Journal of Ethnopharmacology 139 (2012) 712–720

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Journal of Ethnopharmacology

jo urnal homepage: www.elsevier.com/locate/jethpharm

Pharmacological, genotoxic and phytochemical properties of selected South

African medicinal plants used in treating stomach-related ailments

∗

A. Okem, J.F. Finnie, J. Van Staden

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

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: The evaluated medicinal plants are used in South African traditional

Received 3 August 2011

medicine in treating stomach-related ailments.

Received in revised form

Aims of the study: The study aimed at evaluating the pharmacological, genotoxic and phytochemical

10 November 2011

properties of the seven selected medicinal plants used for treating stomach-related ailments.

Accepted 18 November 2011

Materials and methods: : Ethyl acetate (EtOAc), ethanol (EtOH) 70% and water extracts of the selected

Available online 3 December 2011

plant parts were evaluated for their antimicrobial and anthelmintic activities using microdilution

assays. Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus), Gram-negative bac-

Keywords:

Anthelmintic terium (Escherichia coli) and Candida albicans were used for antimicrobial assays. Caenorhabditis elegans

Antimicrobial was used for the anthelmintic assay. Plant extracts were also assayed for their cyclooxygenase-inhibitory

Cyclooxygenase activity against cyclooxygenase-1 and -2 enzymes. The Ames test was used to evaluate the genotoxicity of

Genotoxicity the plant extracts. A spectrophotometric method was used to determine the total phenolics, gallotannins,

Medicinal plants ﬂavonoids and saponins.

Phenolics, Saponin

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 extract

of Tetradenia riparia had the best minimum lethal concentration (MLC) value (0.004 mg/mL) against

Caenorhabditis 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 towards

Salmonella typhimurium tester strains TA98, TA100 and TA1537 without metabolic activation. Phyto-

chemical analysis revealed relatively high amounts of total phenolics, gallotannins and ﬂavonoids in the

evaluated plant extracts.

Conclusions: The general pharmacological activities exhibited by some of the plant extracts in this study

support the traditional uses of the selected plants in treating stomach-related ailments. The Ames test

showed that all the plant extracts were non-mutagenic but cytotoxicity tests are needed to ascertain the

safety for long-term consumption.

1. Introduction patients suffering from different kinds of infectious diseases such

as persistence diarrhoea, gastroenteritis, and candidemia caused by

In traditional medicine, healers often treat symptoms for stom- opportunistic etiologic organisms such as Candida spp and Entero-

ach ailments and these encompass a broad spectrum of disorders coccus faecalis (Drouhent and Dupont, 1988; Hobson, 2003; Kayser,

involving the lower and upper abdominal cavity. In developing 2003). Gastroenteritis is the inﬂammation of the digestive tract,

countries there is a high rate of morbidity and mortality result- involving both the stomach and the small intestines which are char-

ing from co-existing conditions of infectious and parasitic diseases. acterized by symptoms such as stomach pain, diarrhoea, dysentery,

This is attributed to several conditions such as poor hygiene and vomiting, fever, inﬂammatory infections of the colon and abdom-

lack of clean water that makes individuals vulnerable to infections inal cramp (WHO, 2003). Bacteria, viruses and parasitic organisms

(Bi et al., 2004). The recent increase in the number of immuno- were identiﬁed long ago as the major etiologic agents of infectious

suppressed and/or debilitated patients in South Africa resulting diseases that plague man. Escherichia coli and Staphylococcus aureus

from the high rate of HIV/AIDS epidemic, have led to a number of are 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 pains

∗ and cramps (Kloos and Schleifer, 1981; Sleisenger and Fordtrand,

Corresponding author. Tel.: +27 33 2605130; fax: +27 33 2605897.

E-mail address: [email protected] (J. Van Staden). 1993). The menace of diarrhoea and cholera caused by some of

A. Okem et al. / Journal of Ethnopharmacology 139 (2012) 712–720 713

these etiologic agents in tropical and subtropical countries have 2.2. Pharmacological screening

been reported as one of the worst scenarios of disease outbreaks,

as these have claimed lives of millions of people especially children 2.2.1. Antibacterial microdilution assay

and infants (Cohen and Tartasky, 1997; Sarkar et al., 2007). Antibacterial activity of the plant extracts was determined using

The use of plant-based medicine in treating infectious diseases the minimum inhibitory concentration (MIC) technique described

has been in existence for thousands of years and will continue to by Eloff (1998) and as detailed by Ndhlala et al. (2009). Overnight

provide mankind with new remedies (Cragg and Newman, 2007). cultures of a Gram-negative (Escherichia coli ATCC 11775) and two

In South Africa, around 80% of the population relies mainly on tradi- Gram-positive (Staphylococcus aureus ATCC 12600 and Enterococcus

tional medicine for their primary health care needs, largely because faecalis ATCC 19433) bacterial strains were used. Neomycin was

of the high cost of Western medicine, availability of natural prod- used as the positive control and 70% EtOH was used as the negative

ucts and the cultural beliefs of the people (Dauskardt, 1990; Cocks control. The MIC and minimum bactericidal concentration (MBC)

and Møller, 2002). Medicinal plants are a reservoir of important were recorded as the concentration of the last well in which there

biologically active compounds. Some of these have demonstrated was no bacterial growth. The assay was repeated twice in duplicate

potent antimicrobial activities against an array of bacterial, fungal for each extract.

and protozoan organisms, as well as anti-inﬂammatory and anti-

cancer activities (Hernändez et al., 2000; Polya, 2003). Others have 2.2.2. Antifungal microdilution assay

been reported to have antispasmodic effects, delay gastrointesti- The antifungal activity of the plant extracts was evaluated using

nal transit, suppress gut motility, stimulate water adsorption or the microdilution assay described by Eloff (1998) and modiﬁed

reduce electrolyte secretion (Palombo, 2006). Biologically active for fungi by Masoko et al. (2007). An overnight culture of Candida

compounds from medicinal plants have now become the major albicans (ATCC 10231) was used. Amphotericin B (Sigma–Aldrich)

focus for developing new and effective pharmaceuticals. This is as a (0.25 mg/mL) was used as the positive control. The MIC and

result of the side effects and the resistance that pathogenic organ- minimum fungicidal concentration (MFC) were recorded as the

isms develop against the antibiotic agents currently used (Essawi concentration of the last well in which there was no fungal growth.

and Srour, 2000). The screening was done in duplicate and repeated twice for each

In spite of the great advancement in instrumentation and extract.

achievement in the pharmaceutical industries in the search for

drug lead compounds from plants, much of the plant biodiver- 2.2.3. Anthelmintic colorimetric assay

sity still remains unexplored as sources of novel principles (Cragg An in vitro determination of free-living nematode larvae via-

and Newman, 2007). Growing evidence has shown that some of bility, as described by James and Davey (2007) with modiﬁcation

the plant’s secondary metabolites are toxic and/or carcinogenic, as outlined by Aremu et al. (2010), was used to evaluate the MLC

which can induce adverse effects leading to mutation and/or degen- values of the plant extracts. A 3-day-old culture of Caenorhabditis

erative diseases (Popat et al., 2001). Hence, evaluating natural elegans var. Bristol (N2) was used. Levamisole was used as the pos-

products for their efﬁcacy and toxic or genotoxic potential before itive control and 70% EtOH was used as the negative control. The

applying them as therapeutic agents is becoming increasingly assay was done in duplicate and repeated twice for each extract.

important.

The present study evaluates the efﬁcacy of seven South African 2.2.4. Anti-inﬂammatory assay

medicinal plants used in traditional medicine for the treatment Both the cyclooxygenase-1 and -2 assays were conducted as

of stomach-related ailments. The plant extracts were screened described by Jäger et al. (1996) and as outlined by Eldeen and

for antibacterial and antifungal activities against some of the Van Staden (2008). The stock solution of COX-1 and -2 enzymes

◦

␮ −

pathogenic agents known to cause stomach-related ailments in (60 L) (Sigma–Aldrich Germany) stored at 70 C were acti-

␮

immunosuppressed patients. Plant extracts were also screened for vated with 1250 L of a co-factor solution. Plant extracts were

␮

anthelmintic and cyclooxygenase-inhibitory (anti-inﬂammatory) screened at 250 g/mL for the organic solvents and 2 mg/mL for

␮

activities. The genotoxicity test was done to establish the safe use of the water extracts. Three controls were run in each assay (2.5 L

␮

the plant extracts as therapeutic agents. A preliminary phytochemi- ethanol + 17.5 L distilled water). Two were the negative controls:

cal screening was done to determine the presence of different types the background in which the enzyme was inactivated with 2 N HCl

of secondary metabolites. and kept on ice before adding [ C] arachidonic acid, and a solvent

blank. Indomethacin was used as a positive control (5 ␮M for COX-

1 and 200 ␮M for COX-2). Percentage inhibition for the test extracts

2. Materials and methods was calculated using the formula:

−

DMPsample DMPbackground

2.1. Plant collection and extractions COX inhibition (%) = 1 − × 100 − DMPblank DMPbackground

(1)

Plant material was collected from around Pietermaritzburg in

KwaZulu-Natal, South Africa. The plants were identiﬁed and the

voucher specimens were prepared and lodged in the Bews Herbar- 2.2.5. Plate incorporation assay

ium, University of KwaZulu-Natal, Pietermaritzburg (Table 1). Plant The genotoxicity potential of crude plant extracts was deter-

◦

material was oven dried at 50 C and milled into powders using mined using the Salmonella microsome assay, based on the standard

a Retsch ZM 200 ultra centrifugal mill (Germany). The ground plate-incorporation procedure with Salmonella typhimurium tester

material (1 g each) was extracted sequentially with 10 mL of EtOAc, strains TA98, TA100 and TA1537 without metabolic activation

EtOH and water in order to extract both the polar and non-polar (Maron and Ames, 1983; Mortelmans and Zeiger, 2000). The bac-

compounds. The extraction was done in a sonication bath (Julabo terial tester strains were grown overnight in 10 mL Oxoid nutrient

◦

GmbH sonicator) for 1 h. The plant extracts were then ﬁltered using broth No. 2 for 16 h at 37 C. Top agar was melted at the begin-

Whatman No. 1 ﬁlter paper. The ﬁltrates were concentrated using a ning of the assay and supplemented with 10 mL of histidine/biotin

◦

rotary evaporator and then air-dried under a stream of cold air. The (0.5 mM) and kept in a water bath at 50 C. In triplicate, 100 ␮L of

␮

aqueous extracts were freeze-dried. The dried extracts were kept each of the three dilutions (50, 500 and 5000 g/mL) per sample

◦

in the dark at 10 C until ready for use. were added to sterile glass tubes, followed by 500 ␮L of phosphate

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

Table 1

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

Plant family Scientiﬁc name Traditional preparation Reference

(Voucher specimen number)

Crassulaceae Crassula multicava (Lem) Decoction of whole plant is used as strong Hutchings et al. (1996)

A OKEM 3 NU emetic and as love charm

Icacinacea Cassinopsis ilicifolia (Hochst.) Kuntze Leaf and bark infusion is used to treat Watt and Breyer-Brandwijk (1962)

A OKEM 5 NU diarrhoea and inﬂammation of ear

Lamiaceae Tetradenia riparia (Hochst.) Codd Leaf infusion is used to treat gastroenteritis. Van Wyk and Wink (2004)

A OKEM 7 NU The leaf decoctions and infusions are widely

taken for cough and sore throats and as

antimalarial. This plant is also used in treating

livestock diseases

Rubiaceae Canthium spinosum (Klotzsch) Kuntze Leaf infusion is used in treating diarrhoea Hutchings et al. (1996)

A OKEM 4 NU

Rubiaceae Coddia rudis (E. Mey. Ex Harv.) Verdc. Leaf infusion is used to treat diarrhoea and Hutchings et al. (1996)

A OKEM 2 NU unspeciﬁed parts are used to treat malaria and

fever. Pounded root decoction is used in

treating impotency and infertility

Rubiaceae Conostomium natalensis (Stapf) Cufod. Whole plant infusion is used to treat diarrhoea, Hutchings et al. (1996)

A OKEM 1 NU root infusions are used as emetics in the

Transkei

Rubiaceae Lagynia lasiantha (Sond.) bullock Powdered leaf decoction is used to treat Hutchings et al. (1996)

A OKEM 6 NU diarrhoea

buffer (0.1 mM, pH 7.4). Thereafter, 100 ␮L of the overnight bac- values <1 mg/mL as highlighted in bold, were considered as hav-

terial culture were added to each tube. From the supplemented ing good antimicrobial activities (Gibbons, 2005). Of all the plant

top agar, 2 mL were added to the mixture in the sterile tubes. extracts tested only 12 extracts showed good activities against

The contents of the tubes were then mixed with a vortex mixer the bacterial test strains and ﬁve extracts showed good antifun-

and poured onto the labelled minimal agar plates. After the top gal activities against Candida albicans. The EtOAc extracts showed

◦

agar hardened, the plates were inverted and incubated at 37 C for better antimicrobial activities than other solvent extracts. It could

48 h. The revertant colonies were counted using a colony counter. be that the EtOAc extract contained mainly lipophilic (fatty acids)

The assay was performed twice for each bacterial strain and the compounds which are widespread in plants and are known for

results were expressed as the mean (±standard error) number of their potent antimicrobial activities (Heinrich et al., 2004). With

the revertant colonies per plate. 4-Nitroquinoline-N-oxide (4NQO) the exception of Tetradenia riparia that exhibited good activity

(2 ␮g/plate) was used as the positive control and 10% dimelthyl against Gram-negative Escherichia coli all the other extracts only

sulfoxide (DMSO) as the negative control. showed good activities against the Gram-positive (Enterococcus

faecalis and Staphylococcus aureus) bacterial strains. This is an indi-

cation that Gram-negative bacteria are more resistant to antibiotic

2.2.6. Phytochemical analysis

agents than the Gram-positive bacteria (Quesnel and Russell, 1983).

The selected phytochemical compounds were evaluated as

The leaf extract of Tetradenia riparia is known to contain diter-

follows and their absorbent was read using a UV–vis spectropho-

penediol (Van Puyvelde et al., 1986) which could be responsible

tometer (Varian Cary 50, Australia). The amount of total phenolics

for the exceptional activities observed in the extracts of Tetrade-

was evaluated using the Folin Ciocalteu (Folin C) assay as described

nia riparia in the present study. Coddia rudis was the only plant

by Makkar (1999). Total phenolic concentration was expressed

that did not show good antibacterial activities but exhibited good

as gallic acid equivalents (GAE)/g dry matter. Gallotannin con-

activity against Candida albicans. The lack of good activities among

tent was determined using the rhodanine assay as described by

some of the extracts does not mean complete absence of bioac-

Makkar (1999) with slight modiﬁcation as outlined by Ndhlala et al.

tive compounds (Taylor et al., 2001). It could be that the bioactive

(2007). Gallotannin concentrations in the extracts were expressed

compounds are present in a small amount, or their actions were

as GAE/g dry matter. Flavonoid content was determined using the

antagonized by the presence of other compounds. None of the plant

aluminium chloride colorimetric assay as described by Zhishen

extracts that exhibited good MIC (<1 mg/mL) values showed corre-

et al. (1999) and Marinova et al. (2005). Total ﬂavonoid content

sponding good MBC effects on any of the bacterial test strains. This

was expressed as catechin equivalents (CTE).

implies that the observed antibacterial activities were all bacterio-

The froth test was used to determine the presence of saponins

static. The inhibitory activities observed in some of the extracts to

as described by Tadhani and Subhash (2006). The saponins were

some extent support their uses in traditional medicine as antimi-

extracted for plant extracts that tested positive in the froth test

crobial agents in treating stomach-related ailments. Plant extracts

using the method as described by Makkar et al. (2007). The total

that demonstrated good activities against Candida albicans are very

and steroidal saponins were determined using spectrophotometric

promising considering the fact that this organism can develop

assays as described by Hiai et al. (1976) and Baccou et al. (1977)

extensive resistance to commonly used antibiotic agents due to

respectively. Total and steroidal saponins were expressed as dios-

their genetic ﬂexibility (Buwa and Van Staden, 2006). Hence, fur-

genin equivalents (DE).

ther studies are needed to isolate and identify these bioactive

compounds.

3. Results and discussion

3.1. Antimicrobial activity of plant extracts 3.1.1. Anthelmintic activity of plant extracts

The minimum lethal concentration (MLC) values of the investi-

Antimicrobial activities of the investigated medicinal plant gated medicinal plant extracts are presented in Table 3. The plant

extracts are presented in Table 2. Plant extracts that exhibited MIC extracts that exhibited MLC values <1 mg/mL (as highlighted in

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

Table 2

Antimicrobial 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 3.13

EtOAc 3.13 12.5 0.78 6.26 1.56 6.25 0.19 12.5

Water 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 12.5

EtOAc 3.13 3.13 0.19 12.5 1.56 12.5 3.13 6.25

Water 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 6.25

EtOAc 3.13 12.5 0.78 12.5 1.56 12.5 3.13 6.25

Water 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 3.13

EtOAc 12.5 6.25 1.56 6.25 3.13 3.13 0.39 12.5

Water 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 6.25

EtOAc 6.25 6.25 0.09 1.56 3.13 3.13 1.56 6.25

Water 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 1.56

EtOAc 1.56 3.13 0.09 1.56 1.56 3.13 3.13 12.5

Water 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 3.13

EtOAc 1.56 6.25 0.78 3.13 1.56 6.25 6.26 12.5

Water 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 1.56

EtOAc 0.19 6.25 0.04 3.13 1.56 1.56 0.39 1.56

Water 6.25 3.13 6.25 3.13 6.25 6.25 12.5 6.25

MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; MFC, minimum fungicidal concentration; E. c., Escherichia coli; E. f., Enterococcus faecalis;

C. a., Candida albicans; S. a., Staphylococcus aureus; EtOAc, ethyl acetate; EtOH, ethanol; Neomycin at 2 ␮g/mL E. c. = 0.39 ␮g/mL; E. f. = 6.25 ␮mg/mL; S. a. = 0.19 ␮g/mL;

amphotericin B at 0.25 mg/mL; C. a. = 0.97 ␮g/mL.

bold), 1–4 mg/ml and above 4 mg/mL were considered as hav- extracts exhibited high anthelmintic activity and most notably

ing high, moderate and low anthelmintic activity, respectively Tetradenia riparia showed the best anthelmintic activity with an

(Aremu et al., 2010). In the present study Caenorhabditis elegans MLC value of 0.004 mg/mL. 1,8-Cineole and ishelenine (eudes-

was used as the model for detecting novel anthelmintics, this manolide sesquiterpene lactone) have been isolated from the leaf

nematode has been valuable in basic research on anthelmintic extracts of Tetradenia riparia. Terpenes are essential oil fractions,

pharmacology of human and agricultural parasites (Dengg and highly enriched in compounds based on an isoprene structure and

van Meel, 2004). Caenorhabditis elegans is very different to other have been recognized for their potent pharmacological properties

nematodes, because it is easy to culture, making the assay cheap (Polya, 2003).

and rapid and sensitive to the majority of anthelmintic drugs that The observed activities in the present study were randomly dis-

are used for the treatment of parasitic worm infections (Behnke tributed between the aqueous and the organic solvent extracts.

et al., 2008). Although the adult nematode is the major target for For example, the organic extracts of Coddia rudis showed high

chemotherapeutic studies, in vitro tests using free living stages of activity whereas the aqueous extract exhibited moderate activity.

parasitic nematodes (egg and larval stages) have been used to eval- In other cases, the organic extracts of Crassula multicava exhib-

uate the anthelmintic activity of plant compounds (Asase et al., ited high anthelmintic activity but the aqueous extract exhibited

2005) due to difﬁculty in raising nematode parasites in continuous low activity. The EtOAc extract of Conostomium natalensis and

culture (Geary et al., 1999). Laygenia lasiantha showed good activity whereas the EtOH and

In the present study most of the plant extracts yielded promising aqueous extracts of these plants showed low activities. Most inter-

anthelmintic activity against Caenorhabditis elegans. All the EtOAc estingly the EtOAc extract of Tetradenia riparia that exhibited the

Table 3

Anthelmintic activity of medicinal plants used in treating stomach-related ailments in South African traditional medicine.

Plant species Minimum lethal concentration (mg/mL)

Plant part EtOAc EtOH water

Canthium spinosum Leaf 0.260 0.016 0.033

Cassinopsis illicifolia Leaf 0.130 0.033 0.065

Bark 0.270 0.016 0.016

Coddia rudis Leaf 0.520 0.008 2.083

Conostomium natalensis Leaf 0.270 1.042 2.083

Crassula multicava Whole plant 0.008 0.521 8.330

Lagynia lasiantha Leaf 0.065 2.083 8.330

Tetradenia riparia Leaf 0.004 2.083 2.083

MLC value of levamisole against Caenorhabditis elegans at 1 mg/mL = 1.04 ␮g/ml; EtOAc, ethyl acetate; EtOH, ethanol.

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

considered as good inhibition of the COX enzymes (Eldeen and Van

COX-1 Control Staden, 2008). All the EtOAc extracts tested showed percentage

100

EtOAc inhibition in the range of 50.7–94.7% against both COX-1 and COX-

EtOH 2 respectively. The percentage inhibitory activity of EtOAc extracts

water

80 against COX-2 enzyme were generally higher compared to that of

COX-1 at 250 ␮g/mL. This ﬁnding is very important because COX-2

speciﬁc inhibitors have been suggested to potentiate the devel-

opment of non-steroidal anti-inﬂammatory agents due to their

low side-effects and negligible risk of platelet aggregation, gas-

40 tric haemorrhage colitis and gastro-intestinal toxicity (MacAulay

and Blackburn, 2002; Nurtjahja-Tjendraputra et al., 2003; Bertin,

20 2004). It should be noted that anti-inﬂammatory effects of selective

COX-2 inhibitors can only be possible if the dose is not increased

above the levels which can also inhibit COX-1 activity (Li et al.,

2006). The low inhibitory activity against COX-1 isoenzyme in the

COX-2 present study is noteworthy due to the fact that the COX-1 enzyme

100

is constitutively expressed in most tissues of the body, hence, it

Inhibition of prostaglandins (%)

is undesirable to have a remedy that has high COX-1 inhibitory

80 activity because this inhibition can lead to serious adverse effects

(Whittle, 2004; Li et al., 2006). Except for the aqueous extracts of

Tetradenia riparia and Coddia rudis that showed good inhibitory

activities against COX-1 and COX-2 enzymes respectively, all the

other aqueous extracts in this investigation demonstrated poor

inhibitory activity against both COX enzymes. Contrary to a pre-

vious study (Ndhlala et al., 2011) aqueous extracts of Tetradenia

20 riparia exhibited COX-1 inhibitory activity to a greater extent than

the COX-2 enzyme. These differences in activities might be due to

the effect of harvesting time or storage of plant materials. The anti-

inﬂammatory activity exhibited by some of the evaluated medicinal

Control TrL CsL CmW CrL CiL CiB CnL LlL

plants support their uses in traditional medicine in alleviating

Plant species

pain and inﬂammation associated with stomach-related ailments.

Plant extracts that exhibited good anti-inﬂammatory activity espe-

Fig. 1. Percentage inhibitory activity against COX-1 and COX-2 by plant extracts

used in treating stomach-related ailments. TrL, Tetradenia riparia leaf; CsL, Can- cially against the COX-2 enzyme need further studies to determine

thium spinosum leaf; CmW, Crassula multicava whole plant; CrL, Coddia rudis leaf; their inhibitory potential against other pro-inﬂammatory media-

CiL, Cassinopsis ilicifolia leaf; CiB, Cassinopsis ilicifolia bark; CnL, Conostomium natal-

tors such as nuclear transcriptase factors mediated the signalling

ensis leaf; LlL, Lagynia lasiantha leaf. Plant extracts with inhibitory activity above

pathways in immune cells that leads to the production of inducible

50% were considered to be active. Aqueous extracts were evaluated at 2 mg/ml,

® nitric oxygen species, pro-inﬂammatory cytokines and inducible

organic extracts at 250 ␮g/ml. Percentage inhibition by Indomethacin in COX-1

was 57.15 ± 5.2% and COX-2 was 75.5 ± 4.5%, respectively. cyclooxygenase (Polya, 2003).

3.1.3. Genotoxicity properties of plant extracts

best anthelmintic activity demonstrated moderate anthelmintic

The spontaneous reversion responses of the Salmonella

activity in the EtOH and aqueous fractions respectively. The dif-

typhimurium tester strains to different dilutions of plant extracts

ferent levels in anthelmintic activity across the test extracts

are presented in Table 4. The Ames test without metabolic activa-

clearly indicates the importance of testing both the organic and

tion is designed to detect only direct mutagens. A positive response

aqueous extracts, which could lead to the isolation and iden-

in any single bacterial strain either with or without metabolic

tiﬁcation of a diverse range of bioactive compounds in plants.

activation is sufﬁcient to designate a substance as a mutagen

In the present study plant materials were extracted sequentially

(Zeiger, 2001). The results presented in this study indicated that

using organic solvents ﬁrst, and then followed by water extrac-

all the evaluated extracts were non-mutagenic towards Salmonella

tion. The anthelmintic activities observed among the aqueous and

typhimurium tester strains TA98, TA100 and TA1537. None of the

organic extracts support the claim by herbal healers, that some

evaluated plant extracts exhibited a dose dependent increase in

of the studied plants are agents for treating stomach-related ail-

the number of revertants, which were not equal to, or greater than,

ments caused by parasitic helminths. The detection of anthelmintic

two times that of the negative control (Maron and Ames, 1983).

activity in both the organic and aqueous extracts indicates the

This implies that the evaluated plant extracts were devoid of any

presence of more than one type of anthelmintic agent in the

direct mutagenic compounds. The dense background of the plates

same plant (Whitﬁeld, 1996). The low anthelmintic activity among

as compared to the negative control after 48 h showed that all

some of the extracts could mean that such extracts might have

the extracts were non-toxic to the Salmonella typhimurium tester

purgative properties. This investigation indicates the presence of

strains. Several compounds isolated from plants have been iden-

potential anthelmintic compounds in all the studied plant species.

tiﬁed as carcinogens, and some of these compounds such as the

Isolation and identiﬁcation of these compounds could lead to

aromatic amines, furoquinoline alkaloids, isothiocyanates and sev-

developing effective anthelmintic agents that may supplement the

eral polycyclic aromatic hydrocarbons e.g. benzo-[a]-pyrene can

current clinical treatment in the hope of preventing anthelmintic

remain biologically inactive until they are metabolized to active

resistance.

diol epoxide products as the ultimate carcinogen (Rether et al.,

1990; Polya, 2003). Metabolic activation or detoxiﬁcation of xeno-

3.1.2. Anti-inﬂammatory activity of plant extracts biotic compounds in humans mostly take place in the liver, lungs

The percentage inhibitory activity of plant extracts against COX- and kidneys and sometimes the process may result in bioactivation

1 and COX-2 are presented in Fig. 1. Inhibitory activity of <50% was of metabolites capable of damaging DNA (Mortelmans and Zeiger,

A. Okem et al. / Journal of Ethnopharmacology 139 (2012) 712–720 717 2.8 4.4 1.5 1.2 1.2 1.7 2.5 1.5 0.6 1.5 1.5 1.2 2.6 0.7 6.0 1.2 1.7 EtOH,

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

20.0 22.0 16.0 32.0 19.0 18.3 19.7 17.7 19.3 15.3 14.3 16.3 16.3 18.3 14.7 14.3 15.3 acetate;

ethyl

3.3 3.4 6.1 5.0 2.8 6.8 4.1 2.9 2.7 1.8 1.8 4.5 3.7 2.3 1.2 2.7 3.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

EtOAc,

30.7 57.0 29.0 62.0 29.0 30.3 32.1 62.3 57.7 33.7 49.7 29.7 47.3 47.3 48.3 57.7 32.3

sulfoxide;

5.5 1.7 3.4 2.6 2.3 5.4 5.8 2.3 1.8 3.9 3.9 4.9 2.4 3.8 14 4.4 2.3 4.8 1.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

40.0 30.3 39.0 30.5 51.0 28.0 55.7 49.3 58.3 22.7 56.3 29.7 51.7 51.7 59.7 25.7 58.7 84.6 58.7 dimethyl

DMSO,

5.6 6.4 2.5 4.9 4.9 9.0 15 2.1 7.3 7.8 7.8 2.9 6.3 8.4 9.9 4.7 10

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

assay.

170.3 158.0 169.0 153.0 168.0 174.0 174.0 172.0 167.7 134.7 173.7 173.7 182.7 161.3 142.7 197.7 187.3 the

for

5.1 10 2.6 18.2 4.4 10 8.7 11 9.4 7.2 7.2 9.3 9.8 8.6 9.5 7.4 4.4

control

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

197 170.0 176.0 174.0 158.0 152.0 152.0 163.0 181.0 149.6 163.3 187.3 145.7 189.7 171.3 179.7 189.3 positive

the

1.7 11 6.4 8.8 6.9 7.2 6.6 0.7 9.3 1.2 1.2 7.4 8.7 8.1 8.1 2.5 6.1 9.8 5.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

used

was 160.0 160.0 165.0 184.0 158.0 171.0 162.7 166.7 174.7 196.3 164.3 148.3 148.3 185.3 179.3 159.3 124.3 898.7 165.7

extracts.

g/mL

␮

2.0 4.4 1.5 1.2 1.2 1.7 2.5 1.5 0.6 1.5 1.5 1.2 2.6 0.7 6.0 1.2 1.7 bioactive

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

the

20.0 22.0 16.0 14.0 32.0 19.0 18.3 19.7 17.7 19.3 15.3 16.3 16.3 18.3 14.7 14.3 15.3 by

g/mL)

␮ induced (

2.9 0.7 2.5 0.0 1.7 1.3 1.2 2.0 0.9 1.3 1.3 3.5 2.5 4.5 1.2 0.9 3.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

TA1537 15.0 16.0 20.7 23.0 15.7 19.7 15.3 14.3 16.7 18.7 15.3 15.3 17.3 11.3 21.5 11.7 19.7

4-nitroquinoline-oxide

revertants and +

His

4-NQO;

2.2 1.2 3.2 2.5 0.9 0.9 1.9 2.4 1.2 1.8 1.8 2.3 1.8 3.8 0.3 2.6 2.3 10 2.3 of

TA100

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

25.0 16.7 15.7 21.7 19.7 19.7 16.7 sample. TA98,

TA985000 500 50 5000 TA100 500 50 5000 TA1537 500 50 per

strains

triplicates

extracts Number

typhimurium three EtOAc EtOAc EtOAc EtOAc EtOAc EtOHWaterEtOH 16.0 EtOH 17.0 EtOH 17.7 EtOHWater 18.3 EtOHWater 18.3 18.3 17.0 12.3

values

Salmonella plant

part Plant in

mean

Leaf EtOAc 15.7 Leaf EtOAc 18.7 Leaf EtOAc 14.7 Whole Leaf Leaf Leaf Bark observed

revertants/plate: revertants natalensis

+ +

ilicifolia

riparia

spinosum His

His lasiantha

multicava

rudis of of

species Plant

DMSO 4

Plant Tetradenia 10% Canthium Crassula Coddia Cassinopsis Conostomium Laygenia 4-QNO 271.7 Table Number Number ethanol.

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

1.8 0.14

1.6 0.12

1.4 0.10 1.2

0.08 1.0

0.8 0.06

Total phenolics DW) GAE/g (mg 0.6 0.04 Gallotannin concentration (mgGAE/g DW0 0.4 0.02 0.2

0.0 0.00

TrL CsL CmW CrL CiL CiB CnL TrL CsL CmW CrL CiL CiB CnL

Plant samp le Plant sample

Fig. 3. Gallotannin concentrations expressed as gallic acid equivalents, detected in

Fig. 2. Total phenolic compositions expressed as gallic acid equivalents detected in

the investigated medicinal plants. DW, dry weight; GAE, gallic acid equivalents; TrL,

the evaluated medicinal plants. DW, dry weight; GAE, gallic acid equivalents; TrL,

Tetradenia riparia leaﬂ; CsL, Canthium spinosum leaf; CmW, Crassula multicava whole

Tetradenia riparia leaf; CsL, Canthium spinosum leaf; CmW, Crassula multicava whole

plant; CrL, Coddia rudis leaf; CiL, Cassinopsis ilicifolia leaf; CiB, Cassinopsis ilicifolia

bark; CnL, Conostomium natalensis leaf.

2000). Hence, the evaluated medicinal plants which are admin- could be responsible for the observed pharmacological activities in

istered orally in treating stomach-related ailments may result in the present study.

many potential carcinogenic compounds to be metabolized after The highest amount of gallotannins was detected in the leaf

enzymatic activation within the body which could lead to adverse extract of Coddia rudis and Conostomium natalensis (0.12 and

effects. The absence of mutagenic activity shown by the extracts 0.118 mg GAE/g) respectively (Fig. 3). Extracts of Tetradenia riparia

does not indicate absolute safety of these extracts as their metabo- had the lowest amount of gallotannin (0.016 mg GAE/g). Gallotan-

lites could be mutagenic. At this stage, the consumption of these nins have been reported to exhibit signiﬁcant biological activities

plants in traditional medicine could be said to be reasonably safe including antimicrobial, anti-inﬂammatory and anticancer effects

dependent on further studies such as toxicity testing and the use (Bruneton, 1995). The mechanism of anti-inﬂammatory activity of

of metabolizing enzymes that will enhance bioactivation of muta- gallotannin is based on its ability to scavenge free radicals that

genic metabolites that might be present in the extracts. can initiate inﬂammatory responses and the inhibition of various

pro-inﬂammatory mediators, such as the COX-2 enzyme, inducible

3.2. Phytochemical contents nitric oxygen species and prostaglandins (PGs) (Polya, 2003). The

presence of gallotannins at varying concentrations in all the evalu-

Total phenolic composition as depicted in Fig. 2 shows vary- ated medicinal plants may explain the observed antimicrobial and

ing concentrations of phenolics in all the evaluated plant extracts. anthelmintic activities as well as good anti-inﬂammatory effects

The leaf extract of Conostomium natalensis had the highest amount especially against COX-2 by most of the plant extracts. This pro-

of total phenolic compounds (1.7 mg GAE/g) compared to other vides evidence indicating why the selected medicinal plants are

extracts. The extract of Tetradenia riparia had the lowest amount used in traditional medicine in treating stomach pains and cramps

of total phenolic compounds (0.2 mg GAE/g) but exhibited the best associated with stomach-related ailments.

antimicrobial, anthelmintic and cyclooxygenase-inhibitory activ- Relatively high levels of ﬂavonoids were detected in all the

ities. This result suggests that Tetradenia riparia might have a evaluated plant extracts (Fig. 4). The highest concentrations of

quality composition of bioactive compounds better than the other ﬂavonoids were detected in the leaf extracts of Canthium spinosum

extracts evaluated. The observed pharmacological activities exhib- (0.26 mg CTE/g) and Coddia rudis (0.27 mg CTE/g). The anti-

ited among some of the studied plants could be due to other types of inﬂammatory and antimicrobial activities of several ﬂavonoids

phytochemicals such as tannin that were not screened for. Tannin are well known. They inhibit various pro-inﬂammatory media-

has been reported to have anti-diarrhoeal properties, and vasocon- tors such as PGs, COX and lipooxygenase by modulating essential

strictor effects on small superﬁcial vessels. They can also enhance biosynthetic and signal transduction pathways in organisms and

tissue regeneration in the case of superﬁcial wounds or burns by this activity may be directly related to their radical scavenging

preventing ﬂuid losses (Okuda, 2005). capability (Hodek et al., 2002). The presence of ﬂavonoids at con-

Plant phenolics play important roles as defence agents against siderably high levels in all the evaluated plant extracts could partly

pathogens, for attracting pollinating insects and protecting the be responsible for the observed pharmacological activities of some

plant against adverse environmental factors such as ultravio- plant extracts. Flavonoids are known to exhibit numerous beneﬁ-

let radiation and pest attack (Hodek et al., 2002; Makkar et al., cial effects, but care should be exercised in the application of these

2007). Phytochemical compounds (e.g. phenolics) at lower concen- compounds as therapeutic agents, because some metabolites of

trations have signiﬁcant beneﬁcial effects such as antimicrobial, these compounds could be genotoxic which might lead to adverse

antioxidant and anti-inﬂammatory, antiviral, antimutagenic and effects.

chemopreventic effects (Makkar et al., 2007). Higher concentra- The qualitative froth test for the presence of saponins was

tions of phytochemical compounds, on the other hand, have been positive for only two plant samples Canthium spinosum (leaf)

reported to have negative physiological effects such as neurolog- and Cassinopsis ilicifolia (bark) (Table 5). Saponins are structurally

ical disfunction, gastrointestinal toxicity, and reproductive failure diverse bioactive compounds of plant origin. They consist of

(Polya, 2003). The presence of phenolics at varying concentrations non-polar aglycones coupled with one or more monosaccharide

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

Table 5

Saponin 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 ± 4.8

Cassinopsis ilicifolia Leaf − ND ND

Bark + 11.7 ± 12.3 4.5 ± 9.1

Coddia rudis Leaf − ND ND

Conostomium natalensis Leaf − ND ND

Crassula multicava Whole plant − ND ND

Lagynia lasiantha Leaf − ND ND

Tetradenia riparia Leaf − ND ND

Absence of saponins (−), present of saponins (+), diosgenin equivalence (DE), not determined (ND).

0.30 in the evaluated plant species and this might lead to developing

superior drugs with potent pharmacological activities.

0.25 Acknowledgements

0.20

The authors thank Mrs A. Young of the University of KwaZulu-

Natal Botanical Garden and Dr. C. Potgieter (NU Herbarium) for their

0.15

assistance in plant identiﬁcation and voucher specimen prepara-

tions. The National Research Foundation (NRF), Pretoria and the

0.10 University of KwaZulu-Natal are acknowledged for ﬁnancial support. Flavonoid concentration Flavonoid (mgCTE/g DW)

0.05

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