Archives of Medical Research 41 (2010) 324e331

ORIGINAL ARTICLE

Selected South African Honeys and Their Extracts Possess In Vitro Anti-Helicobacter pylori Activity
Christy E. Manyi-Loh,a Anna M. Clarke,a Thilivhali Munzhelele,a Ezekiel Green,a Noxolo F. Mkwetshana,a and Roland N. Ndipa,b
a

Microbial Pathogenicity and Molecular Epidemiology Research Group, Department of Biochemistry and Microbiology, University of Fort Hare, Alice, South Africa b Department of Biochemistry and Microbiology, Faculty of Science, University of Buea, Cameroon Received for publication April 23, 2010; accepted July 30, 2010 (ARCMED-D-10-00193).

Background and Aims. Eradication of Helicobacter pylori by triple therapy often results in a failure rate of 10-20%; thus, there is a need to seek alternative treatments. The aim of this study was to screen selected South African honeys for their anti-H. pylori activity, to extract the antimicrobial components using organic solvents and to determine the minimum inhibitory concentrations (MICs) of the extracts. Methods. Three locally produced honeys from different regions in South Africa were screened for anti-H. pylori activity at four different concentrations using the agar well diffusion technique. Subsequently, Pure honey was extracted using n-hexane, diethyl ether, chloroform and ethyl acetate; extracts were also examined for anti-H. pylori activity by agar well diffusion method. The MICs of the three most active extracts were determined both by visual inspection and spectrophotometric analysis at 620 nm using the broth microdilution method. The results were analyzed by one-way ANOVA at 95% signicance level. Results. All honeys demonstrated anti-H. pylori activity and were most active at 75% v/v. The positive control (clarithromycin) recorded a zone diameter of 18.0 7.4 mm not signicantly different ( p O0.05) from honeys at 75% v/v and solvent extracts. Chloroform extract recorded the lowest MIC95 values that ranged from 0.156-5% v/v conrming this extract to be the most active. Conclusion. All honeys demonstrated anti-H. pylori activity at concentrations $10%, as did the solvent extracts. Therefore, these honeys and solvent extracts possess potential compounds with therapeutic activity that could be further exploited as lead molecules in the treatment of H. pylori infections. 2010 IMSS. Published by Elsevier Inc.
Key Words: Honeys, Solvent extracts, Antimicrobial activity, Helicobacter pylori, Minimum inhibitory concentration.

Introduction Helicobacter pylori remains one of the most common human chronic microbial infections worldwide, with humans being the major reservoir for this organism (1). Despite a vigorous immune response mounted by the hosts system, the bacterium colonizes the gastric mucosa of the

Address reprint requests to: Dr. Anna M. Clarke, Department of Biochemistry and Microbiology, University of Fort Hare, P/B X 1314, Alice, South Africa; Phone: 27(0) 836516892; FAX: 27(0) 866224759; E-mail: aclarke@ufh.ac.za

stomach and is protected by gastric mucus. This may be as a result of molecular mimicry that exists between H. pylori lipopolysaccharide and human Lewis x antigen, making it possible for the organism to evade immunological clearance (2). H. pylori also possess striking characteristics that help it to evade the harsh conditions in the gastrointestinal tract. Consequently, it causes peptic ulcer disease, gastric adenocarcinomas and mucosa-associated lymphoid tissue (MALT) lymphoma (3). However, only about 15% of those colonized eventually develop disease, and pathogenesis depends upon strain virulence, host genetic susceptibility, and environmental cofactors (4).

0188-4409/$ - see front matter. Copyright 2010 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2010.08.002

Honeys and Their Extracts Possess In Vitro Anti-H. pylori Activity

325

Successful eradication of H. pylori infection has been shown to result in ulcer healing and prevention of peptic ulcer recurrence and may also reduce the prevalence of gastric cancer in high-risk populations (5). At present, the rst-line regimen for eradication of the organism includes a triple therapy, which combines two known antibiotics (clarithromycin or amoxicillin and metronidazole) with a proton pump inhibitor (6). However, because triple therapy fails to eradicate infection in 10e20% of patients, quadruple therapy was introduced as a new treatment modality as well as a rescue treatment for antibioticresistant strains of H. pylori (7). Although, most of these therapeutic modalities are |90% effective, they are usually associated with high levels of antibiotic resistance (5,8), undesirable side effects, poor patient compliance (9), and high cost and unavailability of antibiotics especially in rural areas. These, therefore, result in signicant levels of treatment failure and contraindications for some patients. An alternative mode of treatment using natural products, which are relatively nontoxic and cheap, is therefore necessary. This has led to the re-evaluation of the therapeutic use of ancient remedies including honey. Honey-derived remedies constitute a potential source of new compounds that may be useful in the management of H. pylori infections. Honey has been recognized for its medicinal properties since antiqiuty (10). It has been shown to be active against a diverse range of microorganisms including gram-positive and -negative organisms, aerobic and anaerobic bacteria (11,12), and Candida albicans as well as inhibiting the germination of the spores of Bacillus cereus (13). The principal antibacterial factor has been reported to be hydrogen peroxide produced by the oxidation of glucose by the enzyme glucose oxidase, which is activated by successive dilutions of honey (14). Other antimicrobial factors include its osmolarity, acidity, low protein content and nonperoxide components (15). These nonperoxide factors (avonoids and phenolic acids) are derived from plant origin. The amount of these components may be small or diluted in the honey but when extracted with organic solvents (16), they become concentrated and therefore exhibit more activity. Flavonoids, phenolic and organic acids in honey are known to scavenge for free superoxide and other reactive oxygen metabolites liberated during respiratory burst in H. pylori-induced mucosal damage (17). Honeys from different countries and regions have a wide variability in their antimicrobial activity as a result of different vegetative owers and plant species blooming in different seasons (12,18). Interestingly, South Africa has a large oral biodiversity with many unique plants indigenous to the region from which various honeys are being produced. The inhabitants of the country consume these honeys with the belief that it boosts the immune system and is good for wound healing and stomach ailments. However, the observation that honey in New Zealand and

Saudi Arabia at concentrations approximating 20% v/v can inhibit the growth of H. pylori in vitro coupled to the fact that Medihoney and Manuka honeys have been shown to have in vivo activity and are suitable for use in ulcers, infected wounds and burns are important ndings (19), which merits clinical attention. Although several other studies have documented the antimicrobial activity of honey against several pathogens including H. pylori, we are not aware of any study in South Africa that has investigated the antibacterial activity of honey and its organic solvent extracts on H. pylori isolates, especially so when the organism has been reported to be prevalent in our environment (20) with an increasing trend in antibiotic resistant strains (8). Therefore, a survey of different honey varieties (Pure honey, Citrus blossom and Gold crest) may reveal a honey with considerable antimicrobial activity against medically important organisms including H. pylori. The purpose of this study was therefore to screen the selected South African honeys for their anti-H. pylori activity to extract the components responsible for this activity using organic solvents and to determine the MICs of the extracts.

Materials and Methods Bacterial Strains Thirty clinical strains of H. pylori as well as H. pylori NCTC11638 (National Collection of Type Cultures, London, UK) were used. The clinical strains were obtained from gastric biopsy specimens of patients with gastroduodenal pathologies attending the Livingston Hospital, Port Elizabeth (Eastern Cape, South Africa) after informed consent following our previously reported schemes (5,8). Conrmed strains were stored in BHI broth 20% glycerol at 80 C for subsequent bioassays. Preparation of Concentrations of Crude Honey Three honey varieties were used. They included Pure honey, Citrus blossom and Goldcrest. They were purchased from different regions in South Africa. The oral sources of Pure honey and Goldcrest were mainly Citrus limon and Citrus sinesis, whereas the oral source of Citrus blossom was berry orchards. Each of the honeys was purchased directly from the producer as one sample. To the best of our knowledge, these honeys were raw, not processed. Different concentrations of each honey constituting 10% v/v, 20% v/v, 50% v/v and 75% v/v were made in sterile distilled water. This was done by dissolving the respective volumes: 0.1 mL, 0.2 mL, 0.5 mL, 0.75 mL of each honey into corresponding volumes of sterile distilled water to give a 1 mL nal volume. The different dilutions of each honey were sterilized by ltering through a 0.22 mm membrane lter (21) into separate sterile bijou bottles.

326

Manyi-Loh et al./ Archives of Medical Research 41 (2010) 324e331

Susceptibility Testing of Crude Honey The agar well diffusion method was adopted according to the method of Dastouri et al. (22) to assess the antimicrobial activity of the crude honeys. Brain Heart Infusion (BHI) agar (Oxoid, Cambridge, UK) was prepared following the manufacturers instructions, supplemented with 7% laked horse blood (Oxoid) and Skirrows antibiotics (SR 0147E, Oxoid). An inoculum of each clinical strain was prepared from a subculture of bacterial suspension and the turbidity adjusted to 1.8 x 108 CFU/mL (corresponding to 0.5 McFarland standards). A sterile cotton swab was dipped into the standardized bacterial suspension and used to evenly inoculate the BHI agar plates. The plates were allowed to dry for 3e5 min. Five wells were cut in each agar plate with a amed, cooled, cork borer of 6-mm diameter, and the agar plugs removed with a sterile needle. One hundred mL of the different concentrations of each honey was dispensed separately into each well in each plate. Clarithromycin (0.05 mg/mL) was used as the positive control. The plates were incubated at 37 C for 2e5 days under microaerophilic conditions (CampyGen BR0056A, Oxoid). Three replicates were carried out for each strain. After incubation, plates were examined and the diameters (in millimeters) of the zones of inhibition were measured not including the diameter of the well, averaged and the mean values recorded. H. pylori control strain NCTC11638 was included in all the experiments. Extraction of Crude Honey This was carried out using the method of Zaghloul et al. (11). One hundred grams of crude honey was placed in a 500 mL separating funnel, diluted with 150 mL of sterile distilled water and extracted with 150 mL of the different organic solvents (n-hexane, diethyl ether, chloroform and ethyl acetate). This was performed as three successive extractions using 50 mL of solvent each time. The shaking time for each extraction process was 15 min, after which the mixture was allowed to stand to permit the solvent layer to separate. The three layers were collected, mixed and water contaminating extracts was removed by ltration over anhydrous sodium sulfate. The organic solvent extract was concentrated by evaporating the extract under reduced pressure using a rotary evaporator (Steroglass, Strike 202, Padua, Italy) at 40 C for n-hexane, 30 C for diethyl ether, 50 C for chloroform, and 60 C for ethyl acetate. Susceptibility Testing of Honey Extracts All honeys were most active at 75% v/v concentration. Consequently, the different extracts of the most active honey at 75% concentration were tested against the strains by the method reported for crude honeys above. The respective pure solvent used for the extraction of honey was tested side by side with its extract. Diameters of zones of

inhibition of each honey extract were measured; averaged and mean values were recorded in millimeters. Determination of Minimum Inhibitory Concentration (MIC) The MICs of three of the four extracts from Pure honey were determined by broth microdilution in 96-well roundbottom microtiter plates (Grenier Bio-One, Frickenhausen, Germany) according to the method of Ban et al. (23) with modications. Two-fold dilutions were prepared in test wells in BHI broth (Oxoid); the nal honey extract concentration was 0.0098e10% v/v. Similarly, amoxicillin and metronidazole (0.0012e1.25 mg/mL) were two-fold diluted and tested on the same plate with the honey extracts as reference antimicrobials. Control wells were prepared with culture medium only, culture medium with honey extract and culture medium with bacterial isolate only. The inoculum of each strain was diluted tenfold in sterile normal saline. Twenty mL of the bacterial suspension (108 CFU/mL) was aliquoted into each well. The nal volume in each well of BHI broth, extract, and inoculum was 220 mL. The plates were sealed and incubated at 37 C under microaerophilic conditions for 2e3 days. After incubation, 32 mL of resazurin was added per well, coloring them blue. Plates were incubated for an additional 1 h and growth of bacteria in broth was observed by visual inspection and by measuring optical density (OD) using a ELISA microplate reader (Model 680,S/N19138, Bio-Rad, Tokyo, Japan) at 620 nm. The MIC was dened as the lowest concentration of honey extract that prevented resazurin color change from blue to pink. Likewise, spectrophotometrically, it was the lowest concentration of the honey extract that resulted in inhibition of bacterial growth by 95%. Statistical Analysis Diameters of zones of inhibition were expressed as mean standard deviation, calculated using spread sheetsoft ware (Excel). SPSS v.17.0 (Chicago, IL) was used to determine any statistically signicant difference in comparing the zone diameters of different honeys at various concentrations, zone diameters of clarithromycin to different honey extracts and all honeys at different concentrations as well as zone diameters of different honey extracts at 95% signicance level. Results Susceptibility Testing of Crude Honey All honeys at the various concentrations (10, 20, 50, and 75%) demonstrated antibacterial activity against H. pylori isolates (Table 1). The zones of inhibition (mean SD) ranged from 12e16 mm. The percentages of the number of H. pylori isolates inhibited by the different honeys at various concentrations are shown in Figure1. The greatest

Honeys and Their Extracts Possess In Vitro Anti-H. pylori Activity Table 1. Antibacterial activity of selected South African honeys on clinical isolates of H. pylori Zones of inhibition (mm)a Concentration of honey (% v/v) Honey types Goldcrest Citrus blossom Pure honey Clarithromycin (CLR; 0.05 mg/mL)
a

327

Table 2. Zone diameter of inhibition of the various solvent extracts of Pure honey against H. pylori isolates Zone of inhibition (mm)a Solvent extracts of Pure honey

10 13.3 13.3 12.0 18.0

20

50

75

7.8 12.9 7.9 14.4 9.1 13.7 10.0 7.5 12.6 8.0 15.1 8.7 15.5 8.5 8.8 13.2 7.8 12.7 9.9 16.0 7.5 7.3

H. pylori isolates PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE 11A 11C 26A 76A 84C 93A 102C 115A 162C 219C 252C 258C 308C 369A 369C 397C 406C 407C 411C 430A 430C 435A 436A 462A 462C 464A 464C 466A 466C 473A

n-Hexane 29 12 16 23 20 21 13 16 20 13 11 22 12 24 31 12 0 0 21 22 10 13 20 0 12 14 12 21 9 26

Diethyl ether 27 14 17 23 20 23 12 22 24 22 12 29 13 24 39 16 0 0 25 23 11 12 23 16 11 26 12 23 17 27

Chloroform 26 15 16 25 20 20 12 20 21 10 11 25 13 20 29 10 14 15 23 21 11 11 24 0 11 14 12 21 12 26

Ethyl acetate 25 13 20 25 20 22 13 23 22 17 11 23 12 20 30 15 13 0 20 22 11 14 21 0 12 13 13 0 14 26

Mean of triplicate assay standard deviation.

inhibitory effect was demonstrated by Pure honey (22/30, 73.33%) with zone diameter 16.0 7.5 mm followed by Citrus blossom (21/30, 70%) with zone diameter 15.5 8.5 mm and Goldcrest (20/30, 66.7%) with zone diameter 13.7 10.0 mm at 75% v/v, whereas the least was observed with Goldcrest and Pure honeys (16/30, 53.3%) at 20% and 10% concentrations, respectively. However, no statistically signicant difference ( p O0.05) was recorded in comparing the mean zone diameters of the different honeys at various concentrations as well as the mean zone diameters of honeys at 75% v/v concentration to clarithromycin. Susceptibility Testing of Honey Extracts Based on the mean diameter of zones of inhibition and percentage susceptibility, Pure honey was the most active. Subsequently; it was extracted using four solvents used in increasing order of polarity. All solvent extracts of the honey were active against the strains. The zone of inhibition (mean SD) ranged from 15.8e18.8 mm (Table 2). Considering the mean diameter of zones of inhibition and percentage susceptibility, the activity of the extracts was in the following order: diethyl ether, chloroform, ethyl acetate and n-hexane. The most inhibitory activity was demonstrated by diethyl ether extract (21/30, 70%) as shown by an inhibition zone of 18.8 8.3 mm, whereas the least antibacterial effect was observed with the n-hexane extract (16/30, 53.3%) as demonstrated by an inhibition zone of 15.8 7.9 mm. Both the chloroform and ethyl acetate extracts (19/30, 63.3% and 18/30, 60%) were also active with inhibition zones of 16.9 6.6 and 16.3 7.6 mm, respectively. However, comparing the mean zone diameters of extracts, there was no statistically signicant difference ( p O0.05). MIC Determination To determine MICs of the extracts, the values were determined both by visual inspection and using a spectrophotometer. By visual inspection, the MIC values of the extracts ranged from 0.625e10% v/v (Table 3). On the other hand,

Mean SD 15.8 7.9 18.8 8.3 16.9 6.6 16.3 7.6; % susceptibility 53.3% 70% 63.3% 60%. a Mean zone diameter after triplicate assay; zone diameter of sensitive strain $14 mm.

spectrophotometric readings at 95% inhibition (MIC95) gave lower MIC values that ranged from 0.078e10% v/v (Table 4). The chloroform extract recorded the lowest MIC95 values that ranged from 0.156e5% v/v, thus the best activity. MIC95 values for both diethyl ether and ethyl acetate extracts ranged from 0.625e10% v/v and 0.078e10% v/v, respectively. In contrast, the MIC95 values of amoxicillin and metronidazole ranged from 0.002e1.25 mg/mL and 0.002e5 mg/mL, respectively.

Discussion The indiscriminate use of antibiotics has developed many resistant microorganisms creating immense clinical problems in the treatment of infectious diseases such as those caused by H. pylori. Therefore, there is a need to develop

328

Manyi-Loh et al./ Archives of Medical Research 41 (2010) 324e331

Table 3. MIC values determined by visual inspection MIC values in different concentrations Solvent extracts of Pure honey (% v/v) H. pylori isolates PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE 11A 11C 26A 76A 84C 93A 102C 115A 162C 219C 252C 258C 308C 369A 369C 397C 406C 407C 411C 430A 430C 435A 436A 462A 462C 464A 464C 466A 466C 473A Diethyl ether 1.25 1.25 2.5 2.5 2.5 1.25 2.5 2.5 2.5 1.25 0.625 1.25 1.25 1.25 2.5 1.25 1.25 2.5 2.5 2.5 1.25 1.25 1.25 1.25 1.25 1.25 1.25 2.5 1.25 10 Chloroform 1.25 1.25 1.25 2.5 2.5 1.25 2.5 2.5 2.5 1.25 1.25 1.25 1.25 1.25 2.5 1.25 2.5 2.5 2.5 1.25 1.25 1.25 1.25 1.25 1.25 2.5 2.5 2.5 1.25 1.25 Ethyl acetate 1.25 1.25 2.5 2.5 1.25 2.5 1.25 2.5 2.5 2.5 1.25 2.5 1.25 1.25 2.5 2.5 1.25 2.5 2.5 2.5 1.25 1.25 2.5 1.25 2.5 2.5 2.5 2.5 2.5 1.25 Antibiotics (mg/mL) Amoxicillin 0.313 0.078 0.313 0.625 1.25 0.625 1.25 0.625 1.25 0.625 0.625 0.625 0.313 0.313 1.25 0.625 0.313 0.625 0.625 0.313 0.156 0.625 0.625 1.25 1.25 0.625 0.625 1.25 0.313 Metronidazole -

MIC values after triplicate assay; -, MIC values not within susceptible range.

alternative antimicrobial agents for the treatment of these infectious diseases. A nonantibiotic approach to the treatment and prevention of these infections includes the application of honey. Honey is produced from many different oral sources and its antimicrobial activity varies from origin and processing (24). Considering the enormous potential using honey in a clinical setting, it is important that research continues not only using honeys that are commercially available but also those of local origin with a dearth of information on their antimicrobial potential. Therefore, in this study three locally produced honeys were screened for their antimicrobial activity against local strains of H. pylori. Our data showed that all honeys tested demonstrated antibacterial action from concentrations as low as 10% v/v. This corroborates the nding of Al-jabri et al. (25) who reported in vitro antibacterial activity of both Omani and African honeys, with 25% of the honey samples showing excellent activity. Furthermore, our results are in line with the studies of Kumar et al. (26) and Adetuyi et al. (27) that reported potent activity against gram-

positive and -negative bacteria in India and Nigeria, respectively. Although not determined in this study, the antimicrobial activity in honey has been ascribed to factors such as high osmolarity, acidity, hydrogen peroxide and nonperoxide factors (28). The susceptibility of strains to the various honeys at different concentrations was dependent on the type of honey and dilutions. Zones were clearly marked with the 10% concentration. This could be due to a higher dilution factor producing higher levels of hydrogen peroxide, the major antibacterial factor in honey. Hydrogen peroxide is said to be produced by the enzyme glucose oxidase from the oxidation of glucose when it is activated by successive dilutions of honey (14). This may hold true for this study because our honeys were diluted with sterile distilled water. This may suggest that the different honeys contain antimicrobial components active against H. pylori. Although there was no statistically signicant difference between values of zone diameters of the different honeys, variations occurred in the percentage susceptibilities of

Honeys and Their Extracts Possess In Vitro Anti-H. pylori Activity Table 4. MIC values determined by spectrophotometric analysis MIC
95

329

values in different concentrations Antibiotics (mg/mL) Ethyl acetate 10 0.078 2.5 0.156 1.25 1.25 5 1.25 Amoxicillin 0.01 0.313 0.313 0.625 0.625 1.25 0.078 0.01 0.002 0.078 0.625 Metronidazole 0.313 5 0.078 0.625 0.002 0.039 -

Solvent extracts of Pure honey (% v/v) H. pylori isolates PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE 11A 11C 26A 76A 84C 93A 102C 115A 162C 219C 252C 258C 308C 369A 369C 397C 406C 407C 411C 430A 430C 435A 436A 462A 462C 464A 464C 466A 466C 473A Diethyl ether 1.25 0.625 1.25 0.625 1.25 1.25 10 Chloroform 0.156 2.5 5 1.25 0.625 1.25 -

MIC95 values after triplicate assay; -, MIC95 values not within susceptible range.

the strains (Figure 1). These variations may be attributed to the difference in antimicrobial potential because bees are able to obtain nectar from different sources. The distribution of vegetative owers and plant species from which honeybees collect nectar and sweet deposits to produce honeys

Figure 1. Antimicrobial activity of honey varieties at 10% v/v, 20% v/v, 50% v/v, 75% v/v against H. pylori isolates. Zone diameters of sensitive isolates were $14.

are affected by several factors including climatic conditions, among others (18), imparting different properties on honey from different locations due to differences in their chemical composition of fatty acids, lipids, amylases, ascorbic acid, peroxidases, fructose and tetracyclins (29). Alhough not investigated in this study, lysozyme, a well-known antibacterial agent, has been claimed to be present in honey, thus it may be responsible for the variations (22). Our positive control, clarithromycin (CLR), recognized as a key antibiotic for H. pylori (30), demonstrated good activity against the strains (23/30, 76.7%) with an inhibition zone diameter of 18.0 7.4 mm (Table 1). Comparing the zone diameter of CLR to the honeys at various concentrations, using one-way ANOVA, we observed there was no statistically signicant difference in the values ( p O0.05). On the contrary, statistically signicant differences ( p !0.05) were observed comparing CLR to Pure honey and Citrus blossom at 10% and 20% concentrations, respectively. This may suggest that Pure honey, Citrus blossom and Goldcrest honeys at specied concentrations contain substances whose antibacterial activities are highly

330

Manyi-Loh et al./ Archives of Medical Research 41 (2010) 324e331

comparable to CLR. This is in agreement with the study of Agbaje et al. (31). The nonperoxide antibacterial activity of honey is usually attributed to the presence of organic components originating from oral sources. A number of organic compounds with antibacterial activity have been extracted from honey using different solvents (32). In the present study, the most active honey (Pure honey) was extracted with four different solvents of different polarities, namely, n-hexane, diethyl ether, chloroform and ethyl acetate. Compounds such as lipids, free fatty acids, unsaturated or saturated hydrocarbons, esters, waxes (33), pyrrolizidine alkaloids, avonoids, aliphatic alcohols (32), organic acids, and phenolic acids (34) have been reported to be present in extracts of these solvents. The extracts demonstrated much more activity than the crude honey as evidenced by an increase in the zone diameter of inhibition. This may suggest that the antimicrobial components are concentrated in the honey after extraction. The diethyl ether extract was the most active extract against the strains. This is in contrast with the nding of Zagloul et al. (11) that showed a better activity of the ethyl acetate extract against their isolates. However, comparing the zone diameters of the four extracts, no statistically signicant difference was reached ( p O0.005). This may suggest that all extracts contain compounds that are inhibitory, which is in line with the ndings of other investigators (35,36). Guided by the work of Tanih et al. (8) who reported amoxicillin and metronidazole as the most sensitive and resistant antibiotics, respectively, against H. pylori in our environment, we included both antibiotics as reference antimicrobials in the assay to determine the MICs of our extracts. The MIC values determined by visual inspection may not have been accurate because impurities in the honey extracts could result in disturbance and imprecision in the readings. Consequently, we also used spectrophotometry for MIC determination. This is in accordance with the study of Tan et al. (37) that equally reported lower MIC95 values obtained spectrophotometrically at 620 nm for Tualang and Manuka honeys against their isolates. Our results revealed the chloroform extract to be the most active extract with lowest MIC95 values (thus highest activity) that ranged from 0.156e5% v/v. This result is comparable to that of the most sensitive antibiotic amoxicillin in our environment whose MIC95 values ranged from 0.002e1.25 mg/mL. In this study we demonstrated that all the honey varieties have antibacterial activity against our local isolates of H. pylori at concentrations $10%. Also, the four extracts of the most active honey (Pure honey) demonstrated inhibitory activity against our studied strains, with the chloroform extract presenting the most potent activity with a MIC95 value of 0.156e5% v/v. These honeys and their extracts may contain compounds with anti-H. pylori activity and therefore call for more elaborate phytochemical studies to isolate and characterize the compounds.

Acknowledgments
We are grateful to the Govan Mbeki Research and Development Centre, University of Fort Hare, South Africa for funding. Special thanks to Dr. N. Naidoo, Ms. N.F. Tanih and Mr. B.I. Okeleye for technical assistance.

References
1. Salih BA. Helicobacter pylori infection in developing countries: the burden for how long? Saudi J Gastroenterol 2009;15:201e207. 2. Farthing MJG. Helicobacter pylori infection: an overview. Br Med Bull 1998;54:1e6. 3. Chong VH, Lim KC, Rajendran N. Prevalence of active Helicobacter pylori infection among patients referred for endoscopy in Brunei Darussalam. Singapore Med J 2008;49:4246. 4. Atherton JC. The pathogenesis of Helicobacter pylori-induced gastroduodenal diseases. Annu Rev Pathol 2006;1:63e96. 5. Ndip RN, Takang MEA, Ojongokpoko AEJ, et al. Helicobacter pylori isolates recovered from gastric biopsies of patients with gastroduodenal pathologies in Cameroon: current status of antibiogram. Trop Med Int Health 2008;13:848e854. 6. Malfertheiner P, Megraud F, OMorain C, et al. Current concepts in the management of Helicobacter pylori infection: the Maastricht 2 2000 Consensus Report. Aliment Pharmacol Ther 2002;16:167e180. 7. Suerbaum S, Michetti P. Helicobacter pylori infection. N Engl J Med 2002;347:1175e1186. 8. Tanih NF, Okeleye BI, Naido N, et al. Marked susceptibility of South African Helicobacter pylori strains to ciprooxacin and amoxicillin: clinical implication. S Afr Med J 2010;49e52. 9. Bytzer P, OMorain C. Treatment of Helicobacter pylori. Helicobacter 2005;10:40e45. 10. Namias N. Honey in the management of infections. Surg Infect 2003; 4:219e226. 11. Zaghloul AA, El-Shattaw HH, Kassem AA, et al. Honey, a prospective antibiotic: extraction, formulation, and stability. Pharmazie 2001;56: 643e647. 12. Ndip RN, Malange Takang AE, Echakachi CM, et al. In vitro antimicrobial activity of selected honeys on clinical isolates of Helicobacter pylori. Afr Health Sci 2007;7:228e231. 13. El-Toun SK, Yagoub SO. Compression study of anti-microbial activity of honey-bees. Res J Microbiol 2007;2:776e781. 14. Iurlina MO, Fritz R. Characterization of micro-organism in Argentinean honeys from different sources. Int J Food Microbiol 2005;105: 297e304. 15. Malika N, Mohamed F, Chakib El-A. Antimicrobial activities of natural honey from aromatic and medicinal plants on antibioresistant strains of bacteria. Int J Agricult Biol 2004;6:289e293. 16. Aljadi AM, Yusoff KM. Isolation and identication of phenolic acids in Malaysian honey with antibacterial properties. Turk J Med Sci 2003;33:229e236. 17. Li CQ, Pignatelli B, Ohshima H. Increased oxidative and nitrative stress in human stomach associated with cag A Helicobacter pylori infection and inammation. Dig Dis Sci 2001;46:836e844. 18. Basson NJ, Grobler SR. Antimicrobial activity of two South African honeys produced from indigenous Leucospermum cordifolium and Erica on selected micro-organisms. BMC Complement Altern Med 2008;8:41. 19. Lusby PE, Coombes AL, Wilkinson JM. Bactericidal activity of different honeys against pathogenic bacteria. Arch Med Res 2005; 36:464e467. 20. Samie A, Obi CL, Barrett LJ, et al. Prevalence of Campylobacter species, Helicobacter pylori and Arcobacter species in stool samples from the Venda region, Limpopo, South Africa: studies using molecular diagnostic methods. J Infect Dis 2007;54:558e566.

Honeys and Their Extracts Possess In Vitro Anti-H. pylori Activity 21. Al Somal NA, Coley KE, Molan PC, et al. Susceptibility of Helicobacter pylori to the antibacterial activity of manuka honey. J R Soc Med 1994;87:9e12. 22. Dastouri MR, Fakhimzadeh K, Shayeg J, et al. Evaluating antibacterial activity of the Iranian honey through MIC method on some dermal and intestinal pathogenic bacteria. J Anim Vet Adv 2008;7:409e412. 23. Ban E, Scialino G, Monti-Bragadin C. Development of a microdilution method to evaluate Mycobacterium tuberculosis drug susceptibility. J Antimicrob Chemother 2003;52:796e800. 24. Weston R. The contribution of catalase and other natural products to the antibacterial activity of honey: a review. Food Chem 2000;71:235e239. 25. Al-jabri AA, Nzeako B, Mahrooqi Z Al, et al. In vitro antibacterial activity of Omani and African honey. Br J Biomed Sci 2003;60: 1e4. PMID 12680622. 26. Kumar A, Kaushik R, Kashyap A, et al. Indian honey: a natural product with antibacterial activity against antibiotic resistant pathogens, an in vitro study. Pak J Biol Sci 2005;8:190e193. 27. Adetuyi FO, Ibrahim TA, Jude-Ojei, et al. Total phenol, tocopherol and antibacterial quality of honey Apis mellifera sold in Owo community, Ondo state, Nigeria. Afr J Biotechnol 2009;8:1305e1309. 28. Taormina PJ, Niemira BA, Beuchat LR. Inhibitory activity of honey against food-borne pathogens as inuenced by the presence of hydrogen peroxide and level of antioxidant power. Int J Food Microbiol 2001;69:217e225.

331

 _ V, Venskutonis PR, Ceksteryt _ V. Antibacterial activity 29. Baltru saityre e of honey and beebread of different origin against S. aureus and S. epidermidis. Food Technol Biotechnol 2007;45:201e208. graud F. H. pylori antibiotic resistance: prevalence, importance, 30. Me and advances in testing. Gut 2004;53:1374e1384. 31. Agbaje EO, Ogunsanya T, Aiwerioba OIR. Conventional use of honey as antibacterial agent. Ann Afr Med 2006;5:78e81. 32. Weston RJ, Mitchell KR, Allen KL. Antibacterial phenolic components in New Zealand manuka honey. Food Chem 1999;64:295e301. 33. Fro hlich B, Riederer M, Tautz J. Honey bees discriminate cuticular waxes based on esters and polar components. Apidologie 2001;32: 1e10. 34. Aljadi AM, Kamaruddin MY. Evaluation of the phenolic contents and antioxidant capacities of two Malaysian oral honeys. Food Chem 2004;85:513e518. 35. Molan PC, Cooper RA. Honey and sugar as a dressing for wound and ulcers. Trop Doct 2000;30:249e251. PMID:11075670. 36. Lusby PE, Coombes A, Wilkinson JM. Honey: a potent agent for wound healing? J Wound Ostomy Continence Nurs 2002;29: 295e300. 37. Tan HT, Rahman RA, Gan SH, et al. The antibacterial properties of Malaysian tualang honey against wound and enteric microorganisms in comparison to manuka honey. BMC Complement Altern Med 2009;9:34. doi:10.1186/1472e6882e9e34.