Screening for antimicrobial activity of ten medicinal plants used in Colombian folkloric medicine: A possible alternative in the treatment of non-nosocomial infectionsJhon J Rojas, #1 Veronica J Ochoa,#1 Saul A Ocampo,#1 and John F Muñoz#1

1Department of Pharmacy, Universidad de Antioquia, Cll. 67 # 53-108 of. 1-157, Medellín, ColombiaCorresponding author.

#Contributed equally.Jhon J Rojas: jrojas@farmacia.udea.edu.co; Veronica J Ochoa: vochoa@farmacia.udea.edu.co; Saul A Ocampo:socampo@farmacia.udea.edu.co; John F Muñoz: jmuñoz@farmacia.udea.edu.coReceived July 4, 2005; Accepted February 17, 2006.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 This article has been cited by other articles in PMC.

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Abstract

Background

The antimicrobial activity and Minimal Inhibitory Concentration (MIC) of the extracts

ofBidens pilosa L., Bixa orellana L., Cecropia peltata L., Cinchona officinalis L.,Gliricidia

sepium H.B. & K, Jacaranda mimosifolia D.Don, Justicia secunda Vahl.,Piper

pulchrum C.DC, P. paniculata L. and Spilanthes americana Hieron were evaluated against

five bacteria (Staphylococcus aureus, Streptococcus β hemolític,Bacillus

cereus, Pseudomonas aeruginosa, and Escherichia coli), and one yeast (Candida albicans).

These plants are used in Colombian folk medicine to treat infections of microbial origin.

Methods

Plants were collected by farmers and traditional healers. The ethanol, hexane and water

extracts were obtained by standard methods. The antimicrobial activity was found by using

a modified agar well diffusion method. All microorganisms were obtained from the American

Type Culture Collection (ATCC). MIC was determined in the plant extracts that showed

some efficacy against the tested microorganisms. Gentamycin sulfate (1.0 μg/ml),

clindamycin (0.3 μg/ml) and nystatin (1.0 μg/ml) were used as positive controls.

Results

The water extracts of Bidens pilosa L., Jacaranda mimosifolia D.Don, and Piper

pulchrum C.DC showed a higher activity against Bacillus cereus and Escherichia coli than

gentamycin sulfate. Similarly, the ethanol extracts of all species were active

against Staphylococcus aureus except for Justicia secunda. Furthermore, Bixa

orellana L, Justicia secunda Vahl. and Piper pulchrum C.DC presented the lowest MICs

against Escherichia coli (0.8, 0.6 and 0.6 μg/ml, respectively) compared to gentamycin

sulfate (0.9 8g/ml). Likewise, Justicia secunda and Piper pulchrum C.DC showed an

analogous MIC against Candida albicans (0.5 and 0.6 μg/ml, respectively) compared to

nystatin (0.6 μg/ml). Bixa orellana L, exhibited a better MIC againstBacillus cereus (0.2

μg/ml) than gentamycin sulfate (0.5 μg/ml).

Conclusion

This in vitro study corroborated the antimicrobial activity of the selected plants used in

folkloric medicine. All these plants were effective against three or more of the pathogenic

microorganisms. However, they were ineffective

against Streptococcus βhemolytic and Pseudomonas aeruginosa. Their medicinal use in

infections associated with these two species is not recommended. This study also showed

thatBixa orellana L, Justicia secunda Vahl. and Piper pulchrum C.DC could be potential

sources of new antimicrobial agents.

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Background

In developing countries and particularly in Colombia, low income people such as farmers,

people of small isolate villages and native communities use folk medicine for the treatment

of common infections. These plants are ingested as decoctions, teas and juice preparations

to treat respiratory infections[1]. They are also made into a poultice and applied directly on

the infected wounds or burns.

When people from these remote communities get an infectious disease, they are usually

treated by traditional healers and shamans because of their expertise in such procedures as

making diagnoses, treating wounds, setting bones and making herbal medicines. Traditional

healers claim that their medicine is cheaper and more effective than modern medicine.

Patients of these communities have a reduced risk to get infectious diseases from resistant

pathogens than people from urban areas treated with traditional antibiotics. However, if they

are treated in a hospital the chance of contracting a nosocomial infection is increased[2].

One way to prevent antibiotic resistance of pathogenic species is by using new compounds

that are not based on existing synthetic antimicrobial agents[3]. Traditional healers claim

that some medicinal plants such as bixa spp. and bidens spp. are more efficient to treat

infectious diseases than synthetic antibiotics. It is necessary to evaluate, in a scientific

base, the potential use of folk medicine for the treatment of infectious diseases produced by

common pathogens. Medicinal plants might represent an alternative treatment in non-

severe cases of infectious diseases. They can also be a possible source for new potent

antibiotics to which pathogen strains are not resistant. [4].

We chose ten species used in folk medicine to determine their antimicrobial activity:B.

pilosa L. (Asteraceae), B. orellana L. (Bixaceae), C. peltata L. (Moraceae), C. officinalis L.

(Rubiaceae), G. sepium H.B. & K (Fabaceae), J. mimosifolia D.Don(Bignoniaceae), J.

secunda Vahl. (Acanthaceae), P. pulchrum C.DC (Piperaceae),P. paniculata L.

(Polygalaceae), and S. americana Hieron (Asteraceae). In general, these plants are used in

folk medicine in the treatment of pharyngitis, gingivitis, bronchitis, infected wounds, topical

ulcers, and as antiparasitic agents.

The extract of B. pilosa is used in folk medicine as an antihelmintic and protozoacide agent;

it also has antiseptic properties[5]. It contains flavonoids [6]. The ethanol extract of the

leaves of B. orellana possesses antimicrobial activity against Gram (+) microorganisms

and C. albicans [7]. Also, its leaves have been employed to treat malaria and

leishmaniasis[8]. Its seeds contain carotenoids [9]. The ethanol extract of C. Peltata has

been used as an antibilious, cardiotonic and diuretic agent[10]. In addition, its leaves have

been employed against blennorrhea and warts[11,12]. The decoction of the leaves of C.

officinalis is used to treat amebiasis. Its dry bark is active against P. falciparum, and

herpes[13]. It contains quinoline alkaloids[14]. Branches and leaves of G. sepium are used

to reduce fever in children and adults. It has also been used as insecticide and to treat

infections produced by Microsporum canis, Trichophyton mentagrophytes, and Neisseria

gonorrohae [15]. Its leaves contain triterpene saponins (I and II)[16]. The water extract of J.

mimosifolia is active against P. aeruginosa. Its flowers contain flavones and flavonoids[17].

Its leaves have iridoids, triterpenes, flavones, and steroids[18]. J. secunda has been used to

disinfect scorpion wounds[19]. P. pulchrum is used to disinfect snakebites[20]. Other

species exhibit antimicrobial activity against P. aeruginosa andC.albicans [21].Polygala spp.

possesses trypanocidal activity[22,23]. It contains coumarins [24]. Flowers of S.

americana are used to treat mouth infections and some varieties of herpes. It contains

spilantol[25].

Evidently, there are not sufficient scientific studies that confirm the antimicrobial properties

of these plants. This study looks into the in vitro antimicrobial activity of these plants against

six pathogenic microorganisms that cause the most common cases of infectious diseases of

poverished communities in Colombia.

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Methods

Plant material

All the plants were collected by farmers and traditional healers from the Andes and pacific

region of Colombia (Antioquia, Chocó and Huila departments). All the species were

identified by Professor Francisco Roldan (Instituto de Biología) in the Herbarium of the

Universidad de Antioquia (HUA). Voucher numbers and plant organs are shown in Table 1.

Preparation of plant extracts

The plant extracts were prepared using the modified method of Alade & Irobi [26]. Briefly,

three 100 g portions of the dried powdered plant were soaked separately in 500 ml of

distilled water, ethanol (98 %) and η-hexane (99 %), for 72 h. Then, each mixture was

refluxed followed by agitation at 200 rpm for 1 h. The filtrates obtained were concentrated

under vacuum at 40°C to obtain the dry extracts (Table 1). '[seeAdditional file 1]'.

Determination of antimicrobial activity

Microorganisms used

The test organisms (S. aureus ATCC 29737, S. β hemolític ATCC 10389, B. cereusATCC

14603, P. aeruginosa ATCC 25619, E. coli ATCC 10536, and C. albicansATCC 53324)

were obtained from the microbiology laboratory, College of Medicine of the Universidad de

Antioquia.

Culture media

The medium used for the activation of the microorganisms was soybean casein broth

(SBCB). The following selective agar media were used for the antimicrobial test: Baird-

Parker (S. aureus), Cetrimide (P. aeruginosa), McConkey (E. coli), Blood (S. β hemolitic),

Nutritive (B. cereus), and Saboraud-dextrose (C. albicans). All the culture media were

prepared and treated according to the manufacturer guidelines (Becton Dickinson® M.D.

USA).

Inoculum

The microorganisms were inoculated into SBCB and incubated at 35 ± 2°C for 4 h. The

turbidity of the resulting suspensions was diluted with SBCB to obtain a transmittance of

25.0 % at 580 nm. That percentage was found spectrophotometrically comparable to 1

McFarland turbidity standard. This level of turbidity is equivalent to approximately 3.0 ×

108 CFU/ml. The Bausch & Lomb®spectrophotometer, Model Spectronic 20 was used to

adjust the transmittance of the working suspensions.

Agar diffusion assay

The modified agar well diffusion method of Perez et al., [27] was employed. Each selective

medium was inoculated with the microorganism suspended in SBCB. Once the agar was

solidified, it was punched with a six millimeters diameter wells and filled with 25 μL of the

plants extracts and blanks (ethanol, distilled water, and hexane). The concentration of the

extracts employed was 25 μg/ml. Simultaneously, gentamycin sulfate (S. aureus, P.

aeruginosa, E. coli, and B. cereus), clindamycin (S. β hemolytic), and nystatin (C. albicans)

were used as positive controls at a concentration of 1.0, 0.3 and 1.0 μg/ml respectively. The

dilution medium for the positive controls was sterile distilled water. The test was carried out

by triplicate. The plaques were incubated at 35 ± 2°C for 24 h, except for C. albicans which

was incubated at 29 ± 2°C. The antimicrobial activity was calculated by applying the

expression:

Where RIZD is the percentage of relative inhibition zone diameter and IZD is the inhibition

zone diameter (mm). Equation (1) compensates the possible effect of the solvent (blank)

other than water on the IZD. The resulting IZD of the samples were either higher than or

equal to the IZD of the blanks. Therefore, the obtained percentages were positive (Table 1).

'[see Additional file table1]'. The test was considered negative (-) when the IZD of the

sample was equal to the IZD of the blank.

Minimal inhibitory concentration (MIC) evaluation

The MIC was evaluated on plant extracts that showed antimicrobial activity. This test was

performed at four concentrations of each extract (6.3, 12.5, 25, 50 μg/ml) employing the

same modified agar well diffusion method. Calculations of MIC were determined by

regression analysis using the software STATGRAPHICS® v. 4 (Table 2). '[see Additional file

table1]'.

Phytochemical screening

The method of Martinez & Valencia [28] was implemented to identify the general

phytochemical groups of compounds in the extracts (Table 1). '[see Additional file table1]'.

The test for amino acids was conducted by dissolving 10 mg of dry extracts in 1 ml of

ethanol and adding 1 droplet of ninhidrine reagent. For flavonoids, Shimoda's test was

adopted (15 mg of dry extract was dissolved in 1 ml of ethanol, concentrated HCl, and

magnesium turnins were added). Anthocyanins were identified by adding 1 ml of boiling

water, 0.5 ml of 37 % HCl to 10 mg of dry extract. The solution was heated at 100°C, cooled

and added 0.4 ml of amylic alcohol.

The test for phenolic compounds was carried out by dissolving 10 mg of dry extract in 1 ml

of 1 % ferric chloride solution. For tannins, 1 ml of the gelatin reagent was added to 1 ml of

the filtered aqueous extract. Quinones were identified by extracting 10 ml of the aqueous

extract with dichloromethane, evaporating the organic phase, and adding 5 ml of ethanol, 1

ml of hydrogen peroxide 5 % and 1 ml of sulfuric acid 50 % respectively. The solution was

heated, cooled, extracted with benzene and 1 ml of ammonia solution added.

Cardiac glycosides were identified by evaporating 1 ml of the organic phase, dissolving the

residue in 1 ml of ethanol and adding 0.5 ml of Kedde's reagent. For triterpenoids and

steroids, 0.5 ml of acetic anhydride and 1 droplet of 37 % sulfuric acid solution were added

to 0.5 ml of the organic phase. The test for alkaloids was carried out by adding 0.5 ml of the

aqueous extract into four test tubes; boiled, filtered and one droplet of the reagents of

Mayer, Valser, Dragendorff and ammonium Reineckate was added respectively.

Statistical analysis

All values are expressed as means ± standard deviation (Table 2). '[see Additional file

table1]'. The MIC data for each microorganism were analyzed using one-way analysis of

variance (ANOVA) and the differences among group means were analyzed using the

Dunnett's multiple comparisons test. P value < 0.05 was considered as significant. The

software MINITAB® was employed for the statistical analysis.

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Results and discussion

All the plants showed antimicrobial activity in regards to at least three microorganisms

tested (Table 1). '[see Additional file table1]'. The ethanol extracts ofB. orellana (seeds), G.

sepium, J. mimosifolia and P. pulchrum were the most active against the microorganisms

studied. In some cases, the three extracts of the same plant had antimicrobial activity

against the same microorganism. For instance, the three extracts of J. mimosifolia were

active against B. cereus. This possibly means that the compound responsible for the

antimicrobial activity was present in each extract at a different concentration.

All the plants exhibited different kinds of secondary metabolites. In particular, J.

mimosifolia presented the highest yield of extractable substances in the water extract

(15.0%). This result indicates that decoction is a good method to extract anthocyanins,

phenolic compounds, and alkaloids found in this species. These compounds could be

responsible for its antimicrobial activity against B. cereus andE. coli. Former studies of the

water extract revealed antimicrobial activity against P. aeruginosa [17]. However, we did not

find antimicrobial activity against this pathogenic microorganism. Furthermore, the water

extract of J. mimosifolia, and P. pulchrum showed a higher activity (compared to the

gentamycin sulfate) against B. cereus. The water extract of P. pulchrum also showed

antimicrobial activity againstS. aureus.

Ethanol extracts exhibited a higher degree of antimicrobial activity as compared with water

and hexane extracts fractions. This finding is correlated with the medicinal preparations that

use rum and liquor to extract the active plant components.

E. coli, B. cereus and S. aureus were the most susceptible bacteria to all plant extracts. On

the contrary, S. β hemolytic, P. aureginosa and C. albicans were the most resistant

microorganisms. None of the extracts was more active against S. βhemolytic than the

positive control (clindamycin). Likewise, the ethanol extract of J. secunda was the only

extract active against P. aeruginosa. Only three plants (J. secunda, P. pulchrum and P.

paniculata) were the most active against C. albicans. This result is in accord with formers

studies executed on Piper spp. [21].

Steroids and anthocyanins of B. orellana (seeds) could be responsible for their antimicrobial

activity against S. aureus, B. cereus and E. coli. Similarly, the presence of steroids and

amino acids in C. peltata could correspond to its high antimicrobial activity exhibited

against E. coli. B. pilosa showed a low activity against S. aureusand B. cereus. However,

some studies established that the methanol extract of this plant is highly active against S.

aureus, S. epidermidis and B. subtilis [29].

C. officinalis was the species that exhibited the greatest variety of secondary metabolites. It

also showed antimicrobial activity against all the pathogens studied. Similarly, S.

americana presented antimicrobial activity against all the microorganisms studied except

for C. albicans. Alkaloids and steroids found in this plant might account for this. Former

studies associated the alkaloid spilantol with its biological activity [21]. This plant also

showed the lowest yield of extractable solids among all the plants (0.02%).

Table 2 shows that J. secunda and P. pulchrum presented similar MICs against C.

albicans (0.5 and 0.6 μg/ml, respectively) compared to nystatin (0.6 μg/ml). Moreover, these

two plants manifested a better MIC against E. coli (0.6 μg/ml) than gentamycin sulfate (0.9

μg/ml). However, there was no statistical difference between MICs of seven plants and

gentamycin sulfate. J. secunda and B. orellana were the only plants that exhibited a similar

MIC against P. aeruginosa (1.3 and 4.8 μg/ml) compared to gentamycin sulfate (0.3 μg/ml).

B. orellana L, J. secunda and P. pulchrum presented the lowest MICs against E. coli(0.8,

0.6 and 0.6 μg/ml, respectively) compared to gentamycin sulfate (0.9 μg/ml). Likewise, B.

orellana manifested the lowest value of MIC against B. cereus (0.2 μg/ml) compared to

gentamycin sulfate (0.5 μg/ml). However, for B. cereus there was no statistical difference

between MICs of eight plants and gentamycin.

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Conclusion

All the extracts showed varying degrees of antimicrobial activity on the microorganisms

tested. Some of these plants were more effective than traditional antibiotics to combat the

pathogenic microorganisms studied. The chance to find antimicrobial activity was more

apparent in ethanol than water extracts of the same plants. Three species (B. orellana, J.

secunda and P. pulchrum presented the lowest MIC compared to the antibiotic standard.

These plants could be a source of new antibiotic compounds. Further work is needed to

isolate the secondary metabolites from the extracts studied in order to test specific

antimicrobial activity.

This in vitro study demonstrated that folk medicine can be as effective as modern medicine

to combat pathogenic microorganisms. The millenarian use of these plants in folk medicine

suggests that they represent an economic and safe alternative to treat infectious diseases.

However, none of the plants are recommended in the treatment of infections produced by S.

β hemolitic and P. aeruginosa.

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List of abbreviations

ATCC – American Type Culture Collection

B. cereus – Bacillus cereus

B. pilosa – Bidens pilosa L

B. subtilis – Bacillus subtilis

B. orellana – Bixa orellana L.

C. albicans-Candida albicans

C. peltata – Cecropia peltata L.

C. officinalis – Cinchona officinalis L.

E. coli – Escherichia coli

G. sepium – Gliricidia sepium H.B. & K

J. secunda – Justicia secunda Vahl

J. mimosifolia – Jacaranda mimosifolia D.Don

P. aeruginosa – Pseudomonas aeruginosa

P. pulchrum – Piper pulchrum

P. paniculata – Polygala paniculata L.

SBCB-Soybean casein broth

S. β hemolitic – Streptococcus β hemolític

S. aureus – Staphylococcus aureus

S. epidermidis – Staphylococcus epidermidis

S. americana – Spilanthes americana Hieron

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

JJR conceived and designed the study, performed the MIC tests, analyzed the data

obtained and drafted this paper. JFM and SAO interviewed local healers and farmers; they

also performed the phytochemical screening and the antimicrobial tests. VJO Participated in

the extraction, susceptibility testing and analysis of data. All authors read and approved the

final manuscript.

Pre-publication history

The pre-publication history for this paper can be accessed here:

http://www.biomedcentral.com/1472-6882/6/2/prepub

Supplementary Material

Additional file 1

Table 1 – Antimicrobial activity and phytochemicals screening of the plants studied. Table 2

– Minimum Inhibitory Concentration of the plants studied.

Click here for file(53K, rtf)

Acknowledgements

The authors acknowledge the assistance of the technicians Johny Sánchez, Gloria A.

Valencia, and Jorge Arango. We also acknowledge local healers for their assistance in

finding the plant species. Similarly, we acknowledge Professor Francisco Roldán for

assisting us with the identification of plant materials. We thank Professor Carol Severino for

checking the language and style of this manuscript. This work was supported by grants from

the Pharmacy department of the Universidad de Antioquia.

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Antibacterial activity and phytochemical study of six medicinal plants used in Benin.Anago E, Lagnika L, Gbenou J, Loko F, Moudachirou M, Sanni A.

Source

Laboratory of Biochemistry and Molecular Biology, Institute des Sciences Biomédicales Appliquées (ISBA), University of

Abomey-Calavi, Cotonou, Republic of Benin.

AbstractThe ethanol extracts obtained from Psidium guajava, Flacourtia flavescens Boswellia dalzielii, Ficus exasperata, Pavetta corymbosa and Hybanthus enneaspermus, six species traditionally used in Benin to treat several infectious diseases, were evaluated for their in vitro antimicrobial activity against Staphylococcus aureus, Enteroccocus feacalis, Escherichia coli and Pseudomonas aeruginosa. The minimum inhibitory concentration of extracts was determinate using the microplate dilution method. The presence of major phytoconstituents was detected qualitatively. The diphenylpicrylhydrazine radical scavengingactivity was also performed. The extracts exhibited antibacterial activities against the tested bacteria. Boswellia dalzielii,Psidium guayava, Pavetta corymbosa and Flacourtia flavescens exibited the lowest minimum inhibitory concentration values (0.313-2.5 mg mL(-1)). Pseudomonas aeruginosa was the lest sensitive microorganism with MIC values higher than 10 mg mL(-1). In antioxidant assay the crude extracts of B. dalzielii and P. corymbosa appeared to be as potent as quercetol with an inhibition percentage of 83 and 75.3% at 10 microg mL(-1) which is comparable to 75.9% for quercetol at the same concentration.

In-vitro antibacterial activity of selected medicinal plants from Longisa region of Bomet district, KenyaK R Cheruiyot, D Olila, and J KatereggaDepartment of Physiological Sciences, Faculty of Veterinary Medicine, Makerere University, P. O Box 7062, Kampala-UgandaCorresponding author Cheruiyot K. R Email: cheruiyot@vetmed.mak.ac.ug, cheruiyotronald@gmail.com, Mobile Number: +254724601814

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Abstract

Background

Current strategies to overcome the global problem of antimicrobial resistance include

research in finding new and innovative antimicrobials from plants. This study was carried

out to determine the antibacterial activity of plant extracts of Olea africanastem-

bark, Psidium guajava leaves, Vernonia amygdalina leaves, Lantana camaraleaves

and Mangifera indica leaves which are used in folklore medicine to treat infections of

microbial origin in Longisa region of Bomet District, Kenya.

Methods

Methanol extracts were derived and screened. Standard cultures of Escherichia coliATCC

25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureusATCC 25923

were used in the study. The antibacterial tests used were the agar well diffusion assays at

concentration 1gm/ml. Minimum Inhibition Concentration (MIC) was determined in the plant

extract that showed some efficacy against the tested microorganisms. Gentamicin (10µg)

was used as a positive control.

Results

The methanol extracts showed weak antibacterial activity against the study organisms

compared to Gentamicin. All extracts exhibited a significant bactericidal activity against S.

aureus while L. camara and V. amygdalina lacked efficacy againstP.aeruginosa and E. coli.

O. africana and P. guajava presented the lowest MIC against S.aureus (62.5 mg/ml and 250

mg/ml respectively P.guajava and M. indicashowed analogous MICs

against P.aeruginosa (250 mg/ml). P.guajava exhibited a better MIC against E.coli (500

mg/ml).

Conclusions

This in-vitro study corroborated the antimicrobial activity of the selected plants used in

folklore medicine. The plants could be potential sources of new antimicrobial agent.

Key words: Medicinal Plant extracts, antibacterial activity, MIC

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Introduction

Many works have been done which aim at knowing the different antimicrobial and

phytochemical constituents of medicinal plants and using them for the treatment of microbial

as possible alternatives to chemically synthetic drugs to which many infectious

microorganisms have become resistant. Moreover, antibacterial pharmaceuticals are not

accessible to the majority of the communities in the developing countries1. Increase in

resistance calls for new antibacterial drugs, one source of which are traditional medicinal

plants. Plants may provide natural source of antimicrobial drugs that will/or provide novel or

lead compounds that may be employed in controlling some infections globally.

Olea africana (Oleaceae) is a tree of about 3–15 m in height, may assume bushy habit if

stunted. Leaf or Stem bark are used to treat sore throat, kidney problems, backache, colic

or urinary tract infections. Olea africana has been demonstrated scientifically possessing;

antimicrobial activity, antiarrhythmic activity, diuretic activity, anti-inflammatory activity and

hepatic activity 2. Phytochemical tests in laboratories indicated the presence of alkaloids,

saponins and tannins, but not of cardiac or cyanogenic glycosides. Psidium guajava L

(Myrtaceae) is a native plant of tropical America. Different parts of the plants are used in

treatment of various human aliments such as wounds, ulcers, bowels and cholera.

Pharmacological investigations indicated that its bark, fruit, and leaves possess,

hypoglycemic, anti-inflammatory, analgesic, antipyretic, spasmolytic, CNS depressant and

antimicrobial activities. The leaves of P. guajava contain an essential oil rich in cineol,

tannins, triterpenes and flavonoids3–5. Vernonia amygdalina (Compositae) is a shrub, of 2–5

m tall with petiolate green leaves of about 6 mm diameter. The roots and the leaves are

used in ethno medicine to treat fever, hiccups, kidney problems, stomach discomfort and

bacterial infections6. The active components of the plant have been shown to be mainly

sesquiterpene lactones like vernodalin and vernoamygdalin and steroid glycosides like

vernonioside B1 and vernoniol B7. Lantana camara Linn.(Verbenaceae) is a rugged

evergreen strong-smelling herb, native of tropical America, but now naturalized in many

parts of Africa. All the parts of this plant have been used traditionally for several ailments

throughout the world. The leaves are used as a bechic, antitumoral, antibacterial and

antihypertensive agent8. The root of this plant is used for the treatment of malaria,

rheumatism and skin rashes9.Mangifera indica (Anacardiaceae) is a large evergreen tree,

with a heavy, dome-shaped crown. It is found all over the tropical regions of the world

where it is used as a horticultural and medicinal plant. The use of leaf extracts as

antimicrobial in the treatment of burns, scalds, sores, wounds, abscesses and other

infections in humans and animals has been reported in a number of ethnobotanical

surveys10. The leaves have been reported to contain saponins, glycosides, unsaturated

sterols, polyphenols, euxanthin acid, mangiferine, mangin, gallic tannins11. This study looks

into the in vitro antibacterial activity of these plants against three pathogenic

microorganisms (Staphylococus aureus, Pseudomonas aeruginosa and Escherichia coli)

that cause the most common cases of infectious diseases of poverished communities in

Bomet District. Qualitative phytochemical screening of alkaloids, tannins, flavonoids,

anthraquinones, saponins, glycoside or cardiac glycosides was carried out using standard

methods 12, 13.

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Materials and method

Plant collection and Identification

Ethno pharmacological information of several medicinal plants was sourced from

literatures14, 15 and through consultation from elderly people in age bracket of above 50 years

from Siwot Village, Longisa division in Bomet district, Kenya, during the month of January

2008. Plants selected for study was obtained by ranking basing on diseases they treated,

frequency of their use and their availability. Taxonomic identity of voucher specimen was

done by comparing with those of known identity in the Makerere University herbarium in

Kampala.

Processing and Extraction of Plant Material

The plant parts were chopped and shade-dried at room temperature for 2 weeks then

grounded using metallic mortar and pestle to a fine powder for ease of extraction of active

compounds. The grounded samples were then transported for extraction process at the

Pharmacology Laboratory of Faculty of Vet medicine, Makerere University. The grounded

powder were weighed on Satorius balance type BA 610, soaked in known volumes of 95 %

Analytical Methanol and allowed to stand for two days with intermittent shaking. Filtration

through cotton wool was done to remove coarse particles and finely through filter paper

(Whatman® No.1, England) in Buchner funnel. This was followed by concentration on

Rotavapour type Buchi-R-Switzerland at 50 °C to recover the solvent used. Extracts were

dissolved in small drops of Dimethyl sulfoxide (DMSO) and toped up with Physiological

saline and the stock solution kept at 4 °C.

Preparation of the test micro-organisms

This process followed the previously established procedures for testing antimicrobial

agents. Standard cultures of bacteria from the American Type Culture Collection were

obtained from the Microbiology Laboratory,Faculty of Veterinary Medicine of Makerere

University. A Gram positive bacterium; Staphylococcus aureus ATCC 25923, was used as a

wound/skin pathogen and Gram negative bacteria;Escherichia coli ATCC 25922, was used

to represent pathogens that cause gastro enteritis while Pseudomonas aeruginosa ATCC

27853, was used as an environmental pathogen16. Standardized bacterial suspension was

prepared by picking a colony of respective bacteria using sterile wire loop and suspending it

in 5 ml Brain heart infusion liquid media. The dilutions formed the bacterial stock solutions

for use in the agar-well diffusion assays.

Preparation of culture media

Mueller Hinton agar (Becton Dickinson ® M.D USA) was used for direct sensitivity testing.

The media was prepared and treated according to manufacturer's guidelines. Thirty five

(35) g medium was mixed with one litre of distilled water, enclosed in a screw cap container

and autoclaved at 121 °C for 15 minutes. The medium was later dispensed into 90 mm

sterile agar plates and left to set. The agar plates were incubated for 24 hours at 37 °C to

confirm their sterility. When no growth occurred after 24 hours, the plates were considered

sterile.

Agar-Well Diffusion Assay

A concentration of 1 g/ml of the plant extracts was designed from the stock solution for agar

well diffusion assay. Cultures of S.aureus, E.coli and P.aeruginosa were inoculated

separately on the surface of Mueller Hinton agar plates by surface spreading using a sterile

cotton swab and each bacterium evenly spread over the entire surface of agar plate to

obtain a uniform inoculum. The sensitivity testing of the plant extracts was done using the

agar well diffusion method17 whereby, wells of 6 mm diameter and 5 mm depth were made

on the solid agar using a sterile glass borer. About 50 µl of the methanol extract, of the

concentration 1 g/ml, was dispensed into respective wells and 10 µg Gentamycin was used

as a positive control since it is a broad spectrum antibiotic. Physiological saline/Dimethyl

sulfoxide (DMSO) was used as negative control. All the tests were run in triplicates for

quality results. The set up was incubated for 24 hours at 37 °C. Twenty four (24) hours later,

the zones of inhibition were measured using a ruler (AIM®) and a pair of divider then results

reported in millimeters (mm).

Minimum Inhibition Concentration (MIC) Evaluation

The MIC was evaluated on plant extracts that showed antibacterial activity in the agar well

diffusion assay on any organism. This test was performed at five concentration of each

extract (500 mg/ml, 250 mg/ml, 125 mg/ml, 62.5 mg/ml and 31.25 mg/ml) employing

doubling dilutions of plant extract in Brain heart infusion broth up to the fifth dilution. One (1)

ml of the resultant broth was put in test tube and equal amounts of the extracts (1 ml) were

added to the first test tube and serial dilution done with the last 1 ml being discarded. To

complete the test, each organism was separately suspended in 5 ml of Brain heart infusion

broth and incubated overnight, after which 0.1 ml was added to all the test tubes and

preparation incubated at 37 °C for 18 hours. After incubation, a loop full from each tube was

sub cultured on nutrient agar to see if bacteria growth was inhibited (Minimum Bactericidal

Activity). Growth of bacteria on solid media indicated that particular concentration of the

extract was unable to inhibit the bacteria. The MIC was defined as the lowest concentration

of an antimicrobial that inhibited the visible growth of a microorganism after overnight

incubation 18.

Qualitative Phytochemical analysis of the plant extract

This was a qualitative analysis done at the department of Botany Laboratory, Faculty of

Science, Makerere University. A small portion of the extract was used for phytochemical

screening test. Alkaloids, Saponins, Tannins, Flavonoids, Glycosides, Cardiac glycosides

and Anthraquinones was carried out using standard methods12,13

Data Analysis

Microsoft Excel® was used to enter and capture data. Various graphs and tables were

extracted from this data. Data was then exported to SPSS for further analysis. The MIC for

each microorganism was analyzed using one-way analysis of variance (ANOVA). P value <

0.05 was considered as significant. SPSS 15.0 was employed for statistical analysis.

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Results and discussion

Qualitative phytochemical screening indicated that the methanol plant extracts contained

classes of compounds as shown in Table 1. All plants exhibited different kinds of secondary

metabolites. This probably contributed to their antibacterial activity. All extracts showed

presence of Tannins and Flavonoids as seen in Table 1.Mangifera indica Contained all

screened metabolites except Cardiac Glycosides. This result is in accord with former

studies executed on the M. indica ethanol extract of its leaves 10. The bioactive ingredients

could be responsible for antibacterial activity of the plant extracts. Tannins have been found

to form irreversible complexes with proline-rich proteins resulting in the inhibition of the cell

protein synthesis. This activity was exhibited by Mangifera indica and Psidium

guajava against test organisms19. Previous studies show that secondary plant metabolites

constitute an important source of microbicides, pesticides and many pharmaceutical

drugs20, 21.

Table 1Classes of Bioactive compounds identified in the Methanol plant extract

The results showed that plant extracts demonstrated antibacterial activity against

theS.aureus, E.coli and P.aeruginosa. It can be noted from Figure 1 thatStaphylococcus

aureus was the most susceptible of the three organisms and E.colithe least. All the plant

species showed activity against Staphylococcus aureus (Fig 1). Psidium guajava had the

highest zone of inhibition to the other extracts (19.7 mm) against S.aureus, while Olea

africana had the least MIC (62.5 mg/ml) againstS.aureus signifying higher activity. From the

three methanol extracts with activities against E.coli, Psidium guajava was the most active

with a Zone of inhibition of 16 mm and an MIC of 500 mg/ml. This was followed

by Mangifera indica (13 mm), with an MIC of 1000 mg/ml and lastly Olea africana with a

zone of inhibition of 8.3 mm and an MIC of 1000 mg/ml. The most active methanol plant

extract from the study againstPseudomonas aeruginosa was Mangifera indica with a Zone

of Inhibition of 17 mm and an MIC of 250 mg/ml, followed by P.guajava (16 mm) with an

MIC of 250 mg/ml. This was followed by Olea africana with a Zone of Inhibition of 9.8 mm

and an MIC of 1000 mg/ml. The most active methanol plant extract on Staphylococcus

aureus wasPsidium guajava with a zone of inhibition of 19.7 mm and an MIC of 250 mg/ml,

followed by Olea africana and Mangifera indica both with analogous zone of inhibition (18.5

mm) and a better MIC of 62.5 mg/ml and 500 mg/ml respectively. Lastly wereVernonia

amygdalina and Lantana camara both with zone of inhibition of 17 mm and an MIC of 1000

mg/ml. Escherichia coli showed resistance to Vernonia amygdalinaand Lantana

camara while Pseudomonas aeruginosa was resistant to Vernonia amygdalina. According

to statistical analysis results of one way ANOVA ofStaphylococcus aureus the P-value was

0.306, F- calculated was 2.5 and F-critical was 19.2, one way ANOVA for Pseudomonas

aeruginosa; P-value was 0.808, F-calculated was 0.225 and F-critical was 6.94. Lastly one

way ANOVA for Escherichia coli P-value was 0.530, F-calculated 0.455 and F-critical 6.61.

Since P-values were greater than 0.05 (P >0.05). Hence, we fail to reject the null hypothesis

and conclude that there is no significant variation in the MIC at 95% confidence Interval for

each of the test organisms.

Fig 1Chart showing the Zone of inhibition of Plant Methanol Extract

The zones of inhibition produced by the test organisms indicated their susceptibility to the

plant extracts; it was observed that the zones of inhibition varied from one organism to

another and from one plant part extract to another. According to Prescott22 the effect of an

agent varies with target species. The effective use ofP.guajava in diarrhea, dysentery and

gastroenteritis can be related to guava's documented antibacterial properties. Bark and leaf

extracts have shown to have in vitro toxic action against numerous bacteria. In several

studies guava showed significant antibacterial activity against such common diarrhea-

causing bacteria asStaphylococcus, Shigella, Salmonella, Bacillus, E. coli, Clostridium, and

Pseudomonas (3–5). Olea africana had the least MIC (62.5 mg/ml)

against S.aureussignifying higher activity in this study. The plant showed broad spectrum

activity against the tested organisms. This justified the use of plant in treatment of infections

of microbial origin2. Vernonia amygdalina lacked efficacy against P. aeruginosa.

Also, Vernonia amygdalina and Lantana camara extracts did not have measurable activity

against E.coli. These observations are likely to be the result of the differences in the cell

wall structure between gram-negatives and gram-positive bacteria, with gram-negative

outer membrane acting as a barrier to many environmental substances, including

antibiotics23.

Vernonia amygdalina showed no activity against E.coli and P.aeruginosa thus indicating its

narrow spectrum of activity according to the study. A similar study was carried out to

determine the antibacterial potential of V. amygdalina using a panel multidrug resistant

gram-negative and gram-positive bacteria and standard strains:E. coli ATCC 25922

and Staphylococcus aureus ATCC 25923. Aqueous extract of V. amygdalina leaves was

found to produce growth inhibitory zones of 15.6 to 16.1 mm for E. coli and 12.1–12.3 mm

for S.aureus while two out of the eight Pseudomonas aeruginosa isolates tested showed

susceptibility (inhibitory zone diameter of 6.7+0.2 mm) to V. amygdalina by agar well

diffusion only. This result was in contrary to our results and indicated that water could be the

ideal solvent for extraction24. Lantana camara showed activity against Staphylococcus

aureus and was inactive againstE.coli. Related studies on Lantana camara root-bark

prepared with solvents of different polarity, was evaluated by the agar-well diffusion method.

Twelve bacteria, six each of gram-positive and gram-negative strains, were used in the

study. The activity of the chloroform and methanol extracts of L. camara was found to be

more specific towards the gram-positive strains, although gram-negative P. aeruginosawas

also inhibited by the methanol extracts of the plant in a dose dependent manner. The water

extracts of L. camara was found to be inactive25.

In conclusion Methanol extracts of the plant parts showed antibacterial activity against

disease-causing organisms and this suggest that constituents of the plants could be useful

in chemotherapy. From the findings od this study, the following recommendations could be

made; Firstly, there is a need to further isolate the active antibacterial agent (s) and

secondly, it is necessary to determine toxicity of the active constituents, their side effects

and pharmacokinetics effects.

Acknowledgement

I would like to acknowledge my supervisors, Associate prof. Deo Olila and Dr. John

Kateregga, for their professional guidance. Thanks go to my parents, Mr & Mrs. Mutai, for

their support always during research work and all Laboratory technicians and Technologist

for their technical support.

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References

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of Mangifera indica (L.) African Journal of Ecology, Afr J Ecol. 2007;45(Suppl 1):13–16.

11. Ngo T. Contribution to researches on mango leaves raw materials in northern Vietnam. Science Technological

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Chapman and Hall; 1998. pp. 1–198.

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14. Iwu MM. Handbook of African Medicinal Plants. Boca Raton: CRC Press Inc.; 1993. pp. 223–225.

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Medicare Ltd; 2001. Available from: URL: http://www.dweckdata.co.uk/

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Academic Press, Inc; 1990.

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Fig 1

Chart showing the Zone of inhibition of Plant Methanol Extract

Table 1Classes of Bioactive compounds identified in the Methanol plant extract

Compound O. africana P.guajava V.amygdalina L.camara M.indica

Alkaloids ++ − ++ + ++

Tannins + ++ + + +

Saponinns − + − + +

Flavonoids + ++ + + ++

Anthraquinones − NT − − +

Glycosides − + − − ++

Cardiacglycosides NT − − + −Key: NT; Not tested, ++; strongly positive, +; positive, −; Negative

Inhibitory and Bactericidal Potential of Crude Acetone Extracts of Combretum molle (Combretaceae) on Drug-resistant Strains of Helicobacter pyloriCollise Njume,1 Anthony J. Afolayan,2 Amidou Samie,3 and Roland N. Ndip 1,4

1Microbial Pathogenicity and Molecular Epidemiology Research Group, Department of Biochemistry and Microbiology, University of Fort Hare, P/Bag X1314, Alice 5700, South Africa2Phytomedicine Research Group, Department of Botany, Faculty of Science and Agriculture, University of Fort Hare, P/Bag X1314, Alice 5700, South Africa3Department of Microbiology, University of Venda, Thohoyandou 0950, South Africa4Department of Biochemistry and Microbiology, Faculty of Science, University of Buea, Box 63, Buea, Cameroon

Corresponding author.

Correspondence and reprint requests should be addressed to: Prof. Roland N. Ndip Department of Biochemistry and Microbiology Faculty of Science and Agriculture University of Fort Hare PMB X 1314, Alice 5700 South Africa Fax: +27 866224759 E-mail: rndip@ufh.ac.za OR ; Email: ndip3@yahoo.comThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abstract

Infection with Helicobacter pylori is strongly associated with a number of gastroduodenal

pathologies. Antimicrobial resistance to commonly-used drugs has generated a

considerable interest in the search for novel therapeutic compounds from medicinal plants.

As an ongoing effort of this search, the susceptibility of 32 clinical strains of H. pylori and a

reference strain—NCTC 11638—was evaluated against five solvent extracts of Combretum

molle, a plant widely used for the treatment of gastric ulcers and other stomach-related

morbidities in South Africa. The extracts were screened for activity by the agar-well diffusion

method, and the most active one of them was tested against the same strains by micro-

broth dilution and time kill assays. Metronidazole and amoxicillin were included in these

experiments as positive control antibiotics. The solvent extracts all demonstrated anti-

H. pylori activity with zone diameters of inhibition between 0 and 38 mm. The most potent

anti-H. pylori activity was demonstrated by the acetone extract, to which 87.5% of the

clinical strains were susceptible. The minimum inhibitory concentration (MIC90) values for

this extract ranged from 1.25 to 5.0 mg/mL while those for amoxicillin and metronidazole

ranged from 0.001 to 0.94 mg/mL and from 0.004 to 5.0 mg/mL respectively. The acetone

extract was highly bactericidal at a concentration of 2.5 and 5.0 mg/mL, with complete

elimination of the test organisms in 24 hours. Its inhibitory activity was better than that of

metronidazole (p<0.05) as opposed to amoxicillin (p<0.05). The results demonstrate

that C. molle may contain therapeutically-useful compounds against H. pylori, which are

mostly concentrated in the acetone extract.

Key words: Acetone, Antibiotic resistance, Combretum molle, Crude extracts, Helicobacter pylori,

Microbial sensitivity tests, Minimum inhibitory concentration, South Africa

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INTRODUCTION

Helicobacter pylori is a gram-negative microaerophilic spiral-shaped bacillus that affects the

gastric mucosa and can be found attached to epithelial cells of the human stomach (1).

Infection with this organism is strongly associated with chronic gastritis, peptic ulcer,

duodenal ulcer, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT)

lymphoma (2). The stomach of half of the world's population is colonized by this organism

(3). Treatment of H. pylori infection is relatively successful, with up to 90% of patients

exhibiting eradication of the organism with current therapeutic regimens (4). These

regimens typically involve the use of a proton pump inhibitor (PPI) or bismuth compounds in

combination with two antibiotics—most commonly amoxicillin and clarithromycin, or

metronidazole (5). However, H. pylori infection continues to be difficult to eradicate with

failure rates of up to 40% (6). A major factor to this failure is the development of antibiotic-

resistant strains (5). It is, therefore, not uncommon to find other stronger antibiotics,

particularly of the fluoroquinolone group, being part of the treatment regimen (7)

butH. pylori is also developing resistance to these drugs (8). Considering that eradication

therapies can be ineffective and undesirable side-effects may occur (9), the search for

alternative therapeutic sources for the development of new anti-H.pylori compounds is

imperative.

Other factors, including poor compliance by patients, cost of combination therapy, the

location of the organism in the stomach, and the non-availability of drugs in some rural

settings in Africa, are contraindications for some patients (3,10). Equally important is the

increasing prevalence of virulent strains, particularly those expressing the cytotoxin-

associated gene A antigen (CagA) associated with severe pathological conditions (11) that

may be difficult to manage and post-therapeutic antibiotic resistance which has been known

to decrease the cure rate by more than 50% (12). These factors have generated a

considerable interest in the search for alternative treatment regimens against this notorious

pathogen. Alternative therapeutic agents with highly-selective antibacterial activity against

the organism, without the risk of resistance or other untoward effects, are necessary (5).

Medicinal plants are among the attractive sources of new drugs and have been used for

treating gastrointestinal diseases and other ailments, particularly in the developing world

where infectious diseases are endemic and modern health facilities are not always

adequate or accessible (13). Antimicrobial compounds from plants may inhibit bacterial

growth by mechanisms different from presently-used treatment regimens and could,

therefore, be of clinical value in the treatment of resistant bacteria, including H. pylori. In

fact, our previous studies have documented that some medicinal plant extracts have

antibacterial activity against H. pylori (14).

The genus Combretum is mainly tropical and consists of numerous species,

including Combretum

molle, C. woodii, C. erythophyllum, C. Apiculatum, and C.mossambicense (15,16). They

consist of trees, climbers, and shrubs (16). Almost every part of these plants—roots, leaves,

seeds, and stem barks—is used in African traditional medicine for the treatment of parasitic,

bacterial and fungal infections (15,16). The stem bark of C. molle, a small graceful

deciduous tree (3-13 m high) is popularly used in South Africa for the treatment of stomach

pains, dysentery, gastric ulcers, abdominal disorders, and other illnesses (15). Studies on

its antibacterial properties have produced promising results (15). It is found in the traditional

medicine market where it is commercialized for medicinal purposes. Despite its traditional

uses in the treatment of gastric ulcers and other stomach-related morbidities, the activity of

this plant has not been investigated against H. pylori, a major cause of gastric ulcer. This is

surprising particularly as the prevalence of this organism is reported to vary between 50%

and 80% in South Africa (17,18), and an alarming resistance of 95.5% has been reported in

South Africa for metronidazole, one of the antibiotics used in the treatment regimen

of H. pylori infections (4). This study was, therefore, carried out to evaluate the antimicrobial

activity of C. molle on drug-resistant isolates of H. pylori. The aim was to identify the

potential sources of cheap starting materials for the synthesis of new drugs that could be

cheap and readily available to help circumvent the problem of increasing antimicrobial

resistance.

 Other Sections▼

MATERIALS AND METHODS

Bacterial strains

We used 32 resistant strains of H. pylori isolated from gastric biopsies of patients with

recurrent peptic ulcer infection, despite treatment with metronidazole and amoxicillin,

undergoing endoscopy at the Livingstone Hospital, Port Elizabeth. A standard control strain

—NCTC 11638—was also included. Isolation and identification was done following our

previously-reported scheme (4). Briefly, biopsies were homogenized under aseptic

conditions in 0.2 g/L of cysteine and 20% of glycerol in Brain heart infusion (BHI) broth

(Oxoid, England). A loopful of the homogenate was plated on freshly-prepared Columbia

agar base (Oxoid, England) supplemented with 6% horse blood and Skirrow's supplement

(Oxoid, England) containing trimethoprim (2.5 mg), vancomycin (5 mg), cefsulodin (2.5 mg),

and amphotericin (2.5 mg). Inoculated plates were incubated at 37 °C for five days under

microaerophilic conditions (5-6% O2, 10% CO2, and 80-85% N2) (Anaerocult Basingstoke,

Hampshire, England). The isolates were identified based on colony morphology, positive

oxidase, urease, catalase tests, and amplification of the glmM gene. Confirmed isolates

were suspended in eppendorf tubes containing 1 mL of BHI broth and 20% glycerol and

stored at −80 °C until future use. Gastric biopsies were only collected from patients who had

given consent and had not been on antibiotics, PPI, or bismuth salts for at least a week.

Preparation of plant material

The stem bark of C. molle R. Br. Ex G. Don (Combretaceae) was harvested in the vicinity of

the University of Venda, Limpopo province, and transported in plastic bags to the School of

Biological Sciences, University of Fort Hare, where they were identified and vouchers were

deposited in the school's herbarium (CNUFH05). The plant material was washed, air-dried

for two weeks, and ground to fine powder using a blender (ATO MSE mix, England).

Preparation of plant extracts

Exactly 300 g of dried plant material was macerated separately in 600 mL of concentrated

ethyl acetate, acetone, ethanol, and methanol in large glass bottles (SIMAX, Czech

Republic). Aqueous extracts were also prepared by soaking the same amount of plant

material in tap water. The bottles were labelled and put in an orbital shaker for 48 hours.

The plant extracts were centrifuged at 1,006.2 g for five minutes at 4 °C and filtered using a

fritted filter funnel of pore size 60 Å. The procedure was repeated twice, and the three

extracts were combined and evaporated to dryness under vacuum in a rotary evaporator

(BUCHI rota vapour, Flavil/Schweiz, Switzerland). The filtrate obtained from the aqueous

extract was lyophilized (19). The dried crude extracts were collected in clean glass Petri-

dishes and left open in a biosafety class II cabinet (Durban, South Africa) for complete

evaporation of residual solvents. A 2-g sample of each extract was used for the preliminary

bioassay, and where possible, another 2 g or more was put in universal bottles and kept in

the extract bank. Stock solutions were prepared by dissolving the extracts in 10% dimethyl

sulphoxide (DMSO) or 80% acetone (neither DMSO nor acetone was inhibitory to

the H. pylori strains at the tested concentrations).

Screening of crude extracts for anti-H. pylori activity

This was done by the agar-well diffusion method as previously reported (20).

Briefly,H. pylori inocula prepared at McFarland's turbidity standard 2 were plated onto BHI

agar supplemented with 5% horse blood and Skirrow's supplement (Oxoid, England). The

inocula were evenly spread on the plate. The plate was allowed to dry for about 15 minutes.

Wells (6 mm in diameter) were punched into the agar using a sterile stainless steel borer.

The wells were filled with 65 μL of the extract at 100 mg/mL. Sixty-five μL of 0.05 μg/mL of

clarithromycin and 10% DMSO were included in all the experiments as positive and

negative controls respectively. The plates were incubated under microaerophilic conditions

(Anaerocult, Oxoid, UK) at 37 °C for 72 hours after which the diameters of zones of

inhibition were measured in mm. The experiment was repeated once, and the mean zones

were recorded. A zone diameter of ≥14 mm was used as breakpoint susceptibility for

clarithromycin and the extracts; this was also used for calculating their percentage

susceptibilities (1). A plate inoculated with a reference strain (NCTC 11638) of H. pylori was

included in all the experimental runs.

Determination of minimum inhibitory concentration (MIC90)

The acetone extract which was the only extract to which more than 50% of the strains were

susceptible was chosen for further determination of MIC by the micro-broth dilution method

as earlier reported (21). The test was performed in 96-well plates. The test extract was

prepared at a concentration of 5.0 mg/mL and filtered through a 2.0-μm filter (Acrodisc Pall,

MI, USA). Two-fold dilutions of the extract were made in the test wells in BHI broth

supplemented with 5% horse serum and Skirrow's supplement (Oxoid, England). The final

extract concentration ranged from 0.001 to 5.0 mg/mL. Twenty μL of an 18-hour old broth

culture of H. pylori (McFarland's turbidity standard 2) suspension was added to 100 μL of

extract-containing culture medium. Control wells were prepared with culture medium plus

bacterial suspension and broth only respectively. Metronidazole and amoxicillin were run,

alongside the extract at concentration ranges of 0.005-5.0 mg/mL and 0.001-1.25 mg/mL

respectively. An automatic ELISA micro-plate reader (Tokyo, Japan) adjusted to 620 nm

was used for measuring the absorbance of the plates. The plates were incubated at 37 °C

for 72 hours under microaerophilic conditions (Anaerocult Basingstoke, Hampshire,

England), and the absorbance was read again at 620 nm. The initial and the post-incubation

absorbencies were compared to detect an increase or a decrease in bacterial growth. The

lowest concentration of the test extract resulting in inhibition of 90% of bacterial growth was

recorded as the MIC. The strains were considered susceptible to the control antibiotics if

their MIC90 values were <0.002 mg/mL for amoxicillin and <0.008 mg/mL for metronidazole

(1).

Determination of rate of killing

The rate and extent of killing of H. pylori by the acetone extract of C. molle was determined

as described by Akinpelu et al. (22) with slight modifications. The turbidity of an 18-hour old

broth culture of H. pylori was standardized to 108 CFU/mL. One mL of this suspension was

added to 9 mL of BHI broth supplemented with 5% horse serum and Skirrow's reagents

containing the extract at 0.625 mg/mL (MIC/2), 1.25 mg/mL (MIC), 2.50 mg/mL (2 MIC), and

5.0 mg/mL (4 MIC) in McCartney bottles (Oxoid, England). A negative control bottle was

prepared with bacterial suspension and broth only. A 0.1-mL sample was plated from these

bottles before incubation at 37 °C under microaerophilic conditions. Exactly 0.5 mL of each

suspension was withdrawn at a six-hour interval for 72 hours and transferred to 4.5 mL of

BHI broth recovery medium containing 3% ‘Tween 80’ to neutralize the effects of the

antimicrobial extract carry-overs from the test organisms. The suspension was 10-fold

serially diluted in sterile saline (0.9% w/v sodium chloride) and plated in triplicates. The

plates were incubated at 37 °C for 72 hours under microaerophilic conditions, and the viable

counts were determined.

Statistical analysis

Results were expressed as mean±standard deviation using the SPSS software (version

17.0) (Chicago, Illinois, 2009) and Excel. One-way analysis of variance (ANOVA), followed

by Turkey's post-hoc test, was used for comparing the mean difference in inhibitory

activities of extracts and antibiotics. The differences were considered significant at p<0.05.

Ethical approval

The Eastern Cape Department of Health and the Govan Mbeki Research and Development

Centre, University of Fort Hare, approved the study.

 Other Sections▼

RESULTS

Extract yield

The total amount of crude extract obtained with the different solvents showed that methanol

was quantitatively the best solvent for extraction, with a crude extract yield of 5.1 g (1.7%),

followed by acetone 4.6 g (1.5%), ethanol 3.2 g (1.1%), water 3.1 g (1.0%), and ethyl

acetate 1.3 (0.4%). Ethyl acetate, acetone and methanol extracts were dark brown in colour

while ethanol and aqueous extracts appeared as brown to light brown crystals.

Antimicrobial susceptibility testing and determination of minimum inhibitory concentration

All the crude extracts tested in this study demonstrated antimicrobial activity with zone

diameters of inhibition ranging from 0 to 38 mm (Table 1). The highest zone diameter of 38

mm was recorded for the acetone extract, to which 87.5% of the clinical strains were

susceptible (Fig. 1). Zone diameters of inhibition for the acetone extract were significantly

different from the other extracts (p<0.05) as opposed to the control antibiotic (p>0.05)

(Table 1). Eleven (34.4%) and six (18.8%) of the 32 strains tested against the acetone and

ethanol extracts respectively recorded susceptible zones of inhibition as opposed to the

positive control which was resistant but the differences were not significant (p>0.05).

Table 1.Screening of crude extracts of C. molle against H. pylori isolates

Fig. 1.Anti-H. pylori activity of crude extracts of C. molle by agar well diffusion method

The MIC values for the acetone extract ranged from 1.25 to 5.0 mg/mL while those for

amoxicillin and metronidazole ranged from 0.001 to 0.94 mg/mL and 0.004 to 5.0 mg/mL

respectively (Table 2).

Table 2.Minimum inhibitory concentration (90%) of C. molleand control antibiotics (mg/mL)

Bactericidal activity

The acetone extract of C. molle exhibited considerable bactericidal activity against the

isolates at all concentrations tested over a 72-hour period. The test organisms were

completely eliminated at a concentration of 2.5 and 5.0 mg/mL within 24 hours (Fig. 2). No

cells were killed in the first six hours of the experiment at all the extract concentrations

tested.

Fig. 2.Effect of crude acetone extracts of C. molle on the growth of H. pylori

 Other Sections▼

DISCUSSION

Post-therapeutic antibiotic-resistant H. pylori reduces the cure rate of infection by up to 66%

(12). With very few exceptions, the most commonly-recommended treatment regimen (PPI,

amoxicillin, and clarithromycin or metronidazole) now provides unacceptably low treatment

successes (23), with approximately one in five patients requiring second and third-line

therapies due to eradication failure (12). It should, therefore, not be surprising that all the

clinical strains tested in this study were resistant to metronidazole and amoxicillin

considering that they were isolated from patients who had been on treatment regimens

involving these drugs (4). We were, therefore, probably dealing with a small group of

patients with a high level of post-therapeutic resistance confirmed by the high MIC values

observed.

Other studies have reported high metronidazole and amoxicillin resistance in the developing

world (1,4,5,24). Metronidazole resistance is an existing problem in this part of the world

and is probably due to drug pressure because this inexpensive drug is also used in the

treatment of gynaecological problems and protozoa infections (1,4) while amoxicillin

resistance can be attributed to the non-adherence to drug-prescription protocols.

Consequently, an increased eradication failure will be observed with treatment regimens

involving these drugs. Antimicrobial susceptibility testing to establish resistance patterns,

therefore, becomes imperative to guide empiric treatment. Other measures, such as strict

antibiotic restriction practices, education of the population, and pharmacovigilance will go a

long way to improve the management of antibiotic use in the developing world.

Our results showed that methanol was quantitatively the best solvent for extraction.

Different solvents may be employed in the extraction of antimicrobial compounds from

plants; however, the success in the isolation process is largely dependent on the type of

solvent used (25). The good extracting ability of methanol recorded in this study

corroborates findings of other studies (25-27), thus confirming this solvent as a good

extractant of phytochemicals from Combretum species and other plants. However, the

quantity of extract may not always relate proportionately to the activity as revealed by the

low anti-H. pylori activity of the methanol extract recorded herein. Nevertheless, extracts

with little or no activity in vitro may have properties similar to pro-drugs which are

administered in an inactive form. Their metabolites could be active in vivo (14).

The results of this study indicate that the ethanol and methanol extracts were only minimally

active (Fig. 1). However, in a study on rats in Brazil by Nunes et al. (28), the ethanolic

extract of the stem bark of a local Combretum species—C. leprosum—exhibited anti-

ulcerogenic and gastro-protective effects by increasing the volume and pH of gastric juice

while decreasing the acid output (28). Although Nunes et al. did not investigate the

antimicrobial activity of their extracts against H. pylori (28), the effects demonstrated by the

crude ethanolic extract of C. leprosum in the animal model could be useful in preventing the

development of severe pathological conditions in anH. pylori-infected mucosa.

The zone of inhibition diameters and percentage susceptibilities seem to decrease with

increase in polarity of the solvent from ethanol to water (Fig. 1), which may imply that the

isolates were not very sensitive to the polar compounds of this plant or at least, not many

anti-H. pylori compounds were extracted by polar solvents. However, the activity

demonstrated by these extracts indicates their potential as useful bioactive substances.

The acetone extract demonstrated remarkable activity against the test strains in the entire

study. This indicates that the active components of this plant are more soluble in acetone

and have a good antimicrobial potential. Further investigation may lead to the isolation of

potentially-useful compounds for the treatment of H. pylori infections. The activity of this

extract compared favourably with metronidazole, with no significant differences between

their mean MIC values (p>0.05). This is remarkable considering that all the clinical strains

used in this study were resistant to the antibiotics.

The extract was also strongly bactericidal at a concentration of 2.5 and 5.0 mg/mL, with

complete elimination of the organisms in 24 hours (Fig. 2). However, active components in

the crude extract may be acting in synergism to produce greater antimicrobial effects (29).

In any case, the acetone extract of C. molle may be considered a possible new source of

compounds for the management of infections caused by resistant strains of H. pylori. This is

particularly important in the study area given the current trend and ever-evolving nature of

resistant H. pylori (4).

Acetone is used for extracting mostly flavonoids and steroids (15,30,31). Flavonoids are

known to be synthesized by plants in response to microbial infection (32,33), which may

account for their antimicrobial activity against a wide range of organisms.

Several studies have reported on the good antimicrobial activity of the ethyl acetate fraction

of Combretum species (34,35) but H. pylori was not among the organisms tested. Our

findings provide preliminary evidence that the ethyl acetate extract of C.molle has very little

activity against H. pylori. The aqueous extracts of C. molle also showed very little activity

(6.3%) against H. pylori (Fig. 1). Other studies have also reported very low antimicrobial

activity of the aqueous extract of other members of the Combretaceae and other plants

(15,35). Despite its availability and relatively low toxicity, water may still not be a suitable

solvent for the extraction of anti-H. pyloricompounds from C. molle.

Conclusions

The results of the study provide preliminary scientific validation of the traditional medicinal

use of this plant in the treatment of infections symptomatic of H. pylori. This plant may

contain compounds, mostly in the acetone crude extract that could be used as lead

molecules for the synthesis of novel drugs against recurrent and

resistant H. pylori infections. Isolation and characterization of the biologically-active

constituents of the acetone extract of C. molle and a detailed assessment of their in-

vivo potencies will add more value to their potential usefulness as anti-H. pyloriagents.

These aspects are already receiving attention in our group.

ACKNOWLEDGEMENTS

The authors are grateful to the National Research Foundation, South Africa (Grant No.

CSUR 2008052900010) and the Govan Mbeki Research and Development Centre,

University of Fort Hare, South Africa, for funding the study. They thank Dr. Naidoo N, Dr.

Tanih NF, Mr. Okeleye BI, and Mr. Tshikhawe P for technical assistance.

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