The fruit of PN were collected in the morning in Zo-Etélé, Cameroon in September 2011. The identification of the plant was confirmed at the National Herbarium in Yaoundé-Cameroon (reference number of the plant: N° 2136/SRFK). The seeds of the fruits of PN were removed and the fruit-rinds were kept. The plant material (fruit-rinds of PN) was then air dried at room temperature. The dried plant material was ground into a fine powder.
Chemicals and culture media
Mueller Hinton agar and salmonella-shigella agar were purchased from Oxoid, Basingstoke, England; Sabouraud dextrose agar from Merck SA, Sao Paulo, Brazil and Peptone water from HiMedia, Mumbai, India. Antimicrobial susceptibility testing was performed by the disc diffusion method with commercially available disks (Biomerieux F 69260 Charbonnières, France) for Cefoxitin (30 μg), Dibekacine (10 μg) and Clindamycin (10 μg); (Becton, Dickinson and Company Sparks, USA) for Ampicilin (10 μg), Colistin (10 μg), Nitrofurantoine (100 μg), Streptomycin (10 μg), Kanamycin (30 μg), Spectinomycin (25 μg) and Tobramycin (2 μg); (Oxoid, Basingstore, England) for Doxycyclin monohydrate (30 μg), Netilmicin (30 μg), Erythromicin (15 μg), Amoxycillin/Clavulanique (20/10 μg), Amoxicillin (10 μg), Gentamicin (10 μg), Vancomycin (30 μg); (Himedia, Mumbai, India) of ciprofloxacin (5 μg). Gentamicin (Gentamycin injection, 8 mg/2ml, ZMC Hamburg GMBH German), Nystatin (500000 UI, Novadina Pharmaceutical Ltd, London, United Kingdom) and Ciprofloxacin (ZOFLOX, ciprofloxacine 750 mg, Odypharm) used as reference antibiotics for the determination of the MIC and MBC as well as for the in vivo test were purchased from local pharmacy.
This was carried out by soaking the dried powdered plant (250 g) in bottle with 3.5 l of methanol (M) and kept for 72 h. The plant-(M) mixture was then sieved. The filtrate (extract) was concentrated by evaporating under vacuum (M) using a rotating evaporator. The extract was further concentrated by allowing it to stand overnight in an oven at 30°C. The yield of the extraction was calculated by dividing the weight of the extract obtained by the weight of the dried material plant extracted.
Preliminary analysis of chemical constituents such as alkaloids, leucoanthocyanins, flavonoids, saponins, tannins, anthraquinones, polyphenols, coumarins, sterols and/or triterpenes, anthocyanins, cardiac glycosides, reducing sugars, glycosides, anthranoids and steroids was carried out. Those constituents were identified by characteristic colour changes using standard procedures previously described. Each test was qualitatively expressed as negative (−) or positive (+) and the intensity of the characteristic colour was expressed as (++) or (+++).
Test for alkaloids (Meyer reagent)
Fifty milligrams of the tested compound was heated in 10 ml of 2% H2SO4 for 2 min and filtered. To 1 ml of the filtrate, a few drops of the Meyer Reagent was added. The presence of alkaloids was indicated by the obtaining of a white precipitation or turbidity.
Test of flavonoides
Fifty milligrams of the tested compound was introduced in 5 ml of methanol. To it was added some magnesium chip and concentrated hydrochloric acid drop wise. The presence of flavonoids was indicated by the appearance of a violet or brick red coloration with effervescence.
Test for leucoanthocyanines
Fifty milligrams of the tested compound was dissolved in 10 ml distilled water. To 0.5 ml of this mixture was added 2 ml of 2N HCl and heated in a water bath at 100°C. The presence of leucoanthocyanines was indicated by the appearance of red coloration which later changes to violet.
Test for saponins
Fifty milligrams of the tested compound was added to 5 ml of distilled water. After homogenization, the mixture was boiled in 5 min, cooled and filtrated. 5 ml of the filtrate was vigorously agitated for 10 to 30 s. The presence of saponins was indicated by the appearance of foam which persists within 15 min.
Test for tanins
Fifty milligrams of the tested compound was added in 5 ml of distiller water. After mixing, the mixture was boiled for 5 min, cooled and filtrated. 5 ml of 2% NaCl was added to 10 ml of the filtrate and then filtered. The presence of tannins was indicated by the formation of a precipitate after the addition of 1% gelatin to the filtrate.
Test of anthraquinones
Fifty milligrams of the tested compound was diluted in 2 ml of chloroform, homogenized and filtered. Next, 1 ml of 10% NaOH was added to 1 ml of the filtrate. The presence of anthraquinone was indicated by the appearance of a red coloration.
Test of phenols and polyphenols
Fifty milligrams of the tested compound was dissolved in 15 ml distilled water, headed in a water bath for 15 min then cooled and filtered. To 2 ml of the filtrate was added drop wise 1 ml of 1% FeCl3 and 1 ml of 1% K3Fe(CN)6. The presence of polyphenols and phenols was indicated by the apparition of a blue and green precipitate respectively.
Tests of coumarines
On a thin layer chromatographic plate was deposited a spot of the tested compound dissolved in methanol. Next, the plate was exposed to ammoniac vapors. The presence of coumarines was indicated by the appearance of spot whose color varied from blue to purple in the presence of ammoniac.
Test of triterpenes and sterols (Liebermann- burchard test)
Fifty milligrams of the tested compound was dissolved in 2 ml of chloroform and next some drops of acetic anhydride added to the mixture and 1 ml of concentrated H2SO4. The presence of triterpenes was revealed by the apparition of violet-red coloration and sterols by a blue greenish coloration.
Test for anthocyanins
To 50 mg of the tested compound was added 15 ml of a concentrated HCl, the mixture was boiled. The presence of anthocyanins was indicated by the variation of coloration from orange red to orange blue during boiling.
Test for cardiotonic glycosides
In a test tube, 50 mg of the tested compound was dissolved in 2 ml of chloroform. 2 ml of the concentrated H2SO4 was introduced through the walls of the tube. Two phases were formed. The presence of cardiotonic glycosides was indicated by the observation of a brown ring at the interphase.
Test of reducing sugars
Fifty milligrams of the tested compound was dissolved in 10 ml of distilled water, mixed and filtered. In a conical flask containing 4 ml of a Fehling A and B (v/v) liquid mixture, was introduced drop wise of the filtrate. The mixture was boiled; the presence of reducing sugar was indicated by the appearance of a brick red coloration.
Test of glycosides
Fifty milligrams of the tested compound was added to 5 ml of 5% HCl then neutralized with 5 ml 5% NaOH. After homogenization, the mixture was filtered and Fehling liquid test was realized on the filtrate.
Test for anthranoides
Fifty milligrams of the tested compound was added to 2 ml of 0.5 KOH, then 0.5 ml of 5% H2O2 and boiled for 2 min. After cooling, the mixture was filtered. 6 drops of concentrated acetic acid and 3 ml of toluene added to the filtrate. The upper phase (toluene) was transferred to a test tube in which was added 1 ml of 0.5 N KOH. The presence of antranoides was indicated by the appearance of red coloration in the aqueous layer.
Test of steroids
Fifty milligrams of the tested compound was dissolved in 3 ml of chloroform, agitated intermittently for 2 hours and filtered. 1 ml of the filtrate was deposited on a porcelain plate. After evaporation, a drop of sulfuric acid was added, stirred and the color noted. To another portion of the filtrate was added a drop of acetic anhydride and the color noted. The reaction was positive if the same color was obtained for both sulfuric acid and acetic anhydride.
Four weeks old Wistar albino rats (weighing 65–95 g) of both sexes were obtained from the animal house of the Centre for research in Food and Nutrition (CRAN) of the Institute of Medical Research and Medicinal Plant Studies (IMPM), Yaoundé. The rats were given food and water ad libitum. These animals were used for the antimicrobial drug assessment and the study of intestinal transit. Animal housing and the bioassay was conducted in accordance with the internationally accepted principle guidelines of the European Union on Animal Care (CEE Council 86/609). The Cameroon National Ethical Committee approved the protocols of the study (Ref n° FW-IRB 00001954).
The clinical microorganisms included here for the antimicrobial activity screening were the most common strains implicated in gastro-intestinal disorder such as diarrhea and dysentery. There were 15 bacterial and 2 yeast stains : Escherichia coli LMP0101U (E. coli), Klebsiella pneumoniae LMP0210U (K. pneumoniae), Shigella dysenteriae type 1 LMP0504G (S. dysenteriae), Shigella flexneri LMP0313U (S. flexneri), Morganella morganii LMP0904G (M. morganii), Proteus vulgaris LMP0103U (P. vulgaris), Proteus mirabilis LMP0504G (P. mirabilis), Salmonella typhi LMP0209U (S. typhi), Citrobacter freundii LMP0804G (C. freundii), Enterobacter cloacae LMP1104G (E. cloacae), Enterobacter agglomerans LMP1004G (E. agglomerans), Staphylococcus aureus LMP0206U (S. aureus), Streptococcus feacalis LMP0207U (S. feacalis), Pseudomonas aeruginosa LMP0102U (P. aeruginosa), Bacillus cereus T F 3748 (B. cereus T), Candida albicans LMP0204U (C. albicans) and Candida glabrata LMP0205U (C. glabrata). These microorganisms were obtained from Bacteriology and Mycology Laboratories of Centre Pasteur of Yaoundé-Cameroon and Bacillus cereus T was obtained from the Microbiology Laboratory of the Institute of Food Research Reading of Great Britain.
In vitro antimicrobial activity
Disc diffusion assay
The dried plant-extract was dissolved in the same solvent of extraction (M) to a final concentration of 125 mg/ml. Sterile paper disc (6 mm of diameter) prepared from Whatman number one filter paper were impregnated with 10 μl of the crude extract of the plant as described by Edward from a stock of 125 mg/ml. Each disc contains 1.25 mg of the extract. These paper discs were kept in an incubator at 37°C for 24 h to evaporate the solvent. Antimicrobial tests were then carried out by disc diffusion method. 100 μl of the suspension of the tested microorganism (0.5 Mc Farland standard turbidity) containing 108 CFU/ml of bacterial, 106 CFU/ml of yeast prepared from an overnight Mueller Hinton agar culture for bacterial and Sabouraud dextrose agar for yeast was used to seed each prepared and dried Mueller Hinton agar plate for bacteria and Sabouraud dextrose agar for yeast. The discs were arranged and firmly pressed on the agar surface of each seeded plate. These plates, after staying at 4°C for 2 h were incubated aerobically at 37°C for 24 h for bacteria and at 25°C for 24 h for yeast. Similarly, paper discs containing concentration of standard antibiotics (Gentamicin and Nystatin) were prepared and used for the susceptibility test. Negative control was also prepared using the same solvent employed to dissolve the plant extract. Antimicrobial activity was evaluated by measured the zone of inhibition against the tested microorganism and were expressed by – (negative), + (zone of inhibition ≤ 8 mm in diameter), ++ (zone of inhibition > 8 mm and ≤ 20 mm in diameter) and +++ (zone of inhibition > 20 mm in diameter). The result recorded for each bioassay was the average of 3 tests.
Only for S. dysenteriae type 1, a concentration dependent effect of the extract was evaluated using disc diffusion method described above. The reason of this choice was that the strain will later be used to induce bacillary dysentery. The concentrations of the extract tested were 5, 2.5, 1.25 and 0.625 mg. In order to determine their multi-drug resistance profiles, antimicrobial susceptibility testing was performed also against Sd1 by the same method with commercially available disks of Gentamycin 10 μg, Amoxicyclin/clavulanic Acid 20/10 μg, Nitrofurantoin 100 μg, Kanamycin 30 μg, Ampicilin 10 μg, Netilmicin 30 μg, Colistin 10 μg, Erythromycin 15 μg, Ciprofloxacin 5 μg, Amoxycylin 10 μg, Vancomycin 30 μg, Doxycyclin monohydrate 30 μg, Spectinomycin 25 μg, Tobramycin 2 μg, Clindamycin 10 μg, Dibekacin 10 μg, Steptomycin 10 μg and Cefoxitin 30 μg.
Determination of MIC and MBC
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were assessed for the microorganisms that were determined as sensitive to (M) extract of PN in disc diffusion assay using the broth microdilution method. The test was performed in peptone water supplemented with glucose 1% (w/v) with red phenol as a colour indicator (PPG1%). Bacterial strains were cultured overnight at 37°C in Muller Hinton and yeast strains at 25°C in Sabouraud dextrose agar. Test strains were suspended in normal saline (NaCl 9‰), adjusted to 0.5 Mc Farland standard turbidity and suspended in PPG1% to give a final density of 5×105 CFU/ml. For the susceptibility test, the 96-well round bottom sterile plates were prepared by dispensing 180 μl of each inoculated broth into wells. A 20 μl aliquot of the plant extract was added. The concentration of extract used to evaluate the antimicrobial activity was included from 0.39 to 12800 μg/ml. One well was considered as growth control since no extract solution was added. The final volume of each well was 200 μl. The content of each well was mixed and then incubated under normal atmospheric condition at 37°C for 24 h (for bacterial strains) and at 25°C for 24 h (for yeasts strains).
The bacterial and yeasts growth was indicated by the colour change of the well content from the red to yellow. The MIC was defined as the lowest concentration of the extract to inhibit the growth of microorganism. MBC were determined by platting 5 μl sample from red wells on Mueller Hinton agar or on Sabouraud dextrose agar without extract. The MBC was the concentration at which there was not microbial growth. The extract tested in this study was screened three times against each microorganism. Gentamicin for bacteria strains and Nystatin for yeast strains at the concentration range of 0.019 to 320 μg/ml was prepared in (PPG1%) and used as standard drugs for the positive control. The susceptibility of the sd1 to antibiotic Ciprofloxacin which shown the great activity against sd1 in disc diffusion assay was also performed by liquid dilution method and served as positive control.
In vivo antimicrobial activity
Thirty rats were individually kept in metabolic cage during a period of observation of 7 days before diarrheal induction. Diarrhoea was induced in rats using sd1 strains. The turbidity of the sd1 inoculum was matched spectrophotometrically at 450 nm to 4 McFarland standards. After verifying that the rats were not sd1 carriers, they were orally administrated with 1 ml of the saline diluted inoculum. Diarrheic rats were randomly divided into 5 groups, each containing six animals. When diarrhoea appeared, animal were administrated the anti-diarrheic drugs daily by the oral route for 7 consecutive days: The first group (diarrheic control) received the vehicle of the extract (0.5% Tween 80 in distilled water); the second group received the antibiotic ciprofloxacin (20 mg/kg of body weight (bw) and the remaining three groups were given orally 125, 250, and 500 mg/kg bw of M extract of PN.
Animals were observed daily for behavioural changes and mortality patterns once before induction, during induction and up to 7 days after induction. The stools of each animal were also collected daily using a white cloth fixed under the bars supporting the animal in the metabolic cage during all the experiment period. Then, the frequency and weight of normal and diarrheic faeces were recorded. The presence of stools containing mucus or blood was also noted daily at each experimental period. Enumeration of sd1 in faeces was performed before diarrheal induction to make sure that the rats are not sd1 carriers, after the appearance of diarrhoea and once every day during the 7 days of treatment. For this purpose, 0.5 mg of faeces was homogenized in 4.5 ml of sterile saline; serial of dilution were made and 50 μl of each dilution was seeded over salmonella-shigella agar. After 24 h of incubation at 37°C, the number of CFU was determined.
Study of small intestinal transit
This was done according to the method proposed by Mujumdar using charcoal meal as a diet marker. The rats were divided into 4 groups of 6 animals each. The first group (the control group) was orally administered the vehicle. The second and third groups orally received M extract of PN, 250 mg/kg and 500 mg/kg bw respectively. The fourth group also orally received the standard drug, atropine sulphate (5 mg/kg bw). 30 min after administration, each animal was given 1 ml of charcoal meal orally (10% activated charcoal in 5% gum acacia). Also, 30 min after this administration, each animal was sacrificed and the distance covered by the charcoal meal in the intestine, from the pylorus to the caecum was measured and expressed as a percentage of distance moved.
The results are expressed as means ± standard deviation. Bacterial densities were expressed in log10 before analysis. Data were statistically evaluated using the analysis of variance following by the paired t-student’s test. The differences between groups were considered significant at p < 0.05.