Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Activity of medicinal plants from Ghana against the parasitic gut

protist Blastocystis
Charlotte Bremer Christensen a,c, Jens Soelberg a,b, Christen R. Stensvold c, Anna K. Jäger a,n
a
Department of Drug Design and Pharmacology, Universitetsparken 2, DK-2100 Copenhagen, Denmark
b
Museum of Natural Medicine, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark
c
Laboratory of Parasitology, Department of Microbiology and Infection Control, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: The plants tested in this study were examples of plants historically used
Received 10 January 2015 to treat or alleviate several types of stomach disorders manifested by e.g. stomachache, diarrhoea or
Received in revised form dysentery. These plants have been consumed typically as a decoction, sometimes mixed with other
1 March 2015
flavourings. The aim of this study was to evaluate the anti-Blastocystis activity of 24 plant parts from 21
Accepted 3 March 2015
medicinal plants from Ghana.
Materials and methods: The medicinal plants were collected in the Greater Accra region of Ghana. Every
Keywords: plant part was tested in three different extracts; an ethanolic, a warm, and a cold water extract, at a final
Anti-protozoal activity concentration of 1 mg/mL for the initial screening, and in a range from 0.0156 to 1 mg/mL for
Blastocystis
determination of inhibitory concentrations. The obligate anaerobic parasitic gut protist Blastocystis
Mallotus oppositifolius
(subtype 4) was used as a 48 h old subcultivated isolate in the final concentration of 106 cells/mL. Plant
Medicinal plant
extracts inoculated with Blastocystis were incubated at 37 1C for 24 h and 48 h. Both MIC minimum
inhibitory concentration (MIC90) assays and minimal lethal concentration (MLC) assays were performed
after 24 h and 48 h. The half maximal inhibitory concentration (IC50) was derived after 24 h and 48 h.
Antimicrobial activity was tested against two Gram-positive and two Gram-negative bacteria for all 24
plant parts at a final concentration of 1 mg/mL.
Results: Screening of the 24 different plant parts showed significant anti-Blastocystis activity of six of the
ethanolic extracts: Mallotus oppositifolius, IC50, 24 h 27.8 mg/mL; Vemonia colorata, IC50, 24 h 117.9 mg/mL;
Zanthoxylum zanthoxyloides, cortex IC50, 24 h 255.6 mg/mL; Clausena anisata, IC50, 24 h 314.0 mg/mL; Z.
zanthoxyloides, radix IC50, 24 h 335.7 mg/mL and Eythrina senegalensis, IC50, 24 h 527.6 mg/mL. The
reference anti-protozoal agent metronidazole (MTZ) had an IC50, 24 h of 7.6 mg/mL. Only C. anisata
showed antimicrobial activity at a concentration of 800 mg/mL.
Conclusion: Six ethanolic plant extracts showed significant anti-parasitic activity against Blastocystis.
M. oppositifolius showed nearly as good activity as the reference anti-protozoal drug MTZ. Historically,
the active plants found in this study have been used against dysentery, diarrhoea or other stomach
disorders. Nowadays they are not used specifically for dysentery, but they are being used as medicinal
plants against various stomach disorders.
& 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
The present study of anti-protozoal activity in medicinal plants
from Ghana is part of a larger research collaboration investigating
the use of historical and contemporary medicinal plants in Ghana
(Soelberg et al., 2015). The plants tested in this study are presently
used or have been used historically to treat or alleviate many types
of stomach disorders manifested by e.g. stomachache, diarrhoea or
dysentery (Petiver, 1697; Bowdich, 1819; Schumacher, 1827;
n
Corresponding author. Tel.: þ 45 35336339; fax: þ45 35336041.
E-mail address: anna.jager@sund.ku.dk (A.K. Jäger).
Soelberg et al., 2015). Traditionally, the plants have been con-
sumed as a decoction, sometimes mixed with other flavourings.
Parasitic infections take a toll on human health and can affect
all people, not only in the tropics, but also in regions with
temperate climates (Centers for Disease Control and Prevention,
2014). Some parasites such as the intestinal protozoon Entamoeba
dispar appear harmless (Centers for Disease Control and
Prevention, 2012), whereas others may cause fatal infections, for
instance Entamoeba histolytica, a cause of dysentery, and one of the
parasites causing malaria, Plasmodium falciparum (Centers for
Disease Control and Prevention, 2010). Fortunately, most parasitic
diseases are treatable with modern medicines, but several cases of
resistance towards these medicines are emerging for a number of

http://dx.doi.org/10.1016/j.jep.2015.03.006
0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: Bremer Christensen, C., et al., Activity of medicinal plants from Ghana against the parasitic gut protist
Blastocystis. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.006i
2 C. Bremer Christensen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

parasitic protists, e.g. E. histolytica, Giardia lamblia and Blastocystis
(Debnath et al., 2014; Upcroft and Upcroft, 2001). There has been a
great focus on resistance towards antibiotics, but it is clear that
focus now also should be on the increasing resistance towards
anti-protozoal medicines (Borst and Ouellette, 1995; Khaw and
Panosian, 1995; Sinha et al., 2014).
The obligate anaerobic parasitic gut protist Blastocystis is an
intestinal unicellular parasite of humans and a vast variety of non-
human hosts. The genus exhibits extensive genetic diversity, and
to date, nine ribosomal lineages (subtypes), arguably species, have
been identified in humans (Alfellani et al., 2013; Clark et al., 2013;
Stensvold et al., 2007). Blastocystis is one of the most widespread
and common intestinal parasites of humans (Alfellani et al., 2012;
Alfellani et al., 2013; Clark et al., 2013). It is estimated that
between 1 and 2 billion people are infected with this parasite
(Scanlan and Stensvold, 2013). Studies have shown a prevalence
ranging from 0.5% in industrialised countries and up to 60% in
developing countries. Prevalence depends on the identification
technique of the parasite and can therefore vary when testing the
same population. Generally, developing countries show higher
prevalence figures because of poor hygiene and the ingestion of
contaminated food and water (Tan, 2008).
The role of Blastocystis in human health and disease remains
controversial (Scanlan and Stensvold, 2013). Many consider the
parasite harmless due to asymptomatic carriage being common,
but there is some evidence to suggest that the parasite might be
related to irritable bowel syndrome and/or cause irritable bowel
syndrome-like symptoms (Alfellani et al., 2012; Coyle et al., 2012;
El Deeb et al., 2012; Engsbro et al., 2014; Roberts et al., 2014;
Stensvold et al., 2009; Yamamoto-Furusho and Torijano-Carrera,
2010). Symptoms associated with Blastocystis infections are:
abdominal pain, diarrhoea, vomiting, nausea, flatulence, bloating
and anorexia (Roberts et al., 2014; Tan, 2008). Potential patho-
genicity may be subtype-related according to several studies (e.g.
Alfellani et al., 2012; Ramírez et al., 2014; Roberts et al., 2014;
Stensvold et al., 2011).
There is no consensus regarding treatment of Blastocystis infection
—or whether the parasite should be treated at all (Coyle et al., 2012;
Engsbro et al., 2014; Roberts et al., 2014). Nonetheless, eradicating
Blastocystis from the human intestine appears to be challenging.
Metronidazole (MTZ) is the most common anti-protozoal drug of
Table 1
Plant species tested for anti-parasitic activity against Blastocystis.
choice, even though the efficacy ranges from 0% to 100% (Stensvold
et al., 2010). Resistance towards MTZ and difficulties in treating
Blastocystis are also becoming a problem (Roberts et al., 2014; Sekar
and Shanthi, 2013). The different subtypes also show different
susceptibility towards antimicrobial drugs (Roberts et al., 2014). The
usual adult dose of MTZ recommended to treat a Blastocystis infection
is 500–750 mg thrice daily for 10 days or 1.5 g daily for 7 days (Sekar
and Shanthi, 2013). Treatment failure may in principle be due to drug
resistance, poor drug efficacy, or reinfection (Stensvold et al., 2010).
Experience with alternative ways of treating an infection with
Blastocystis is limited, but other medicines could be paromomycin,
trimethroprim–sulfamethoxazole or nitazoxanide (Khaw and
Panosian, 1995; Roberts et al., 2014; Sekar and Shanthi, 2013;
Stensvold et al., 2010). The use of so many different compounds also
indicate the challenge of eradicating Blastocystis.
Previous studies have shown inhibitory effect against Blastocystis
of plant extracts of Coptis chinensis and Brucea javanica (Yang et al.,
1996), Thymus vulgaris, Serenoa repens, Vitis vinifera and Curcubita
pepo (Grabensteiner et al., 2008), Allium sativum (Yakoob et al.,
2011), Ferula asafoetida (El Deeb et al., 2012) and Quercus infectoria
and Achillea millefolium (Özbilgin et al., 2013).
The aim of the present study was to find new ways of treating
infections with Blastocystis by using medicinal plants as a platform
for the design of novel anti-protozoal drugs.
2. Materials and methods
2.1. Blastocystis
Blastocystis (subtype 4) was cultured from a faecal sample from
a voluntary staff member at Statens Serum Institut, Copenhagen,
Denmark. The culture, which was xenic (i.e. containing bacteria),
used Jones medium and the isolate was propagated at 37 1C by
performing subculture every two–three days.
2.2. Plant material and extracts
Medicinal plants were collected during the period November
2013–January 2014 in the Greater Accra region of Ghana. The
plants were identified and authenticated by ethnobotanist Jens

Plant species Family Voucher number Plant part

Boerhavia diffusa L. Nyctaginaceae JS 281 Herba

Clausena anisata (Willd.) Hook. f. ex Benth. Rutaceae JS 214R Radix
Deinbollia pinnata Schumach. & Thonn. Sapindaceae JS 202 Herba
Erythrina senegalensis DC. Fabaceae JS 231 Cortex
Flacourtia flavescens Willd. Salicaceae JS 249 Folium
Flueggea virosa (Roxb. ex Willd.) Royle Phyllantaceae JS 252 Folium
Gardenia ternifolia Schumach. & Thonn. Rubiaceae JS 246 Folium
Launaea taraxacifolia (Willd.) Amin ex C.Jeffrey Asteraceae JS 212 Folium
Mallotus oppositifolius (Geisel.) Müll.-Arg. Euphorbiaceae JS 208 Herba
Newbouldia laevis (P.Beauv.) Seem. Bignoniaceae JS 216 Folium
Paullinia africana R.Br. ex Tedlie Sapindaceae JS 219 Herba
Phyllanthus amarus Schumach. & Thonn. Phyllantaceae JS 237 Herba con radix
Premna quadrifolia Schumach. & Thonn. Verbenaceae JS 283 Herba
Pupalia lappacea (L.) Juss. Amaranthaceae JS 239 Herba
Senna occidentalis (L.) Link Fabaceae JS 234H Herba
Senna occidentalis (L.) Link Fabaceae JS 234R Radix
Spathodea campanulata P.Beauv. Bignoniaceae JS 230 Cortex
Stylosanthes erecta P.Beauv. Fabaceae JS 271 Herba
Tapinanthus bangwensis (Engl. & K.Krause) Danser Loranthaceae JS 210 Herba
Thonningia sanguinea Vahl Balanophoraceae JS 296 Herba
Vernonia colorata subsp. colorata (Willd.) Drake Asteraceae JS 268FF Foliumþflos
Vernonia colorata subsp. colorata (Willd.) Drake Asteraceae JS 268R Radix
Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler Rutaceae JS 243C Cortex
Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler Rutaceae JS 243R Radix

Please cite this article as: Bremer Christensen, C., et al., Activity of medicinal plants from Ghana against the parasitic gut protist
Blastocystis. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.006i
 C. Bremer Christensen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Soelberg, University of Copenhagen, Denmark. Botanical voucher
specimens were deposited at the Herbaria at University of Ghana,
Ghana and University of Copenhagen, Denmark. Voucher numbers
are given in Table 1.
Dried plant parts were grinded in a coffee grinder. Ethanolic
and cold water extracts were prepared by extracting the plant
material with 10 mL ethanol or 10 mL cold demineralised water
and then placing them in an ultrasonic bath for 30 min. Next,
extracts were filtrated using filter paper in a funnel into 20 mL
vials. Warm water extracts were generated using 10 mL deminer-
alised water in round-bottomed flasks in a boiling water bath
under reflux for 30 min. Eventually, extracts were filtrated using
filter paper in a funnel into 20 mL weighed vials. Filtered extracts
were evaporated to dryness under reduced pressure at 35 1C by a
Savant SPD121P speed vacuum concentrator (Thermo Scientific,
Waltham, MA). The extracts of the 24 different plant parts
(Table 1) were screened for their anti-Blastocystis activity at a final
concentration of 1 mg/mL.
2.3. Bürker-Türk counting chamber
A Bürker-Türk haemocytometer (Brand, Germany) was used to
measure the cell concentration of Blastocystis microscopically. This
type of counting chamber has shown good precision properties
compared to other counting chambers and was therefore chosen
for this experiment (Christensen et al., 2005).
2.4. Anti-Blastocystis activity assay
Initial experiments showed that Blastocystis did not survive
when using dimethyl sulfoxide (DMSO) as a solvent; however, the
isolate was able to survive in the presence of ethanol in a final
concentration of 0.5%.
The reference anti-protozoal drug metronidazole (MTZ) was
tested against Blastocystis using twelve different concentrations
(0.98–2000.00 mg/mL). All experiments were performed in Jones
Medium and in duplicate.
The anti-Blastocystis assay was performed as follows: A 48 h old
sub-cultured Blastocystis isolate was allowed to incubate for 48 h
at 37 1C. In the screening process of the plants, 5.0 mg of each
plant extract was weighed into a 1.5 mL Eppendorf tube. For
ethanolic extracts, 0.5% ethanol was used as a solvent. A total of
25 mL ethanol was used to dissolve the ethanolic extracts and
475 mL Jones Medium was added to give the final concentration of
5 mg/500 mL. In the warm and cold water extracts 500 mL Jones
Medium was added to dissolve the extracts. 100 mL dissolved plant
extract in Jones Medium were pipetted into 2 mL Eppendorf tubes
for testing. A final concentration of approximately 106 cells/mL of
Blastocystis in Jones Medium was made for all plant extracts tested
and for the growth control. Jones Medium was added to yield a
final volumen of 1000 mL. Plant extracts inoculated with Blastocys-
tis were incubated at 37 1C for 24 h and 48 h. Each plant extract
was tested in duplicate and the anti-Blastocystis activity was
assessed by counting cells in a Bürker-Türk counting chamber
after both 24 h and 48 h.
The minimum inhibitory concentration was defined as the
lowest concentration of the plant extract that significantly inhibited
(490%) the growth of Blastocystis (MIC90). The MIC90 assay was
performed as follows: the active plant extracts found in the screen-
ing process were re-tested against Blastocystis using eight different
concentrations (15.6–900.00 mg/mL) to identify MIC90. The final
concentration of Blastocystis was approximately 106 cells/mL
. Inhibition (after 24 h and 48 h) was calculated as the percentage
of the mean growth cell count of the control isolate which
contained Jones Medium and Blastocystis at a concentration of 106
cells/mL, corresponding to 100% growth of the Blastocystis. The
Please cite this article as: Bremer Christensen, C., et al., Activity of medicinal plants from Ghana against the parasitic gut protist
Blastocystis. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.006i
3
experiment was performed as a two-fold serial dilution and con-
ducted in two stages; the concentrations of 700 and 900 mg/mL
were later added.
The minimal lethal concentration (MLC) was defined as the
lowest concentration of plant extract where no Blastocystis were
detected after 24 h and 48 h of incubation. The MLC assay was
performed as follows: 10 mL of the active test samples from the
MIC90 assay, which showed significant inhibition, were inoculated
into 2 mL Eppendorf tubes containing 1 mL Jones Medium to
assess viability. No plant extract was added. MLC could then be
confirmed microscopically after 24 h and 48 h by checking for any
presence of viable parasites.
2.5. Antimicrobial activity assay
The Blastocystis culture used for testing was xenic, indicating
the existence of Blastocystis in the presence of bacteria, on which
the parasite might depend. In order to identify whether any effect
of the extracts on Blastocystis was direct (anti-protozoal) or
indirect (anti-bacterial), extracts of the 24 different plant parts
were also tested for their anti-bacterial activity against the two
gram-negative bacteria Escherichia coli (ATCC 1229), Pseudomonas
aeruginosa (ATCC 9027) and the two gram-positive bacteria
Bacillus subtilis (ATCC 6633) and Staphylococcus aureus (ATCC
6538). Extracts were tested at a final concentration of 1 mg/mL,
and for MIC determination at two-fold concentrations between
0.025 and 0.8 mg/mL. Mueller-Hinton Bouillon was used as med-
ium and all experiments were done in 96-wells microtiterplates
and evaluated spectrophotometrically for antimicrobial activity.
2.6. Statistical analyses
All data were entered in Microsoft Excel and analysed. Means of
duplicate data were calculated and used as the final result. IC50
values were generated by non-linear, 4 parameter fit using GraFit
version 5, (1989–2001) from Erithacus Software.
3. Results and discussion
Screening of the 24 different plant parts revealed significant
anti-Blastocystis activity of six ethanolic plant extracts; Mallotus
oppositifolius, Clausena anisata, Erythrina senegalensis, Vernonia
colorata, and for Zanthoxylum zanthoxyloides in both cortex and
radix extracts, whereas none of the other extracts showed any
significant activity.
Results of MIC90 and MLC assays of the six plant extracts showing
anti-Blastocystis activity are displayed in Table 2, which also shows
the MIC90 and MLC values of MTZ, the anti-protozoal drug used for
reference. M. oppositifolius and V. colorata showed the highest anti-
Blastocystis activity of the plant extracts. The cortex and radix extract
of Z. zanthoxyloides together with C. anisata exhibited moderate
activity and the extract of E. senegalensis had the lowest activity.
Table 3 shows the IC50 values of the active plant extracts and MTZ.
The inhibitory activity of the plant extracts were clearly dose
dependent. The results showed that M. oppositifolius with an IC50
of 27.8 mg/mL after 24 h showed a good elimination of Blastocystis in
comparison with MTZ with an IC50 of 7.6 mg/mL. The results of
MIC90, MLC assay and IC50 are specified below.
It is difficult to define when a plant exhibits pharmacologically
relevant activity against Blastocystis or parasites in general. There-
fore, the fact that some plant extracts exhibit in vitro activity
merely suggests that there might be pharmacologically relevant
in vivo anti-protozoal activity of the plants.
In order to control for indirect anti-protozoal action through
antimicrobial action (i.e. elimination of bacteria subsequently
4 C. Bremer Christensen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

Table 2
MIC and MLC values of the plant species, which showed activity against Blastocystis.

Plant species MIC90 assay MLC assay

Conc. Blastocystis  104 cells/mL after 24 h Blastocystis  104 cells/mL after 48 h Conc. (lL/ Blastocystis Blastocystis
(lg/mL) (% inhibition) (% inhibition) mL) after 24 h after 48 h

Clausena anisata 1000 2(97) 1(99) 10 þþ þþ

900 1(100) 1(99) 10 þþ þ
700 3(99) 4(98) 10 þþ þ
500 29(86) 155(40) 10 n n
250 114(44) 256(2) 10 n n
125 152(25) 297(0) 10 n n
62.5 172(15) 294(0) 10 n n
31.3 164(19) 283(0) 10 n n
15.6 169(17) 263(0) 10 n n
Erythrina senegalensis 1000 2(98) 1(99) 10 þþ þþ
900 4(98) 1(100) 10 þþ þþ
700 20(90) 19(92) 10 þþ þþ
500 72(61) 47(84) 10 n n
250 130(29) 212(29) 10 n n
125 151(18) 276(8) 10 n n
62.5 161(12) 285(5) 10 n n
31.3 166(9) 292(3) 10 n n
15.6 176(4) 310(0) 10 n n
Mallotus oppositifolius 1000 6(95) 1(99) 10 þþ þþ
900 1(100) 1(99) 10 þþ þþ
700 0(100) 0(100) 10 þþ þþ
500 2(99) 0(100) 10 þþ þþ
250 2(99) 1(100) 10 þ þþ
125 3(98) 7(98) 10 þ þ
62.5 8(96) 40(87) 10 n n
31.3 51(70) 36(88) 10 n n
15.6 119(30) 75(75) 10 n n
Vernonia colorata subsp. 1000 6(93) 1(99) 10 þþ þþ
colorata
900 7(93) 0(100) 10 þþ þþ
700 3(97) 1(99) 10 þþ þþ
500 3(96) 0(100) 10 þþ þþ
250 6(94) 9(94) 10 þþ þþ
125 40(56) 79(46) 10 þ þ
62.5 71(23) 129(11) 10 n n
31.3 74(19) 137(6) 10 n n
15.6 87(6) 150(0) 10 n n
Zanthoxylum 1000 7(92) 9(91) 10 þþ þþ
zanthoxyloides
Cortex 900 1(98) 2(98) 10 þþ þþ
700 3(94) 5(95) 10 þþ þþ
500 9(84) 9(90) 10 þþ þ
250 28(47) 38(56) 10 þ þ
125 47(11) 81(7) 10 n n
62.5 52(0) 93(0) 10 n n
31.3 63(0) 87(0) 10 n n
15.6 55(0) 101(0) 10 n n
Zanthoxylum 1000 5(95) 6(93) 10 þþ þþ
zanthoxyloides
Root 900 10(89) 6(96) 10 þ þþ
700 14(85) 18(88) 10 þ þ
500 25(73) 27(81) 10 þ þ
250 56(39) 110(24) 10 n n
125 83(10) 152(0) 10 n n
62.5 81(12) 168(0) 10 n n
31.3 91(1) 158(0) 10 n n
15.6 81(12) 152(0) 10 n n
Metronidazole 2000 6(91) 2(98) 10 þþ þþ
1000 12(83) 6(94) 10 þþ þþ
500 12(83) 4(96) 10 þþ þþ
250 13(81) 4(96) 10 þþ þþ
125 14(79) 4(96) 10 þ þþ
62.5 9(87) 3(97) 10 þ þþ
31.3 7(91) 4(96) 10 þþ þ
15.6 6(91) 2(98) 10 þþ þ
7.81 33(52) 47(48) 10 n n
3.91 54(21) 132(0) 10 n n
1.95 62(10) 132(0) 10 n n
0.98 59(15) 138(0) 10 n n

( þ þ) Blastocystis eliminated; no viable parasites. ( þ) Blastocystis alive. (*) Not determined due to o90% inhibition in the MIC assay.

Please cite this article as: Bremer Christensen, C., et al., Activity of medicinal plants from Ghana against the parasitic gut protist
Blastocystis. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.006i
 C. Bremer Christensen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
leading to elimination of Blastocystis), plant extracts were tested
for antimicrobial activity using the same concentration (1 mg/mL)
as used in the anti-Blastocystis study. Only one extract, the
ethanolic root extract of C. anisata, showed antimicrobial activity,
at a concentration of 800 mg/mL, and the anti-Blastocystis activity
of this extract therefore might be indirect only.
No studies have been done previously on the plants identified
as active against Blastocystis in this experiment; however, the
results will be compared to data from studies of the respective
plants tested on other protozoan parasites.
The anti-protozoal drug MTZ was tested against Blastocystis
and showed a MIC90 of 15.6 mg/mL after 48 h (Table 2). MLC was
250 mg/mL after 24 h and 62.5 mg/mL after 48 h (Table 2). IC50
values were 7.6 mg/mL after 24 h and 7.8 mg/mL after 48 h. Pre-
viously, IC50 values for metronidazole against Blastocystis have
been determined for various subtypes; 1.9 71.32 μg/ml (subtype
7), 5.5 72.89 μg/ml (subtype 4), 32.5 73.4 μg/ml (subtype 7,
isolate B) and higher than 100 μg/ml (subtype 7, isolate E)
(Mirza et al., 2011). The value obtained in the present study for
subtype 4 is equivalent to the value for subtype 4 obtained by
Mirza et al. (2011). MTZ showed better inhibitory activity against
Blastocystis than any of the tested plant extracts.
Among the plant extracts, M. oppositifolius showed the highest
activity against Blastocystis at a MIC90 of 62.5 mg/mL and 125 mg/mL
after 24 h and 48 h, respectively. MLC was 500 and 250 mg/mL after
24 h and 48 h, respectively (Table 2). M. oppositifolius was the plant
with the lowest IC50 value of 27.8 mg/mL after 24 h (Table 3). An
earlier study of M. oppositifolius showed that aqueous extracts at a
dose of 240 mg/kg had an in vivo antidiarrhoeal effect on rats
infected with Shigella dysenteriae A1 without showing toxicity. This
study concluded that the aqueous extracts had an in vitro bacterial
MIC of 1172 mg/mL and a MLC of 9375 mg/mL (Kamgang et al., 2006).
Another study showed that a methanolic extract of leaves from M.
oppositifolius was highly active against beta-lactam-resistant Gram-
positive cocci (MIC r0.3–2.50 mg/mL). These tests were performed
using the disc diffusion and agar dilution assays (Gangoué-Piéboji et
al., 2009). The antimicrobial activity of the plant could explain why it
was previously used against dysentery in Ghana, which was not
necessarily caused by a parasitic infection. In this study it was tested
if the ethanolic extract of M. oppositifolius showed antimicrobial
activity at the concentration of 1 mg/mL, which was, however, a
lower concentration than the one tested in the study of Kamgang
et al. (2006) mentioned above. There was no indication of antimi-
crobial activity in the extract at this concentration. This could suggest
that the anti-Blastocystis activity might not be due to an antimicrobial
activity. Another study showed that ethanolic extracts of M. opposi-
tifolius showed strong activity against the protozoon Plasmodium
falciparum. The compounds responsible for the antimalarial activity
were the bioactive dimeric phloroglucinols mallotojaponins B and C
(Harinantenaina et al., 2013).
C. anisata had a MIC90 of 700 mg/mL both after 24 h and 48 h
and a MLC of 700 mg/mL after 24 h and 1000 mg/mL after 48 h
(Table 2). IC50 values were 314 mg/mL after 24 h (Table 3). The plant
has traditionally been used for parasitic infections such as malaria,
Table 3
IC50 of the plant species, which showed activity against Blastocystis.
Plant species IC50 (24 h) (lg/mL)
Clausena anisata 314.0 7 18.0
Erythrina senegalensis 519.0 7 122.4
Mallotus oppositifolius 27.8 7 2.2
Vernonia colorata subsp. colorata 117.9 7 9.2
Zanthoxylum zanthoxyloides, cortex 255.6 7 9.8
Zanthoxylum zanthoxyloides, radix 335.77 40.7
Metronidazole 7.6 7 0.5
Please cite this article as: Bremer Christensen, C., et al., Activity of medicinal plants from Ghana against the parasitic gut protist
Blastocystis. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.006i
5
inflammation, dysentery and for alleviating pain among other
things (Moshi et al., 2005; Okokon et al., 2012; Senthilkumar
and Venkatesalu, 2009). A study on the in vivo antiplasmodial
activity of the leaf extract of C. anisata showed significant activity
against Plasmodium berghei in a dose dependent manner in mice
(Okokon et al., 2012). When the twigs and leaves of C. anisata were
tested against P. falciparum it showed an IC50 ranging from 18 to
4100 mg/mL, and it was not classified as a highly active anti-
plasmodial plant (Clarkson et al., 2004). According to a study on
the leaves and twigs from C. anisata it showed a MIC value of
1.563–6.25 mg/mL against the Gram-positive bacteria S. aureus
and Gram-negative P. aeruginosa (Amoo et al., 2012). The essential
oil of the leaves of C. anisata had significant anti-bacterial activity
against Salmonella typhimurium and P. aeruginosa with MIC values
of 62.5 and 125 mg/mL, respectively (Senthilkumar and
Venkatesalu, 2009). The isolation of two carbazole alkaloids,
clausenol and clausenine, isolated from an alcoholic extract from
the stem bark of C. anisata showed antimicrobial activity. Espe-
cially clausenol showed high activity against both Gram-positive
and Gram-negative bacteria (Chakraborty et al., 1995). The present
study also confirmed antimicrobial activity of C. anisata at a
concentration of 800 mg/mL, so this could have an influence on
the anti-Blastocystis activity in the present study.
E. senegalensis showed to be effective against Blastocystis at
700 mg/mL–both in terms of MIC90 and MLC (Table 2). With IC50
values of 519 mg/mL after 24 h, this was the plant with the highest
IC50 value (Table 3). An in vivo study on the aqueous extracts of the
stem bark of E. senegalensis against the parasite P. berghei showed
only slight antiplasmodial activity. At concentrations of 50 and
100 mg/kg parasite intensity was reduced, but not significantly in
comparison to the reference compound chloroquine. Median
lethal dose (LD50) of the extract was estimated to be
450 724 mg/kg i.p. in the mice (Saidu et al., 2000). The MIC value
of the leaf extracts of the plant was found to be 125 mg/mL against
S. aureus (Magassouba et al., 2007). An IC50 as low as 12 mg/mL
from ethanolic extracts of the roots against Gram-positive bacteria
has been demonstrated (Koné et al., 2004). The possible active
anti-bacterial compounds from the plant, 2,3-dihydroauriculatin,
showed moderate activity against oral microbial organisms (Taylor
et al., 1986). The flavonoid Erybraedin A, another compound
isolated from E. senegalensis, is also known as an antimicrobial
agent, active against multiresistant S. aureus (MRSA) (Koné et al.,
2004; Sato et al., 2004; Wanjala et al., 2002). The root of the plant
is highly active against Staphylococcus pneumonia (Koné et al.,
2007). Isoflavonoid 6–8-diprenylgenistein, which was isolated
from the stem bark, showed activity against 36 different bacteria
at concentrations of 25–200 mg/mL (Dastidar et al., 2004). An
in vivo experiment on the oral toxicity of aqueous extracts of the
stem bark of E. senegalensis showed that there is a large margin of
safety when it comes to the therapeutic use of the plant in doses
lower than 7.5 g/kg body weight. LD50 was greater than 12.5 g/kg
and the plant was considered relatively safe of toxicity (Atsamo et
al., 2011).
Z. zanthoxyloides showed activity of both cortex and root
extracts. The cortex extract had a MIC90 of 700 and 500 mg/mL
after 24 h and 48 h, respectively. MLC of the cortex extract was
500 mg/mL after 24 h and 48 h incubation gave the result of
700 mg/mL. The root extract had a MIC90 and a MLC of 1000 mg/mL
after 24 h and a MIC90 and a MLC of 900 mg/mL after 48 h
(Table 2). IC50 values were 255.8 mg/mL for the cortex extract and
335.7 mg/mL for the root extract after 24 h (Table 3). In a study of
methanolic extracts, some isolated compounds and the essential
oil of the fruits of Z. zanthoxyloides were found to have anti-
bacterial activity. The essential oil exhibited high activity towards
Gram-negative S. typhimurium (MIC 0.26 mL/mL) and Gram-
positive B. subtilis (MIC 0.52 mL/mL). The study suggests that the
6 C. Bremer Christensen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
activity of the essential oil could be caused by the high content of
oxygenated monoterpenes and may be also the alcohols geraniol
and citronellol. The methanolic extracts were antibacterially active
at concentrations above 500 mg/mL (Misra et al., 2013). Dichlor-
omethane extracts of root bark of Z. zanthoxyloides showed
significant antiparasitic activity on the intracellular form of the
intracellular protozoon Leishmania major. Less than 10% of L. major
survived the exposure to a concentration of 17.5 mg/mL of the
dichloromethane extract. Also the aqueous extract of the plant
showed moderate activity (under 20% parasite survival). However,
the aqueous extract showed toxicity against macrophages in the
toxicity assay (Ahua et al., 2007). Crude extracts from bark of the
trunk of Z. zanthoxyloides were tested in vitro against the multi-
resistant strain P. falciparum (W2). The greatest activity was found
in the alkaloid extract with an IC50 of 1.2 mg/mL. The selectivity
index (ratio between cytotoxic and antiparasitic activity) showed
good selective antiplasmodial activity (Gansané et al., 2010). A
study concluded that fagaronine could be the active antimalarial
compound in root extracts with an IC50 as low as 0.018 mg/mL
(Kassim et al., 2005). Methanolic extracts from the stem bark of Z.
zanthoxyloides showed in vivo trypanostatic effect, but could not
completely kill the parasite Trypanosoma brucei brucei. The same
study performed a phytochemical analysis on crude extract of the
plant and found that it consisted of tannins, glycosides, saponins,
terpenes, resins, balsam and flavonoids. Methanolic extracts
showed toxicity in doses above 100 mg/kg (Mann et al., 2011).
V. colorata showed the second-highest anti-parasitic activity
against Blastocystis with an identical MIC90 and MLC of 250 mg/mL
both after 24 h and 48 h (Table 2). The IC50 value of 117.9 mg/mL
showed inhibiting activity (Table 3). Another study showed that
the chloromethylenic root extract of V. colorata had antiplasmodial
activity with an IC50 of 3 mg/mL when tested in vitro against P.
falciparum (W2). The plant also had a low cytotoxicity according to
the selectivity index (Kaou et al., 2008). Another study found that
two sesquiterpenes (vernodalol and 11β,13-dihydrovernodalin)
were responsible for the strong in vitro antiplasmodial activity
(Kraft et al., 2003). Vernodalin have cytotoxic effects on cells
derived from human carcinoma of the nasopharynx. In that study
the lipophilic extracts of the leaves of V. colorata gave an in vitro
IC50 of 12.1 and 17.8 mg/mL against P. falciparum chloroquin-
sensitive and chloroquin-resistant clone Dd2, respectively (Kraft
et al., 2003; Kupchan et al., 1969; Rabe et al., 2002). Aqueous
extracts of V. colorata were also tested against the resistant strain P.
falciparum (W2), but showed lower activity with an IC50 of 78.8 mg/
mL and substantial toxicity (Ilboudo et al., 2013). An in vitro
experiment on the antimicrobial activity and stability of ethanolic
extracts of V. colorata was performed, and the MIC against B. subtilis,
S. aureau, E. coli and K. pneumonia were 0.78, 1.56, 1.56 and 0.78 mg/
mL, respectively (Stafford et al., 2005). MIC results from isolated
compounds from the plant showed MIC values of 0.1 mg/mL
of the compound vernolide and vernodalin against B. subtilis.
However, the control antibiotic drug neomycin showed markedly
better results (MIC value of 1.8  10  4) (Rabe et al., 2002). Another
study showed that V. colorata was active against P. aeruginosa
(Kelmanson et al., 2000).
It is very difficult to compare the results in the present study
with previous studies on plant extracts, as the results depend on
the Blastocystis subtype. In the present study, the most active
species, M. oppositifolium, had an IC50 value four times higher than
MTZ. Coptis chinensis and Brucea javanica showed high inhibition
at a concentration 10 and 50 times higher than MTZ, respectively
(Yang et al., 1996). For a menthanolic extract of Achillea millefolium,
the EC50 was 1000 times higher than for MTZ (Özbilgin et al.,
2013). Other studies have tested at high assay concentrations.
MLCs between 2.5–5 mg/mL was obtained for Thymus vulgaris,
Serenoa repens, Vitis vinifera and Curcubita pepo (Grabensteiner et
Please cite this article as: Bremer Christensen, C., et al., Activity of medicinal plants from Ghana against the parasitic gut protist
Blastocystis. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.006i
al., 2008), the MLC for metronidazole used in the study was not
given. In the study by El Deeb et al. (2012) an extract of Ferula
asafoetida caused complete inhibition of Blastocystis growth at 16
or 40 mg/mL, depending on Blastocystis subtype.
4. Conclusion
Most of the plants, which have been found to have anti-
Blastocystis activity in this study, have historically been used
against dysentery, diarrhoea or other stomach disorders. Some of
these plants are still used against various stomach disorders. All
the active plants are still considered medicinal in Ghana today, but
none are apparently used specifically for dysentery.
Six ethanolic extracts of medicinal plants collected in Ghana
showed significant activity against the anaerobic intestinal para-
site Blastocystis. The MIC90 and MLC assays after 48 h of the six
active plant extracts showed that M. oppositifolius showed as good
an eradication of Blastocystis as MTZ. The highest anti-Blastocystis
activity was seen in M. oppositifolius and V. colorata. The cortex and
radix extract of Z. zanthoxyloides together with the C. anisata
extract showed moderate activity and the E. senegalensis extract
showed the lowest activity.
Of the six plants only C. anisata showed antimicrobial activity at
a concentration of 800 mg/mL and this indicates that for the other
five plant extracts it is not an antimicrobial activity that indirectly
inhibits the Blastocystis by killing the bacteria Blastocystis may be
dependent on.
Although this study concludes that five of the medicinal plants
show anti-Blastocystis activity further in vivo studies needs to be
done in order to investigate the plants' mechanism of action,
toxicity and the identity of the anti-parasitic compounds.
Acknowledgements
The project was funded by the Cand. pharm. Povl M. Assens
Foundation, the Carlsberg Foundation (2012-01-0118) and Hjælpe-
fonden for undergraduates, graduate students and graduates from
School of Pharmaceutical Sciences at Faculty of Health and Medical
Sciences. Thanks are given to Dr. Alex Asase and Mr. J.Y. Amponsah
at Department of Botany, University of Ghana for their assistance.
The authors wish to thank the staff of Laboratory of Parasitol-
ogy, Department of Microbiology and Infection Control at Statens
Serum Institut, Denmark for their help and guidance.
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