in vitro activities of plant extracts on human loa loa isolates and cytotoxicity for eukaryotic...

ORIGINAL PAPER

In vitro activities of plant extracts on human Loa loa isolatesand cytotoxicity for eukaryotic cells

Line-Edwige Mengome & Jean Paul Akue &

Alain Souza & Guy Raymond Feuya Tchoua &

Edouard Nsi Emvo

Received: 20 March 2010 /Accepted: 30 April 2010 /Published online: 22 May 2010# Springer-Verlag 2010

Abstract Loa loa, a filarial worm, can cause fatal encepha-litis in humans. In an attempt to find alternatives to the standardtreatments (ivermectin and diethylcarbamazine citrate), wetested 12methanolic extracts of nine traditional plant remedies.The extracts (100–0.09 µg/ml) were incubated with 20 Loa loamicrofilariae isolated from patients at 37°C with 5% CO2 inmodified Eagle’s medium supplemented with 10% fetalserum and antibiotics. Activity was evaluated 120 h later bycounting live microfilariae under a microscope. Cytotoxicityfor eukaryotic cells was estimated by measuring 3-[4,5-dimethylthiazol-2-yl]-2-5 diphenyl tetrazolium bromide trans-formation to formazan at 450 nM in a spectrophotometer. Theplants tested were Lophira alata, Greenwayodendron sua-veolens, Uapaca togoensis, Zanthoxylum heitzii, Peperomiapellucida, Piptadeniastrum africanum, Petersianthus macro-carpus, Vernonia conferta, and Vernonia hymenolepis.Chemical screening showed that most of the extractscontained reducing sugars, tannin or polyphenols, sterols ortriterpenes, saponosides, and alkaloids. None containedcarotinoids and few contained flavonoids. The 50% lethal

concentration ranged from 0.22 to 70.28 μg/ml, while the50% inhibitory concentration for eukaryotic cells (IC50)ranged from 8.52 to 119.52 μg/ml. Extracts of P. macro-carpus (selectivity index=72.16), P. africanum (13.69), Z.heitzii (12.11), and L. alata (9.26) were highly selective for L.loa.

Introduction

Loa loa is a filarial parasite endemic in the West Africanforest block, where 200 million persons are at risk and morethan 13 million are infected. L. loa filariasis is the thirdcause of medical consultations in this region, after malariaand lung disease (Boulestiex and Carme 1986). L. loa is themain obstacle to the success of the WHO Global Programto Eliminate Lymphatic Filariasis based on mass chemo-therapy (WHO 1997), which has been successful in WestAfrica (Boatin et al. 1998). The drugs used for this programare diethylcarbamazine citrate (DEC) and ivermectin. Al-though both are very active on microfilariae, they can havesevere adverse effects in individuals with more than 8,000 L.loa microfilariae/ml of blood (Chippaux et al. 1996;Boussinesq et al. 1997). In contrast, adverse effects are lesscommon in areas endemic for Wuchereria bancrotfi, Brugiamalayi, or single Onchocerca volvulus infection (Guderian etal. 1997). In addition, long-term use and repeated dosingwith these drugs may result in parasite resistance, as alreadyobserved in veterinary settings (Kaplan 2004) and someindication in the field (Awadzi et al. 2004; Bourguinat et al.2007). Genetic basis for such phenomenon has beendemonstrated (Feng et al. 2002; Prichard 2007). There isthus a need for new effective and well-tolerated drugs.

One possible source of such drugs is traditional plantremedies. Here we examined the anti-L. loa activities and

L.-E. Mengome :A. SouzaInstitut de Pharmacopée et de MédecineTraditionnelle (IPHAMETRA),Libreville, Gabon

G. R. Feuya Tchoua : E. Nsi EmvoUnité de Recherches de Chimie,Université des Sciences et Technique de Masuku (USTM),Franceville, Gabon

J. P. Akue (*)Centre International de RecherchesMédicales de Franceville (CIRMF),BP 769 Franceville, Gabone-mail: [email protected]

Parasitol Res (2010) 107:643–650DOI 10.1007/s00436-010-1910-2

eukaryotic cytotoxicity of extracts of plants used in localtraditional medicine.

Materials and methods

Ethnobotanical survey and plant collection

We conducted a survey of plants used to treat a variety ofillnesses by families in northern Gabon (Bissock district) andby traditional practitioners in Estuaire province. The plantswere identified and authenticated by botanists of the NationalGabonese Herbarium (HNG) at IPHAMETRA. The herbar-ium specimens are deposited at HNG under the followingreferences: Lophira alata Banks ex C. F. Gaertn (Ochnaceae)(1454); Greenwayodendron suaveolens (Engl. & Diels)Verdc. (Annonaceae) (656); Uapaca togoensis Pax. (Euphor-biaceae) (4717); Zanthoxylum heitzii (Aubrév. & Pellegr.) P.G. Waterman (Rutaceae) (139); Peperomia pellucida (L.)Kunth (Fam.) (Piperaceae) (927); Vernonia hymenolepis A.Rich. (Asteraceae) (118); Vernonia conferta Benth. var.Conferta (Compositae) (3530); Piptadeniastrum africanum(Hook. f.) Brenan (Mimosaceae) (2876); and Petersianthusmacrocarpus (Beauv.) Liben (Lecythidaceae) (1614).

Preparation of plant extracts

Selected parts of the plants were dried at room temperature andcrushed to a powder. Then 100 g of each sample was maceratedwith 400 ml of dichloromethane or methanol for 48 h, withagitation, before evaporation in a Rotavapor (Laborotor 4000Heidolph). The resulting powders were dried then furthermacerated in 400 ml of methanol or dichloromethane for 48 hwith agitation. The solutions obtained were evaporated,yielding a dry methanolic extract or dichloromethane extract.

Chemical screening of plant extracts

The presence of the following families of chemicalconstituents was examined as follows:

Alkaloids 0.5 g of each extract was agitated with 5 ml ofhydrochloric acid in a steam bath, then 1-ml aliquots offiltrate were treated with a few drops of Mayer’s reagent orDragendorff’s reagent. The presence of a precipitate aftertreatment with either reagent is a preliminary indicator ofthe presence of alkaloids (Harborne 1973; Trease and Evans1989). To remove non-alkaloid compounds that could leadto false-positive reactions, part of the extract was alkalin-ized with 40% ammonia solution then treated twice withchloroform. The second chloroform extract was concentrat-ed and then retested with the Mayer and Dragendorffreagents (Bruneton 1999).

Sterols and triterpenes These families of compounds wereidentified by using the Lieberman–Bruchard reaction.Briefly, 0.5 g of extract was dissolved in 0.5 ml ofchloroform with 0.5 ml of acetic anhydride, and cooledon ice before carefully adding sulfuric acid. A change incolor from purple to blue indicates the presence of sterols,while a green or purple-red color indicates the presence oftriterpenes (Bruneton 1999).

Tannins and polyphenols Boiled aqueous extract (1 ml)was mixed with 1% ferric chloride. A black-blue colorindicates the presence of gallic tannins and a dark greencolor tannin catechists. When both were detected in thesame extract, they were separated with Styasny’s reagent. Adrop of the extract was placed on a slab of silica gel andeluted in an atmosphere saturated with chloroform/aceticacid/formic acid (5:4:1). Then the plates were sprayed with10 ml of methanol solution at 5% nitrous acid and heated inan oven at 80°C for 10 min. The presence of tannins isrevealed by the appearance of blue spots, while polyphe-nols are revealed by a violet-blue, pink-orange, pink-violet,or red coloration (Bruneton 1999).

Flavonoids were detected by using the Shibata reactionor cyanide test (Bruneton 1999). Briefly, 3 ml of extractwas evaporated and the residue was dissolved in 2 ml of50% methanol, then a few magnesium shavings and a fewdrops of concentrated hydrochloric acid were added. Thedevelopment of a red-orange or purplish color indicates thepresence of flavones aglycones.

Carotinoids were revealed by evaporating 10 ml of theextract and treating the residue with a few drops of Carr–Price’sreagent or concentrated sulfuric acid. A change from blue to redindicates the presence of carotinoids (Bruneton 1999).

Saponosides 1% of each sample decoction was returnedgradually in 10-ml test tubes for a final volume of 10 ml.After two vigorous shakes, the tubes were left to stand for15 min and the height of foam was measured.

Reducing sugars One milliliter of extract was dissolved in2 ml of distilled water and 1 ml of Fehling liquor and boiledfor 30 min. The formation of a brick-red precipitateindicates the presence of reducing sugars.

The color intensity in each of the above reactions wasscored as follows: +++, very intense; ++, intense; +, weak;and −, absent.

In vitro assay of antifilarial activity

L. loa microfilariae were isolated from patients with highparasite burdens, as described by Van Hoegaerden andIvanoff (1986). A 1 mg/ml stock solution of each plant

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extract was prepared in dimethylsulfoxide (DMSO) thendiluted for testing at 100, 25, 6.25, 1.5, 0.39, and 0.09 μg/ml.First, five freshly isolated microfilariae were incubated witheach concentration of each extract in 96-well culture platescontaining modified Eagle’s medium with 10% fetal calfserum, penicillin (200 U/ml), and streptomycin (200 μg/ml).The plates were incubated at 37°C in 5% CO2 atmosphere for120 h. The effects were determined microscopically bycounting motile filarial, with respect to a negative control(medium alone) and positive controls [DEC and ivermectin(Ivomec 1 g/ml, Merck Sharp and Dohme BV, The Nether-lands; H41275 Merial)] at the same concentration as theplant extracts. The tests were then repeated using 20microfilariae per well in duplicate. Antifilarial activity wasexpressed as the median lethal concentration for 50% of

microfilariae (LC50) in micrograms per milliliter. Schneider-Orelli’s formula (1947) was used to adjust the resultsobtained with the extracts when the number of dead filarialin control wells exceeded 10%.

In vitro cytotoxicity assay

Monkey kidney cells (named vero cells) derived from aconfluent culture were incubated at 37°C with 5% CO2 for120 h with each plant extract and control (medium alone andDMSO) at each concentration (100, 25, 6.25, and 1.56 μg/ml).On day 5, 20 μl of 3-[4,5-dimethylthiazol-2-yl]-2-5 diphenyltetrazolium bromide (MTT, SigmaChemical Co., St Louis,MO,USA) was added to each well for 4 h. The supernatant was thenremoved and formazan dissolved in 100 μl of MTT solubiliza-

Table 1 Plant families and traditional uses

Species, Genus, Family Origin Traditional uses

Lophira alata Banks ex C.F. Gaertn (Ochnaceae) Equatorial Africa Decoction used for kidney pain

Greenwayodendron suaveolens (Engl. & Diels)Verdc. (Annonaceae)

Guinea–Congo Sterility, weight loss, paralysis

Uapaca togoensis Pax. (Euphorbiaceae) Sub-Saharan Africa Bark enema used as an emetic; lotion forskin disorders

Zanthoxylum heitzii (Aubrév. & Pellegr.) P.G.Waterman (Rutaceae)

Gabon and Cameroon Rheumatism, muscle pain, palpitations, worms,and “renal cleansing”

Peperomia pellucida (L.) Kunth Fam. (Piperaceae) Imported from SouthAmerica and Asia

Fever, hypertension

Vernonia hymenolepis A. Rich. (Asteraceae) Guinea to Angola Hypertension, neonatal respiratory disorders

Vernonia conferta Benth. var. conferta (Compositae) Guinea to Angola Pain, stimulation of lactation, abscess, asthma

Piptadeniastrum africanum (Hook. f.) Brenan(Mimosaceae)

Senegal, Angola, Uganda Decoction used as ocytocitic (enema); bark usedfor toothache (gargling)

Petersianthus macrocarpus (Beauv.) Liben(Lecythidaceae)

Guinea, Angola, Congo Venereal disease

Table 2 Lethal concentration (LC50) of different plant extracts

Plants Parts Solvents LC50 (µg/ml)

Lophira alata Banks ex C.F. Gaertn (Ochnaceae) Bark Methanol 5.29

Greenwayodendron suaveolens (Engl. & Diels) Verdc. (Annonaceae) Bark Methanol 18.47

Uapaca togoensis Pax. (Euphorbiaceae) Bark Methanol 9.3

Zanthoxylum heitzii (Aubrév. & Pellegr.) P.G. Waterman (Rutaceae) Bark Methanol 12.08

Peperomia pellucida (L.) Kunth (Fam.) (Piperaceae) Leaf Methanol 70.28

Vernonia hymenolepis A. Rich. (Asteraceae) Leaf Methanol 12.63

Piptadeniastrum africanum (Hook. f.) Brenan (Mimosaceae) Bark Methanol 8.73

Petersianthus macrocarpus (Beauv.) Liben (Lecythidaceae) Bark Methanol 0.22

Vernonia conferta Benth. var. conferta (Compositae) Bark Methanol 3.78

Petersianthus macrocarpus (Beauv.) Liben (Lecythidaceae) Root Methanol 11.37

Piptadeniastrum africanum (Hook. f.) Brenan (Mimosaceae) Root Methanol 15.78

Piptadeniastrum africanum (Hook. f.) Brenan (Mimosaceae) Leaf Methanol 1.82

Diethylcarbamazine Tablet DMSO 0.385

Ivermectin Solution DMSO 32.74

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tion solution (Sigma, M8910) was added. The intensity ofcolored formazan was measured at 450 nm as an index of cellviability, relative to negative control wells. This index wasexpressed as an IC50 (concentration of extract capable ofinhibiting 50% of cell activity) in micrograms per milliliter.

Data analysis

Probit analysis (Finney 1952) implemented with SPSSsoftware was used for the calculation of the LC50 and IC50

values. The selectivity (SI) of the extracts for filaria wasdetermined as IC50/LC50 ratio

Results

Survey results

Nine plants belonging to nine families were identified(Table 1). They were tested as bark (Ochnaceae, Annona-ceae, Euphorbiaceae, Rutacea, Mimosaceae, Compositae,Lecythideaceae), leaf (Piperacea, Asteracea, Mimosacea),or root extracts (Lecythidacea, Mimosacea). Methanolicextracts of these parts were tested for antifilarial activity.One (P. pellucida) was an imported spice widely used inSouth America and Asia.

Fig. 1 Concentration-dependent antifilarial activity (percentage ofdead microfilariae in duplicate wells). The response against the sameconcentration of extract or control drug was determined and plottedagainst the percentage of dead microfilariae. Extract from Lophiraalata (LAE), Piptadeniastrum africanum (PAR), and Petersianthusmacrocarpus (PME). DMSO dimethylsulfoxide, MEM modifiedEagle’s medium, DEC diethylcarbamazine

Table 3 Inhibitory concentration (IC50) for different extracts

Plants Parts Solvents IC50 (µg/ml)















Fig. 2 Concentration-dependent cytotoxicity. Percentage inhibition induplicate wells by comparison with medium alone plotted against thecorresponding dose of extract or standard drug. LAE Lophira alata,PAR Piptadeniastrum africanum, PME Petersianthus macrocarpus,DMSO dimethylsulfoxide, MEM modified Eagle’s medium, DECdiethylcarbamazine

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In vitro activity on L. loa

P. macrocarpus bark extract was the most active (LC50=0.22 μg/ml), followed by P. africanum (Hook. f.) leafextract (1.82 μg/ml), V. conferta bark (3.78 μg/ml), and L.alata ex C.F. Gaetn bark (5.29 μg/ml). P. pellucida barkwas the least active (70.28 μg/ml). The LC50 of ivermectinand the DEC was 32.74 and 0.385 μg/ml, respectively(Table 2). The activity of these extracts was concentrationdependent, as shown in Fig. 1. Furthermore, the percentageof dead microfilariae in control pur medium alone variesbetween 0% and 18% after 120-h observation. In the caseof percentage higher or equal to 10%, Schneider-Orelli’sformula was applied and the corrected percentage was used

in all subsequent analysis for the specific effect of plantextracts on microfilariae.

In vitro cytotoxicity

Z. heitzii had the highest IC50 (146.32 μg/ml), followed byP. africanum (119.52 μg/ml). U. togoensis had the lowestIC50 (8.52 μg/ml). The IC50 values of ivermectin and DECwere 20.32 and 182.90 μg/ml, respectively (Table 3). Thecytotoxicity of the extracts was also concentration depen-dent, as shown in Fig. 2. The reduction of cell activity orcell proliferation varies from 96% at high dose of extract to0% for low dose (Table 4). These fluctuations were plantextracts dependent. Thus, P. africanum (root), P. macro-

Table 4 Percentage of inhibition of eukaryotic cell activities according to plant extract concentrations

Plants 100μg/ml 25μg/ml 6.25μg/ml 1.56μg/mla

Lophira alata Banks ex C.F. Gaertn (bark) 68b 23 47 49

Greenwayodendron suaveolens (Engl. & Diels) Verdc. (bark) 83 21 15 0

Uapaca togoensis Pax. (bark) 89 77 60 3

Zanthoxylum heitzii (Aubrév. & Pellegr.) P.G. Waterman (bark) 43 19 13 0

Peperomia pellucida (L.) Kunth (Fam.) (leaf) 72 16 0 3

Vernonia hymenolepis A. Rich. (leaf) 67 0 0 0

Piptadeniastrum africanum (Hook. f.) Brenan (bark) 65 0 0 0

Petersianthus macrocarpus (Beauv.) Liben (bark) 88 69 25 0

Vernonia conferta Benth. var. conferta (bark) 93 50 0 0

Petersianthus macrocarpus (Beauv.) Liben (root) 94 75 29 0

Piptadeniastrum africanum (Hook. f.) Brenan (root) 96 78 0 0

Piptadeniastrum africanum (Hook. f.) Brenan (leaf) 88 45 5 −

a Numbers in this line represent different plant extract concentrationsb Numbers in this column are percentages (%) of inhibition of cell activity or proliferation; − not done

Fig. 3 Selectivity indices (SI). The ratio of inhibition versusantifilarial activity was used as selective indices (SI) and a histogramwas plotted for each extract. LAE Lophira alata, GSE Greenwayoden-dron suaveolens, UTE Uapaca togoensis, ZHE Zanthoxylum heitzii,

PPH Peperomia pellucida, PAE Piptadeniastrum africanum, PMEPetersianthus macrocarpus, VCE Vernonia conferta, PMR Petersian-thus macrocarpus, PAR Piptadeniastrum africanum, PAL Piptadeniaafricanum, VHE Vernonia hymenolepis A. Rich. (Asteraceae)

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carpus (root), V. conferta (bark) induced high percentage ofinhibition at 100 μg/ml (96%, 94%, and 93%, respectively)and at 25 μg/ml. Others like Z. heitzii (bark) did not reach50% inhibition even at high dose (maximum 43% at100 μg/ml).

Selectivity

The selectivity index (Fig. 3) was highest for P. macrocarpus(75.63), followed by P. africanum (13.69), Z. heitzii (12.11),and L. alata (9.26). The lowest indexes were for P. pellucida(0.87), U. togoensis (0.91), P. macrocarpus (1.16), and P.africanum root (1.21). The ivermectin and DEC SI valueswere 0.62 and 474.06, respectively (Table 5).

Influence of the solvent

The antifilarial activity and the SI of P. pellucida were 20and six times higher with the dichloromethane extract thanwith the methanolic extract (4.14 vs. 70.28 μg/ml and 6.08vs. 0.87, respectively), while the IC50 value was higher withthe methanolic extract than with the dichloromethaneextract. Similar trend or inverse can be seen with V.hymenolepis, V. conferta, and P. africanum in Table 6.

Chemical constituents

All the extracts except P. macrocarpus contained alkaloids,steroids/triterpenes, tannins/polyphenols, and reducing sug-ars, while a few contained flavonoids (4/9 extracts) andcarotinoids (1/9), and half of the extracts contained sapono-sides (Table 7). No link was found between the chemicalconstituents and antifilarial activity or cytotoxicity.

Discussion

This is the first study of the effect of plant extracts onhuman L. loa isolates. Plants from nine families commonlyused by local populations were tested, and some werefound to be active on L. loa microfilaria. Some belonged tofamilies also active on other human filaria such as B.malayi (Sahare et al. 2008a, b). This was the case of U.togoensis (Euphorbiacea family) and Z. heitzii (Rutaceafamily). V. conferta, which has strong microfilaricidalactivity, belongs to the Compositae family, of whichanother member, Neurolaena lobata, has shown activityagainst Brugia pahangi macro- and microfilariae (Fujimakiet al. 2005). Microfilariae have been shown to be reliable

Table 5 Selectivity index of different extracts

Plants Parts Solvents SI=IC50/LC50















Dichloromethane Methanol

LC50 IC50 SI LC50 IC50 SI

Peperonia pellucida 4.144 25.22 6.08 70.28 61.46 0.87

Vernonia hymenolepis 21.791 23.272 1.067 12.63 78.72 6.23

Vernonia conferta 22.685 20.709 0.91 3.78 28.37 7.49

Piptadiniastrum africanum 36.878 65.421 1.77 8.73 119.52 13.69

Table 6 Influence of solventon lethal/inhibitory activity andselectivity of extracts

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for pre-screening of filaricidal plant extracts and newcandidate drugs (Murthy and Chatterjee 1999). Based onour previous studies of the in vitro viability of microfilariae(Van Hoegaerden and Ivanoff 1986), we chose an observa-tion period (5 days) compatible with the average lifespan invitro (21 days). P. macrocarpus bark (0.22), P. africanumleaf (1.82), V. conferta bark (3.78), and L. alata bark (5.29)extracts had lower LC50 values than ivermectin (32.74)suggesting the presence of highly active antifilarial com-pounds. P. africanum leaves had a very low LC50 (1.82)compared to the roots (15.78) and bark (8.73). The patternwas similar for the IC50 values, suggesting that the activityvaries with the part of the plant used. Very low IC50 valueswere obtained with U. togoensis (8.52 μg/ml), suggestinghigh cytotoxicity, whereas Z. heitzii bark had a very highIC50 (146.3), suggesting low toxicity for eukaryotic cells.The least selective extract was P. pellucida leaf (0.87).Ivermectin was far less selective than DEC (0.62 vs.475.06) and has previously been shown to be cytotoxicfor mammalian cells in vitro (Molinari et al. 2009). DEC,known to be inactive on microfilariae in vitro, was veryactive on L. loa microfilariae. These data are supported byresults obtained on microscopic observation of microfilariaeexposed to DEC which show strong damage of parasitesuggesting that this drug is active on microfilariae in vitro(Alves et al. 2009). The concentration used in this studywas compatible with therapeutic values (10 μg/ml).Alternatively, DEC is not equally active against all filaria(Peixoto et al. 2003), and neither is ivermectin (Devaneyand Howells 1984). Our results suggest that dichloromethaneand methanol may release different ratios of cytotoxic andantifilarial compounds. It was interesting to notice theabundance of some chemical compounds in different extractslike reducing sugar and sterol/triterpene, while others werealmost absent or scarce like carotinoid or flavonoids. But no

association was established between any specific constituentand antifilarial activity. However, some arguments andexperiences by others suggest that some of the chemicalcompounds have been implicated in antifilarial activity. Thus,it is known that the increase in flavonoid is associated withapoptosis. A Daflon-micronized purified flavonoid fraction ofRutaceae aurantiae is active on edema induced by filaria(Das et al. 2003), while saponin has a detergent action onbiomembranes. Synthetic alkaloids have been reported tohave antifilarial activity in vivo (Singh et al. 1997). Sugarbase molecules have also shown macrofilaricidal activity(Chatterjee et al. 1992). Further work will characterize theseconstituents and their potential role as antifilarial molecules.

Acknowledgments CIRMF is sponsored by the state of Gabon,Total Gabon, and Ministère Français des Affaires Etrangères. Part ofthis work was supported by IPHAMETRA/CENAREST Gabon. Allprocedures in this study are in accordance with the Gabonese laws andethical rules. We declare no conflict of interest.

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Table 7 Chemical constituents in plant extracts

Plants Alkaloids Sterols/triterpenes

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Flanonoids Reducingsugar

Carotinoids Saponosides

Lophira alata Banks ex C.F. Gaertn +++ +++ ++ + +++ − −Greenwayodendron suaveolens(Engl. & Diels) Verdc.

+++ +++ +++ − +++ − +++

Uapaca togoensis Pax. +++ +++ ++ ++ +++ − −Zanthoxylum heitzii (Aubrév. & Pellegr.)P.G. Waterman

+++ +++ +++ − +++ − +++

Peperomia pellucida (L.) Kunth (Fam.) +++ +++ ++ − ++ − +

Vernonia hymenolepis A. Rich. ++ +++ +/− − PF ++ PF

Piptadeniastrum africanum (Hook. f.)Brenan

+ +++ ++ + +++ − +++

Petersianthus macrocarpus (Beauv.) Liben − +++ +++ + +++ − +++

+++ very intense, ++ intense, + weak, − absent, PF not done

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