Communication in Physical Sciences 2020, 5(4): 437-445

Chemical Information from GCMS Analysis of Acetone Extract of

Piper guineense Leaves. Part 1

Onyeije Ugomma Chibuzo* and Imeh Okop

Received 25 June 2020/Accepted 29 July 2020/Published online: 30 July 2020
Abstract Applications of plant leaves for various Imeh Okop
purposes is based on its chemical constituents which Department of Chemistry
may include proximate, elemental, phytochemical, Akwa Ibom State University
toxicant, amino acid and other toxicants. Ikot Akpaden, Akwa Ibom State, Nigeria
Knowledge of phytochemical constituents is signifi- Email: [email protected]
cant for their pharmaceutical/medicinal values. This Orcid id: 0000-0002-1487-6711
study was carried out to investigate the chemical 1.0 Introduction
constituents of acetone extract of Piper guineense Piper guineense is an erect herbaceous climbing li-
leaves through phytochemical screening and GCMS ana native to tropical Africa, P. guineense fruits are
analysis. Results obtained from phytochemical widely applied externally as a counter-irritant or in
screening indicated the major constituents (those a stimulating ointment, internally as a stomachic and
whose concentrations were greater than 1%) to in- carminative.The leaves are useful in the treatment of
clude ,6-dimethyloxazolo(5,4-c)pyridazin-4-amine wounds while the stems and twigs are for the treat-
(31.80 %), 3-(1-methylethyl)-cyclohexene (20.99 ment of coughs and bronchitis (Owolabi et al. 2013).
%), 4-methoxy-N-(4-nitrobenzyl)-benzamide (12.82 Food and food materials can be assessed based on
%), alpha bisabolene (7.33%), 1,2,3,4,4a,5,6,8a-oc- its chemical composition (Ekop and Eddy, 2006).
tahydro-7-methyl-4-methylene-1-(1-methylethyl)- According to Imo et al. (2018), Piper guin-
napthalene (4.42 %), 3,7-diacetamidophenoxathin eense seeds have higher percentage of dry mat-
(4.10 %), 1,3,3-trimethyl tricyclo{2,2,1]heptane ter (94.03±0.21), crude lipid (4.06±0.12) and carbo-
(2.98%), 3H-indazol-3-one (2.11%), 1H-indene,oc- hydrates (65.46±0.85) than the leaves
tahydro-1,7a-dimethyl-4-(1-methylethenyl)-1,4- while the leaves have higher percentage mois-
methano-1H-indene (1.98%), piperidine ture (6.11±0.01), protein (15.17±0.39), crude fibre
(1.97%),2,4-disopropenyl-1-methyl-1-vinyl (20.99±0.16) and ash (11.98±0.03) than the seeds.
(1.70%), n-hexadecanoic acid (1.68%), eudesdma- Several extract of Piper guineense have been found
4[14],11-diene (1.27%). The pharmaceutical values to exhibit pharmaceutical and medicinal values. For
of the identified constituents were also analsed. The example, methanol extract of Piper guineense was
study reveals that acetone extract of Piper guneense found to offered protection against infection that is
contains constituents that are not visible with some comparable to that of Livolin forte with better effi-
other solvents cacy when pre-treated with 400 mg/kg for 14 days
prior to CCl4-exposure (Oyinloye et al., 2017).
Key Words: Piper guineense leaves, Chemical con- Ekudayo et al. (1988) identified elemicin as the ma-
stituents, phytochemicals, GCMS, phytochemical jor essential oil constituent of the plant and stated
screening that the plant has significant medicinal applications.
Olonisakin et al. (2006) identified (1s)-(-1)-β-
Onyeije Ugomma Chibuzo pinene (43.9%), D-Limonene (7.7%), caryophyllene
Department of Chemistry (6.9%), car-2-ene (5.4%) and 1,6,10-dodecetrien-z-
Rhema University, 153-155 Aba Owerri Road ol, 3, 7, 11-trimetyl (2.9%) and found that they did
P. M. B. 7021, Abia State, Nigeria not display any antimicrobial activity against Esch-
Email: [email protected] erichia coli , Serratia , Salmonella typhi , Klebsiella
Orcid id: 0000-0003-2832-7948 sp., Citrobacter and Pseudomonas aeruginosa due
to the solvent he used. Chinwendu et al. (2016) iden-
tified alkaloids (0.86%), HCN (8.87%), saponins
Communication in Physical Sciences 2020, 5(4): 437-445
Available at https://journalcps.com/index.php/volumes
Communication in Physical Sciences 2020, 5(4): 437-445 438

(1.87%) and phenols (0.66%) in ethanol extract of kPa), total flow (6.2 ml/minute), linear velocity
Piper guineense leaves while Ebenso et al. (2008) (46.3 cm/sec), purge flow (3.0ml/min) and split ratio
also identified the presence of alkaloids, tannins, (1.0). The high-pressure injection, carrier gas server
saponins, flavonoids, hydrogen cyanides and phe- and splitter hold functions were switch off. The ini-
nols in ethanol extract of the leaves. They observed tial rate of oven temperature program was 5 C/min
that, Piper guineense (Uziza leave) contains some and was gradually increased to 140C after which
considerable amount of anti-nutrients which have the temperature was increased to 280 C at a rate of
medicinal benefits. 10 C/minute. Some heat unit and detector functions
GCMS analysis of plant extract has been found to be were checked in order to ensure consistency. These
one of the most powerful tool that is useful for iden- included column oven, SPL2, MS, SPL2 carrier,
tifying chemical constituents of plants (Eddy et al., SPL2 purge and were ensured to be on. However,
2011a,b; Ikpeazu et al., 2020). Ojinnaka et al. the APC setting was turned off.
(201,6) identified 22 peaks from the GCMS spec- Other setting functions of the machine were ion
trum of ethanol extract of Piper guineense leaves source temperature (200 C), interface temperature
and found that the spectrum was dominated by acids (250 C), solvent cut time ((2.50 minutes), detector
and hydrocarbon while alcohol and ester were the gain mode (relative), detector gain (0.00kV), thresh-
least constituents. Recent study conducted by old (1000). The analytical start time was 3 minutes
Usaman et al. (2020) indicated that the component and the machine run for 45 minutes using ACQ scan
extracted from plant parts depends on the type of mode at a scan speed of 769. However, mass/charge
solvent. However, most studies on Piper guineense started at 50 and ended with 400 units.
leaves are done with aqueous and ethanol extract. Gas chromatogram and mass spectrum were auto-
Therefore, the present study is aimed at identifying matically plotted and suggested chemical structures
the chemical constituents of acetone extract of Piper were obtained using the National Science Technol-
guneense leaves using GCMS analysis. ogy library installed in the machine. Percentage con-
2.0 Materials and Methods centrations of each identified component was calcu-
Samples of Piper guineense leaves were purchase lated using area normalization.
from Ikot Ekpene main market and transported to 3.0 Results and Discussion
the Chemistry laboratory of the Michael Okpara Fig. 1 presents the GCMS of acetone extract of
University of Agriculture, Umudike. They were Piper guineene while chemical names, retention
thoroughly washed with distilled water and allowed time, molar mass and percentage concentrations of
to dry. The leaves were sun dried for a week until compounds deduced from each peak in the spectrum
the moisture content was reduced to minimum. The are recorded in Table 1. Fig. 2 shows the mass spec-
dried leaves were grounded to a powder form and trum of the compounds.
soaked in acetone solution. The solvent was recov- The chemical constituents observed from the GCMS
ered using cold extractor, leaving behind, acetone spectrum of the plant leaves were 3,6-dimethyloxa-
extract of Piper guineense leaves. zolo(5,4-c)pyridazin-4-amine (31.80 %), 3-(1-meth-
The produced extract was used for GCMS analysis ylethyl)-cyclohexene (20.99 %), 4-methoxy-N-(4-
using spectroscopically pure acetone solvent. The nitrobenzyl)-benzamide (12.82 %), alpha bisabo-
GCMS-QP2010 PLUS Schimadzu (made in Japan) lene (7.33%), 1,2,3,4,4a,5,6,8a-octahydro-7-me-
instrument was used for the analysis. The analytical thyl-4-methylene-1-(1-methylethyl)-napthalene
steps taken were plugger speed (high), syringe injec- (4.42 %), 3,7-diacetamidophenoxathin (4.10 %),
tion speed (high), viscosity/compression time (0.2 1,3,3-trimethyl tricyclo{2,2,1]heptane (2.98%), 3H-
second), injection mode (normal), pumping time indazol-3-one (2.11%), 1H-indene,octahydro-1,7a-
(5), injection port dwell time (0.3 second), termi- dimethyl-4-(1-methylethenyl)-1,4-methano-1H-in-
nated air cap (No), plugger washing speed (high), dene (1.98%), piperidine (1.97%),2,4-disopropenyl-
washing volume (8µl), syring suction position (0), 1-methyl-1-vinyl (1.70%), n-hexadecanoic acid
syringe injection position (0) and used three solvent (1.68%), eudesma-4[14],11-diene (1.27%), tri-
vial (3). The operational setting of the GCMS instru- cosenoic acid (0.77%), alpha cubebene (H-
ment were column oven temperature (60C), injec- ctclopenta[1,3]cyclopropal[1,2]benzene (0.71%),
tion temperature (200C), injection mode (split), 2,6,6-trimethyl-3-(phenylthio)cyclohept-4-enol
flow control mode (linear velocity), pressure (100.2
Communication in Physical Sciences 2020, 5(4): 437-445 439

(0.69%), bicyclo[7,2,0]undec-4-ene,4,11,11-trime- tetramethyl-globulol (0.47%) and 2,6-dimethoxy-

thyl-8-methylene (caryophyllene (0.54%), 1H-cy- toluene (0.30%).
cloprop€azulen-4-ol,decahydro-1,1,4,7-

Fig. 1: GCMS of acetone extract of Piper guineense

Table 1: Characteristics of compounds in acetone extract of Piper guineense leaves

Line Name Mass Retention Molar Concentration

No peak time (mi- mass (%)
nute) (g/mol)
1 2,6-dimethoxytoluene 32 15.333 152 0.30
2 alpha cubebene (H-ctclopenta[1,3]cyclo- 40 17.517 204 0.71
propal[1,2]benzene
3 2,4-disopropenyl-1-methyl-1-vinyl 62 19.308 204 1.70
4 1,3,3-trimethyl tricyclo{2,2,1]heptane (cyclo- 45 20.033 136 2.98
fenchene)
5 bicyclo[7,2,0]undec-4-ene,4,11,11-trimethyl-8- 65 20.108 204 0.54
methylene (caryophyllene)
6 Cis alpha bisabolene 82 21.133 204 7.33
7 eudesma-4[14],11-diene 69 21.308 204 1.27
8 1,2,3,4,4a,5,6,8a-octahydro-7-methyl-4-methylene- 73 22.125 204 4.42
1-(1-methylethyl)-napthalene (gamma murolene)
9 1,2,3,5,6,7,8,8a-octahydro-1,4dimethyl-7-(1-meth- 74 22.65 204 1.37
ylethenyl)-azulene
10 3-(1-methylethyl)-cyclohexene 114 25.658 124 20.99
11 1H-cycloprop(e) azulen-4-ol,decahydro-1,1,4,7- 72 25.775 222 0.47
tetramethyl-globulol
12 1H-indene,octahydro-1,7a-dimethyl-4-(1-meth- 80 27.375 204 1.98
ylethenyl)-1,4-methano-1H-indene
13 2,6,6-trimethyl-3-(phenylthio)cyclohept-4-enol 72 28.65 262 0.69
14 n-hexadecanoic acid 89 32.283 256 1.68
15 Tricosenoic acid 94 34.067 352 0.77
Communication in Physical Sciences 2020, 5(4): 437-445 440

16 3H-indazol-3-one 61 36.075 134 2.11

17 4-methoxy-N-(4-nitrobenzyl)-benzamide 139 37.95 286 12.82
18 3,6-dimethyloxazolo(5,4-c)pyridazin-4-amine 153 39.667 164 31.80
19 piperidine 130 40.467 285 1.97
20 3,7-diacetamidophenoxathin 172 43.75 314 4.10

2,6-dimethoxytoluene

Alpha cubebene

2,4-disopropenyl-1-methyl-1-vinyl

1,3,3-trimethyl tricyclo{2,2,1]heptane (cyclofenchene)

caryophyllene

Cis-alpha bisabolene
Communication in Physical Sciences 2020, 5(4): 437-445 441

Eudema-4[14]-11-diene

Gamma murolene

1,2,3,5,6,7,8,8a-octahydro-1,4dimethyl-7-(1-methylethenyl)-azulene

3-(1-methylethyl)-cyclohexene

1H-cycloprop(e) azulen-4-ol,decahydro-1,1,4,7-tetramethyl-globulol

1H-indene,octahydro-1,7a-dimethyl-4-(1-methylethenyl)-1,4-methano-1H-indene
Communication in Physical Sciences 2020, 5(4): 437-445 442

2,6,6-trimethyl-3-(phenylthio)cyclohept-4-enol

n-hexadecanoic acid

Tricosenoic acid

3H-indazol-3-one

4-methoxy-N-(4-nitrobenzyl)-benzamide
Communication in Physical Sciences 2020, 5(4): 437-445 443

3,6-dimethyloxazolo(5,4-c)pyridazin-4-amine

Piperidine

3,7-diacetamidophenoxathin
Fig. 2: Mass spectrum and chemical structures of compounds in acetone extract of Piper guneense
The mass spectrum of 3,6-dimethyloxazolo(5,4- leaves to the presence of cubebin. Haznedarogku et
c)pyridazin-4-amine was shown in Fig. 2. The com- al. (2001) found that the essential oil of Salvia to-
pound is also call octocrinsghpfl-uhfffaoysa-N. mentosa contain 1,8-cineol (17%), β-caryophyllene
Little is known of its bioactivity or other usefulness. (11%), cyclofenchene (10%) and δ-cadinene (6%).
However, the 5,4 derivatives of the compound has They attributed the antimicrobial activity of the es-
been implicated in the treatment of cancer tumor. sential oil to these constituents, which also inhibited
Caryophyllene identified in line 5 (of the GCMS the growth of tested Gram-positive and Gram-nega-
spectrum) contains several biological activities that tive bacteria except for Pseudomonas aeruginosa.
are attributed to betacaryophyllene, such as anti-in- Alpha-cubebene, camphene, geraniol, limonene,
flammatory, antibiotic, antioxidant, anticarcinogen- myrcene, palmitic acid and sabinen were found to
icc and local anaesthetic. Piperidine (peak 19) has exhibit antioxidant (DPPH assay), anti-inflamma-
been confirmed to be active as antibacterial, analge- tory (5-lipoxygenase assay), antimicrobial (disk
sic and also exhibited anti-inflammatory activity diffusion) and anti-mosquito properties (insecti-
(Mohammed et al., 2016). Alpha and beta cubebene cidal, larvicidal and repellency assays) by Naidoo
were also reported in essential oils of Annona salz- et al. (2009). α-farnesene and bisabolene are known
mannii and A. pickelii (Annonaceae) by Coataa et al. flavour ingredients and their catalytic hydrogenation
(2011) and were found to exhibit strong anti-bacte- gives the hydrocarbons, farnesane and bisabolane,
rial activity. Boligon et al. (2012) also attributed an- respectively. These saturated derivatives are pro-
timicrobial activity of Scutia buxifolia Reissek spective industrial products as they have been
Communication in Physical Sciences 2020, 5(4): 437-445 444

singled out among the most promising biofuel can- L. & Pratae, A. P. D. (2011). Chemical composi-
didates (Clarke, 2008). Sun et al. (2005) isolated tion and antioxidant, antimicrobial, and larvi-
four eudema and found that the compounds showed cidal activities of the essential oils of Annona
glucose consumption activity with an IC value of salzmannii and A. pickelii (Annonaceae). Natu-
10.7 microg/mL in a C2C12 muscle cell assay. The ral Product Communication, 6 , 6, pp. 907 – 912.
MIC value of this compound (100 mg/kg) in a db/db Ebenso, E. E., Eddy, N. O. & Odiongenyi, A. O.
mice model was found to be equivalent to that of (2008). Corrosion inhibitive properties and ad-
metformin in vivo. Limberger et al. (2001) found sorption behaviour of ethanol extract of Piper
gamma murolene (identified in ine 8) in Blepharo- guinensis as a green corrosion inhibitor for mild
calyx salicifolius and linked it to some biological ac- steel in H2SO4. African Journal of Pure and Ap-
tivities. Silva et al. (2009) also reported that gamma plied Chemistry, 4, 11, pp. 107-115.
murolene is active against Bacillus subtilis and Can- Eddy, N. O,,Awe, F. E., Siaka, A., Magaji, L. &
dida tropicalis, including clinical strains. Tan et al. Ebenso, E. E. (2011b). Chemical infor-
(2008) investigated. and obtained results which in- mation from GC-MS studies of ethanol extract of
dicated that globulol (identified in line 11) is the Andrographis paniculata and their corrosion in-
main antimicrobial compound in the ethanol extract hibition potentials on mild steel in HCl solution.
of E. globulus fruits. Hexadecanoic acid (palmitic International Journal of Electrochemical Sci-
acid), was identified in line 14 of the spectrum. It is ences, 6, pp. 4316-4328.
a saturated long-chain fatty acid with a 16-carbon Ekop, A. S. & Eddy, N. O. (2006). Comparative
backbone which has been reported to have potential studies of the lipid characteristics and industrial
antioxidant, antitumor, anti-inflammatory, antibac- potential of Coula edulis (African walnut and
terial and antifungal activities (Vasudevan et al., Terminalia catappa (indian almond) seeds.
2012). Global Journal of Pure and Applied Sciences,
4.0 Conclusion 12, 1, pp. 65-67.
GCMS of acetone extract of Piper guineense leaves Eddy, N. O., Ameh, P., Gimba, C. E. & Ebenso, E.
reveals that the plant leaves contain organic acids, E. (2011a). GCMS studies on Anogessus leocar-
hydrocarbon, alcohol, ester, ketone and other aro- pus (AL) gum and their corrosion inhibition po-
matic compounds. Twenty compounds were identi- tentials for mild steel in 0.1 M HCl. International
fied and some of the compounds found in the spec- Journal of Electrochemical Sciences, 6, pp.
trum differs from those reported by others. Most of 5815-5829.
the the identified compounds have industrial appli- Ekundayo, O., Laakso, I., Adegbola, R.M., Ogun-
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