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Journal of Ethnopharmacology 139 (2012) 712– 720

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

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harmacological, genotoxic and phytochemical properties of selected Southfrican medicinal plants used in treating stomach-related ailments

. Okem, J.F. Finnie, J. Van Staden ∗

esearch Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa

r t i c l e i n f o

rticle history:eceived 3 August 2011eceived in revised form0 November 2011ccepted 18 November 2011vailable online 3 December 2011

eywords:nthelminticntimicrobialyclooxygenaseenotoxicityedicinal plants

henolics, Saponin

a b s t r a c t

Ethnopharmacological relevance: The evaluated medicinal plants are used in South African traditionalmedicine in treating stomach-related ailments.Aims of the study: The study aimed at evaluating the pharmacological, genotoxic and phytochemicalproperties of the seven selected medicinal plants used for treating stomach-related ailments.Materials and methods: : Ethyl acetate (EtOAc), ethanol (EtOH) 70% and water extracts of the selectedplant parts were evaluated for their antimicrobial and anthelmintic activities using microdilutionassays. Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus), Gram-negative bac-terium (Escherichia coli) and Candida albicans were used for antimicrobial assays. Caenorhabditis eleganswas used for the anthelmintic assay. Plant extracts were also assayed for their cyclooxygenase-inhibitoryactivity against cyclooxygenase-1 and -2 enzymes. The Ames test was used to evaluate the genotoxicity ofthe plant extracts. A spectrophotometric method was used to determine the total phenolics, gallotannins,flavonoids and saponins.Results: Twelve extracts exhibited minimum inhibitory concentration (MIC) <1 mg/mL against the bacte-rial test strains, and five extracts exhibited MIC <1 mg/mL against Candida albicans. The EtOAc extractof Tetradenia riparia had the best minimum lethal concentration (MLC) value (0.004 mg/mL) againstCaenorhabditis elegans. All the EtOAc extracts exhibited percentage inhibition in the range of 50.7–94.7%against COX-1 and -2 enzymes at 250 �g/mL. All the plant extracts were non-mutagenic towardsSalmonella typhimurium tester strains TA98, TA100 and TA1537 without metabolic activation. Phyto-

chemical analysis revealed relatively high amounts of total phenolics, gallotannins and flavonoids in theevaluated plant extracts.Conclusions: The general pharmacological activities exhibited by some of the plant extracts in this studysupport the traditional uses of the selected plants in treating stomach-related ailments. The Ames testshowed that all the plant extracts were non-mutagenic but cytotoxicity tests are needed to ascertain thesafety for long-term consumption.

. Introduction

In traditional medicine, healers often treat symptoms for stom-ch ailments and these encompass a broad spectrum of disordersnvolving the lower and upper abdominal cavity. In developingountries there is a high rate of morbidity and mortality result-ng from co-existing conditions of infectious and parasitic diseases.his is attributed to several conditions such as poor hygiene andack of clean water that makes individuals vulnerable to infections

Bi et al., 2004). The recent increase in the number of immuno-uppressed and/or debilitated patients in South Africa resultingrom the high rate of HIV/AIDS epidemic, have led to a number of

∗ Corresponding author. Tel.: +27 33 2605130; fax: +27 33 2605897.E-mail address: rcpgd@ukzn.ac.za (J. Van Staden).

378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.11.034

© 2011 Elsevier Ireland Ltd. All rights reserved.

patients suffering from different kinds of infectious diseases suchas persistence diarrhoea, gastroenteritis, and candidemia caused byopportunistic etiologic organisms such as Candida spp and Entero-coccus faecalis (Drouhent and Dupont, 1988; Hobson, 2003; Kayser,2003). Gastroenteritis is the inflammation of the digestive tract,involving both the stomach and the small intestines which are char-acterized by symptoms such as stomach pain, diarrhoea, dysentery,vomiting, fever, inflammatory infections of the colon and abdom-inal cramp (WHO, 2003). Bacteria, viruses and parasitic organismswere identified long ago as the major etiologic agents of infectiousdiseases that plague man. Escherichia coli and Staphylococcus aureusare mostly associated with food poisoning. Escherichia coli is known

to produce enterotoxins that induce watery diarrhoea and abdom-inal tissue damage resulting in acute or chronic abdominal painsand cramps (Kloos and Schleifer, 1981; Sleisenger and Fordtrand,1993). The menace of diarrhoea and cholera caused by some of

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A. Okem et al. / Journal of Ethn

hese etiologic agents in tropical and subtropical countries haveeen reported as one of the worst scenarios of disease outbreaks,s these have claimed lives of millions of people especially childrennd infants (Cohen and Tartasky, 1997; Sarkar et al., 2007).

The use of plant-based medicine in treating infectious diseasesas been in existence for thousands of years and will continue torovide mankind with new remedies (Cragg and Newman, 2007).

n South Africa, around 80% of the population relies mainly on tradi-ional medicine for their primary health care needs, largely becausef the high cost of Western medicine, availability of natural prod-cts and the cultural beliefs of the people (Dauskardt, 1990; Cocksnd Møller, 2002). Medicinal plants are a reservoir of importantiologically active compounds. Some of these have demonstratedotent antimicrobial activities against an array of bacterial, fungalnd protozoan organisms, as well as anti-inflammatory and anti-ancer activities (Hernändez et al., 2000; Polya, 2003). Others haveeen reported to have antispasmodic effects, delay gastrointesti-al transit, suppress gut motility, stimulate water adsorption oreduce electrolyte secretion (Palombo, 2006). Biologically activeompounds from medicinal plants have now become the majorocus for developing new and effective pharmaceuticals. This is as aesult of the side effects and the resistance that pathogenic organ-sms develop against the antibiotic agents currently used (Essawind Srour, 2000).

In spite of the great advancement in instrumentation andchievement in the pharmaceutical industries in the search forrug lead compounds from plants, much of the plant biodiver-ity still remains unexplored as sources of novel principles (Craggnd Newman, 2007). Growing evidence has shown that some ofhe plant’s secondary metabolites are toxic and/or carcinogenic,hich can induce adverse effects leading to mutation and/or degen-

rative diseases (Popat et al., 2001). Hence, evaluating naturalroducts for their efficacy and toxic or genotoxic potential beforepplying them as therapeutic agents is becoming increasinglymportant.

The present study evaluates the efficacy of seven South Africanedicinal plants used in traditional medicine for the treatment

f stomach-related ailments. The plant extracts were screenedor antibacterial and antifungal activities against some of theathogenic agents known to cause stomach-related ailments in

mmunosuppressed patients. Plant extracts were also screened fornthelmintic and cyclooxygenase-inhibitory (anti-inflammatory)ctivities. The genotoxicity test was done to establish the safe use ofhe plant extracts as therapeutic agents. A preliminary phytochemi-al screening was done to determine the presence of different typesf secondary metabolites.

. Materials and methods

.1. Plant collection and extractions

Plant material was collected from around Pietermaritzburg inwaZulu-Natal, South Africa. The plants were identified and theoucher specimens were prepared and lodged in the Bews Herbar-um, University of KwaZulu-Natal, Pietermaritzburg (Table 1). Plant

aterial was oven dried at 50 ◦C and milled into powders using Retsch® ZM 200 ultra centrifugal mill (Germany). The groundaterial (1 g each) was extracted sequentially with 10 mL of EtOAc,

tOH and water in order to extract both the polar and non-polarompounds. The extraction was done in a sonication bath (Julabo

mbH sonicator) for 1 h. The plant extracts were then filtered usinghatman No. 1 filter paper. The filtrates were concentrated using a

otary evaporator and then air-dried under a stream of cold air. Thequeous extracts were freeze-dried. The dried extracts were keptn the dark at 10 ◦C until ready for use.

acology 139 (2012) 712– 720 713

2.2. Pharmacological screening

2.2.1. Antibacterial microdilution assayAntibacterial activity of the plant extracts was determined using

the minimum inhibitory concentration (MIC) technique describedby Eloff (1998) and as detailed by Ndhlala et al. (2009). Overnightcultures of a Gram-negative (Escherichia coli ATCC 11775) and twoGram-positive (Staphylococcus aureus ATCC 12600 and Enterococcusfaecalis ATCC 19433) bacterial strains were used. Neomycin wasused as the positive control and 70% EtOH was used as the negativecontrol. The MIC and minimum bactericidal concentration (MBC)were recorded as the concentration of the last well in which therewas no bacterial growth. The assay was repeated twice in duplicatefor each extract.

2.2.2. Antifungal microdilution assayThe antifungal activity of the plant extracts was evaluated using

the microdilution assay described by Eloff (1998) and modifiedfor fungi by Masoko et al. (2007). An overnight culture of Candidaalbicans (ATCC 10231) was used. Amphotericin B (Sigma–Aldrich)(0.25 mg/mL) was used as the positive control. The MIC andminimum fungicidal concentration (MFC) were recorded as theconcentration of the last well in which there was no fungal growth.The screening was done in duplicate and repeated twice for eachextract.

2.2.3. Anthelmintic colorimetric assayAn in vitro determination of free-living nematode larvae via-

bility, as described by James and Davey (2007) with modificationas outlined by Aremu et al. (2010), was used to evaluate the MLCvalues of the plant extracts. A 3-day-old culture of Caenorhabditiselegans var. Bristol (N2) was used. Levamisole was used as the pos-itive control and 70% EtOH was used as the negative control. Theassay was done in duplicate and repeated twice for each extract.

2.2.4. Anti-inflammatory assayBoth the cyclooxygenase-1 and -2 assays were conducted as

described by Jäger et al. (1996) and as outlined by Eldeen andVan Staden (2008). The stock solution of COX-1 and -2 enzymes(60 �L) (Sigma–Aldrich Germany) stored at −70 ◦C were acti-vated with 1250 �L of a co-factor solution. Plant extracts werescreened at 250 �g/mL for the organic solvents and 2 mg/mL forthe water extracts. Three controls were run in each assay (2.5 �Lethanol + 17.5 �L distilled water). Two were the negative controls:the background in which the enzyme was inactivated with 2 N HCland kept on ice before adding [14C] arachidonic acid, and a solventblank. Indomethacin® was used as a positive control (5 �M for COX-1 and 200 �M for COX-2). Percentage inhibition for the test extractswas calculated using the formula:

COX inhibition (%) =(

1 − DMPsample − DMPbackground

DMPblank − DMPbackground

)× 100

(1)

2.2.5. Plate incorporation assayThe genotoxicity potential of crude plant extracts was deter-

mined using the Salmonella microsome assay, based on the standardplate-incorporation procedure with Salmonella typhimurium testerstrains TA98, TA100 and TA1537 without metabolic activation(Maron and Ames, 1983; Mortelmans and Zeiger, 2000). The bac-terial tester strains were grown overnight in 10 mL Oxoid nutrientbroth No. 2 for 16 h at 37 ◦C. Top agar was melted at the begin-

ning of the assay and supplemented with 10 mL of histidine/biotin(0.5 mM) and kept in a water bath at 50 ◦C. In triplicate, 100 �L ofeach of the three dilutions (50, 500 and 5000 �g/mL) per samplewere added to sterile glass tubes, followed by 500 �L of phosphate

714 A. Okem et al. / Journal of Ethnopharmacology 139 (2012) 712– 720

Table 1Selected South African medicinal plants used in traditional medicine in treating stomach-related ailments.

Plant family Scientific name(Voucher specimen number)

Traditional preparation Reference

Crassulaceae Crassula multicava (Lem)A OKEM 3 NU

Decoction of whole plant is used as strongemetic and as love charm

Hutchings et al. (1996)

Icacinacea Cassinopsis ilicifolia (Hochst.) KuntzeA OKEM 5 NU

Leaf and bark infusion is used to treatdiarrhoea and inflammation of ear

Watt and Breyer-Brandwijk (1962)

Lamiaceae Tetradenia riparia (Hochst.) CoddA OKEM 7 NU

Leaf infusion is used to treat gastroenteritis.The leaf decoctions and infusions are widelytaken for cough and sore throats and asantimalarial. This plant is also used in treatinglivestock diseases

Van Wyk and Wink (2004)

Rubiaceae Canthium spinosum (Klotzsch) KuntzeA OKEM 4 NU

Leaf infusion is used in treating diarrhoea Hutchings et al. (1996)

Rubiaceae Coddia rudis (E. Mey. Ex Harv.) Verdc.A OKEM 2 NU

Leaf infusion is used to treat diarrhoea andunspecified parts are used to treat malaria andfever. Pounded root decoction is used intreating impotency and infertility

Hutchings et al. (1996)

Rubiaceae Conostomium natalensis (Stapf) Cufod.A OKEM 1 NU

Whole plant infusion is used to treat diarrhoea,root infusions are used as emetics in the

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uffer (0.1 mM, pH 7.4). Thereafter, 100 �L of the overnight bac-erial culture were added to each tube. From the supplementedop agar, 2 mL were added to the mixture in the sterile tubes.he contents of the tubes were then mixed with a vortex mixernd poured onto the labelled minimal agar plates. After the topgar hardened, the plates were inverted and incubated at 37 ◦C for8 h. The revertant colonies were counted using a colony counter.he assay was performed twice for each bacterial strain and theesults were expressed as the mean (±standard error) number ofhe revertant colonies per plate. 4-Nitroquinoline-N-oxide (4NQO)2 �g/plate) was used as the positive control and 10% dimelthylulfoxide (DMSO) as the negative control.

.2.6. Phytochemical analysisThe selected phytochemical compounds were evaluated as

ollows and their absorbent was read using a UV–vis spectropho-ometer (Varian Cary 50, Australia). The amount of total phenolicsas evaluated using the Folin Ciocalteu (Folin C) assay as described

y Makkar (1999). Total phenolic concentration was expresseds gallic acid equivalents (GAE)/g dry matter. Gallotannin con-ent was determined using the rhodanine assay as described by

akkar (1999) with slight modification as outlined by Ndhlala et al.2007). Gallotannin concentrations in the extracts were expresseds GAE/g dry matter. Flavonoid content was determined using theluminium chloride colorimetric assay as described by Zhishent al. (1999) and Marinova et al. (2005). Total flavonoid contentas expressed as catechin equivalents (CTE).

The froth test was used to determine the presence of saponinss described by Tadhani and Subhash (2006). The saponins werextracted for plant extracts that tested positive in the froth testsing the method as described by Makkar et al. (2007). The totalnd steroidal saponins were determined using spectrophotometricssays as described by Hiai et al. (1976) and Baccou et al. (1977)espectively. Total and steroidal saponins were expressed as dios-enin equivalents (DE).

. Results and discussion

.1. Antimicrobial activity of plant extracts

Antimicrobial activities of the investigated medicinal plantxtracts are presented in Table 2. Plant extracts that exhibited MIC

af decoction is used to treat Hutchings et al. (1996)

values <1 mg/mL as highlighted in bold, were considered as hav-ing good antimicrobial activities (Gibbons, 2005). Of all the plantextracts tested only 12 extracts showed good activities againstthe bacterial test strains and five extracts showed good antifun-gal activities against Candida albicans. The EtOAc extracts showedbetter antimicrobial activities than other solvent extracts. It couldbe that the EtOAc extract contained mainly lipophilic (fatty acids)compounds which are widespread in plants and are known fortheir potent antimicrobial activities (Heinrich et al., 2004). Withthe exception of Tetradenia riparia that exhibited good activityagainst Gram-negative Escherichia coli all the other extracts onlyshowed good activities against the Gram-positive (Enterococcusfaecalis and Staphylococcus aureus) bacterial strains. This is an indi-cation that Gram-negative bacteria are more resistant to antibioticagents than the Gram-positive bacteria (Quesnel and Russell, 1983).The leaf extract of Tetradenia riparia is known to contain diter-penediol (Van Puyvelde et al., 1986) which could be responsiblefor the exceptional activities observed in the extracts of Tetrade-nia riparia in the present study. Coddia rudis was the only plantthat did not show good antibacterial activities but exhibited goodactivity against Candida albicans. The lack of good activities amongsome of the extracts does not mean complete absence of bioac-tive compounds (Taylor et al., 2001). It could be that the bioactivecompounds are present in a small amount, or their actions wereantagonized by the presence of other compounds. None of the plantextracts that exhibited good MIC (<1 mg/mL) values showed corre-sponding good MBC effects on any of the bacterial test strains. Thisimplies that the observed antibacterial activities were all bacterio-static. The inhibitory activities observed in some of the extracts tosome extent support their uses in traditional medicine as antimi-crobial agents in treating stomach-related ailments. Plant extractsthat demonstrated good activities against Candida albicans are verypromising considering the fact that this organism can developextensive resistance to commonly used antibiotic agents due totheir genetic flexibility (Buwa and Van Staden, 2006). Hence, fur-ther studies are needed to isolate and identify these bioactivecompounds.

3.1.1. Anthelmintic activity of plant extractsThe minimum lethal concentration (MLC) values of the investi-

gated medicinal plant extracts are presented in Table 3. The plantextracts that exhibited MLC values <1 mg/mL (as highlighted in

A. Okem et al. / Journal of Ethnopharmacology 139 (2012) 712– 720 715

Table 2Antimicrobial activity of the investigated medicinal plant extracts as determined using microdilution techniques.

Plant species Plant Part Extracts Antibacterial activity MIC (mg/mL) Antifungal activity(mg/mL)

E. c. E. f. S. a. C. a.MIC MBC MIC MBC MIC MBC MIC MFC

Canthium spinosum Leaf EtOH 3.13 12.5 1.56 12.5 0.39 6.25 1.56 3.13EtOAc 3.13 12.5 0.78 6.26 1.56 6.25 0.19 12.5Water 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5

Cassinopsis ilicifolia Leaf EtOH 3.13 6.25 3.13 3.13 0.39 3.13 1.56 12.5EtOAc 3.13 3.13 0.19 12.5 1.56 12.5 3.13 6.25Water 3.13 6.25 1.56 12.5 3.13 3.13 12.5 12.5

Bark EtOH 3.13 6.25 3.13 12.5 1.56 12.5 1.56 6.25EtOAc 3.13 12.5 0.78 12.5 1.56 12.5 3.13 6.25Water 6.25 6.25 3.13 6.25 3.13 6.25 12.5 12.5

Coddia rudis Leaf EtOH 6.25 3.13 6.25 6.25 6.25 12.5 3.13 3.13EtOAc 12.5 6.25 1.56 6.25 3.13 3.13 0.39 12.5Water 3.13 6.25 1.56 12.5 3.13 6.25 6.25 6.25

Conostomium natalensis Leaf EtOH 6.25 12.5 3.13 3.13 3.13 3.13 0.39 6.25EtOAc 6.25 6.25 0.09 1.56 3.13 3.13 1.56 6.25Water 6.25 12.5 6.25 6.25 6.25 6.25 3.13 12.5

Crassula multicava Whole plant EtOH 3.13 12.5 0.78 6.25 1.56 6.25 0.19 1.56EtOAc 1.56 3.13 0.09 1.56 1.56 3.13 3.13 12.5Water 6.25 6.25 3.13 3.13 3.13 6.13 6.25 6.25

Lagynia lasiantha Leaf EtOH 3.13 6.25 1.56 3.13 1.56 3.13 1.56 3.13EtOAc 1.56 6.25 0.78 3.13 1.56 6.25 6.26 12.5Water 12.5 12.5 6.25 12.5 6.25 6.25 6.25 12.5

Tetradenia riparia Leaf EtOH 1.56 6.25 1.56 6.25 0.39 3.13 1.56 1.56EtOAc 0.19 6.25 0.04 3.13 1.56 1.56 0.39 1.56Water 6.25 3.13 6.25 3.13 6.25 6.25 12.5 6.25

M ; MFCC hanola

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IC, minimum inhibitory concentration; MBC, minimum bactericidal concentration. a., Candida albicans; S. a., Staphylococcus aureus; EtOAc, ethyl acetate; EtOH, etmphotericin B at 0.25 mg/mL; C. a. = 0.97 �g/mL.

old), 1–4 mg/ml and above 4 mg/mL were considered as hav-ng high, moderate and low anthelmintic activity, respectivelyAremu et al., 2010). In the present study Caenorhabditis elegansas used as the model for detecting novel anthelmintics, thisematode has been valuable in basic research on anthelminticharmacology of human and agricultural parasites (Dengg andan Meel, 2004). Caenorhabditis elegans is very different to otherematodes, because it is easy to culture, making the assay cheapnd rapid and sensitive to the majority of anthelmintic drugs thatre used for the treatment of parasitic worm infections (Behnket al., 2008). Although the adult nematode is the major target forhemotherapeutic studies, in vitro tests using free living stages ofarasitic nematodes (egg and larval stages) have been used to eval-ate the anthelmintic activity of plant compounds (Asase et al.,005) due to difficulty in raising nematode parasites in continuous

ulture (Geary et al., 1999).

In the present study most of the plant extracts yielded promisingnthelmintic activity against Caenorhabditis elegans. All the EtOAc

able 3nthelmintic activity of medicinal plants used in treating stomach-related ailments in So

Plant species Minimum lethal concentration (mg

Plant part

Canthium spinosum Leaf

Cassinopsis illicifolia Leaf

Bark

Coddia rudis Leaf

Conostomium natalensis Leaf

Crassula multicava Whole plant

Lagynia lasiantha Leaf

Tetradenia riparia Leaf

LC value of levamisole against Caenorhabditis elegans at 1 mg/mL = 1.04 �g/ml; EtOAc, e

, minimum fungicidal concentration; E. c., Escherichia coli; E. f., Enterococcus faecalis;; Neomycin at 2 �g/mL E. c. = 0.39 �g/mL; E. f. = 6.25 �mg/mL; S. a. = 0.19 �g/mL;

extracts exhibited high anthelmintic activity and most notablyTetradenia riparia showed the best anthelmintic activity with anMLC value of 0.004 mg/mL. 1,8-Cineole and ishelenine (eudes-manolide sesquiterpene lactone) have been isolated from the leafextracts of Tetradenia riparia. Terpenes are essential oil fractions,highly enriched in compounds based on an isoprene structure andhave been recognized for their potent pharmacological properties(Polya, 2003).

The observed activities in the present study were randomly dis-tributed between the aqueous and the organic solvent extracts.For example, the organic extracts of Coddia rudis showed highactivity whereas the aqueous extract exhibited moderate activity.In other cases, the organic extracts of Crassula multicava exhib-ited high anthelmintic activity but the aqueous extract exhibitedlow activity. The EtOAc extract of Conostomium natalensis and

Laygenia lasiantha showed good activity whereas the EtOH andaqueous extracts of these plants showed low activities. Most inter-estingly the EtOAc extract of Tetradenia riparia that exhibited the

uth African traditional medicine.

/mL)

EtOAc EtOH water

0.260 0.016 0.0330.130 0.033 0.0650.270 0.016 0.0160.520 0.008 2.0830.270 1.042 2.0830.008 0.521 8.3300.065 2.083 8.3300.004 2.083 2.083

thyl acetate; EtOH, ethanol.

716 A. Okem et al. / Journal of Ethnopharm

COX-2

Plant speciesLlLCnLCiBCiLCrLCmWCsLTrLControl

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0

20

40

60

80

100

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0

20

40

60

80

100Control EtOAcEtOHwater

Fig. 1. Percentage inhibitory activity against COX-1 and COX-2 by plant extractsused in treating stomach-related ailments. TrL, Tetradenia riparia leaf; CsL, Can-thium spinosum leaf; CmW, Crassula multicava whole plant; CrL, Coddia rudis leaf;CiL, Cassinopsis ilicifolia leaf; CiB, Cassinopsis ilicifolia bark; CnL, Conostomium natal-ensis leaf; LlL, Lagynia lasiantha leaf. Plant extracts with inhibitory activity above5ow

bafcatIutoomapssppIdcr

3

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0% were considered to be active. Aqueous extracts were evaluated at 2 mg/ml,rganic extracts at 250 �g/ml. Percentage inhibition by Indomethacin® in COX-1as 57.15 ± 5.2% and COX-2 was 75.5 ± 4.5%, respectively.

est anthelmintic activity demonstrated moderate anthelminticctivity in the EtOH and aqueous fractions respectively. The dif-erent levels in anthelmintic activity across the test extractslearly indicates the importance of testing both the organic andqueous extracts, which could lead to the isolation and iden-ification of a diverse range of bioactive compounds in plants.n the present study plant materials were extracted sequentiallysing organic solvents first, and then followed by water extrac-ion. The anthelmintic activities observed among the aqueous andrganic extracts support the claim by herbal healers, that somef the studied plants are agents for treating stomach-related ail-ents caused by parasitic helminths. The detection of anthelmintic

ctivity in both the organic and aqueous extracts indicates theresence of more than one type of anthelmintic agent in theame plant (Whitfield, 1996). The low anthelmintic activity amongome of the extracts could mean that such extracts might haveurgative properties. This investigation indicates the presence ofotential anthelmintic compounds in all the studied plant species.

solation and identification of these compounds could lead toeveloping effective anthelmintic agents that may supplement theurrent clinical treatment in the hope of preventing anthelminticesistance.

.1.2. Anti-inflammatory activity of plant extractsThe percentage inhibitory activity of plant extracts against COX-

and COX-2 are presented in Fig. 1. Inhibitory activity of <50% was

acology 139 (2012) 712– 720

considered as good inhibition of the COX enzymes (Eldeen and VanStaden, 2008). All the EtOAc extracts tested showed percentageinhibition in the range of 50.7–94.7% against both COX-1 and COX-2 respectively. The percentage inhibitory activity of EtOAc extractsagainst COX-2 enzyme were generally higher compared to that ofCOX-1 at 250 �g/mL. This finding is very important because COX-2specific inhibitors have been suggested to potentiate the devel-opment of non-steroidal anti-inflammatory agents due to theirlow side-effects and negligible risk of platelet aggregation, gas-tric haemorrhage colitis and gastro-intestinal toxicity (MacAulayand Blackburn, 2002; Nurtjahja-Tjendraputra et al., 2003; Bertin,2004). It should be noted that anti-inflammatory effects of selectiveCOX-2 inhibitors can only be possible if the dose is not increasedabove the levels which can also inhibit COX-1 activity (Li et al.,2006). The low inhibitory activity against COX-1 isoenzyme in thepresent study is noteworthy due to the fact that the COX-1 enzymeis constitutively expressed in most tissues of the body, hence, itis undesirable to have a remedy that has high COX-1 inhibitoryactivity because this inhibition can lead to serious adverse effects(Whittle, 2004; Li et al., 2006). Except for the aqueous extracts ofTetradenia riparia and Coddia rudis that showed good inhibitoryactivities against COX-1 and COX-2 enzymes respectively, all theother aqueous extracts in this investigation demonstrated poorinhibitory activity against both COX enzymes. Contrary to a pre-vious study (Ndhlala et al., 2011) aqueous extracts of Tetradeniariparia exhibited COX-1 inhibitory activity to a greater extent thanthe COX-2 enzyme. These differences in activities might be due tothe effect of harvesting time or storage of plant materials. The anti-inflammatory activity exhibited by some of the evaluated medicinalplants support their uses in traditional medicine in alleviatingpain and inflammation associated with stomach-related ailments.Plant extracts that exhibited good anti-inflammatory activity espe-cially against the COX-2 enzyme need further studies to determinetheir inhibitory potential against other pro-inflammatory media-tors such as nuclear transcriptase factors mediated the signallingpathways in immune cells that leads to the production of induciblenitric oxygen species, pro-inflammatory cytokines and induciblecyclooxygenase (Polya, 2003).

3.1.3. Genotoxicity properties of plant extractsThe spontaneous reversion responses of the Salmonella

typhimurium tester strains to different dilutions of plant extractsare presented in Table 4. The Ames test without metabolic activa-tion is designed to detect only direct mutagens. A positive responsein any single bacterial strain either with or without metabolicactivation is sufficient to designate a substance as a mutagen(Zeiger, 2001). The results presented in this study indicated thatall the evaluated extracts were non-mutagenic towards Salmonellatyphimurium tester strains TA98, TA100 and TA1537. None of theevaluated plant extracts exhibited a dose dependent increase inthe number of revertants, which were not equal to, or greater than,two times that of the negative control (Maron and Ames, 1983).This implies that the evaluated plant extracts were devoid of anydirect mutagenic compounds. The dense background of the platesas compared to the negative control after 48 h showed that allthe extracts were non-toxic to the Salmonella typhimurium testerstrains. Several compounds isolated from plants have been iden-tified as carcinogens, and some of these compounds such as thearomatic amines, furoquinoline alkaloids, isothiocyanates and sev-eral polycyclic aromatic hydrocarbons e.g. benzo-[a]-pyrene canremain biologically inactive until they are metabolized to activediol epoxide products as the ultimate carcinogen (Rether et al.,

1990; Polya, 2003). Metabolic activation or detoxification of xeno-biotic compounds in humans mostly take place in the liver, lungsand kidneys and sometimes the process may result in bioactivationof metabolites capable of damaging DNA (Mortelmans and Zeiger,

A.

Okem

et al.

/ Journal

of Ethnopharm

acology 139 (2012) 712– 720

717

Table 4Number of His+ revertants observed in Salmonella typhimurium strains TA98, TA100 and TA1537 induced by the bioactive extracts.

Plant species Plant part Plant extracts Number of His+ revertants (�g/mL)

TA98 TA100 TA1537

5000 500 50 5000 500 50 5000 500 50

Tetradenia riparia Leaf EtOAc 14.7 ± 2.2 15.7 ± 2.9 22.0 ± 2.0 165.0 ± 1.7 176.0 ± 5.1 170.3 ± 5.6 30.3 ± 5.5 30.7 ± 3.3 22.0 ± 2.8

Canthium spinosum Leaf EtOAc 15.7 ± 1.2 19.7 ± 0.7 18.3 ± 4.4 184.0 ± 11 174.0 ± 10 167.7 ± 6.4 39.0 ± 1.7 32.1 ± 3.4 18.3 ± 4.4EtOH 16.0 ± 3.2 15.0 ± 2.5 19.7 ± 1.5 162.7 ± 6.4 149.6 ± 2.6 158.0 ± 2.5 55.7 ± 3.4 62.3 ± 6.1 19.7 ± 1.5Water 17.0 ± 2.5 15.3 ± 0.0 17.7 ± 1.2 166.7 ± 8.8 197 ± 18.2 169.0 ± 4.9 49.3 ± 2.6 57.7 ± 5.0 17.7 ± 1.2

Crassula multicava Whole plant EtOAc 16.7 ± 0.9 16.0 ± 1.7 19.3 ± 1.2 174.7 ± 6.9 163.3 ± 4.4 153.0 ± 4.9 30.5 ± 2.3 33.7 ± 2.8 19.3 ± 1.2EtOH 17.7 ± 0.9 14.3 ± 1.3 15.3 ± 1.7 160.0 ± 7.2 170.0 ± 10 168.0 ± 9.0 58.3 ± 5.4 57.0 ± 6.8 15.3 ± 1.7

Coddia rudis Leaf EtOAc 15.7 ± 1.9 16.7 ± 1.2 16.0 ± 2.5 196.3 ± 6.6 187.3 ± 8.7 174.0 ± 15 22.7 ± 5.8 29.0 ± 4.1 16.0 ± 2.5EtOH 18.3 ± 2.4 18.7 ± 2.0 20.0 ± 1.5 164.3 ± 0.7 158.0 ± 11 174.0 ± 2.1 56.3 ± 2.3 49.7 ± 2.9 20.0 ± 1.5

Cassinopsis ilicifolia Leaf EtOAc 21.7 ± 1.2 20.7 ± 0.9 14.0 ± 0.6 158.0 ± 9.3 145.7 ± 9.4 134.7 ± 7.3 29.7 ± 1.8 29.7 ± 2.7 14.3 ± 0.6EtOH 18.3 ± 1.8 15.3 ± 1.3 16.3 ± 1.5 148.3 ± 1.2 152.0 ± 7.2 173.7 ± 7.8 51.7 ± 3.9 47.3 ± 1.8 16.3 ± 1.5EtOH 18.3 ± 1.8 15.3 ± 1.3 16.3 ± 1.5 148.3 ± 1.2 152.0 ± 7.2 173.7 ± 7.8 51.7 ± 3.9 47.3 ± 1.8 16.3 ± 1.5Water 19.7 ± 2.3 17.3 ± 3.5 18.3 ± 1.2 185.3 ± 7.4 189.7 ± 9.3 182.7 ± 2.9 59.7 ± 4.9 62.0 ± 4.5 18.3 ± 1.2

Bark EtOAc 19.7 ± 1.8 11.3 ± 2.5 14.7 ± 2.6 179.3 ± 8.7 171.3 ± 9.8 161.3 ± 6.3 25.7 ± 2.4 29.0 ± 3.7 14.7 ± 2.6EtOH 17.0 ± 3.8 21.5 ± 4.5 14.3 ± 0.7 159.3 ± 8.1 163.0 ± 8.6 142.7 ± 8.4 51.0 ± 3.8 48.3 ± 2.3 14.3 ± 0.7Water 12.3 ± 0.3 11.7 ± 1.2 32.0 ± 6.0 124.3 ± 8.1 181.0 ± 9.5 197.7 ± 9.9 58.7 ± 14 57.7 ± 1.2 32.0 ± 6.0

Conostomium natalensis Leaf EtOAc 18.7 ± 2.6 19.7 ± 0.9 15.3 ± 1.2 171.0 ± 2.5 179.7 ± 7.4 187.3 ± 4.7 28.0 ± 4.4 30.3 ± 2.7 15.3 ± 1.2

Laygenia lasiantha Leaf EtOAc 16.7 ± 2.3 23.0 ± 3.7 19.0 ± 1.7 160.0 ± 6.1 189.3 ± 4.4 172.0 ± 10 40.0 ± 2.3 32.3 ± 3.2 19.0 ± 1.7

4-QNO 271.7 ± 10 898.7 ± 9.8 84.6 ± 4.810% DMSO 25.0 ± 2.3 165.7 ± 5.5 58.7 ± 1.5

Number of His+ revertants/plate: mean values of three triplicates per sample. 4-NQO; 4-nitroquinoline-oxide at 5 �g/mL was used as the positive control for the assay. DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; EtOH,ethanol.

718 A. Okem et al. / Journal of Ethnopharmacology 139 (2012) 712– 720

Plant samp le

TrL CsL Cm W CrL CiL CiB Cn L

Tota

l phe

nolic

s (m

g G

AE/g

DW

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Fig. 2. Total phenolic compositions expressed as gallic acid equivalents detected inthe evaluated medicinal plants. DW, dry weight; GAE, gallic acid equivalents; TrL,Tpb

2imeedlpdog

3

iToeoaiqeiphstp

ppl2tactri(

Plant sample

CnLCiBCiLCrLCmWCsLTrL

Gal

lota

nnin

con

cent

ratio

n(m

gGA

E/g

DW

0

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Fig. 3. Gallotannin concentrations expressed as gallic acid equivalents, detected inthe investigated medicinal plants. DW, dry weight; GAE, gallic acid equivalents; TrL,

etradenia riparia leaf; CsL, Canthium spinosum leaf; CmW, Crassula multicava wholelant; CrL, Coddia rudis leaf; CiL, Cassinopsis ilicifolia leaf; CiB, Cassinopsis ilicifoliaark; CnL, Conostomium natalensis leaf.

000). Hence, the evaluated medicinal plants which are admin-stered orally in treating stomach-related ailments may result in

any potential carcinogenic compounds to be metabolized afternzymatic activation within the body which could lead to adverseffects. The absence of mutagenic activity shown by the extractsoes not indicate absolute safety of these extracts as their metabo-

ites could be mutagenic. At this stage, the consumption of theselants in traditional medicine could be said to be reasonably safeependent on further studies such as toxicity testing and the usef metabolizing enzymes that will enhance bioactivation of muta-enic metabolites that might be present in the extracts.

.2. Phytochemical contents

Total phenolic composition as depicted in Fig. 2 shows vary-ng concentrations of phenolics in all the evaluated plant extracts.he leaf extract of Conostomium natalensis had the highest amountf total phenolic compounds (1.7 mg GAE/g) compared to otherxtracts. The extract of Tetradenia riparia had the lowest amountf total phenolic compounds (0.2 mg GAE/g) but exhibited the bestntimicrobial, anthelmintic and cyclooxygenase-inhibitory activ-ties. This result suggests that Tetradenia riparia might have auality composition of bioactive compounds better than the otherxtracts evaluated. The observed pharmacological activities exhib-ted among some of the studied plants could be due to other types ofhytochemicals such as tannin that were not screened for. Tanninas been reported to have anti-diarrhoeal properties, and vasocon-trictor effects on small superficial vessels. They can also enhanceissue regeneration in the case of superficial wounds or burns byreventing fluid losses (Okuda, 2005).

Plant phenolics play important roles as defence agents againstathogens, for attracting pollinating insects and protecting thelant against adverse environmental factors such as ultravio-

et radiation and pest attack (Hodek et al., 2002; Makkar et al.,007). Phytochemical compounds (e.g. phenolics) at lower concen-rations have significant beneficial effects such as antimicrobial,ntioxidant and anti-inflammatory, antiviral, antimutagenic andhemopreventic effects (Makkar et al., 2007). Higher concentra-

ions of phytochemical compounds, on the other hand, have beeneported to have negative physiological effects such as neurolog-cal disfunction, gastrointestinal toxicity, and reproductive failurePolya, 2003). The presence of phenolics at varying concentrations

Tetradenia riparia leafl; CsL, Canthium spinosum leaf; CmW, Crassula multicava wholeplant; CrL, Coddia rudis leaf; CiL, Cassinopsis ilicifolia leaf; CiB, Cassinopsis ilicifoliabark; CnL, Conostomium natalensis leaf.

could be responsible for the observed pharmacological activities inthe present study.

The highest amount of gallotannins was detected in the leafextract of Coddia rudis and Conostomium natalensis (0.12 and0.118 mg GAE/g) respectively (Fig. 3). Extracts of Tetradenia ripariahad the lowest amount of gallotannin (0.016 mg GAE/g). Gallotan-nins have been reported to exhibit significant biological activitiesincluding antimicrobial, anti-inflammatory and anticancer effects(Bruneton, 1995). The mechanism of anti-inflammatory activity ofgallotannin is based on its ability to scavenge free radicals thatcan initiate inflammatory responses and the inhibition of variouspro-inflammatory mediators, such as the COX-2 enzyme, induciblenitric oxygen species and prostaglandins (PGs) (Polya, 2003). Thepresence of gallotannins at varying concentrations in all the evalu-ated medicinal plants may explain the observed antimicrobial andanthelmintic activities as well as good anti-inflammatory effectsespecially against COX-2 by most of the plant extracts. This pro-vides evidence indicating why the selected medicinal plants areused in traditional medicine in treating stomach pains and crampsassociated with stomach-related ailments.

Relatively high levels of flavonoids were detected in all theevaluated plant extracts (Fig. 4). The highest concentrations offlavonoids were detected in the leaf extracts of Canthium spinosum(0.26 mg CTE/g) and Coddia rudis (0.27 mg CTE/g). The anti-inflammatory and antimicrobial activities of several flavonoidsare well known. They inhibit various pro-inflammatory media-tors such as PGs, COX and lipooxygenase by modulating essentialbiosynthetic and signal transduction pathways in organisms andthis activity may be directly related to their radical scavengingcapability (Hodek et al., 2002). The presence of flavonoids at con-siderably high levels in all the evaluated plant extracts could partlybe responsible for the observed pharmacological activities of someplant extracts. Flavonoids are known to exhibit numerous benefi-cial effects, but care should be exercised in the application of thesecompounds as therapeutic agents, because some metabolites ofthese compounds could be genotoxic which might lead to adverseeffects.

The qualitative froth test for the presence of saponins was

positive for only two plant samples Canthium spinosum (leaf)and Cassinopsis ilicifolia (bark) (Table 5). Saponins are structurallydiverse bioactive compounds of plant origin. They consist ofnon-polar aglycones coupled with one or more monosaccharide

A. Okem et al. / Journal of Ethnopharmacology 139 (2012) 712– 720 719

Table 5Saponin composition of South Africa medicinal plants used in treating stomach-related ailments.

Plant species Plant part Froth test Total saponin (mg DE/g) Steroidal saponin (mg DE/g)

Canthium spinosum Leaf + 14.2 ± 6.2 13.9 ± 4.8Cassinopsis ilicifolia Leaf − ND ND

Bark + 11.7 ± 12.3 4.5 ± 9.1Coddia rudis Leaf − ND NDConostomium natalensis Leaf − ND NDCrassula multicava Whole plant − ND NDLagynia lasiantha Leaf − ND NDTetradenia riparia Leaf −

Absence of saponins (−), present of saponins (+), diosgenin equivalence (DE), not determ

Plant sa mple

TrL CsL Cm W CrL CiL CiB Cn L

Flav

onoi

d co

ncen

tratio

n(m

gCTE

/g D

W)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Fig. 4. Flavonoid concentration, as catechin equivalents, detected in the evaluatedmedicinal plants. DW, dry weight; CTE, catechin equivalent; TrL, Tetradenia riparialeaf; CsL, Canthium spinosum leaf; CmW, Crassula multicava whole plant; CrL, Coddiart

miispiitm2

4

tuodttSitPmbba

cology. Veterinary Parasitology 84, 275–295.

udis leaf; CiL, Cassinopsis ilicifolia leaf; CiB, Cassinopsis ilicifolia bark; CnL, Conos-omium natalensis leaf.

oieties and are well known for their numerous properties whichnclude pharmacological activities, sweetness and bitterness, foam-ng and emulsifying properties (Sparg et al., 2004). There could be aynergism in the pharmacological activities observed among theselant extracts that tested positive to saponins. Saponins are found

n most parts of plants that are vulnerable to fungal or bacterial andnsect attacks (Wina et al., 2005). This suggests their roles in plantso act as a chemical barrier against potential pathogens, which

ay explain their potent pharmacological activities (Vincken et al.,007).

. Conclusions

The results of pharmacological screening presented in this studyo some extent validate the efficacy of the selected medicinal plantssed in treating stomach-related ailments. However, other aspectsf stomach-related ailments such as gastric ulcers, motility disor-ers, indigestion and the infections of the lower gastro-intestinalract were not covered in this study. The genotoxicity test showedhat none of the plant extracts were mutagenic towards thealmonella tester strains. However, further studies such as toxic-ty and the use of metabolizing enzymes are needed to ascertainhe safety of the plant metabolites on long-term consumption.hytochemical analysis revealed different classes of secondaryetabolites in the plant species studied. This indicates the type of

ioactive compounds that could be responsible for the antimicro-ial, anthelmintic and anti-inflammatory activities. Further studiesre needed to isolate and identify the bioactive compounds present

ND ND

ined (ND).

in the evaluated plant species and this might lead to developingsuperior drugs with potent pharmacological activities.

Acknowledgements

The authors thank Mrs A. Young of the University of KwaZulu-Natal Botanical Garden and Dr. C. Potgieter (NU Herbarium) for theirassistance in plant identification and voucher specimen prepara-tions. The National Research Foundation (NRF), Pretoria and theUniversity of KwaZulu-Natal are acknowledged for financial sup-port.

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