Hindawi
International Journal of Microbiology
Volume 2020, Article ID 8645691, 5 pages
https://doi.org/10.1155/2020/8645691
Research Article
Cytotoxic andAntioxidantEffectsofAntimalarialHerbalMixtures
Isaac Dadzie ,1 Shaibu Adams Avorgbedo,1 Regina Appiah-Opong,2 and Obed Cudjoe3
1Department of Medical Laboratory Science, School of Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana
2Department of Clinical Pathology, Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana
3Department of Microbiology and Immunology, School of Medical Sciences, University of Cape Coast, Cape Coast, Ghana
Correspondence should be addressed to Isaac Dadzie; idadzie@ucc.edu.gh
Received 29 January 2019; Revised 1 January 2020; Accepted 20 January 2020; Published 10 February 2020
Academic Editor: Clemencia Chaves-López
Copyright © 2020 Isaac Dadzie et al. (is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Many developing countries depend on herbal mixtures as the first line and cost-effective therapy for malaria. (ese mixtures with
such curative tendencies may also be a source of toxicity to host cells. On the other hand, these mixtures may have anticancer
potential activity characterized by cytotoxicity to cancer cells.(e aim of the study was to determine the cytotoxic and antioxidant
effects of five different antimalarial herbal mixtures. Five antimalarial herbal mixtures commonly used in Ghana (coded as STF,
SMH, SMM, SGM, and STT) were purchased and freeze-dried. (e dried samples were tested on human acute T-cell leukemia
(Jurkat) and breast adenocarcinoma (MCF-7) cell lines. Cytotoxicity was assessed using the tetrazolium-based colorimetric
(MTT) assay while antioxidant activity was determined using DPPH free-radical scavenging assay. Among the mixtures, SMM
and SGM exhibited the strongest cytotoxicity towards Jurkat cells (IC50 values 59.17 μg/ml and 49.57 μg/ml, respectively), whereas
STTshowed the weakest cytotoxicity (IC50 � 244.94 μg/ml). Cytotoxic effect of SMMwas also strongest towardsMCF-7 cells whilst
the least cytotoxic sample was SGM (IC50> 1000 μg/ml). SMM had the highest antioxidant percentage (EC50 �1.05mg/ml). (e
increasing order of antioxidant percentage among the five herbal mixtures is SMM> SMH> STT> STF> SGM. (e herbal
mixtures may be potential sources of toxic agents to host cells. (erefore, further toxicity studies must be performed to safeguard
health of the public. Interestingly, cytotoxicities exhibited by SMM and SGM suggest the presence of anticancer constituents in
them which warrant further studies.
1. Introduction With the increasing levels of drug resistance and the high
cost of treatment with conventional antimicrobial drugs,
Numerous strategies have been adopted to control the in- herbal mixtures readily serve as the most assessable and
cidence of malaria; however, an estimated 216 million cases affordable means of treatment for many illnesses in many
of malaria were recorded worldwide in 2017, with 445,000 parts of the world [4, 5]. (e majority of the population in
deaths. Approximately 90% of all malaria deaths occur in many developing countries, where malaria is endemic, de-
Africa [1]. (e resistance of the Plasmodium parasite to the pends on herbal mixtures as therapy for malaria [6, 7].
available antimalarial drugs is a major challenge to the Medicinal plants, such as Uvaria chamae, Strophanthus
control of malaria [2]. Previously, chloroquine was the hispidus, Psidium guajava, Cassia abbreviata, Aristolochia
drug of choice in Ghana and other endemic African albida, Annona muricata, Mangifera indica, and Azadir-
countries for treatment of uncomplicated malaria caused achta indica have been documented and are used in most
by P. falciparum, the parasite responsible for most malaria African countries to treat malaria and other ailments, either
cases. Artemisinin combination therapies (ACTs) are alone or in combination with other plant medicines
currently the drugs of choice for the treatment of un- [6, 8–10].
complicated malaria in Ghana. (e emergence of drug- (e preference for herbal preparation has been partly
resistant strains therefore necessitates intensive search for informed by the fact that these herbs are of natural origin
new drugs [3]. and are therefore considered safe to use as compared to the
2 International Journal of Microbiology
synthetic or the orthodox drugs. However, not all of such microplate reader (Tecan Infinite M200, Austria). (e effect
natural products are safe. Herbal remedies associated with of the herbal mixtures on cell viability was calculated using
adverse effects have been reported and continue to engage the following formula:
the attention of researchers [11–14]. (ere are so many
A − A
herbal mixtures available and sold in Ghana for treatment of viability(%) � 0 1 − B × 100, (1)
malaria. However, safety profile of the majority of these A0
antimalarial mixtures has not yet been scientifically inves- where A0 is the mean absorbance of wells with untreated
tigated. Hence, this study sought to determine the cytotoxic cells (vehicle), A1 is the absorbance of test wells, and B is the
effect of five different commonly used antimalarial herbal absorbance of blank wells (extract control, cell free). (e
mixtures sold on the Ghanaian market on cultured human concentration of the test sample that resulted in 50% de-
cell lines. crease in the cell number, i.e., 50% inhibitory concentration
(IC50), as compared with that of the control cultures (un-
2. Materials and Methods treated cells) was then determined.
Fetal bovine serum (FBS), RPMI culture medium, penicillin-
streptomycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- 2.4. Determination of Antioxidant Activity: DPPH Assay.
nyltetrazolium bromide (MTT) were obtained from Sigma (e scavenging activities of the powdered herbal mixtures
Chemical Company (St. Louis, MO, USA). Human acute on the stable free-radical DPPH (2,2-diphenyl-1-picryl-
T-cell leukemia (Jurkat) and breast adenocarcinoma (MCF- hydrazyl-hydrate) were assayed according to a method
7) cell lines were obtained from RIKEN BioResource Center described, with slight modification [17, 18]. Serial dilutions
Cell Bank (Japan). All other reagents and chemicals used for of each sample were prepared (0.027–20mg/ml). Each re-
the work were of analytical grade and obtained from action mixture comprised 100 μl of 0.5mM DPPH solution
standard suppliers. (in methanol) and 100 μl of the sample in 96-well plates.
Butylated hydroxytoluene (BHT) was used as positive
2.1. Sample Preparation. Five (5) commonly used herbal control. Triplicate experiments were performed. (e plates
antimalarial mixtures from different manufacturers were were incubated for 20min at room temperature in the dark,
purchased from the market in Accra. (ese samples were and the absorbance was read using a microplate reader
coded (as STF, SMH, SMM, SGM, and STT), frozen at 20°C (Tecan Infinite M200, Austria) at 517 nm. (e percentage of−
and freeze-dried. inhibition was calculated using the equation:
A − A
inhibition(%) � 0 1 × 100, (2)
2.2. Cell Culture. Jurkat cells were cultured in RPMI me- A0
dium supplemented with 10% FBS and 1% penicillin- where A0 is the absorbance of the control and Ai is thestreptomycin, whilst MCF-7 cells were cultured in DMEM absorbance of the samples. EC50, which is the concentrationwith the same supplements. (e cells were maintained in a where 50% of the free-radical activity of DPPH is quenched,
humidified incubator with 5% CO2 at 37°C and subcultured was extrapolated from a graph of percent antioxidant ac-
when they were about 80% confluent. tivity versus sample concentration [18].
2.3. Cell Viability (MTT) Assay. Determination of cytotox- 3. Results
icity of the herbal extracts was carried out in vitro using the
MTT assay as described in [15, 16]. Five different concen- 3.1. Cytotoxicity. For evaluation of cytotoxicity, the cells
trations (62.5 to 1000 μg/ml) of each of the herbal mixtures were exposed to increasing concentrations of the herbal
were prepared by serial dilution. Hundred microliters extracts with curcumin serving as positive control. Figures 1
(100 μl) cell suspension was added into each well in a 96-well and 2 show the dose-dependent effect of the herbal mixtures
microtitre plate, and 10 μl of extract dilution was added. on Jurkat cell lines, with their corresponding IC50 values
Triplicate experiments were performed. Curcumin was used (Table 1). (e antimalarial STT showed the least cytotoxic
as positive control. Negative control (vehicle, water) and effect towards Jurkat cells with an IC50 value of 244.92 μg/ml,
extract control experiments were also set up.(e plates were whilst SGM gave the strongest cytotoxic effects
incubated in a humidified incubator with 5% CO2 at 37°C for (IC50 � 49.57mg/ml) on the cell lines. On the other hand,
72 h. Twenty microliters (20 μl) of 2.5mg/ml MTT solution among the herbal mixtures, SGM showed the least cyto-
(in PBS) was added to each well of the plates, and incubation toxicity towards MCF-7 cell lines (IC50> 1000mg/ml),
was continued for 4 h. After 4 hours, 150 μl of acidified whereas SMM gave the strongest activity (IC50 � 40.82mg/
isopropanol was added to each well, and the plates were ml) on the cells. (e herbal mixtures STF and SMH
incubated in the dark at room temperature overnight. (e exhibited weak cytotoxic activities towards both human cell
MTT assay spectrophotometrically measures the purple- lines (IC50> 100 μg/ml). Curcumin, which was used as the
coloured formazan resulting from the reduction of the positive control exhibited a higher cytotoxicity against both
yellow tetrazolium salt by metabolically active cells. Ab- cell lines as expected, with an IC50 value of 3.84 μg/ml and
sorbance was read at the wavelength of 570 nm using a 7.45 μg/ml for Jurkat and MCF-7 cells, respectively.
International Journal of Microbiology 3
120 Table 1: In vitro cytotoxic effects of the herbal mixtures on human
cells.
100 Herbal mixtures IC50 values of JURKAT cells andMCF-7 cells (μg/ml)
80 STF 110.6± 8.46 432.78± 5.77
SMH 143.99± 13.22 196.55± 2.88
SMM 59.17± 11.54 40.82± 1.52
60 SGM 49.57± 4.10 1000± 4.35
STT 244.94± 10.41 97.95± 1.15
40 CUR 3.80± 0.15 7.45± 0.11
(e values represent the mean of three independent experiments. CUR:
20 curcumin, positive control.
0 Table 2: Antioxidant activity of the herbal mixtures.
0 100 200 300 400 500 600 700 800 900 1000 1100 Herbal mixtures EC50 (mg/ml)
Extract concentration (μg/ml)
STF 2.69± 0.35
CUR SMM SMH 1.45± 0.23
STF SGM SMM 1.05± 0.21
SMH STT SGM 3.58± 0.31
Figure 1: Cytotoxic effects of the herbal mixtures on Jurkat leu- STT 1.65± 0.38
kemia cells. Each plotted point represents the mean of three in- BHT 0.23± 0.02
dependent experiments, and the bars are standard deviations. BHT, butylated hydroxytoluene, a known potent antioxidant was used as
positive control.
120
activity. (is was followed by SMH and STT which gave
100 similar activities (EC50 values of 1.45mg/ml and 1.65mg/ml,
respectively). (e highest EC50 value was found for SGM.80
60 4. Discussion
40 Traditional herbal medicines have been used to treat malaria
for years, and they are the source of major groups (arte-
20 misinin and quinine derivatives) of potent modern anti-
0 malarial drugs [20]. Herbal preparations may achieve the
desired curative purpose when administered but may also
–20 contain other ingredients that may have toxic effects on
human. We assessed the cytotoxic effects of five known
–40
0 100 200 300 400 500 600 700 800 900 1000 1100 potent antimalarial herbal mixtures on human cell lines
Extract concentration (μg/ml) Jurkat and MCF-7. Antioxidant activities of the mixtures
were also assessed to determine the potential of the products
CUR SMM to protect the body against oxidative stress. (e herbal
STF SGM mixtures that were considered in this study are commonly
SMH STT used mixtures claimed to be very effective antiplasmodial
Figure 2: Cytotoxic effects of the herbal mixtures on MCF-7 breast agents.
cancer cells. Each plotted point represents the mean of three in- Results obtained show that the five antimalarial herbal
dependent experiments, and the bars are standard deviations. mixtures tested had varying cytotoxic activities toward the
human cell lines compared to the positive control (curcu-
3.2. Antioxidant Activity. Protection against oxidative min), which was highly cytotoxic to both cells. Possibly, the
damage is one of the most widely described attributes of cytotoxic effect of individual components has been diluted
plant extracts and relates to their radical scavenging activity. out in the mixture. Alternatively, the observed cytotoxicity
(e scavenging properties of the herbal mixtures were could be an additive or synergistic effect from the various
evaluated by the DPPH radical scavenging assay. (e EC50 components of the mixtures. In vitro analysis of potential
values are a parameter widely used to measure antioxidant toxic, mutagenic, and carcinogenic effects of herbal medi-
activity [19]. (e lower the EC50 value, the higher the an- cines has been established on both normal and cancer cells
tioxidant activity. Table 2 shows the EC50 values of the herbal by many scientific studies [21–25]. (ese medicines have
mixtures and BHT, which was used as positive control. been reported to have therapeutic synergistic effect or may
Among the mixtures, SMM gave the lowest EC50 value also be antagonistic for the side effects observed [26, 27].
(1.05mg/ml) which indicates the highest antioxidant Assessment of the effects of other commercial herbal
Cell viability (%) Cell viability (%)
4 International Journal of Microbiology
preparations has been done in South Africa, where 6
commonly used preparations were tested for their effects on Table 3: Herbal antimalarial mixtures and their plant components.
isolated human platelets [28]. Similar studies by Mothibe Herbal Mass of dried Plant component
et al. on a herbal body-healing mixture observed variable mixtures extract (mg)∗
stimulatory and inhibitory effects on human neutrophils STF 0.34 Azadirachta indica, Alstonia
[29]. boonei
(e inhibitory effects of a herbal mixture in general have SMH 0.69 Cryptolepis sanguinolenta
been attributed to the total activity of the crude extract, SMM 0.17 Cryptolepis sanguinolenta
rather than that of a single major component of the mixtures SGM 0.07 Cryptolepis sanguinolenta,
[30], although this may not always be the case. Cancer cells Morinda lucida, Nauclea latifolia
are frequently used for cytotoxicity studies because of the STT 0.03 Carapa procera, Cryptolepissanguinolenta
rapid growth rate compared to normal cells, thus facilitating ∗
quick acquisition of in vitro data on test samples. More so, Mass of the extract obtained after freeze drying 30ml of the herbalmixtures.
whatever harms cancer cells will also usually harm normal
healthy cells in the body.
Figures 1 and 2 show the inhibitory effect of the herbal herbal mixtures, as testified by consumers, could partly be
mixtures on the two cell lines. In all instances, the percentage attributed to the relatively high antioxidant activity of these
cell viability decreased in a dose-dependent manner with mixtures.
increasing concentrations of the mixture. It could also be It is worth noting that SMM had the highest percentage
seen that toxic effect of mixtures on cells varied depending antioxidant activity among the five herbal medicines as well
on the cell line used. Although both SMH and SMM were as have the highest cytotoxic effect on the two cancer cells
produced from the same plant component (Cryptolepis that we used. (e high antioxidant activity of SMM, coupled
sanguinolenta) (Table 3), they showed different degrees of with its strong effect on cancer cells, could be explored in the
cytotoxicity on the cells tested. (is difference may possibly treatment of cancer. (e active components of this herbal
be due to different extraction methods employed by the two preparation (Cryptolepis sanguinolenta) could be a subject
different manufacturers.(e activities may also partly be due for further investigation as far as treatment of cancer is
to the fact that these two mixtures have other plant com- concerned.
ponents with cytotoxic effect which have not been stated on In conclusion, although these herbal mixtures are
their respective labels. claimed to be potent antimalarial agents they could be
Four out of the five herbal mixtures considered in this potentially toxic to host cells. (erefore, comprehensive
study were either made from Cryptolepis sanguinolenta or toxicity studies are warranted to safeguard public health.
had the plant as a component of the mixture. Indeed, an- Also, close attention must be paid to the dosage and the
timalarial activity of extracts of this plant has been long patient’s health history since certain diseases could be
established in Africa [31–33]. Cytotoxicity of aqueous ex- secondary factors in the generation of toxicities in the
tract of Cryptolepis sanguinolenta on different cell lines has presence of these herbal mixtures.
also been reported by Ansah and Goodman [34]. Except for
SMM which was cytotoxic even at a lower concentration, Data Availability
cytotoxicity increased with increasing concentrations of the
mixtures. In most rural communities, extract of these plants (e data used to support the findings of this study are
may be the only alternative for the treatment of malaria.(is available from the corresponding author upon request.
raises the concern on howmuch of these mixtures one needs
to consume to achieve the desired curative purpose without
experiencing significant toxic effect. Disclosure
Plant-based medicines generally contain a significant (e research was performed as part of the employment of
amount of phyto-antioxidants which prevent oxidative the authors from the University of Cape Coast.
damage to the hosts such as that which is caused by the
Plasmodium parasite from malaria infection. We also tested
whether these mixtures have antioxidant effects. All the Conflicts of Interest
herbal mixtures had high antioxidant activities compared to (e authors declare that there are no conflicts of interest
the positive control, with SMM recording the strongest regarding the publication of this paper.
activity (EC50 �1.05mg/ml). (e importance of antioxidant
activity is to slow down or prevent the oxidative damage to
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