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

Contents lists available at ScienceDirect

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 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 b

art ic l e i nf o

a b s t r a c t

Article history: Received 10 January 2015 Received in revised form 1 March 2015 Accepted 3 March 2015

Ethnopharmacological relevance: The plants tested in this study were examples of plants historically used to treat or alleviate several types of stomach disorders manifested by e.g. stomachache, diarrhoea or dysentery. These plants have been consumed typically as a decoction, sometimes mixed with other flavourings. The aim of this study was to evaluate the anti-Blastocystis activity of 24 plant parts from 21 medicinal plants from Ghana. Materials and methods: The medicinal plants were collected in the Greater Accra region of Ghana. Every plant part was tested in three different extracts; an ethanolic, a warm, and a cold water extract, at a final concentration of 1 mg/mL for the initial screening, and in a range from 0.0156 to 1 mg/mL for determination of inhibitory concentrations. The obligate anaerobic parasitic gut protist Blastocystis (subtype 4) was used as a 48 h old subcultivated isolate in the final concentration of 106 cells/mL. 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.

Keywords: Anti-protozoal activity Blastocystis Mallotus oppositifolius Medicinal plant

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: [email protected] (A.K. Jäger).

Soelberg et al., 2015). Traditionally, the plants have been consumed 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

C. Bremer Christensen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

2

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 nonhuman 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 pathogenicity 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

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

Table 1 Plant species tested for anti-parasitic activity against Blastocystis. Plant species

Family

Voucher number

Plant part

Boerhavia diffusa L. Clausena anisata (Willd.) Hook. f. ex Benth. Deinbollia pinnata Schumach. & Thonn. Erythrina senegalensis DC. Flacourtia flavescens Willd. Flueggea virosa (Roxb. ex Willd.) Royle Gardenia ternifolia Schumach. & Thonn. Launaea taraxacifolia (Willd.) Amin ex C.Jeffrey Mallotus oppositifolius (Geisel.) Müll.-Arg. Newbouldia laevis (P.Beauv.) Seem. Paullinia africana R.Br. ex Tedlie Phyllanthus amarus Schumach. & Thonn. Premna quadrifolia Schumach. & Thonn. Pupalia lappacea (L.) Juss. Senna occidentalis (L.) Link Senna occidentalis (L.) Link Spathodea campanulata P.Beauv. Stylosanthes erecta P.Beauv. Tapinanthus bangwensis (Engl. & K.Krause) Danser Thonningia sanguinea Vahl Vernonia colorata subsp. colorata (Willd.) Drake Vernonia colorata subsp. colorata (Willd.) Drake Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler

Nyctaginaceae Rutaceae Sapindaceae Fabaceae Salicaceae Phyllantaceae Rubiaceae Asteraceae Euphorbiaceae Bignoniaceae Sapindaceae Phyllantaceae Verbenaceae Amaranthaceae Fabaceae Fabaceae Bignoniaceae Fabaceae Loranthaceae Balanophoraceae Asteraceae Asteraceae Rutaceae Rutaceae

JS JS JS JS JS JS JS JS JS JS JS JS JS JS JS JS JS JS JS JS JS JS JS JS

Herba Radix Herba Cortex Folium Folium Folium Folium Herba Folium Herba Herba con radix Herba Herba Herba Radix Cortex Herba Herba Herba Foliumþflos Radix Cortex Radix

281 214R 202 231 249 252 246 212 208 216 219 237 283 239 234H 234R 230 271 210 296 268FF 268R 243C 243R

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 demineralised 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 Blastocystis 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 screening 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

3

experiment was performed as a two-fold serial dilution and conducted 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 medium 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 antiBlastocystis 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. Therefore, 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

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 ∎ (∎∎∎∎) ∎∎∎–∎∎∎

4

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

MIC90 assay Conc. Blastocystis  104 cells/mL after 24 h (lg/mL) (% inhibition)

Clausena anisata

1000 900 700 500 250 125 62.5 31.3 15.6 Erythrina senegalensis 1000 900 700 500 250 125 62.5 31.3 15.6 Mallotus oppositifolius 1000 900 700 500 250 125 62.5 31.3 15.6 Vernonia colorata subsp. 1000 colorata 900 700 500 250 125 62.5 31.3 15.6 Zanthoxylum 1000 zanthoxyloides Cortex 900 700 500 250 125 62.5 31.3 15.6 Zanthoxylum 1000 zanthoxyloides Root 900 700 500 250 125 62.5 31.3 15.6 Metronidazole 2000 1000 500 250 125 62.5 31.3 15.6 7.81 3.91 1.95 0.98

MLC assay Blastocystis  104 cells/mL after 48 h (% inhibition)

Conc. (lL/ mL)

Blastocystis after 24 h

Blastocystis after 48 h

2(97) 1(100) 3(99) 29(86) 114(44) 152(25) 172(15) 164(19) 169(17) 2(98) 4(98) 20(90) 72(61) 130(29) 151(18) 161(12) 166(9) 176(4) 6(95) 1(100) 0(100) 2(99) 2(99) 3(98) 8(96) 51(70) 119(30) 6(93)

1(99) 1(99) 4(98) 155(40) 256(2) 297(0) 294(0) 283(0) 263(0) 1(99) 1(100) 19(92) 47(84) 212(29) 276(8) 285(5) 292(3) 310(0) 1(99) 1(99) 0(100) 0(100) 1(100) 7(98) 40(87) 36(88) 75(75) 1(99)

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

þþ þþ þþ n n n n n n þþ þþ þþ n n n n n n þþ þþ þþ þþ þ þ n n n þþ

þþ þ þ n n n n n n þþ þþ þþ n n n n n n þþ þþ þþ þþ þþ þ n n n þþ

7(93) 3(97) 3(96) 6(94) 40(56) 71(23) 74(19) 87(6) 7(92)

0(100) 1(99) 0(100) 9(94) 79(46) 129(11) 137(6) 150(0) 9(91)

10 10 10 10 10 10 10 10 10

þþ þþ þþ þþ þ n n n þþ

þþ þþ þþ þþ þ n n n þþ

1(98) 3(94) 9(84) 28(47) 47(11) 52(0) 63(0) 55(0) 5(95)

2(98) 5(95) 9(90) 38(56) 81(7) 93(0) 87(0) 101(0) 6(93)

10 10 10 10 10 10 10 10 10

þþ þþ þþ þ n n n n þþ

þþ þþ þ þ n n n n þþ

10(89) 14(85) 25(73) 56(39) 83(10) 81(12) 91(1) 81(12) 6(91) 12(83) 12(83) 13(81) 14(79) 9(87) 7(91) 6(91) 33(52) 54(21) 62(10) 59(15)

6(96) 18(88) 27(81) 110(24) 152(0) 168(0) 158(0) 152(0) 2(98) 6(94) 4(96) 4(96) 4(96) 3(97) 4(96) 2(98) 47(48) 132(0) 132(0) 138(0)

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

þ þ þ n n n n n þþ þþ þþ þþ þ þ þþ þþ n n n n

þþ þ þ n n n n n þþ þþ þþ þþ þþ þþ þ þ n n 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. Previously, 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 Grampositive 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 antimicrobial 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. oppositifolius 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 Erythrina senegalensis Mallotus oppositifolius Vernonia colorata subsp. colorata Zanthoxylum zanthoxyloides, cortex Zanthoxylum zanthoxyloides, radix Metronidazole

314.0 7 18.0 519.0 7 122.4 27.8 7 2.2 117.9 7 9.2 255.6 7 9.8 335.77 40.7 7.6 7 0.5

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 antiplasmodial 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. Especially 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 antibacterial activity. The essential oil exhibited high activity towards Gram-negative S. typhimurium (MIC 0.26 mL/mL) and Grampositive B. subtilis (MIC 0.52 mL/mL). The study suggests that 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

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). Dichloromethane 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 multiresistant 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 chloroquinsensitive 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

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 antiBlastocystis 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 parasite 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 Hjælpefonden 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 Parasitology, Department of Microbiology and Infection Control at Statens Serum Institut, Denmark for their help and guidance.

References Ahua, K.M., Ioset, J.R., Ioset, K.N., Diallo, D., Mauël, J., Hostettmann, K., 2007. Antileishmanial activities associated with plants used in the Malian traditional medicine. J. Ethnopharmacol. 110, 99–104. Alfellani, M.A., Stensvold, C.R., Vidal-Lapiedra, A., Onuoha, E.S.U., Fagbenro-Beyioku, A.F., Clark, C.G., 2012. Variable geographis distribution of Blastocystis subtypes and its potential implications. Acta Trop. 126, 11–18. Alfellani, M.A., Taner-Mulla, D., Jacob, A.S., Imeede, C.A., Yoshikawa, H., Stensvold, C. R., et al., 2013. Genetic diversity of Blastocystis in livestock and zoo animals. Protist 164, 497–509. Amoo, S.O., Aremu, A.O., Moyo, M., Staden, J.V., 2012. Assessment of long-term storage on antimicrobial and cyclooxygenase-inhibitory properties of South African medicinal plants. Phytother. Res. 27, 1029–1035. Atsamo, A.D., Nguelefack, T.B., Datté, J.Y., Kamanyi, A., 2011. Acute and subchronic oral toxicity assessment of the aqueous extract from the stem bark of Erythrina senegalensis DC (Fabaceae) in rodents. J. Ethnopharmacol. 134, 697–702. Borst, P., Ouellette, M., 1995. New mechanisms of drug resistance in parasitic protozoa. Annu. Rev. Microbiol. 49, 427–460. Bowdich, T.E., 1819. Mission from Cape Coast Castle to Ashantee. Frank Cass & Co. Ltd., London. Centers for Disease Control and Prevention. 2010. Malaria. 〈http://www.cdc.gov/ malaria/about/biology/parasites.html〉. (accessed 09.07.14.).

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 ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Centers for Disease Control and Prevention. 2012. Parasites—nonpathogenic (harmless) intestinal protozoa. 〈http://www.cdc.gov/parasites/nonpathprotozoa/〉. (accessed 09.07.14.). Centers for Disease Control and Prevention. 2014. Parasites.〈http://www.cdc.gov/ parasites/about.html#protozoa〉. (accessed 09.07.14.). Chakraborty, A., Chowdhury, B.K., Bhattacharyya, P., 1995. Clausenol and clausenine —two carbazole alkaloids from Clausena anisata. Phytochemistry 40, 295–298. Christensen, P., Stryhn, H., Hansen, C., 2005. Discrepancies in the determination of sperm concentration using Bürker-Türk, Thomas and Makler counting chambers. Theriogenology 63, 992–1003. Clark, C.G., van der Giezen, M., Alfellani, M.A., Stensvold, C.R., 2013. Recent developments in Blastocystis research. Adv. Parasitol. 82, 1–32. Clarkson, C., Maharaj, V.J., Crouch, N.R., Grace, O.M., Pillay, P., Matsabisa, M.G., et al., 2004. In vitro antiplasmodial activity of medicinal plants native to or naturalised in South Africa. J. Ethnopharmacol. 92, 177–191. Coyle, C.M., Varughese, J., Weiss, L.M., Tanowitz, H.B., 2012. Blastocystis: to treat or not to treat. Clin. Pract. 54, 105–110. Dastidar, S.G., Manna, A., Kumar, K.A., Mazumdar, K., Dutta, N.K., Chakra-barty, A.N., 2004. Studies on the antibacterial potential of isoflavones. Int. J. Antimicrob. Agents 23, 99–102. Debnath, A., Shahinas, D., Bryant, C., Hirata, K., Miyamoto, Y., Hwang, G., 2014. Hsp90 inhibitors as new leads to target parasitic diarrheal diseases. Antimicrob. Agents Chemother. 58 (7), 4138–4144. El Deeb, H.K., Khadrawy, F.M.A., El-Hameid, A.K.A., 2012. Inhibitory effect of Ferula asafoetida L. (Umbelliferae) on Blastocystis sp. subtype 3 growth in vitro. Parasitol. Res. 111, 1213–1221. Engsbro, A.L., Stensvold, C.R., Nielsen, H.V., Bytzer, P., 2014. Prevalence, incidence, and risk factors of intestinal parasites in Danish primary care patients with irritable bowel syndrome. Scand. J. Infect. Dis. 46, 204–209. Gangoué-Piéboji, J., Eze, N., Djintchui, A.N., Ngameni, B., Tsabang, N., Pegnyemb, D. E., et al., 2009. The in-vitro antimicrobial activity of some traditionally used medicinal plants against beta-lactam-resistant bacteria. J. Infect. Dev. Ctries. 3, 671–680. Gansané, A., Sanon, S., Ouattara, L.P., Traoré, A., Hutter, S., Ollivier, E., et al., 2010. Antiplasmodial activity and toxicity of crude extracts from alternative parts of plants widely used for the treatment of malaria in Burkina Faso: contribution for their preservation. Parasitol. Res. 106, 335–340. Grabensteiner, E., Liebhart, D., Arshad, N., Hess, M., 2008. Antiprotozoal activities determined in vitro and in vivo of certain plant extracts against Histomonas meleagridis, Tetratrichomonas gallinarum and Blastocystis sp. Parasitol. Res. 103, 1257–1264. Harinantenaina, L., Bowman, J.D., Brodie, P.J., Slebodnick, C., Callmander, M.W., Rakotobe, E., et al., 2013. Antiproliferative and antiplasmodial dimeric phloroglucinols from Mallotus oppositifolius from the Madagascar dry forest. J. Nat. Prod. 76, 388–393. Ilboudo, D.P., Basilico, N., Parapini, S., Corbett, Y., D’Alessandro, S., Dell’Agli, M., et al., 2013. Antiplasmodial and anti-inflammatory activities of Canthium henriquesianum (K. Schum), a plant used in traditional medicine in Burkina Faso. J. Ethnopharmacol. 148, 763–769. Kamgang, R., Kamgne, E.V.P., Fonkoua, M.C., Beng, V.P.N., Sida, M.B., 2006. Activities of aqueous extracts of Mallotus Oppositifolium on Shigella Dysenteriae A1induced diarrhoea in rats. Clin. Exp. Pharmacol. Physiol. 33, 89–94. Kaou, A.M., Mahiou-Leddet, V., Hutter, S., Aïnouddine, S., Hassani, S., Yahaya, I., et al., 2008. Antimalarial activity of crude extracts from nine African medicinal plants. J. Ethnopharmacol. 116, 74–83. Kassim, O.O., Loyevsky, M., Elliott, B., Geall, A., Amonoo, H., Gordeuk, V.R., 2005. Effects of root extracts of Fagara zanthoxyloides on the in vitro growth and stage distribution of Plasmodium falciparum. Antimicrob. Agents Chemother. 49 (1), 264–268. Kelmanson, J.E., Jäger, A.K., Staden, J.V., 2000. Zula medicinal plants with antibacterial activity. J. Ethnopharmacol. 69, 241–246. Khaw, M., Panosian, C.B., 1995. Human antiprotozoal therapy: past, present, and future. Clin. Microbiol. Rev. 8, 427–439. Koné, W.M., Atindehou, K.K., Terreaux, C., Hostettmann, K., Traoré, D., Dosso, M., 2004. Traditional medicine in North Côte–d’Ivoire: screening of 50 medicinal plants for antimicrobial activity. J. Ethnopharmacol. 93, 43–49. Koné, W.M., Atindehou, K.K., Kacou-N’Douba, A., Dosso, M., 2007. Evaluation of 17 medicinal plants from northern Côte d’Ivoire for their in vitro activity against Streptococcus pneumonia. Afr. J. Tradit. CAM 4, 17–22. Kraft, C., Jenett-Siems, K., Siems, K., Jakupovic, J., Mavi, S., Bienzle, U., et al., 2003. In vitro antiplasmodial evaluation of medicinal plants from Zimbabwe. Phytother. Res. 17, 123–128. Kupchan, S.M., Hemingway, R.J., Karim, A., Werner, D., 1969. Tumor Inhibitors. XLVII. Vernodalin and vernomygdin, two new cytotoxic sesquiterpene lactones from Vernonia amygdalina Del. J. Org. Chem. 34, 3908–3911. Magassouba, F.B., Diallo, A., Kouyaté, M., Mara, F., Mara, O., Bangoura, O., et al., 2007. Ethnobotanical survey and antibacterial activity of some plants used in Guinean traditional medicine. J. Ethnopharmacol. 114, 44–53. Mann, A., Ifarajimi, O.R., Adewoye, A.T., Ukam, C., Udeme, E.E., Okorie, I.I., et al., 2011. In vivo antitrypanosomal effects of some ethnobotanical plants from Nupeland of North Central Nigeria. J. Tradit. Complement. Altern. Med. 8, 15–21.

7

Mirza, H., Teo, J.D.W, Upcroft, J., Tan, K.S.W., 2011. A rapid, high-throughput viability assay for Blastocystis spp. reveals metronidazole resistance and extensive subtype-dependent variations in drug susceptibilities. Antimicrob. Agents Chemother. 55, 637–648. Misra, L.N., Wouatsa, N.A.V., Kumar, S., Kumar, R.V., 2013. Antibacterial, cytotoxic activities and chemical composition of fruits of two Cameroonian Zanthoxylum species. J. Ethnopharmacol. 148, 74–80. Moshi, M.J., Kagashe, G.A.B., Mbwambo, Z.H., 2005. Plants used to treat epilepsy by Tanzanian traditional healers. J. Ethnopharmacol. 97, 327–336. Okokon, J.E., Etebong, E.O., Udobang, J.A., Essien, G.E., 2012. Antiplasmodial and analgesic activities of Clausena anisata. Asian Pasific J. Trop. Med., 214–219. Özbilgin, A., Durmuskahya, C., Kilicioglu, A.A., Kayalar, H., Kurt, O., Ermis, V.O., Tabak, T., Ostan, I., 2013. In vitro efficacy of Quercus infectoria Oliv. and Acillea millefolium L. extracts against Blastocystis spp. isolates. Kafkas Univ. Vet. Fak. Derg. 19, 511–516. Petiver, J., 1697 (1753). A catalogue of some guinea-plants, with their native names and virtues. J. Philos. Trans. 19, 677–686. Rabe, T., Mullholland, D., Staden, J.V., 2002. Isolation and identification of antibacterial compounds from Vernonia colorata leaves. J. Ethnopharmacol. 80, 91–94. Ramírez, J.D., Sánchez, L.V., Bautista, D.C., Corredor, A.F., Flórez, A.C., Stensvold, C.R., 2014. Blastocystis subtypes detected in humans and animals from Colombia. Infect. Genet. Evol. 22, 223–228. Roberts, T., Stark, D., Harkness, J., Ellis, J., 2014. Update on the pathogenic potential and treatment options for Blastocystis sp. Gut Pathog. 6, 17–25. Saidu, K., Onah, J., Orisadipe, A., Olusola, A., Wambebe, C., Gamaniel, K., 2000. Antiplasmodial, analgesic, and anti-inflammatory activities of the aqueous extract of the stem bark of Erythrina senegalensis. J. Ethnopharmacol. 71, 275–280. Sato, M., Tanaka, H., Oh-Uchi, T., Fukai, T., Etoh, H., Yamaguchi, R., 2004. Antibacterial activity of phytochemicals isolated from Erythrina zeyheri against vancomycin-resistant enterococci and their combinations with vancomycin. Phytother. Res. 18, 906–910. Scanlan, P.D., Stensvold, C.R., 2013. Blastocystis: getting to grips with our guileful guest. Trends Parasitol. 29, 523–529. Schumacher, C.F., 1827. Beskrivelse af guineiske planter, som ere fundne af danske botanikere, især af etatsraad thonning. hartv. Frid. Popps Bogtrykkerie, Copenhagen. Sekar, U., Shanthi, M., 2013. Blastocystis: consensus of treatment and controversies. Trop. Parasitol. 3, 35–39. Senthilkumar, A., Venkatesalu, V., 2009. Phytochemical analysis and antibacterial activity of the essential oil of Clausena anisata (Willd.) Hook. f. Ex Benth. Int. J. Integr. Biol. 5, 116–120. Sinha, M., Dola, V.R., Agarwal, P., Srivastava, K., Haq, W., Puri, S.K., et al., 2014. Antiplasmodial activity of new 4-aminoquinoline derivatives against chloroquine resistant strain. Bioorganic Med. Chem. 22, 3573–3586. Soelberg, J., Asase, A., Akwetey, G., Jäger, A.K., 2015. Historical versus contemporary medicinal plant uses in Ghana. J. Ethnopharmacol. 160, 109–132. Stafford, G.I., Jäger, A.K., Staden, J.V., 2005. Effect of storage on the chemical composition and biological activity of several popular South African medicinal plants. J. Ethnopharmacol. 97, 107–115. Stensvold, C.R., Suresh, G.K., Tan, K.S.W., Thompson, R.C.A., Traub, R.J., Viscogliosi, E., et al., 2007. Terminology for Blastocystis subtype—a consensus. Trends Parasitol. 23 (3), 93–96. Stensvold, C.R., Lewis, H.C., Hammerum, A.M., Porsbo, L.J., Nielsen, S.S., Olsen, K.E.P., et al., 2009. Blastocystis: unravelling potential risk factors and clinical significance of a common but neglected parasite. Epidemiol. Infect. 137, 1655–1663. Stensvold, C.R., Smith, H.V., Nagel, R., Olsen, K.E.P., Traub, R.J., 2010. Eradication of blastocystis carriage with antimicrobials: reality of delusion? J. Clin. Gastroenterol. 44, 85–90. Stensvold, C.R., Christiansen, D.B., Olsen, K.E.P., Nielsen, H.V., 2011. Blastocystis sp. subtype 4 is common in Danish Blastocystis-positive patients presenting with acute diarrhea. Am. J. Trop. Med. Hyg. 84, 883–885. Tan, K.S.W., 2008. New insights on classification, identification, and clinical relevance of Blastocystis spp. Clin. Microbiol. Rev. 21, 639–665. Taylor, R.B., Corley, D.G., Tempesta, M.S., Fomum, Z.T., Ayafor, J.F., Wandji, J., et al., 1986. Erythrina studies. Part 7. 2,3-Dihydroauriculatin, a new prenylated isoflavanone from Erythrina senegalensis. Application of the selective INEPT technique. J. Nat. Prod. 49, 670–673. Upcroft, P., Upcroft, J.A., 2001. Drug targets and mechanisms of resistance in the anaerobic protozoa. Clin. Microbiol. Rev. 14, 150–164. Yakoob, J., Abbas, Z., Beg, M.A., Naz, S., Awan, S., Hamid, S., Jafri, W., 2011. In vitro sensitivity of Blastocystis hominis to garlic, ginger, white cumin, and black pepper used in diet. Parasitol. Res. 109, 379–385. Wanjala, C.C.W., Juma, B.F., Bojase, G., Gashe, B.A., Majinda, R.R.T., 2002. Erythrinaline alkaloids and antimicrobial flavonoids from Erythrina latissima. Planta Med. 68, 640–642. Yamamoto-Furusho, J.K., Torijano-Carrera, E., 2010. Intestinal protozoa infections among patients with ulcerative colitis: prevalence and impact on clinical disease course. Digestion 82, 18–23. Yang, L.Q., Singh, M., Yap, E.H., Ng, G.C., Xu, H.X., Sim, K.Y., 1996. In vitro response of Blastocystis hominis against traditional Chinese medicine. J. Ethnopharmacol. 55, 35–42.

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