Screening for antimicrobial activity of ten medicinal plants used in Colombian folkloric medicine: A possible alternative in the treatment

of non-nosocomial infections
Jhon J Rojas,
1

#1

Veronica J Ochoa,#1 Saul A Ocampo,#1 and John F Muoz#1

Department of Pharmacy, Universidad de Antioquia, Cll. 67 # 53-108 of. 1-157, Medelln, Colombia Corresponding 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 Muoz: jmuoz@farmacia.udea.edu.co Received 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 hemoltic,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 g/ml), clindamycin (0.3 g/ml) and nystatin (1.0 g/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 g/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 g/ml, respectively) compared to nystatin (0.6 g/ml). Bixa orellana L, exhibited a better MIC againstBacillus cereus (0.2 g/ml) than gentamycin sulfate (0.5 g/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 hemolytic 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 nonsevere 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, Choc and Huila departments). All the species were

identified by Professor Francisco Roldan (Instituto de Biologa) 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 -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 40C to obtain the dry extracts (Table 1). '[seeAdditional file 1]'.

Determination of antimicrobial activity

Microorganisms used
The test organisms (S. aureus ATCC 29737, S. hemoltic 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: BairdParker (S. aureus), Cetrimide (P. aeruginosa), McConkey (E. coli), Blood (S. 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 2C 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 & Lombspectrophotometer, 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 L of the plants extracts and blanks (ethanol, distilled water, and hexane). The concentration of the extracts employed was 25 g/ml. Simultaneously, gentamycin sulfate (S. aureus, P.

aeruginosa, E. coli, and B. cereus), clindamycin (S. hemolytic), and nystatin (C. albicans) were used as positive controls at a concentration of 1.0, 0.3 and 1.0 g/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 2C for 24 h, except for C. albicans which was incubated at 29 2C. 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 g/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 100C, 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. hemolytic, P. aureginosa and C. albicans were the most resistant microorganisms. None of the extracts was more active against S. hemolytic 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 g/ml, respectively) compared to nystatin (0.6 g/ml). Moreover, these two plants manifested a better MIC against E. coli (0.6 g/ml) than gentamycin sulfate (0.9 g/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 g/ml) compared to gentamycin sulfate (0.3 g/ml). B. orellana L, J. secunda and P. pulchrum presented the lowest MICs against E. coli(0.8, 0.6 and 0.6 g/ml, respectively) compared to gentamycin sulfate (0.9 g/ml). Likewise, B. orellana manifested the lowest value of MIC against B. cereus (0.2 g/ml) compared to gentamycin sulfate (0.5 g/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. 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. hemolitic Streptococcus hemoltic 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 Snchez, Gloria A. Valencia, and Jorge Arango. We also acknowledge local healers for their assistance in finding the plant species. Similarly, we acknowledge Professor Francisco Roldn 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 Biomdicales Appliques (ISBA), University of Abomey-Calavi, Cotonou, Republic of Benin.

Abstract
The 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, Kenya
K R Cheruiyot, D Olila, and J Kateregga
Department of Physiological Sciences, Faculty of Veterinary Medicine, Makerere University, P. O Box 7062, KampalaUganda Corresponding 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 africanastembark, 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 (10g) 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 315 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 flavonoids35. Vernonia amygdalina (Compositae) is a shrub, of 25 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 l of the methanol extract, of the concentration 1 g/ml, was dispensed into respective wells and 10 g 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 1 Classes 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 1 Chart 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 Prescott 22 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 diarrheacausing bacteria asStaphylococcus, Shigella, Salmonella, Bacillus, E. coli, Clostridium, and Pseudomonas (35). 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 12.112.3 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
1. World Health Organisation, author. Global strategy for containment of antibiotic resistance.Geneva: WHO; 2001. p. 99. 2. Anesini C, Perez C. Screening of plants used in Argentinian folk medicine for antimicrobial activity.Journal of Ethnopharmacology. 1993;39(2):119128. [PubMed] 3. Olajide O A, Awe S O, Makinde J M. Pharmacological studies on the leaf of Psidium guajava.Fitoterapia. 1999;70:2531. 4. Begum S, Hassan S I, Siddiqui B S, Shaheen F, Ghayur M N, Gilani A H. Triperpenoids from the leaves from Psidium guajava. Phytochemistry. 2002;61:399403. [PubMed] 5. Gonalves FA, Andrade Neto M, Bezerra JN, Macrae A, Sousa OV, Fonteles-Filho AA, et al. Antibacterial activity of GUAVA, Psidium guajava Linnaeus, leaf extracts on diarrhea-causing enteric bacteria isolated from Seabob shrimp, Xiphopenaeus kroyer (Heller) Rev Inst Med Trop Sao Paulo.2008;50(1):1115. [PubMed] 6. Banso A, Adeyemo SO, Jeremiah P. Antimicrobial properties of Vernonia amygdalina extract. J Applied Sci Magt. 1999;3:911. 7. Kupcham SM, Hernichway RJ, Wermer D. Vernodalin and Vernomygdin. Two new sesquiterpene lactones from Vernonia amygdalina del. J Org Chem. 1969;3:39083909. 8. Taoubi K, Fauvel MT, Gleye J, Moulis C. Phenylpropanoid glycosides from Lantana camara andLippia multiflora. Planta Med. 1997;63:192193. [PubMed] 9. Chharba SC, Mahunnah RLA, Mshiu EN. Plants used in traditional medicine in eastern Tanzania. J Ethnopharmacol. 1993;39:83103. [PubMed] 10. Odyek O, Bbosa G S, Waako P, Kyegombe D B, Bukenya-Ziraba R, Ogwal-Okeng J. Antibacterial activity of Mangifera indica (L.) African Journal of Ecology, Afr J Ecol. 2007;45(Suppl 1):1316. 11. Ngo T. Contribution to researches on mango leaves raw materials in northern Vietnam. Science Technological Publish. 2001:563565. 12. Harbone JB. Phytochemical Methods. A Guide to Modern Techniques of plant Analysis. 3rd edition. Newyork: Chapman and Hall; 1998. pp. 1198. 13. Evans WC. Trease and Evans Pharmacognosy. 14th edition. WB Saunders Company Limited; 1998. pp. 1516. 14. Iwu MM. Handbook of African Medicinal Plants. Boca Raton: CRC Press Inc.; 1993. pp. 223225. 15. Dweck AC. Article for cosmetics & toiletries magazine ethnobotanical plants from Africa. Iltshire, UK: Black Medicare Ltd; 2001. Available from: URL: http://www.dweckdata.co.uk/

16. Carter G R, Cole J R. Diagnostic Procedures in Vet. Bacteriology and Mycology. 5th Edn. London and Newyork: Academic Press, Inc; 1990. 17. Irobi ON, Moo-Young M, Daramola SO. In J Pharmacognosy. 1996;34(2):8790. 18. Andrew JM. Determination of MIC. Journal of Antimicrobial Chemotherapy. 2001;48(Suppl 1):516. [PubMed] 19. Subhuti Dharmananda. Gallnuts and the Uses of Tannins in Chinese Medicine. 2003. A paper delivered at Institute for Traditional Medicine, Portland, Oregon. 20. Ibrahim MB, Owonubi MO, Onaopo JA. Antibacterial effect of extract of leaf, stem and roof bark of Anogeissus leiocarpus on some bacterial organisms. J Pharm Res Dev. 1997;2(1):2023. 21. Kolapo AL, Ogundiya MO, Okunade MB. Antimicrobial activities of some Nigerian chewing stick.2007. Available from: URL: http://www.siu.odu/webl/leaflets/ogun diya hfm. 22. Prescott LMH, arley JP, Klein D. Microbiology (International Edition) Fifth edition. Mc Graw Hill Book Company; 2002. pp. 809819. 23. Tortora GJ, Funke BR, Case CL. Microbiology: An introduction. San Franscisco: Benjamin Cummings; 2001. 24. Iwalokun B A, Bamiro S B, Durojaiye O O. An antimicrobial evaluation of Vernonia amygdalina(Compositae) against gram-positive and gram-negative bacteria from Lagos, Nigeria. West African Journal of Pharmacology and Drug Research. 2004;19 25. Basu Subhalakshmi, Ghosh Abhijit, Hazra Banasri. Evaluation of the antibacterial activity ofVentilago madraspatana Gaertn., Rubia cordifolia Linn. and Lantana camara Linn.: isolation of emodin and physcion as active antibacterial agents. Calcutta: Department of Pharmaceutical Technology, Jadavpur University; 2002. 700 032, India.

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

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

Compound Alkaloids Tannins Saponinns Flavonoids Anthraquinones Glycosides Cardiacglycosides

O. africana ++ + + NT

P.guajava ++ + ++ NT +

V.amygdalina ++ + +

L.camara + + + + +

M.indica ++ + + ++ + ++

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 pylori
Collise Njume,1 Anthony J. Afolayan,2 Amidou Samie,3 and Roland N. Ndip
1

1,4

Microbial Pathogenicity and Molecular Epidemiology Research Group, Department of Biochemistry and Microbiology, University of Fort Hare, P/Bag X1314, Alice 5700, South Africa 2 Phytomedicine Research Group, Department of Botany, Faculty of Science and Agriculture, University of Fort Hare, P/Bag X1314, Alice 5700, South Africa 3 Department of Microbiology, University of Venda, Thohoyandou 0950, South Africa 4 Department 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.com

This 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 strainNCTC 11638was 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 microbroth dilution and time kill assays. Metronidazole and amoxicillin were included in these experiments as positive control antibiotics. The solvent extracts all demonstrated antiH. 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 antibioticsmost 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 antibioticresistant 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 cytotoxinassociated 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 plantsroots, leaves, seeds, and stem barksis 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.

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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 strainNCTC 11638was 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 . 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 Petridishes 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 L of the extract at 100 mg/mL. Sixty-five L of 0.05 g/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-m 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 L of an 18-hour old broth culture of H. pylori (McFarland's turbidity standard 2) suspension was added to 100 L 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 meanstandard 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.

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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

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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 speciesC. leprosumexhibited antiulcerogenic 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 invivo 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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