Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2021, Article ID 4051555, 11 pages
https://doi.org/10.1155/2021/4051555
Research Article
Antiproliferative Activities ofMethanolic Extract and Fractions of
Tetrapleura Tetraptera Fruit
AnastasiaRosebudAikins ,1PeggyAfuaBirikorang ,1MaryChama ,2EuniceDotse ,3
Abigail Anning,3 and Regina Appiah-Opong 3
1West African Centre for Cell Biology of Infectious Pathogens, Department of Biochemistry, Cell and Molecular Biology,
University of Ghana, Accra, Ghana
2Department of Chemistry, School of Physical and Mathematical Sciences, University of Ghana, Accra, Ghana
3Department of Clinical Pathology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana
Correspondence should be addressed to Anastasia Rosebud Aikins; araikins@ug.edu.gh
Received 27 April 2021; Accepted 5 July 2021; Published 19 July 2021
Academic Editor: Chan-Yen Kuo
Copyright © 2021 Anastasia Rosebud Aikins et al. 'is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in anymedium, provided the original work is
properly cited.
Most of the current cancer chemotherapeutics are associated with harsh and undesirable side effects, including toxicity and
chemoresistance, driving the need for safer and more effective alternatives. In this study, the antiproliferative activities of the
methanolic extract of Tetrapleura tetraptera fruits and nine different fractions (C1–C9) from the column chromatographic
separation of the extract against leukemia (Jurkat) and human breast cancer (MCF-7) cell lines were investigated using a
tetrazolium-based colorimetric assay. Phytochemical screening of the extract and fractions found alkaloids, carbohydrates,
flavonoids, glycosides, phenols, saponins, steroids, tannins, and terpenoids in the methanolic extract. Most of the fractions
exhibited antiproliferative activity (>100 μg/mL) with the Jurkat cells being more susceptible than the MCF-7 cells. Four of the
collected fractions C4, C3, C5, and C2 had good selective indices in decreasing order of activity, in the case of Jurkat cells. Liquid
chromatography-mass spectrometry analysis of all samples (except for C4 and C9) revealed that C1, C2, C3, and C5 each had a
single component. More importantly, fractions C2, C3, and C5, which were selective to Jurkat cells, also had the same retention
time of 1.846min. Fractions C6 and C8 had two components, with C7 having four components. 'is study serves as a basis for
further work to isolate and characterize potential anticancer agents from the fractions of extracts of T. tetraptera fruits.
1. Introduction Tetrapleura tetraptera is a perennial plant that is
widespread in tropical Africa, particularly Ghana, where it is
Cancer remains a global health challenge, with about 18.1 foundmostly in the northern part of the country and its fruit
million new cases and 9.6 million deaths recorded an- is locally known as “prekese.” 'e most common use of the
nually [1]. Most of the available therapies or interventions fruit in Ghana is adding it to food as a spice because of its
for cancer treatment are accompanied by side effects such unique aroma. T. tetraptera is one of the most medicinally
as hair loss, numbness or chronic pain in some parts of beneficial plants because of its several biological activities.
the body, damage to vital organs, chemoresistance, and Notable among them are its antioxidant [5, 6], anti-in-
tumor reoccurrence [2, 3]. Cancer research, therefore, flammatory [7], antimicrobial [8], hypotensive [9], hypo-
continues to be focused on the development of new glycemic, and antidiabetic activities [10].
therapies with little to no side effects and/or improve- Studies involving the anticancer effects of T. tetraptera
ment of existing therapies. Exploring the use of natural have mainly focused on crude extracts. For instance, studies
products, especially plant parts, is a major area of focus have shown that the methanolic extract of T. tetraptera fruit
[4]. exhibits potent anticancer activity against human breast
2 Evidence-Based Complementary and Alternative Medicine
cancer cell lines, BT-549 and BT-20, and the T-lympho- 2.6. Carbohydrates. Two milliliters of Fehling’s solutions A
blastic leukemia cell line, Jurkat [11]. In addition, Kuete et al. and B was added to 2mL of the crude extract or fractions and
(2011) showed that the methanolic extract had anticancer heated until boiling. 'e presence of carbohydrates was
activity against MiaPaCa-2, a human pancreatic cancer cell indicated by the formation of brick-red precipitates.
line, and the leukemia cell lines, CCRF-CEM and CEM/
ADR5000 [12]. 'e ethanolic extract of T. tetraptera fruit 2.7. Carotenoids. A few drops of chloroform followed by
also exhibited strong antiproliferative activities against sulfuric acid were added to 2mL of the crude extract or
Ehrlich ascites carcinoma both in vitro and in vivo [13]. fractions. 'e blue color formation at the interface showed
Anticancer studies involving fractionation of the extracts of the presence of carotenoids.
T. tetraptera would help better elucidate the components of
the crude extract with antiproliferative activity.
In this study, we investigated the antiproliferative ac- 2.8. Flavonoids. 'ree drops of ferric chloride solution were
tivities of the crude extract and column chromatography- added to 2mL aliquots of the test samples, and the presence
derived fractions of T. tetraptera fruit against two human of flavonoids was ascertained by the formation of a blackish-
cancer cell lines as a measure of anticancer activity. red color.
2. Materials and Methods 2.9.Glycosides. Concentrated sulfuric acid (2mL) was added
2.1. Plant Collection and Preparation. T. tetraptera fruits to 2mL of each test sample. A reddish-brown coloration
were purchased from the Madina market in Accra, Ghana, indicated the presence of glycosides.
and authenticated by a taxonomist at the University of
Ghana Herbarium, Department of Plant and Environmental 2.10. Phenols. A few drops of lead acetate were added to
Biology. 'e fruits were washed thoroughly and oven-dried 2mL of the crude extract or fractions, and the formation of a
at 65°C for 4 h.'e dried fruits were pulverized and stored at yellow precipitate showed the presence of phenols.
4°C in an airtight container until use.
2.11. Saponins. Two milliliters of the extract or fractions was
2.2. Extraction and Fractionation of the Crude Methanolic mixed with approximately 3mL of water. Upon shaking, the
Extract. Five hundredmilliliters of 99%methanol was used to formation of foam, which was stable for 15min, showed the
extract the compounds in 100 g of the pulverized fruit using presence of saponins.
the Soxhlet extraction method for 10 h. 'e resulting extract
was filtered and concentrated using a rotary evaporator.
2.12. Steroids. Two milliliters of chloroform was added to
2.3. Fractionation of the Extract Using Column 2mL of each sample, and then 2mL of chloroform was
Chromatography. A glass column preloaded with a slurry of added, followed by a few drops of concentrated sulfuric acid.
silica gel and chloroform was loaded with the methanolic 'e development of a red layer in the test tube confirmed the
extract. Beginning with 100% chloroform, the ratio of presence of steroids.
chloroform to methanol was varied as the columns were
progressively run until 100%methanol was used for the final 2.13. Tannins. A few drops of basic lead acetate solution
elution. Volumes of 20mL eluates were collected in test were added to 2mL of the crude extract or fractions, and the
tubes. Based on the thin layer chromatography of the formation of a white precipitate indicated the presence of
fractions, they were combined and concentrated to dryness tannins.
using rotary evaporation under vacuum. 'e resulting
residues were reconstituted in dimethyl sulfoxide (DMSO)
and stored at − 4°
2.14. Terpenoids. Two milliliters of chloroform was added to
C until use. 2mL aliquots of the sample, and then 2mL of chloroform
was added.'e resultant mixture was evaporated to dryness,
2.4. Qualitative Phytochemical Analysis. Qualitative phyto- and a few drops of sulfuric acid were added and heated for
chemical screening was performed on the crude extract and approximately 2min. 'e development of a gray color in-
fractions as described by Trease and Evans [14]. 'e phy- dicates the presence of terpenoids.
tochemicals that were tested were terpenoids, flavonoids,
alkaloids, glycosides, carotenoids, tannins, saponins, phe- 2.15. Cell Culture. 'e cell lines used in this study were the
nols, steroids, and carbohydrates. 'e methods used to human breast cancer cell line, MCF-7, T-lymphoblastic
ascertain the presence or absence of these phytochemicals leukemia cell line, Jurkat, and Chang liver cells (derived from
are briefly described below. HeLa cells but used in place of normal cells due to their slow
proliferative properties). Jurkat and Chang liver cell lines
2.5.Alkaloids. A few drops of a saturated picric acid solution were cultured in RPMI-1640 medium, andMCF-7 cells were
were added to 2mL of a solution of the crude extract or cultured in DMEM. Both culture media were supplemented
fractions. 'e formation of yellow precipitates indicates the with 10% fetal bovine serum (FBS) and 1% penicillin-
presence of alkaloids. streptomycin. 'e cells were maintained in a humidified
Evidence-Based Complementary and Alternative Medicine 3
incubator at 37°C and 5% CO2 with periodic changes in 2.17.CytotoxicityAssay. 'e3-(4, 5-dimethylthiazol-2-yl)-2,
media and passaging until they were ready for use. 'ese 5-diphenyltetrazolium bromide (MTT) assay was performed
cells were seeded into 96-well microtiter plates at a density of tomeasure the effect of the crude extract and fractions on the
1× 104 cells per well in a 100 μL volume for 24 h prior to proliferation of MCF-7 and Jurkat cells. 'e effect of the
treatment. extracts was assessed using Chang liver cells to calculate the
selectivity index. After 72 h of incubation of treated cells,
2.16. Treatment of SeededCells. Five different concentrations 20 μL of 2.5mg/mLMTTwas added to each well in a 96-well
of the crude extract and each of the fractions were prepared culture plate and further incubated in the dark for 4 h. 'e
from stock solutions using 1% DMSO as the solvent. 'ey reactions were stopped by adding 150 μL of acidified iso-
were added to the seeded wells at a final concentration of propanol. Subsequently, the cells were incubated in the dark
1000, 500, 250, 125, and 62.5 μg/mL in triplicate. Curcumin at room temperature (26
°C) overnight. Absorbance was read
was used as a standard compound for the positive control. at a wavelength of 570 nm using a fluorescence microplate
For the wells designated for use as a positive control, 10 μL of reader (Tecan Infinite M200, Austria).
five different concentrations of curcumin was added to From the absorbance values obtained, percentage cell
obtain final concentrations ranging from 2 to 38 μg/mL. viabilities were calculated for the various concentrations of
DMEM containing 1% DMSO was used as the negative the fractions, crude extract, and curcumin using the fol-
control. 'e treated cells were then incubated for 72 h. lowing formula:
absorbance of treatedwells − absorbance of color control
percentage cell viability � . (1)
absorbance of untreatedwells − absorbance of blank
Graphs of percentage viability against concentration 4.50min. Mass spectra were collected from m/z 100–800 in
were plotted for each fraction, crude extract, and curcumin both the negative and positive modes.
for the three cell lines. Inhibition concentrations at 50%
(IC50) were obtained from these graphs.'e IC50 value is the
concentration of the test compound required to reduce cell 2.19. Statistical Analysis. 'e data were analyzed using
viability by 50%.'ese IC values were used to calculate the GraphPad Prism version 8 and the 2013 version of Microsoft50
selectivity indices using the following formula: Excel. 'e data are expressed as the mean± SD.
IC50 of treated normal cell linesselectivity index 2 3. Results� . ( )
IC50 of treated cancer cells 3.1. Qualitative Phytochemical Screening of Crude Extract and
'e selectivity index (I) is a measure of the cytotoxic Fractions. All phytochemicals tested, including alkaloids,
selectivity of drugs or extracts, which implies their ability to carbohydrates, flavonoids, flavonoids, glycosides, phenols,
differentiate and target cancer cells with little to no harm to saponins, steroids, tannins, and terpenoids, were present in
nonmalignant cells. Drug candidates with SI ≥2 were con- the crude extract, except for carotenoids, which were also
sidered to have good therapeutic abilities. absent in the fractions (Table 1). 'e first fraction, C1, did
not indicate the presence of any phytochemicals tested.
Among the fractions, steroids were only present in C2,
2.18. Liquid Chromatography-Mass Spectrometry (LC-MS) of terpenoids were present in C3 and C5, and phenols were
theCrudeExtract andFractions. Low-resolution ESi-MS data present in C3 and C6. Flavonoids and saponins were present
were acquired on an Agilent 1260 Infinity HPLC system in fractions C6–C9.
(Agilent® 1260 Infinity Binary Pump, Agilent® 1260 InfinityDiode Array Detector (DAD), Agilent 1290 Infinity Column
Compartment, and Agilent 1260 In®finity Standard Auto- 3.2.Cytotoxicity (MTT)Assay. 'e effect of the crude extract
sampler) coupled to an Agil®ent 6120 Quadrupole MS system and fractions of T. tetraptera fruit on the proliferation of the
and Peak Scientific® Genius 1050 nitrogen generator. A cell lines was determined using the MTT assay. Dose-re-Phenomenex Kinetex 2.6μm EVO C18 100 Å (30 2.1mm) sponse curves plotted as percentage viability against the×
reverse-phase analyti®cal column was used. 'e chromato- concentration of fractions and crude extract are shown in
graphic method included a column temperature of 40°C, an Figure 1. Fractions C5, C6, and C7 exhibited higher cyto-
injection volume of 2 μL, a flow rate of 0.7mL/min, and toxicity in the three cell lines, similar to curcumin, whereas
maximum column backpressure set at 600 bars. 'e mobile fraction C1 did not have much effect on the cell lines even at
phase consisted of 10mM NH OAc in water (A) and 10mM a concentration of 1000 μg/mL (Figure 1).4
NH4OAc in methanol (B). 'e LC run is a 4.50min duration
beginning with 15% a of B from 0 to 0.30min, followed by a 3.3. Inhibition Concentration at 50% (IC50) Values. 'e IC50
speedy increase in the gradient to 100% B over 0.90min. 'e values of the extracts and fractions are presented in Table 2.
mobile phase composition at 100% B was maintained at 'e crude extract and fractions were more cytotoxic to
4 Evidence-Based Complementary and Alternative Medicine
Table 1: Phytochemical constituents of crude extract and fractions of T. tetraptera fruit.
Phytochemicals Crude extract C1 C2 C3 C4 C5 C6 C7 C8 C9
Alkaloids + − + − − − + − + +
Carbohydrates + − − + − + + − + +
Carotenoids − − − − − − − − − −
Flavonoids + − − − − − + + + +
Glycosides + − + − + + − − − −
Phenols + − − + − − + − − −
Saponins + − − − − − + + + +
Steroids + − + − − − − − − −
Tannins + − + − − − + − + +
Terpenoids + − − + − + − − − −
A plus sign (+) denotes presence, whereas a minus sign (− ) denotes the absence of phytochemicals.
120 140 120
100 120 100
80 100 80
60 8060 60
40 40 40
20 20 20
0 0 0
0 10 20 30 40 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200
[Curcumin] µg/mL [Whole extract] µg/mL [C1] µg/mL
CHANG CHANG CHANG
JURKAT JURKAT JURKAT
MCF–7 MCF–7 MCF–7
(a) (b) 120 (c)
120 120 100
100 100 80
80 80
60 60 60
40 40 40
20 20 20
0 0 0
–20 200 400 600 800 1000 1200 –20 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200
[C2] µg/mL [C3] µg/mL [C4] µg/mL
CHANG CHANG CHANG
JURKAT JURKAT JURKAT
MCF–7 MCF–7 MCF–7
(d) (e) (f )
120 120 120
100 10080 100
80 60 80
60 4020 60
40 0 40
20 –20 200 400 600 800 1000 1200–40 20[C6] µg/mL
0 –60 0
0 200 400 600 800 1000 1200 CHANG 0 200 400 600 800 1000 1200
[C5] µg/mL JURKAT [C7] µg/mL
CHANG MCF–7 CHANG
JURKAT JURKAT
MCF–7 MCF–7
(g) (h) (i)
Figure 1: Continued.
% cell viability % cell viability % cell viability
% cell viability % cell viability % cell viability
% cell viability % cell viability % cell viability
Evidence-Based Complementary and Alternative Medicine 5
140 120
120 100
100 80
80
60 60
40 40
20 20
0 0
0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200
[C8] µg/mL [C9] µg/mL
CHANG CHANG
JURKAT JURKAT
MCF–7 MCF–7
(j) (k)
Figure 1: Dose-response curves of percentage viability against the concentration of T. tetraptera whole extract and its fractions and
curcumin on Chang liver, Jurkat, and MCF-7 cell lines.
Table 2: IC50 (μg/mL± SD) values of crude methanolic extract and chromatographic fractions of T. tetraptera fruit on Chang liver, Jurkat,
and MCF-7 cells.
Treatment Chang liver Jurkat MCF-7
Crude extract 294.37± 10.91 340.37± 6.58 380.12± 29.66
C1 >1000 >1000 >1000
C2 693.09± 3.20 222.57± 9.14 593.78± 15.43
C3 576.55± 10.80 115.48± 5.63 302.84± 10.62
C4 967.91± 39.34 184.42± 2.04 663.65± 22.45
C5 185.91± 4.29 51.38± 3.64 190.40± 8.58
C6 31.86± 0.31 35.85± 2.29 70.07± 0.75
C7 100.98± 4.37 70.56± 3.89 152.73± 24.14
C8 >1000 634.56± 60.16 933.06± 16.19
C9 >1000 866.64± 31.24 878.63± 15.61
Curcumin 11.39± 0.59 6.96± 1.08 12.87± 1.33
'e values are expressed as the mean± standard deviation. Curcumin was used as the standard compound.
Jurkat thanMCF-7 cells, showing lower IC50 values in Jurkat ability. 'ese fractions are 4, 3, 5, and 2 in decreasing order
than MCF-7 cells. of activity.'e crude extract had SI values less than 2 in both
Fraction 1 (C1) was inactive because it had IC50 values Jurkat andMCF-7 cell lines. Fraction 6, which had the lowest
>1000 μg/mL in all cell lines. For both Jurkat and MCF-7 IC50 values in both cell lines, had the lowest SI values in both
cells, fraction 6 had the strongest cytotoxic activity, with the cancer cell lines, indicating poor therapeutic potential.
lowest IC50 values of 35.85± 2.29 μg/mL and 70.07± 0.75 μg/
mL, respectively, in Jurkat and MCF-7 cells. In Chang liver
cells, fraction 6 was also cytotoxic with an IC50 value of 3.5. LC-MS Analysis of Crude Methanolic Extract and
31.86± 0.31 μg/mL. Fractions. Fractions C1–C3 and C5–C8 obtained from the
chromatographic separation of the crude methanolic extract
were analyzed for their various components using liquid
3.4. Selectivity Indices. From the IC50 values obtained for chromatography-mass spectrometry (LC-MS). Each of the
the crude extract and fractions against the three cell lines, fractions C1–C3 and C5 had single components with C1
selectivity indices (SI) were calculated as a measure of their eluted at a retention time of 1.839min and the rest at
ability to differentiate malignant from nonmalignant cells 1.846min (Table 3 and Figure 3). 'e same retention times
in exerting their antiproliferative effects. Fractions with SI and fragment ions were obtained for components C2, C3,
values above the 2.0 threshold were considered to have and C5 suggesting that these three fractions are the same.
therapeutic activity, whereas those below were considered Fractions C6 and C8 had two components each, whereas
toxic. fraction C7 had four components. 'e component in
Figure 2 shows that none of the fractions or crude extract fraction C6 eluted at 1.839min (87.3%) was also eluted in C1
had therapeutic activity against MCF-7 cells, as they had SI (100%) with similar fragmentation, while the one at
values less than 2. However, against Jurkat cells, four 10.145min (12.7%) was present in C7 (9.69%). 'e crude
fractions had SI values greater than 2, indicating therapeutic extract showed a peak at 1.853min (100%).
% cell viability
% cell viability
6 Evidence-Based Complementary and Alternative Medicine
6
5
4
3 Threshold of 2.0
2
1
0
JURKAT
MCF-7
Figure 2: Selectivity indices of crude extract (crude), fractions, and curcumin.
Table 3: Percentage content of crude and fractions from liquid chromatograph-mass spectrometry.
Crude/fraction RT/min % content Some fragment ions
C1 1.839 100 101.0, 146.1, 179.1, 241.1, 319.1, 373.1
C2 1.846 100 101.1, 146.1, 179.1, 202.3, 241.1, 319.1, 373.1
C3 1.846 100 101.1, 146.1, 179.1, 241.1, 319.1, 373.1
C5 1.846 100 101.1, 146.1, 179.1, 202.3, 241.1, 319.1, 373.1
C6 1.839 87.3 101.1, 146.1, 179.1, 241.1, 319.1, 373.110.145 12.7 111.1, 161.2, 210.2, 261.2, 349.2, 397.3, 535.1
1.372 10.28 111.1, 161.2, 223.1, 245.2, 263.2, 349.2, 393.2
C7 1.595 6.35 89.1, 111.1, 161.2, 203.1, 245.2, 290.2, 349.2, 383.2, 435.11.839 79.68 101.0, 146.1, 179.1, 241.1, 319.1, 373.0
10.145 9.69 111.1, 161.2, 210.2, 261.2, 349.2, 397.3, 535.0
C8 1.609 24.38 111.1, 161.1, 203.1, 290.2, 349.2, 383.2, 445.21.861 74.62 101.0, 146.1, 179.0, 241.1, 319.1, 373.0, 533.4
Crude extract 1.853 100 101.1, 111.1, 161.1, 163.1, 205.1, 223.1, 261.1, 279.2 349.2, 535.0
RT: retention time.
4. Discussion fruit of T. Tetraptera, derived from column chromatography,
against human MCF-7 breast cancer and Jurkat leukemia
One of the major limitations of most cancer therapies used cell lines. T. tetraptera is known to contain various phy-
in recent times is the accompanying side effects, including tochemicals. Most phytochemicals are produced by plants
damage to vital organs and tumor reoccurrence. Research for protective roles, thereby conferring one or more bio-
into the efficacy of natural compounds, especially products logical activities to the plant.
from plants as anticancer agents, continues to be of para- Phytochemicals found in the fruit of T. tetraptera include
mount importance [15]. Over the years, research has shown alkaloids, saponins, tannins, flavonoids, reducing sugars,
that some plants have potent anticancer activities. For in- glycosides, terpenoids, phenols, steroids, and anthraqui-
stance, the crude extract from the Allium wallichii plant has nones [6, 18, 19]. Two oleanane-type saponins, tetraptero-
anticancer activity against prostate, breast, and cervical side A and tetrapteroside B, have been isolated from the stem
cancer cell lines [16]. 'e extract from the stem bark of bark of plants [20]. 'e leaves and stems also contain
Zanthoxylum alatum is also cytotoxic against human lung stigmasterol, stigma-5, 22-diene-3-O-β-D-glucopyranoside,
and pancreatic cancer cell lines [17]. 3-O-β-D-glucopyranosyl-2ʹ-acetamido-2ʹ -deoxy]-oleanolic
Our study sought to investigate the antiproliferative acid, pheophytin, and tetracosanol [21]. 'e high saponin
potential of the crude methanolic extract and fractions of the content, for instance, confers potent antimicrobial activity
Selectivity indices (SI)
Curcumin
Crude
C2
C3
C4
C5
C6
C7
C8
C9
Evidence-Based Complementary and Alternative Medicine 7
Fragmentor voltage 0 Collision energy 0 Ionization mode ESI
×109 +ESI TIC scan CF = 0.000 DF = 0.000 1679-PES2-20.d
1 ∗1.839 C1 1
1.2
1
0.8
0.6
0.4
0.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Counts vs. acquisition time (min)
(a)
Fragmentor voltage 0 Collision energy 0 Ionization mode ESI
×109 +ESI TIC scan CF = 0.000 DF = 0.000 1680-PES2-20.d1 ∗1.846 C2 1
1.2
1
0.8
0.6
0.4
0.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Counts vs. acquisition time (min)
(b)
Fragmentor voltage 0 Collision energy 0 Ionization mode ESI
×109 +ESI TIC scan CF = 0.000 DF = 0.000 1681-PES2-20.d
1.4 1 ∗1.846 C3 1
1.2
1
0.8
0.6
0.4
0.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Counts vs. acquisition time (min)
(c)
Fragmentor voltage 0 Collision energy 0 Ionization mode ESI
×109 +ESI TIC scan CF = 0.000 DF = 0.000 1682-PES2-20.d
1.4 1 ∗1.846 C5 1
1.2
1
0.8
0.6
0.4
0.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Counts vs. acquisition time (min)
(d)
Figure 3: Continued.
8 Evidence-Based Complementary and Alternative Medicine
Fragmentor voltage 0 Collision energy 0 Ionization mode ESI
×109 +ESI TIC scan CF = 0.000 DF = 0.000 1683-PES2-20.d1 ∗1.839 C6 1
1.4
1.2 ∗10.145
1
0.8
0.6
0.4
0.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Counts vs. acquisition time (min)
(e)
Fragmentor voltage 0 Collision energy 0 Ionization mode ESI
×109 +ESI TIC scan CF = 0.000 DF = 0.000 1684-PES2-20.d
1 ∗1.839 C7 1
1.4
1.2 ∗10.145
1
0.8
0.6
0.4
0.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Counts vs. acquisition time (min)
(f )
Fragmentor voltage 0 Collision energy 0 Ionization mode ESI
×109 +ESI TIC Scan CF = 0.000 DF = 0.000 1685-PES2-20.d
1 ∗1.861 C8 1
1.2
1
0.8
0.6
0.4
0.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Counts vs. acquisition time (min)
(g)
Fragmentor voltage 0 Collision energy 0 Ionization mode ESI
×109 +ESI TIC scan CF = 0.000 DF = 0.000 1686-PES2-20.d
1 ∗1.853 1
1
0.8
0.6
0.4
0.2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Counts vs. acquisition time (min)
(h)
Figure 3: (a) LC-MS chromatogram of fraction C1 of T. tetraptera. (b) LC-MS chromatogram of fraction C2 of T. tetraptera. (c) LC-MS
chromatogram of fraction C3 of T. tetraptera. (d) LC-MS chromatogram of fraction C5 of T. tetraptera. (e) LC-MS chromatogram of
fraction C6 of T. tetraptera. (f ) LC-MS chromatogram of fraction C7 of T. tetraptera. (g) LC-MS chromatogram of fraction C8 of T.
tetraptera. (h) LC-MS chromatogram of the crude methanolic extract of T. tetraptera.
Evidence-Based Complementary and Alternative Medicine 9
because saponins are produced to deter foreign pathogens 'ree out of four fractions with good selectivity indices, C2,
from attacking and destroying the plant [22]. 'e flavonoids C4, and C5, contained glycosides, another member of the
scopoletin, 2ʹ, 4, 4ʹ-trihydroxychalcone isoliquiritigenin, 2ʹ, polyphenol group. Indeed, some glycosides have been re-
3, 4, 4ʹ-tetrahydroxychalcone-butein, and 4ʹ, 5, 7-trihy- ported to exhibit potent anticancer activities. Schneider et al.
droxyflavanone-naringenin have also been isolated from the reported that a group of glycosides, referred to as cardiac
fruits of T. tetrapleura [23, 24]. Furthermore, phytic acids, glycosides, have potent anticancer activities against various
oxalates, and cyanogenic glycosides are also present in the types of human cancers, including breast cancer [40].
fruit [25]. Despite its potent antiproliferative activity, C6 is cyto-
In our study, apart from carotenoids that were not found toxic to cancer cells and hence not suitable for consideration
in the crude extract and fractions, the other phytochemicals in chemotherapeutic development. Phytochemicals present
were tested; alkaloids, carbohydrates, flavonoids, glycosides, in the most cytotoxic fraction, C6, are flavonoids, alkaloids,
phenols, saponins, steroids, tannins, and terpenoids were tannins, saponins, phenols, and carbohydrates. However, the
present, consistent with the findings of other studies [26, 27]. fractions that were most selective to the cells (C2, C3, C4,
Various phytochemicals, especially polyphenols, in- and C5) did not contain flavonoids and saponins. Further
cluding phenolic acids, flavonoids, and stilbenes, have been studies are needed to determine whether the absence of
shown to possess potent anticancer activities [28]. For in- flavonoids and saponins accounts for the good selectivity
stance, resveratrol (a stilbenoid) has potent anticancer ac- indices of these fractions.
tivity against various cancer types, including human From the LC-MS analysis, the same retention time (RT)
colorectal cancer [29], while curcumin is potent against of 1.846min was obtained for fractions C2, C3, and C5,
various cancers, including human liver cancer [30]. Epi- suggesting that they may be the same, which explains why
gallocatechin-3-gallate, one of the most important tannins they were all selective against Jurkat cells. Further studies to
found in green tea, has also been reported to have potent elucidate the active compounds in these fractions might
apoptotic, antiproliferative, and antimetastatic effects in contribute to the development of improved agents for
various human cancers, such as liver, lung, and ovarian cancer, especially leukemia.
cancers [31–33]. Similarly, flavonoids, such as quercetin, Altogether, our findings underscore the importance of
have been reported to exhibit anticancer effects in prostate fractionating crude extracts to help isolate compounds that
cancer [34]. 'us, the phytochemicals in the crude extract are selective only for cancer cells to minimize toxicity against
and fractions might be responsible for the observed anti- normal cells.
proliferative effect on the cancer cells in this study.
'e crude extract had antiproliferative activity against 5. Conclusion
both MCF-7 cells and the acute T cell leukemia cell line,
Jurkat, exhibiting a more cytotoxic effect on Jurkat than In conclusion, our findings are consistent with those from
MCF-7 cells. 'is finding is consistent with the findings of other studies on the prospects of T. tetraptera in the quest for
other studies. For instance, the methanolic extract of natural and less harmful therapies for cancer. More im-
T. tetraptera fruit showed strong cytotoxicity against two portantly, our findings showed that some fractions from the
human breast cancer cell lines: BT-549 and BT-20 as well as methanolic extract had better antiproliferative activity than
Jurkat with IC50 values of 9.1, 23.1, and 37.5 μg/mL, re- the crude extract. Our findings also underscore the im-
spectively [11]. It has also been shown that extracts from the portance of fractionating the crude extract to help isolate
fruit had cytotoxic effects against CCRF-CEM leukemia cells compounds that are selective only for cancer cells to min-
and MDA-MB-231-pcDNA3 breast cancer cells as well as imize toxicity against normal cells.
their drug-resistant variants, CEM/ADR5000 and MDA- In the present study, identification of the main com-
MD-231-BCRP, with IC50 values of 10.27, 20.47, 17.16, and ponents in the fractions with good selectivity indices against
15.75 μg/mL, respectively [35]. In another study, the leu- cancer cells would have provided better insights into the
kemia cell lines, CCRF-CEM and CEM/ADR5000, and the therapeutic potential of T. tetraptera. However, this was not
pancreatic cancer cell line, MiaPaCa-2, showed cytotoxicity, investigated, which is a limitation of this study, and future
although it was not as potent as some of the other plants studies would be targeted at investigating this.
tested [12]. T. tetraptera fruit has lower amounts of tannins, 'is is important because the identification and isolation
saponins, phenols, and sterols with higher contents of al- of the compounds in the fractions responsible for the
kaloids, flavonoids, and hydrogen cyanide, with flavonoids antiproliferative activity and with good selectivity indices
being the most abundant [36].'e overall anticancer activity would be beneficial for pharmacological purposes.
of the samples that showed antiproliferative activity in this
study might be due to the abundance of flavonoids. Data Availability
Compounds with promising therapeutic value must be
able to selectively target cancer cells without affecting All the data supporting the results of this study are available
normal cells [37]. An anticancer agent is considered to have from the corresponding author upon request.
pharmacological or therapeutic activity if it has an SI value
greater than or equal to 2 [38, 39]. Compounds or drugs with Conflicts of Interest
SI values less than 2 are therefore considered toxic as they
harm normal cells as much as or more than cancerous cells. 'e authors declare there are no conflicts of interest.
10 Evidence-Based Complementary and Alternative Medicine
Acknowledgments Journal of Ethnopharmacology, vol. 134, no. 3, pp. 803–812,
2011.
'e authors acknowledge Professor Takuhiro Uto of [13] M. Ozaslan, I. D. Karagoz, R. A. Lawal et al., “Cytotoxic and
Nagasaki International University who kindly donated the anti-proliferative activities of the Tetrapleura tetraptera fruit
cell lines used for the work. Reagents for the study were extract on ehrlich ascites tumor cells,” International Journal of
provided by the Department of Biochemistry, Cell and Pharmacology, vol. 12, no. 6, pp. 655–662, 2016.
Molecular Biology, University of Ghana, and the Depart- [14] W. C. Evans, Trease and Evans’ Pharmacognosy, SaundersLtd,
ment of Clinical Pathology, Noguchi Memorial Institute for Sixteenth Edition, 2009.
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