American Journal Of Multidisciplinary Research & Review (AJMRR) September-2024
American Journal of Multidisciplinary Research & Review (AJMRR)
Volume-03, Issue-09,
PP-01-12
[Link]
Research Paper Open Access
Extraction, Characterization and confirmation the structure of Lycopsamine from Ageratum conyzoides used
for Tooth ache, Fever and Viral activity in Sierra Leone
*Lahai Koroma a and Ronnie Freezer-Williams b
*a
Department of Environmental Sciences Eastern Technical University of Sierra Leone, Kenema, Sierra Leone,
West Africa
b
Department of Chemistry, Fourah Bay College, University of Sierra Leone, Freetown, Sierra Leone, West Africa
ABSTRACT: The research work on extraction, characterization and confirmation the structure of Lycopsamine from
Ageratum conyzoides used treatment of Fever, tooth ache, and viral activity has been carried in Sierra Leone. 316 mg
of alkaloid labelled Sample LK004 was isolated from 300g of the dried powdered leaves of Ageratum conyzoides
using standard procedures. Solubility tests was carried out on Sample LK004 using water, ethanol, chloroform, tested
with litmus paper and subjected to Wet Chemical Methods of Analysis and sent to USA and UK for Elemental and
Instrumental Analysis.
The Sample LK004 was slightly soluble in water, Ethanol, Chloroform during solubility tests and the as [ (7r,8r)-7-
Hydroxy-5,6,7,8-Tetrahydro-3h-Pyrrolizin-1-Yl] Methyl (2s)-2-Hydroxy-2- [ (1s)-1-Hydroxyethyl]-3-
Methylbutanoate known as Lycopsamine which is one of the compounds responsible for the use of Ageratum
Conyzoides in Traditional Medicine in Sierra Leone.
Key Words: Crystalline, Traditional, Solubility, Standard procedures and Basified
I. Introduction
The extraction, characterization and confirmation the structure of the alkaloid, Lycopsamine isolated from
Ageratum conyzoides used traditionally for, tooth ache, fever and viral activity in Sierra Leone is reported in this
research work
Ageratum conyzoides L. [family Asteraceae] [Burkill, (1985); Abbiw DK. (1990), Daziel (1937)].
Kingdom: Plantae
Phylum: Angiospermatophyta
Scientific Name: Ageratum conyzoides
Local names in Sierra Leone [Deighton, 1957]
Mende: YANI-gBɛ
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Creole: WET – ED- LIF
Temne: -Ka-BAL-Ka-YAN
Kono: YANI
Kissi: YANIYO
Ageratum conyzoides is an environmental weed growing rampantly in all the regions of Sierra Leone. The plant is
commonly known as goat weed, native also to Tropical regions of America, Brazil and in west Africa as an invasive
weed. It is reported as a herb that is 0.5–1 m. high, with ovate leaves 2–6 cm long, and colour of the flowers vary
from purple to white and widely used by many traditional cultures, against dysentery and diarrhoea [Panda et al.,
(2018)], an insecticide and nematicide [Panda et al., (2018); Ming (1999)].
A. conyzoides has been reported to cause liver lesions and tumors [Sani and Bahri, (1994); Fu, et al. (2002);
Mohammad Fahim Kadir, et al., 2014] and mass poisoning incident in Ethiopia as a result of contamination of
grain with A. conyzoides [Wiedenfeld, H. (2011)]. The presence of Pyrrolizidine alkaloids including lycopsamine
[Riaz et al., (1991)] and echinatine [Mondal et al., (1998)] among many has been reported [Fu, et al. (2002),
Wiedenfeld, H. (2011)]. The toxicity of this plant has not been well studied however, the essential oil obtained from
the plant by steam distillation has been reported to have a powerful nauseating odor [Sood, (1973)]. Folk remedies
reported include, the use as purgative, febrifuge, for ophthalmia, colic, treatment of ulcers, wound dressing,
antienteralgic and antipyretic [Kerharo and Adam (1974)]; treatment of mental and infectious diseases as well as
headaches and dyspnea properties of the plant [Adjanohoun et al., (1988)]
II. Methodology
2.1.0. Collection and preparation of dried plant materials
Fresh Leaves with the flowers of Ageratum conyzoides were collected from Kenema City, Eastern Region
of Sierra Leone, dried under shade and grounded using a laboratory mill and stored in specially sealed plastic
container.
A voucher specimen of the plant organ investigated was deposited in the Herbarium of the Botany Department,
Fourah Bay College, University of Sierra Leone.
2.2.0. Experimental
2.2.1. Extraction of alkaloids from plant sample
300g of the dried powdered leaves of Ageratum conyzoides was moistened with water, mixed with 1000ml of
saturated solution of calcium (II) hydroxide [Ca(OH) 2] and allowed to stand for forty-eight hours. The mixture was
filtered using a Buchner funnel and a vacuum pump. The filtrate was extracted with petroleum (60 – 80oC) three
times and concentrated. The remaining solution was then shaken with aqueous 1% Hydrochloric acid and allowed to
separate. Alkaloid salt in in the aqueous layer was separated from the mixture using a separating funnel. The
resulting solution was basified with NaHCO3 and extracted into chloroform. The chloroform layer was allowed to
evaporate at room temperature to produce 316mg of white crystalline flakes labelled Sample LK004.
Figure 2.0.0.: Showing the flow chart for the extraction of Sample LK004 from
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Ageratum conyzoides
Sample LK004 was dissolved water, ethanol and chloroform, tested with litmus paper and subjected to wet chemical
methods of analysis. 100mg of each of Sample LK004 was sent to USA and UK for Elemental and Instrumental
Analysis.
2.2.0. Wet Chemical Methods of Analysis. (Lahai Koroma, et al., 2021; Lahai Koroma and
Kamara, et. al., 2020)
Elemental analysis was carried out on Sample LK004 isolated from the traditional medicinal plants investigated for
the presence of carbon, hydrogen, oxygen, nitrogen, Sulphur and halogens using The Middleton’s Test.
i) Test for carbon, hydrogen and oxygen.
The Sample LK004was initially dried in an oven and heated strongly with excess of pure copper (I) oxide in a test
tube until no further change occurred and test for water and carbon dioxide carried out.
ii) Test for nitrogen, Sulphur and halogens
The Middleton’s Test was carried out on Sample LK004 to ascertain the presence of Nitrogen, Sulphur and
Halogens.
5mg of sample LK004, was mixed with 1g of Middleton’s mixture [i.e., 2 parts of zinc powder to 1 part of
anhydrous Sodium Trioxocarbonate (IV)] in a small test tube and heated strongly for 2-3 minutes in a hot Bunsen
flame. The red-hot tube was the plunged into 50 ml of water in a beaker. The whole mixture obtained was boiled to
dissolve the sodium salts formed and filtered. The filtrate was used to carry out the following analysis below;
iii) Test for cyanide ions
To one portion of the filtrate was added some crystals of Iron (II) tetraoxosulphate (VI) salt and few drops of 2.0M
Sodium hydroxide and the whole mixture boiled for one minute to form hexacyanoferrate (II) ions. 10 ml of Iron (II)
chloride solution was added with sufficient concentrated hydrochloric acid to dissolve any hydroxide present and
filtered.
iv) Test for sulphide ions
The second portion of the filtrate, was tested for the presence of sulphide ions, where fresh solution of sodium
nitroprusside was added and observed.
v) Test for halide ions
To the third portion of the filtrates was boiled with 2MHNO3 in a fume cupboard to expel hydrogen cyanide and/or
hydrogen sulphide. To half of the portions freed from cyanide and sulphide ions, was added few drops of 2M HNO 3
and 2-3 drops of 0.05M AgNO3 solution.
vi) Acid test
A solution of Sample LK004 was tested with blue litmus paper and with Sodium hydrogen Trioxocarbonate (IV)
solution.
vii) Phenol test
1% iron (III) Chloride solution was added to aqueous solution of Sample LK004 the compounds and the colour
change observed.
viii) Test for unsaturation
1% KMnO4 solution was added drop wise to a solution of the Sample LK004 in a test tube and colour change
observed.
ix) Test for Aromaticity – action of heat.
A portion of solid crystals of the Sample LK004 was tested for aromaticity using the flame test.
The results of each test are reported in the Result Chapter.
2.3.0. LC-MS and NMR Spectrophotometry for Sample LK004 (USA &UK)
2.3.1. Instrumental analyses (USA) of Sample LK004(Lahai Koroma, et al., 2021; Lahai
Koroma and Kamara, [Link]., 2020)
1
H and 13C NMR spectra were acquired on an Agilent DirectDrive2 500 MHz NMR spectrometer equipped with a
One-Probe operating at 500 MHz for 1H NMR and 126 MHz for 13C NMR in CDCl3, deuterated DMSO, (CD3)2CO,
D2O or toluene-d8 and recorded at 25 °C. 1H-NMR spectra were recorded with 8 scans, a relaxation delay of 1s, and
a pulse angle of 45° and referenced to the various NMR solvents as necessary. 13C-NMR spectra were collected with
254 scans, a relaxation delay of 0.1 s, and a pulse angle 45°. Thin-layer chromatography (TLC) was performed on
plates of EMD 250-µm silica 60-F254. High-resolution mass spectroscopy was performed with APCI mass spectra
recorded on Finnigan LCQ Deca (Thermo-Quest) technologies with LC/MS/MS (quadrupole/time-of-flight) and
Waters Xevo G2-XS UPLC/MS/MS inert XL MSD with SIS Direct Insertion Probe. Melting points for all products
were measured with a Thomas HOOVER capillary uni-melt melting point apparatus and are uncorrected.
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2.3.2. Instrumental Analyses of Sample LK004 (UK)
A set of procedures for routine characterization of Sample LK004 that require a high level of confidence to assign
purity by reverse phase Ultra High-Performance liquid chromatography (RP-UHPLC) under acidic mobile phase
conditions were carried out. All compounds sent for analysis within and from outside the chemistry departments
need to comply with an agreed analytical specification.
The Sample LK004 was labelled as MSQ3AB_15NOV2019SLK_004 with file name EV-SLK_004and MS file
number IM-METCR-AB101-PosNeg, inlet file Number METCR-AB101 using the Open-Lynx equipment.
This specification typically comprised a test for the determination of the compound's identity and a test for the
determination of the compound's purity with a high degree of confidence. Elemental analysis, UHPLC-MS, coupled
with other ancillary detectors, were the predominant method of analyses used.
Common Apparatus and Reagents used were
0.1% Formic acid in water – Mobile phase “A”
0.1% Formic acid in acetonitrile – Mobile phase “B”
Waters ACQUITY UPLC CSH C18 Column, 130Å, 1.7 µm, 2.1 mm X 100 mm column
UHPLC system that is capable of gradient elution with UV or diode array detection with other detectors as required
(e.g., MS, ELS) were used in the instrumental analysis.
No test sample was used to confirm operational performance of the system during daily setup of the system as the
sample was a natural product. If the test solution data does not conform to set criteria regarding peak shape, response
and retention time, a trouble-shooting procedure was followed by carrying out a number of test samples.
2.6.0. Instrument parameters UK
Specific instrument parameters were varied depending upon the make and model of equipment used. For the LC
Conditions; a flow rate = 0.6 ml/min; Column temperature = 40ºC in 5.82 minutes. UV detection was typically
performed at a selected wavelength or over a scan range. MS detection was typically performed over a mass range to
include target masses and other ions of interest. Additional detectors such as ELS can also be included to meet
specific project requirements. Acquired data was processed automatically using Open-Lynx Software, the data was
then distributed electronically and read using the Open-Lynx data browser application.
III. Results and Discussions
Sample LK004 from the alkaloid extract of Ageratum conyzoides used traditionally for Headache, cold, fever and
viral activity
Nature = white crystalline flakes
Solubility: Slightly soluble in water, Ethanol, Chloroform
[Link] of Wet Chemical Methods of Analysis[Lahai Koroma, et al., 2021;
Lahai Koroma and Kamara, [Link]., 2020]
Table 1.0.0.: Showing the results of wet chemical analysis on Sample LK004
Test Observation Inference
a. Acid Test – Solutions of sample Red litmus paper turned blue Sample LK004 is basic
LK004 was tested with Litmus
paper
Sample + Ethanoic acid Smell of ester observed Contains OH group
Solution of Sample LK004 + No reaction observed Sample LK001 is not acidic
NaHCO3
b. Phenol Test No reaction observed Sample LK004 does not contain
Phenolic compound
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c. Test for unsaturation The colour of 0.1M KMnO4 solution
changes from purple to colourless Sample LK004 is unsaturated
ci. Test for Aromaticity smoky flame Sample LK004 is aromatic
d. Carbohydrate
Portion of Sample LK004 was Sample LK004 turned black with a
strongly heated in a boiling tube colourless gas and droplets of Probably carbohydrate present
until no further change occurred. colourless liquid at the mouth of the
i. Gas + Lime water test tube. Presence of Carbon dioxide
ii. Liquid + CuSO4 Turns lime water milky. Presence of water
Colour changes from white to blue Hence Sample LK004 contains
Carbon, Hydrogen and Oxygen
e. The Middleton’s test
5mg of Sample LK004 was mixed
with 1g of Middleton’s mixture in
small test tube and heated for two
minutes in a hot Bunsen flame.
The red-hot test tube was plunged
into 50ml of water in a beaker.
whole mixture was boiled to
dissolve the sodium salts formed,
filtered and the filtrate divided
into three portions
i. Test for cyanide ions Specks of Prussian blue precipitated Sample LK004 contained nitrogen
seen on the filter paper atoms.
ii Test for sulphide ions No visible reaction seen Sulphide ions are absent.
iii. Test for halides ions No visible reaction seen Halides ions are absent
Sample LK004 has been qualitatively shown through wet chemical methods of analysis to contain to Carbon (C),
Hydrogen (H), Oxygen (O) and Nitrogen. Sulphide and halide ions were absent. The compound was basic but gave
negative test for phenol indicating that Sample LK004 is a Pyrrolizidine-alkaloids.
[Link] of Instrumental Methods of Analysis(Lahai Koroma, et al., 2021; Lahai
Koroma and Kamara, [Link]., 2020)
3.2.1. Elemental Analysis
The Table below indicates the results of elemental analysis
Table 2.0.0.: Showing the results of elemental composition on Sample LK004
Symbol Element Atomic weight Atoms Mass percent
C Carbon 12.0107 15 60.1813 %
H Hydrogen 1.00794 25 8.4174 %
N Nitrogen 14.0067 1 4.6788 %
O Oxygen 15.9994 5 26.7224 %
The expected molecular formula of Sample LK004 = C15H25NO5
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Expected Molecular mass: 299.36
Expected structure is as shown below;
H3C CH3
H O O H
O
H3C
O H O H
N
Figure 3.0.0.: Showing the proposed structure of Sample LK004
The Proposed name of Sample LK004 is Lycopsamine (a Pyrrolizidine-alkaloid) and its presence in Ageratum
conyzoides has been reported [Riaz et al., (1991)]. The above Structure was confirmed from the Instrumental
analysis results sent to USA and UK, by its physical properties and by proton and LCMS/Mass Spectroscopic
analysis shown below
[Link] of Proton NMR Spectroscopy (USA) for Sample LK004[Lahai
Koroma, et al., 2021; Lahai Koroma and Kamara, [Link]., 2020]
Figure 3.1.0.: 1H NMR Spectrum for Sample LK004
The 1H NMR Spectrum for Sample LK004 is worthy of some comments according to Figure 3.0.0., the
interpretations of δ – values (ppm) for aH – O, bH3C, and c H – C drawn above are shown below;
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Figure 3.1.1.: Showing the proposed structure of Sample LK004 and the
three different proton environments around it
The interpretations of δ – values (ppm) shifts drawn above are shown below[Lahai
Koroma, et al., 2021; Lahai Koroma and Kamara, [Link]., 2020]
a
a. H – O= 8.50 ppm
b
b. H3C = 4.2 – 4.5 ppm
c
c. H – C = 3.0 ppm
Solvent Peak 1 = HDO = 3.34 ppm
Solvent Peak 2 = DMSO = 2.40 – 2.5 ppm as illustrative in the figure above
Figure 3.1.2.: Proposed location of fragments on the proton NMR for Sample LK004
The above table clearly indicates the position of Hydrogen atoms in the different environments in the proposed
structure of Sample LK004. The confirmation of the structure can also be obtained from the Mass Spectrum
obtained from the UK as shown below.
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Full Spectrum LK 004
Figure 3.2.0.: Showing the Mass Spectrum of Sample LK004
3.4.0. RESULTS OF LCMS/MASS SPECTROSCOPY OF SAMPLE LK004
Table 3.0.0.: ID and Description of mass spectrum of Sample LK004
Sample Vial ID File Date Time Description
4 01:13 A5 MSQ3AB_15NOV2019SLK_004 15-Nov-19 [Link] EV-SLK_004
Figure 3.2.1.: LCMS/Mass spectroscope of Sample LK004
A total of eighteen (18) peaks were obtained from the Blind research as shown in the data below,[Lahai Koroma, et
al., 2021; Lahai Koroma and Kamara, [Link]., 2020]
Table 3.1.0.: Number of Peaks used in obtaining Fragments of Sample LK004[Lahai
Koroma, et al., 2021; Lahai Koroma and Kamara, [Link]., 2020]
Peak Area
Number Vial Function Trace BPI Area Abs. %BP Width Height State
MS ES+: Diversity
1 01:13 1:MS ES+ TIC 6.78E+05 3.00E+05 25.69 0.05 11832889 fail
MS ES+: Diversity
2 01:13 1:MS ES+ TIC 6.61E+05 1.00E+05 11.43 0.033 6345449.5 fail
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MS ES+: Diversity
3 01:13 1:MS ES+ TIC 2.34E+05 3.00E+04 2.44 0.02 2506302 fail
MS ES+: Diversity
4 01:13 1:MS ES+ TIC 1.36E+05 3.00E+04 2.62 0.053 1263809.5 fail
MS ES+: Diversity
5 01:13 1:MS ES+ TIC 4.52E+05 8.00E+04 6.33 0.067 3951286.75 fail
Diversity
6 01:13 1:MS ES+ DAD: 215 3.68E+05 5.00E+02 34.21 0.057 37466.469 fail
Diversity
7 01:13 1:MS ES+ DAD: 215 1.69E+05 2.00E+03 100 0.063 62524.141 fail
Diversity
8 01:13 1:MS ES+ DAD: 215 2.24E+05 1.00E+02 7 0.033 5893.8 fail
Diversity
9 01:13 1:MS ES+ DAD: 215 3.57E+04 2.00E+02 13.13 0.047 14433.71 fail
Diversity
10 01:13 1:MS ES+ DAD: 215 3.56E+04 7.00E+01 4.21 0.052 3332.64 fail
MS ES+: Diversity
11 01:13 1:MS ES+ TIC 6.30E+04 4.00E+04 3.12 0.03 2666277.5 fail
Diversity
12 01:13 1:MS ES+ DAD: 215 2.49E+04 7.00E+01 4.38 0.047 4265.71 fail
Diversity
13 01:13 1:MS ES+ DAD: 215 5.25E+04 7.00E+01 4.25 0.037 4304.91 fail
Diversity
14 01:13 1:MS ES+ DAD: 215 1.24E+05 7.00E+02 41.93 0.053 41825.25 fail
MS ES+: Diversity
15 01:13 1:MS ES+ TIC 9.97E+06 1.00E+06 100 0.093 58832740 fail
Diversity
16 01:13 1:MS ES+ DAD: 215 9.95E+06 2.00E+02 14.21 0.037 12191.16 fail
Diversity
17 01:13 1:MS ES+ DAD: 215 2.20E+04 5.00E+02 30.62 0.053 27709.131 fail
Diversity
18 01:13 1:MS ES+ DAD: 215 5.14E+04 6.00E+01 3.58 0.042 3401.28 fail
The following results are obtained from eighteen peaks of Mass Spectroscopy with respect to the fragments that
could be possibly obtained from Sample LK004 and by McLafferty Rearrangement with ID. NO of the
MSQ3AB_15NOV2019SLK_004 and interpreted as shown below;
Figure 3.3.0.: Showing the Fragments that can possibly be obtained from Proposed
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structure of Sample LK004 [Lahai Koroma, et al., 2021; Lahai Koroma and Kamara, [Link]., 2020]
Table 3.1.1.: Usual fragmentation patterns and by Maclerfty Rule shown in Figure 3.3.0. above
corresponding to molecular ions [M]+ in the various peak spectrums [Lahai Koroma, et al., 2021; Lahai
Koroma and Kamara, [Link]., 2020]
Fragmentation Expected Actual
ion Molecular mass Molecular mass Intensity Spectrum
Number
[M]+ 299.36 299.38 774.30 4
[M]+ 299.17 696.38 13
[M]+ 299.77 452.99 14
[M_ 15] + 284 284.99 51020.21 1
[M+15] + 314 314.68 1333.19 11
[M+15] + 314 314.13 3093.55 4
[M +15] + 314 314.73 2596.80 7
[M _ 30] + 269 269.12 900.06 8
[M _ 30] + 269.17 144.96 15
[M+ 30] + 329 329.17 2654.997 14
[M- 45] + 254 254.20 3082.07 8
[M- 45] + 254.63 3128.95 15
[M+45] + 344 344.19 1767.90 8
[M+ 45] + 344.40 3743.05 15
[M + 50] + 349.36 349.19 23011 1
[M _ 74] + 225 225.24 16283.94 8
[M _ 74] + 225.05 6738.98 14
[M +74] + 373 373.33 494.07 8
[M +74] + 373.11 750.08 14
[M _ 72] + 227 227.18 1660.97 8
[M +72] + 371 371.48 566.96 15
[M _ 83] + 216 216.03 943.02 8
[M +83] + 382 382.07 441.08 8
[M +83] + 382.50 320.03 15
[M_ 87] + 212 212.26 3099.04 15
[M +87] + 386 386.78 662.03 15
[M _ 109] + 190 190.28 2219.06 8
[M _ 109] + 190.06 2352.03 14
[M +109] + 480 480.12 1942.15 11
[M +109] + 480.90 2054.93 14
[M+83 + 87] + 469 469.20 2030.03 14
469.21 512.96 15
Table 3.1.1; above showing the fragmentation patterns detected from the mass spectrometer corresponds to the
fragments of the proposed structure of sample LK004 thus confirming the structure below;
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Figure 3.3.1.: Showing the confirmed structure of Sample LK004
Lycopsamine, A Pyrrolizidine Alkaloid and a potential Antitumor Agent[Zalkow, et al.(1985); Wiedenfeld, et al.,
(2009)]. The Structure of Sample Lk004 was Identified as [ (7r,8r)-7-Hydroxy-5,6,7,8-Tetrahydro-3h-Pyrrolizin-
1-Yl] Methyl (2s)-2-Hydroxy-2- [ (1s)-1-Hydroxyethyl]-3-Methylbutanoate Known as Lycopsamine[Lexichem
(2021)]which is one of the compounds responsible for the Traditional Medicinal use of Ageratum Conyzoides in
Sierra Leone.
IV. Conclusion and Recommendations
4.1.0. Conclusion
The Sample LK004 isolated from the alkaloid extract of dried powdered leaves of Ageratum Conyzoides
was characterized using both wet chemical and instrumental methods of analysis. It has been qualitatively shown
through wet chemical methods of analysis that Sample LK004 to contains to Carbon (C), Hydrogen (H), Oxygen (O)
and Nitrogen. The compound was basic but gave negative test for phenol indicating that Sample LK004 is a
Pyrrolizidine-alkaloids. The structure of Sample LK004 was identified and confirmed using fragmentation patterns
from LCMS, Proton NMR spectroscopy and by McLafferty rearrangement as [ (7r,8r)-7-Hydroxy-5,6,7,8-
Tetrahydro-3h-Pyrrolizin-1-Yl] Methyl (2s)-2-Hydroxy-2- [ (1s)-1-Hydroxyethyl]-3-Methylbutanoate known as
Lycopsamine which is one of the compounds responsible for the Traditional Medicinal use of Ageratum Conyzoides
in Sierra Leone.
4.2.0. Recommendations
This research work has shown that the alkaloid extract of dried powdered leaves of Ageratum Conyzoides contains
Lycopsamine which is one of the compounds responsible for the Traditional Medicinal use of Ageratum Conyzoides
in Sierra Leone. It is therefore recommended that further methods be used to synthesis Lycopsamine for use in
Modern Medicine.
4.3.0. Sources of Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit
sectors.
4.3.1. Conflict of Interest
The authors have declared that no competing interests exist.
V. Acknowledgment
The authors are grateful to Dr. Lukulay Sombay (UK) and Mr. Mohamed Barrie (USA) for LCMS/GCMS,
elemental analysis and spectral analysis of Sample sent to them, Laboratory technicians of the Departments of
Chemistry and Botany, Fourah Bay College, University of Sierra Leone and the Vice Chancellor/Principal Eastern
Technical University of Sierra Leone, Kenema for providing financial assistance
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