical compounds were identified
in the methanolic extract of P. nigrum. The identification of phytochemical compounds is based on the peak area, retention
time molecular weight, molecular formula, chemical structure, MS Fragment ions and pharmacological actions. GC-MS
analysis of P. nigrum revealed the existence of the Propanedioic acid , dimethyl ester, Bicyclo[3.1.1]heptane,6,6-dimethyl-
2-methylene-,(1S), 3-Carene, Cyclohexene, 1-methyl-5-(1-methylethenyl)-,(R), 1,6-Octadien-3-ol,3,7-dimethyl, 2-
Methyl-1-ethylpyrrolidine, 2-Isopropenyl-5-methylhex-4-enal, L-α-Terpineol, (R)-lavandulyl acetate, Pyrrolizin-1,7-
dione-6-carboxylic acid , methyl(ester), 7-epi-cis-sesquisabinene hydrate, Phenol, 2-methoxy-4-(1-propenyl)-,(Z),
Eugenol, Alfa.Copaene, Naphthalene,1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methyl), Epiglobulol, Caryophyllene,
1,4,7-Cycloundecatriene, 1,5,9,9-tetramethyl-,Z,Z,Z, α- ylangene, ß-copaene, Cedran-diol,8S,13, Isocalamendiol, Cinnami
acid , 4-hydroxy -3-methoxy-,{5-hydroxy-2-hydroxymethyl, (-)-Spathulenol, 1-Heptatriacotanol, Desacetylanquidine, 5-
Isopropyl-2,8-dimethyl-9-oxatricyclo[4.4.0.0.(2,8)]decan-7-one, Estra-1,3,5(10)-trien-17ß-ol, Trans-1,2-
Diaminocyclohexane-N,N,N´,N´-tetraacetic acid, Phytol, Piperidine,1-(1-oxo-3-phenyl-2-propenyl)-, Eicosanoic acid , 2-
(acetyloxy0-1-[(acetyloxy)methyl]ethyl ester, 2,5,5,8a-Tetramethyl-6,7,8,8a-tetrahydro-5H-chromen-8-ol, Z-5-methyl-6-
heneicosen-11-one, 2H-1,2-Benzoxazine-3-carbonitrile,2- cyclohexyloctahydro-4a,8a-d, Indoxazin-4-one ,4,5,6,7-
tetrahydro-3-undecyl, 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol,(3ß,5Z,7E), 3-Oxo-10(14)-epoxyguai-11(13)-en-
6,12-olide, 7-[2-(Ethoxycarbonyl)-3α,5ß-dimethoxycyclopentyl -1]-heptanoic acid, 2H-Benzo[f]oxireno[2,3-
E]benzofuran-8,(9H)-one,9-[[(1,3-benzodio, Nalorphine, 2-Cyclohexen -3-ol-1-one , 2-[1-iminotetradecyl]-, Piperine,
Fenretinide, 11-Dehydrocorticosterone, 5H-Cyclopropa[3,4]benz[1,2-e]azulen-5-one,1,1a,1b,4,4a,7a,7b, 17a-Ethyl-3ß-
methoxy-17a-aza-D-homoandrost-5-ene-17-one, Bufa-20,22-dienolide m,14,15-epoxy-3,11-dihydroxy-,(3ß,5ß,11α,15, 9-
Desoxo-9-x-acetoxy-3,8,12-tri-O-acetylingol, Retinal ,9-cis-, 6-ß-Naltrexol, Piperine, Ursodeoxycholic acid, 5α-Cholan-
24-oic acid , 12α-hydroxy-3,7-dioxo-,methyl ester and Stigmasterol. The FTIR analysis of P. nigrum leaves proved the
presence of Alkenes, Aliphatic fluoro compounds, Alcohols, Ethers, Carboxlic acids, Esters and Nitro Compounds.
Methanolic extract of bioactive compounds of P. nigrum was assayed for in vitro antibacterial activity against Escherichia
coli, Pseudomonas aerogenosa, Proteus mirabilis, Staphylococcus aureus and Klebsiella pneumonia by using the diffusion
method in agar. The zone of inhibition were compared with different standard antibiotics. The diameters of inhibition zones
ranged from 5.00±0.16 to 0.40±0.12 mm for all treatments.

Keywords: FT-IR, GC-MS analysis, Piper nigrum.

5
INTRODUCTION . The main pungent principle in the green berries of pepper
Piper nigrum L. is a flowering vine in the family of P. nigrum L. is piperine. Generally the piperine content of
piperaceae, therefore an important medicinal plant is used black or white peppercorns lies within the range of 3-8
in traditional medicine in Asia and Pacific islands g/100 g, whereas the content of minor alkaloids piperyline
especially in Indian medicine1. Pepper aroma and flavor and piperettine have been estimated as 0.2-0.3 and 0.2-1.6
due to their chemical substances especially the volatile oil. g/100g respectively. The bioavailabilityenhancing
Black pepper oil has medicinal values. It can be used to property of piperine indicates its potential to be used as an
help in treatment of pain relief, rheumatism, chills, flu, adjuvant with therapeutic drugs in chronic ailments, to
colds exhaustion, muscular aches, physical and emotional reduce the effective dose of the drug and, hence,
coldness, fever as nerve tonic and to increase circulation2-

*Author for Correspondence

 Ghaidaa et al. / Antibacterial and Phytochemical …

Table 1: Major phytochemical compounds identified in methanolic extract of Piper nigrum.

S. Phytochemical RT Formula Mol. Exact Chemical structure MS P’cological
No. compound (min) Wt. Mass Fragment- actions
ions
1. Propanedioic 3.150 C5H8O4 132 132.042 59,74,101,1 Anti-tumor and
acid , dimethyl 258 32 antioxidant
ester activity

2. Bicyclo[3.1.1] 3.339 C10H16 136 136.125 53,69,79,93 Anti-

heptane,6,6- 2 ,107,121,13 Helicobacter
dimethyl-2- 6 pylori activity
methylene- and Anti-
,(1S)- Candida
activity
3. 3-Carene 3.693 C10H16 136 136.125 53,67,79,93 Several
2 ,105,121,13 biological
6 activities such
as anti-
inflammatory
4. Cyclohexene , 3.997 C10H16 136 136.125 53,68,79,93 Anti-microbial
1-methyl-5-(1- 2 ,107,121,13 agents
methylethenyl) 6
-,(R)-
5. 1,6-Octadien- 5.067 C10H18O 154 154.135 55,71,80,83 Pharmacologic
3-ol,3,7- 765 ,93,107,121 al effects such
dimethyl- ,127,136,15 as: anti-
4 inflammation

6. 2-Methyl-1- 5.696 C7H15N 113 113.120 56,69,84,98 Anti-tumor

ethylpyrrolidin 4495 ,113 activity.
e

7. 2-Isopropenyl- 6.108 C10H16O 152 152.120 69,84,95,10 Antihyperglyc

5-methylhex- 115 9,123,137 emic and
4-enal anticariogenic

8. L-α-Terpineol 6.303 C10H18O 154 154.135 55,59,67,81 Anti-

765 ,93,107,121 inflammatory
,136

9. (R)-lavandulyl 6.629 C12H20O2 196 196.146 53,69,80,93 Anti-

acetate 33 ,107,121,13 inflammatory
6,154

10. Pyrrolizin-1,7- 7.562 C9H11NO4 197 197.068 55,69,84,98 Anti-Viral and

dione-6- 808 ,110,142,19 Anti-Tumor
carboxylic acid 7 Activity
, methyl(ester)

11. 7-epi-cis- 8.357 C15H26O 222 222.198 55,69,82,93 Antioxidant

sesquisabinene 365 ,105,119,13 effects and
hydrate 3,147,161,1 anti-mutagenic
75,189,204, action
222

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12. Phenol , 2- 8.494 C10H12O2 164 164.083 51,55,65,77 Anti-

methoxy-4-(1- 73 ,91,103,115 inflammatory
propenyl)-,(Z)- ,121,131,14 effects
9,164
13. Eugenol 8.523 C10H12O2 164 164.083 51,55,65,77 Anti-
73 ,91,103,115 Inflammatory
,121,131,13 agents and
7,149,164 antioxidants
14. Alfa.Copaene 8.740 C15H24 204 204.187 55,69,77,91 Analgesic and
8 ,105,119,13 anti-
3,147,161,1 inflammatory
75,189,204 activities
15. Naphthalene,1, 8.958 C15H24 204 204.187 55,67,79,10 Anti-bacterial
2,3,5,6,7,8,8a- 8 7,119,133,1
octahydro- 61,175,189,
1,8a-dimethyl- 204
7-(1-methyl
16. Epiglobulol 9.181 C15H26O 222 222.198 55,69,82,93 Antioxidant
365 ,109,121,14 activity,
7,161,204,2 antimicrobial,
22 anti-
inflammatory
17. Caryophyllene 9.456 C15H24 204 204187 55,69,79,93 Several
8 ,105,120,13 biological
3,147,161,1 activities are
75,189,204 attributed to
beta-
caryophyllene,
such as anti-
inflammatory,
antibiotic,
antioxidant,
anticarcinogeni
c and local
anaesthetic
18. 1,4,7- 9.810 C15H24 204 204.187 55,67,80,93 Anti-aging,
Cycloundecatri 8 ,107,121,13 anti-
ene, 1,5,9,9- 6,147,161,1 hyperlipidemia
tetramethyl- 75,189,204 and
,Z,Z,Z- antimicrobial
activities
19. α- ylangene 10.00 C15H24 204 204.187 55,67,77,93 Anti-
5 8 ,105,119,13 inflammatory
3,147,161,1 properties
89,204
20. ß-copaene 10.08 C15H24 204 204.187 55,79,105,1 Anti-
5 8 61,204 neuroinflamma
tory and
neuroprotectiv
e
21. Cedran- 10.46 C15H26O2 238 238.193 55,74,93,10 Anti-fungal
diol,8S,13- 8 28 7,119,149,1 activity
61,190,238

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22. Isocalamendiol 10.85 C15H26O2 238 238.193 55,81,111,1 Anti- fertility

2 28 59,202,223, activity and
238 anti-bacterial
activity
23. Cinnami acid , 12.38 C31H40O15 652 652.236 55,77,91,10 Anti-
4-hydroxy -3- 5 72 5,121,151,1 inflammatory
methoxy-,{5- 68,194,215, properties
hydroxy-2- 296,330,35
hydroxymethyl 4,386,418
-
24. (-)-Spathulenol 13.57 C15H24O 220 220.182 55,69,91,11 Antioxidant
0 715 9,159,187,2 and anti-
05,220 inflammatory
activities

25. 1- 13.92 C37H76O 536 536.589 55,81,95,14 Antioxidant,

Heptatriacotan 4 62 7,161,190,2 anticancer, anti
ol 29,244,257 inflammatory
and sex
hormone
activity
26. Desacetylanqui 14.47 C17H24O6 324 324.157 55,67,91,10 New chemical
dine 4 288 5,124,145,1 compound
59,187,205,
264,281

27. 5-Isopropyl- 15.24 C14H23O2 222 222.161 55,69,81,95 Anti-termitic

2,8-dimethyl- 0 98 ,109,123,15 Activity
9- 1,162,194,2
oxatricyclo[4.4 22
.0.0.(2,8)]deca
n-7-one
28. Estra- 15.81 C18H24O 256 256.182 57,73,85,97 Anti-
1,3,5(10)-trien- 8 714 ,129,157,18 osteoporosis
17ß-ol 5,213,241,2 activity
56

29. Trans-1,2- 16.25 C14H22N2O8 346 346.137 55,81,96,11 Unknown

Diaminocycloh 3 615 0,153,181,1
exane- 97,225,241,
N,N,N´,N´- 270
tetraacetic
acid,
30. Phytol 16.66 C20H40O 296 296.307 57,71,81,95 Antioxidant
5 917 ,111,123,13 activity
7,196,221,2
49,278
31. Piperidine,1- 16.88 C14H17NO 215 215.131 51,84,103,1 Antibacterial,
(1-oxo-3- 3 014 31,149,172, analgesic and
phenyl-2- 198,215 anti-
propenyl)- inflammatory
activity
32. Eicosanoic 18.14 C27H50O6 470 470.360 57,71,84,98 Anti-
acid , 2- 7 739 ,117,137,15 inflammatory,
(acetyloxy0-1- 9,171,227,2 and anti-
[(acetyloxy)me 69,295 melasma
thyl]ethyl ester properties

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33. 2,5,5,8a- 18.53 C13H20O2 208 208.146 57,91,106,1 Anticancer and

Tetramethyl- 1 33 34,175,190, antioxidant
6,7,8,8a- 208 activity
tetrahydro-5H-
chromen-8-ol
34. Z-5-methyl-6- 18.55 C22H42O 322 322.323 55,71,83,97 Anti-
heneicosen-11- 9 566 ,139,169,19 Mycobacteriu
one 7,241,307,3 m tuberculosis
22 Activity, anti-
inflammatory,
analgesic and
antimicrobial
activities
35. 2H-1,2- 18.74 C17H28N2O 276 276.220 55,67,83,96 Anti-
Benzoxazine- 8 163 ,108,134,15 inflammatory
3- 0,165,178,1
carbonitrile,2- 91,206,216,
cyclohexylocta 234,249,27
hydro-4a,8a-d 6
36. Indoxazin-4- 18.94 C18H29NO2 291 291.219 55,69,83,12 New chemical
one ,4,5,6,7- 3 83 3,138,151,1 compound
tetrahydro-3- 64,178,206,
undecyl- 234,262,29
1
37. 9,10- 19.24 C27H44O3 416 416.329 55,118,136, Biocides and
Secocholesta- 6 044 158,176,20 anti-corrosion
5,7,10(19)- 7,253,383,4 agents
triene-3,24,25- 16
triol,(3ß,5Z,7E
)-
38. 3-Oxo-10(14)- 19.37 C15H18O4 262 262.120 53,81,91,10 Anti fungal
epoxyguai- 7 508 5,135,174,1 activity
11(13)-en- 93,244,262
6,12-olide

39. 9- 20.75 C22H43NO 337 337.334 55,69,100,1 Antimicrobial,

Octadecenami 1 465 28,184,210, anti-
de , n-butyl- 252,280,29 inflammatory
4,308,337 and antioxidant
40. 7-[2- 20.96 C19H34O6 358 358.235 265,280,29 New chemical
(Ethoxycarbon 8 538 7,311,326 compound
yl)-3α,5ß-
dimethoxycycl
opentyl-1]-
heptanoic acid
41. 2H- 21.24 C23H29NO5 399 399.204 68,77,91,10 Antimicrobial
Benzo[f]oxiren 3 573 5,135,150,1 and antifungal
o[2,3- 83,202,236, activities
E]benzofuran- 264
8,(9H)-one,9-
[[(1,3-
benzodio
42. Nalorphine 21.27 C19H21NO3 311 311.152 81,91,115,1 Anti-pruritic
1 143 28,152,174, agent against
188,200,22 morphine
6,241,311

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43. 2-Cyclohexen - 21.73 C20H35NO2 321 321.266 55,83,110,1 Antiulcer,

3-ol-1-one , 2- 4 779 25,153,166, hypolipidemic,
[1- 180,194,20 antiatheroscler
iminotetradecy 8,236,278,3 otic and anti-
l]-, 04,321 HIV
44. Piperine 22.31 C17H19NO3 285 285.136 63,84,115,1 Anti-pyretic
3 494 43,159,173, and analgesic
201,229,25 activities
6,285
45. Fenretinide 22.78 C26H33NO2 391 391.251 58,69,81,95 Anti-
2 13 ,109,135,14 proliferative
8,161,202,2 activity
13,255,268

46. 11- 24.67 C21H28O4 344 344.198 55,67,79,91 Anti-

Dehydrocortic 0 76 ,105,121,14 proliferative
osterone 7,189,227,2 activity
67,285,313,
344
47. 5H- 24.83 C20H28O5 348 348.193 55,83,173,1 Biocides, anti-
Cyclopropa[3, 6 674 99,217,312, corrosion
4]benz[1,2- 330,348 agents, drying
e]azulen-5- agents, and
one,1,1a,1b,4,4 coating
a,7a,7b
48. 17a-Ethyl-3ß- 24.91 C22H35NO2 345 345.266 55,71,91,10 Anti- cancer
methoxy-17a- 6 779 5,138,193,2 activity
aza-D- 09,241,296,
homoandrost- 330,345
5-ene-17-one
49. Bufa-20,22- 25.11 C42H32O5 400 400.224 55,67,79,91 Cell growth
dienolide 1 974 ,107,135,18 inhibitory
m,14,15- 7,213,231,2 activity, and
epoxy-3,11- 49,278,349, antitumor
dihydroxy- 364,382,40
,(3ß,5ß,11α,15 0
50. 9-Desoxo-9-x- 25.52 C28H40O10 536 536.262 55,69,122,2 Biocide and
acetoxy- 3 146 07,236,297, anti-fungal
3,8,12-tri-O- 357,417,47
acetylingol 7

51. Retinal ,9-cis- 25.96 C20H28O 284 284.214 55,69,79,95 Anti-oxidant

3 016 ,119,133,17 role
3,199,213,2
41,255
52. 6-ß-Naltrexol 27.19 C20H25NO4 343 343.178 55,84,98,11 Potential anti-
4 358 0,128,161,2 angiogenic
02,228,246, activity
288,302,32
4,343
53. Ursodeoxychol 28.34 C24H40O4 392 392.292 55,67,81,93 Antiinflammat
ic acid 4 66 ,145,213,25 ory effect and
5,302,356,3 anti-
74,392 inflammatory
activity

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54. 5α-Cholan-24- 28.51 C25H38O5 418 418.271 55,95,219,2 Having

oic acid , 12α- 0 925 45,285,345, biological
hydroxy-3,7- 382,400,41 activity (insect
dioxo-,methyl 8 moulting
ester hormones)

55. Stigmasterol 29.16 C29H48O 412 412.370 69,83,133,2 Anti-

8 516 13,255,300, inflammatory
351,369,41 and anti-
2 parasitic
effects

Table 2: FT-IR peak values of methanolic seeds extract of Piper nigrum.

S. No. Peak (Wave Intensity Bond Functional group assignment Group frequency
number cm-ˡ)
1. 650.1 57.535 - Unknown -
2. 688.59 59.347 C-H Alkenes 675-995
3. 704.02 60.135 C-H Alkenes 675-995
4. 759.95 64.754 C-H Alkenes 675-995
5. 827.46 67.689 C-H Alkenes 675-995
6. 852.54 65.098 C-H Alkenes 675-995
7. 894.97 68.412 C-H Alkenes 675-995
8. 927.76 60.301 C-H Alkenes 675-995
9. 999.13 38.637 - Unknown -
10. 1014.56 38.696 C-F stretch Aliphatic fluoro compounds 1000-10150
11. 1076.28 54.627 C-O Alcohols, Ethers, Carboxlic acids, Esters 1050-1300
12. 1134.14 61.967 C-O Alcohols, Ethers, Carboxlic acids, Esters 1050-1300
13. 1149.57 63.780 C-O Alcohols, Ethers, Carboxlic acids, Esters 1050-1300
14. 1195.87 71.015 C-O Alcohols, Ethers, Carboxlic acids, Esters 1050-1300
15. 1249.87 55.761 C-O Alcohols, Ethers, Carboxlic acids, Esters 1050-1300
16. 1292.87 72.884 C-O Alcohols, Ethers, Carboxlic acids, Esters 1050-1300
17. 1361.74 68.851 NO2 Nitro Compounds 1300-1370
18. 1417.68 64.723 C-H Alkanes 1340-1470
19. 1436.97 59.046 C-H Alkanes 1340-1470
20. 1489.05 65.725 - Unknown -
21. 1506.41 66.871 - Unknown -
22. 1593.20 66.461 - Unknown -
23. 1616.35 64.515 - Unknown -
24. 1635.64 61.408 - Unknown -
25. 2665.62 88.750 - Unknown -
26. 2854.65 79.246 C-H Alkanes 2850-2970
27. 2926.01 75.034 C-H Alkanes 2850-2970

Table 3: Zone of inhibition (mm) of test bacterial strains to Piper nigrum bioactive compounds and standard antibiotics.
/ Piper nigrum Antibiotics Bacteria
Staphylococcus Escherichia Proteus Klebsiella Pseudomonas
aureus coli mirabilis pneumonia eurogenosa
Piper nigrum 4.00±0.31 4.90±0.13 5.00±0.16 4.63±0.41 4.12±0.11
Rifambin 1.01±0.10 0.77±0.41 0.98±0.11 1.00±0.30 1.05±0.42
Streptomycin 0.91±0.27 1.60±0.29 1.90±0.10 0.96±0.47 0.87±0.20
Kanamycin 0.42±0.18 1.12±0.46 0.40±0.12 1.00±0.10 0.90±0.47
Cefotoxime 0.87±0.95 0.96±0.27 0.93±0.25 0.92±0.18 0.71±0.13

subsequent adverse effects6-12 Recently, biochemical hepatoprotective activities21,22. Studies have revealed
activities of some important medicinal plants including anticonvulsant and bioavailability-enhancing properties of
Piper species and their metabolites have been described13- the drug. The fruits contain 1.0–2.5% volatile oil, 5–9%
20
. Pharmacological and clinical studies have revealed that alkaloids, of which the major ones are piperine, chavicine,
piperine has CNS depressant, antipyretic, analgesic, anti- piperidine, and piperetine, and a resin23. The present study
inflammatory (Ratner et al., 1991), antioxidant, and involves an assessment using GC-MS and FT-IR

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Figure 1: GC-MS chromatogram of methanolic extract of Piper nigrum.

spectroscopic techniques to investigate the chemical when the injection was made (initial time) to when elution
composition methanolic fruit extract of P. nigrum. occurred is referred to as the retention time (RT). While
the instrument was run, the computer generated a graph
MATERIALS AND METHODS from the signal called chromatogram. Each of the peaks in
Plant Material and Preparation of Extracts the chromatogram represented the signal created when a
P. nigrum dried fruits were purchased from local market in compound eluted from the gas chromatography column
Hilla city, middle of Iraq. after thorough cleaning and into the detector. The x-axis showed the RT and the y-axis
removal foreign materials, the fruits were stored in airtight measured the intensity of the signal to quantify the
container to avoid the effect of humidity and then stored at component in the sample injected. As individual
room temperature until further use. About 30 g of the plant compounds eluted from the gas chromatographic column,
sample powdered were soaked in 100 mL of methanol for they entered the electron ionization (mass spectroscopy)
16 h in a rotatory shaker24,25. Whatman No.1 filter paper detector, where they were bombarded with a stream of
was used to separate the extract of plant. The filtrates were electrons causing them to break apart into fragments. The
used for further phytochemical analysis. It was again fragments obtained were actually charged ions with a
filtered through sodium sulphate in order to remove the certain mass28. The M/Z (mass / charge) ratio obtained was
traces of moisture. calibrated from the graph obtained, which was called as the
Identification of component by gas chromatography – Mass spectrum graph which is the fingerprint of a
mass spectrum analysis molecule. Before analyzing the extract using gas
The physicochemical properties of the essential oil of P. chromatography and mass spectroscopy, the temperature
nigrum L. (Black Pepper) are presented in Table1. of the oven, the flow rate of the gas used and the electron
Interpretation of mass spectroscopy (GC-MS) was gun were programmed initially. The temperature of the
conducted using data base of the National Institute oven was maintained at 100°C. Helium gas was used as a
Standard and Technology (NIST) having more than 62000 carrier as well as an eluent. The flow rate of helium was
patterns. The spectrum of the unknown component was set to 1mL per min29-33. The electron gun of mass detector
compared with the spectrum of the known component liberated electrons having energy of about 70eV.The
stored in the NIST library. The identity of the components column employed here for the separation of components
in the extracts was assigned by the comparison of their was Elite 1(100% dimethyl poly siloxane).
retention indices and mass spectra fragmentation patterns Fourier transform infrared spectrophotometer (FTIR)
with those stored on the computer library and also with The powdered sample of P. nigrum specimen was treated
published literatures26,27. The GC-MS analysis of the plant for FTIR spectroscopy (Shimadzu, IR Affinity 1, Japan).
extract was made in a (Agilent 789 A) instrument under The sample was run at infrared region between 400 nm and
computer control at 70 eV. About 1μL of the methanol 4000 nm31.
extract was injected into the GC-MS using a micro syringe Determination of antibacterial activity of crude bioactive
and the scanning was done for 45 min. As the compounds compounds of Piper nigrum.
were separated, they eluted from the column and entered a The test pathogens (Pseudomonas aeruginosa, Klebsiella
detector which was capable of creating an electronic signal pneumoniae, E. coli, and Staphylococcus aureus) were
whenever a compound was detected. The greater the swabbed in Müller-Hinton agar plates. 60 L of plant
concentration in the sample, bigger was the signal obtained extract was loaded on the bored wells. The wells were
which was then processed by a computer. The time from

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Figure 2: Structure of Cyclohexene, 1-methyl-5-(1- Figure 3: Structure of 1,6-Octadien-3-ol,3,7-dimethyl

methylethenyl)-,(R) present in the methanolic present in the methanolic seeds extract of Piper nigrum by
seeds extract of Piper nigrum by using GC-MS analysis. using GC-MS analysis.

Figure 4: Structure of 2-Methyl-1-ethylpyrrolidine Figure 5: Structure of Propanedioic acid , dimethyl ester

present in the methanolic seeds extract of Piper nigrum present in the methanolic seeds extract of Piper nigrum by
by using GC-MS analysis. using GC-MS analysis.

Figure 6: Structure of Bicyclo[3.1.1]heptane, 6,6- Figure 7: Structure of 3-Carene present in the methanolic
dimethyl-2-methylene-,(1S) present in the methanolic seeds extract of Piper nigrum by using GC-MS analysis.
seeds extract of Piper nigrum by using GC-MS analysis.

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Figure 8: Structure of Pyrrolizin-1,7-dione-6-carboxylic Figure 9: Structure of 7-epi-cis-sesquisabinene hydrate

acid, methyl(ester) present in the methanolic seeds present in the methanolic seeds extract of Piper nigrum by
extract of Piper nigrum by using GC-MS analysis. using GC-MS analysis.

Figure 10: Structure of Phenol, 2-methoxy-4-(1- Figure 11: Structure of 2-Isopropenyl-5-methylhex-4-enal

propenyl)-,(Z) present in the methanolic seeds extract of present in the methanolic seeds extract of Piper nigrum by
Piper nigrum by using GC-MS analysis. using GC-MS analysis.

Figure 12: Structure of L-α-Terpineol present in the Figure 13: Structure of (R)-lavandulyl acetate present in
methanolic seeds extract of Piper nigrum by using GC- the methanolic seeds extract of Piper nigrum by using GC-
MS analysis. MS analysis.

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Figure 14: Structure of Epiglobulol present in the Figure 15: Structure of Caryophyllene present in the
methanolic seeds extract of Piper nigrum by using GC- methanolic seeds extract of Piper nigrum by using GC-MS
MS analysis. analysis.

Figure 16: Structure of 1,4,7-Cycloundecatriene, 1,5,9,9- Figure 17: Structure of Eugenol present in the methanolic
tetramethyl-,Z,Z,Z present in the methanolic seeds seeds extract of Piper nigrum by using GC-MS analysis.
extract of Piper nigrum by using GC-MS analysis.

Figure 18: Structure of Alfa.Copaene present in the Figure 19: Structure of Naphthalene,1,2,3,5,6,7,8,8a-
methanolic seeds extract of Piper nigrum by using GC- octahydro-1,8a-dimethyl-7-(1-methyl present in the
MS analysis. methanolic seeds extract of Piper nigrum by using GC-MS
analysis.

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Figure 20: Structure of Isocalamendiol present in the Figure 21: Structure of Cinnami acid , 4-hydroxy -3-
methanolic seeds extract of Piper nigrum by using GC- methoxy-,{5-hydroxy-2-hydroxymethyl present in the
MS analysis. methanolic seeds extract of Piper nigrum by using GC-MS
analysis.

Figure 22: Structure of (-)-Spathulenol present in the Figure 23: Structure of α- ylangene present in the
methanolic seeds extract of Piper nigrum by using GC- methanolic seeds extract of Piper nigrum by using GC-MS
MS analysis. analysis.

Figure 24: Structure of ß-copaene present in the Figure 25: Structure of Cedran-diol,8S,13present in the
methanolic seeds extract of Piper nigrum by using GC- methanolic seeds extract of Piper nigrum by using GC-MS
MS analysis. analysis.

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Figure 26: Structure of Estra-1,3,5(10)-trien-17ß-ol Figure 27: Structure of Trans-1,2-Diaminocyclohexane-

present in the methanolic seeds extract of Piper nigrum N,N,N´,N´-tetraacetic acid present in the methanolic seeds
by using GC-MS analysis. extract of Piper nigrum by using GC-MS analysis.

Figure 28: Structure of Phytol present in the methanolic Figure 29: Structure of 1-Heptatriacotanol present in the
seeds extract of Piper nigrum by using GC-MS analysis. methanolic seeds extract of Piper nigrum by using GC-MS
analysis.

Figure 30: Structure of Desacetylanquidine present in the Figure 31: Structure of 5-Isopropyl-2,8-dimethyl-9-
methanolic seeds extract of Piper nigrum by using GC- oxatricyclo[4.4.0.0.(2,8)]decan-7-one present in the
MS analysis. methanolic seeds extract of Piper nigrum by using GC-MS
analysis.

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Figure 32: Structure of Z-5-methyl-6-heneicosen-11-one Figure 33: Structure of 2H-1,2-Benzoxazine-3-

present in the methanolic seeds extract of Piper nigrum carbonitrile,2- cyclohexyloctahydro-4a,8a-d present in the
by using GC-MS analysis. methanolic seeds extract of Piper nigrum by using GC-MS
analysis.

Figure 34: Structure of Indoxazin-4-one ,4,5,6,7- Figure 35: Structure of Piperidine,1-(1-oxo-3-phenyl-2-

tetrahydro-3-undecyl present in the methanolic seeds propenyl) present in the methanolic seeds extract of Piper
extract of Piper nigrum by using GC-MS analysis. nigrum by using GC-MS analysis.

Figure 36: Structure of Eicosanoic acid , 2-(acetyloxy0- Figure 37: Structure of 2,5,5,8a-Tetramethyl-6,7,8,8a-
1-[(acetyloxy)methyl]ethyl ester present in the tetrahydro-5H-chromen-8-ol present in the methanolic
methanolic seeds extract of Piper nigrum by using GC- seeds extract of Piper nigrum by using GC-MS analysis.
MS analysis.

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Figure 38: Structure of 7-[2-(Ethoxycarbonyl)-3α,5ß- Figure 39: Structure of 2H-Benzo[f]oxireno[2,3-

dimethoxycyclopentyl-1]-heptanoic acid present in the E]benzofuran-8,(9H)-one,9-[[(1,3-benzodio present in the
methanolic seeds extract of Piper nigrum by using GC- methanolic seeds extract of Piper nigrum by using GC-MS
MS analysis. analysis.

Figure 40: Structure of Nalorphine present in the Figure 41: Structure of 9,10-Secocholesta-5,7,10(19)-
methanolic seeds extract of Piper nigrum by using GC- triene-3,24,25-triol,(3ß,5Z,7E)present in the methanolic
MS analysis. seeds extract of Piper nigrum by using GC-MS analysis.

Figure 42: Structure of 3-Oxo-10(14)-epoxyguai-11(13)- Figure 43: Structure of 9-Octadecenamide , n-butyl

en-6,12-olide present in the methanolic seeds extract of present in the methanolic seeds extract of Piper nigrum by
Piper nigrum by using GC-MS analysis. using GC-MS analysis.

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Figure 44: Structure of 11-Dehydrocorticosterone present Figure 45: Structure of 5H-Cyclopropa[3,4]benz[1,2-

in the methanolic seeds extract of Piper nigrum by using e]azulen-5-one,1,1a,1b,4,4a,7a,7b present in the
GC-MS analysis. methanolic seeds extract of Piper nigrum by using GC-MS
analysis.

Figure 46: Structure of 17a-Ethyl-3ß-methoxy-17a-aza- Figure 47: Structure of 2-Cyclohexen -3-ol-1-one , 2-[1-
D-homoandrost-5-ene-17-one present in the methanolic iminotetradecyl] present in the methanolic seeds extract of
seeds extract of Piper nigrum by using GC-MS analysis. Piper nigrum by using GC-MS analysis.

Figure 48: Structure of Piperine present in the methanolic Figure 49: Structure of Fenretinide present in the
seeds extract of Piper nigrum by using GC-MS analysis. methanolic seeds extract of Piper nigrum by using GC-MS
analysis.

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Figure 50: Structure of 6-ß-Naltrexol present in the Figure 51: Structure of Piperine present in the methanolic
methanolic seeds extract of Piper nigrum by using GC- seeds extract of Piper nigrum by using GC-MS analysis.
MS analysis.

Figure 52: Structure of Ursodeoxycholic acid present in Figure 53: Structure of Bufa-20,22-dienolide m,14,15-
the methanolic seeds extract of Piper nigrum by using epoxy-3,11-dihydroxy-,(3ß,5ß,11α,15present in the
GC-MS analysis. methanolic seeds extract of Piper nigrum by using GC-MS
analysis.

Figure 54: Structure of 9-Desoxo-9-x-acetoxy-3,8,12-tri- Figure 55: Structure of Retinal ,9-cis present in the
O-acetylingol present in the methanolic seeds extract of methanolic seeds extract of Piper nigrum by using GC-MS
Piper nigrum by using GC-MS analysis. analysis.

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Figure 56: Structure of Stigmasterol present in the Figure 57: Structure of 5α-Cholan-24-oic acid , 12α-
methanolic seeds extract of Piper nigrum by using GC- hydroxy-3,7-dioxo-,methyl ester present in the methanolic
MS analysis. seeds extract of Piper nigrum by using GC-MS analysis.

Figure 58: FT-IR profile of Piper nigrum.

bored in 0.5cm in diameter. The plates were incubated at Vaccenic acid, Phenanthrenol,4b,5,6,7,8,8a,9,10-
37ºC for 24 h and examined. After the incubation the octahydro-4b,8,8-trimethyl-1, Galanthamine,
diameter of inhibition zones around the discs was Dibenz[a,c]cyclohexane,2,4,7-trimethoxy, 2,4a,7-
measured. Trihydroxy-1-methyl-8-methyleneqibb-3-ene. 1,10-
carboxylic acid, Retinoic acid, 7,8,12-Tri-O-acetyl-3-
RESULTS AND DISCUSSION desoxy-ingol-3-one, 4,6-Androstadien-3β-ol-17-one,
Gas chromatography and mass spectroscopy analysis of acetate. (Figure 4-57). FTIR analysis of dry methanolic
compounds was carried out in methanolic fruits extract of extract of Rosmarinus oficinalis leaves proved the
P. nigrum, shown in Table1. The GC-MS chromatogram presence of Alkenes, Aliphatic fluoro compounds,
of the 55 peaks of the compounds detected was shown in Alcohols, Ethers, Carboxlic acids, Esters and Nitro
Figure 1. Chromatogram GC-MS analysis of the methanol Compounds which shows major peaks at 688.59, 827.46,
extract of P. nigrum showed the presence of fifty five 927.76, 1014.56, 1249.87, 1361.74, 1417.68, 1506.41,
major peaks and the components corresponding to the 2665.62, 2854.65 and 2926.01 (Table 2; Figure 58).
peaks were determined as follows. The first set up peak Boutekedjiret et al. (2003)32 studied the constituents of
were determined to be α-pinene Figure 2. The second peak rosemary essential oil from Algeria. They reported 1, 8-
indicated to be Camphene Figure 3. The next peaks cineole, camphor, β-pinene, and α-Pinene as the major
considered to be Eucalyptol, 2-Methoxy-4-vinylohenol, 1- constituents in the oil. Viuda-Martos et al. (2007)33
Oxaspiro [4,5] deca-3,6-diene,2,6,10,10-tetramethyl, 1- investigated chemical composition of the essential oil of
Oxaspiro [4,5]deca-3,6-diene,2,6,10,10-tetramethyl, anther sample of rosemary leaves from Spain. The major
Neocurdione, Isoaromadendrene epoxide, 1b,4a-Epoxy- constituents identified were α-pinene, camphor, 1,8-
2H-cyclopenta[3,4]cyclopropa[8,9] cycloundec., Cis- cineole and camphene. The main components detected in

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the oils were: α-pinene, 1, 8-cineole, camphene, camphor, fourier-transform infrared spectroscopy. Journal of
myrcene and broneol34. GC and GC-MS analysis of oils Pharmacognosy and Phytotherapy. 2015b;7(8): 132-
from rosemary leave samples from India revealed the 163.
presence of camphor, 1,8-cineole and α-pinene as major 9. Mitsuhashi M, Tanaka A, Fujisawa C, Kawamoto K,
constituents in the oils35-37. In this study five clinical Itakura A. Necessity of thromboxane A2for initiation
pathogens selected for antibacterial activity namely, of platelet-mediated contact sensitivity: dual activation
(staphylococcus aeureus, klebsiella pneumoniae, ofplatelets and vascular endothelial cells. J. Immunol.
pseudomonas aeroginosa, E. coli. and Proteus mirabilis. 2001; 166: 617–623.
Maximum zone formation against Proteus mirabilis, Table 10. Dorsam RT, Kim S, Murugappan S, Rachoor S,
3. Shankar H, Jin J, Kunapuli SP. Differential
requirements for calcium and Src family kinases in
CONCLUSION platelet GPIIb/IIIa activation and thromboxane
P. nigrum is native plant of Iraq. It contain chemical generation downstream of different G-protein path
constitutions which may be useful for various herbal ways. Blood. 2005; 105: 2749–2756.
formulation as anti-inflammatory, analgesic, antipyretic, 11. Huang JS, Ramamurthy SK, Lin X, Le Breton GC. Cell
cardiac tonic and antiasthamatic. signaling through thromboxane A2 receptors. Cell
Signal. 2009; 16: 521–533.
ACKNOWLEDGEMENT 12. Hameed IH, Hussein HJ, Kareem MA, Hamad NS.
I thank Dr. Abdul-Kareem Al-Bermani, Lecturer, Identification of five newly described bioactive
Department of Biology, for valuable suggestions and chemical compounds in methanolic extract of Mentha
encouragement. viridis by using gas chromatography-mass
spectrometry (GC-MS). Journal of Pharmacognosy and
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