Acute toxicity studies , antioxidant and in vitro antibacterial activities of extract from the barks of Ricinodendron heudoletti ( Euphorbiaceae )

Many bacteria are involved in infectious diseases. Most of these bacteria become resistant to the most commonly used synthetic drugs. In Cameroon, natural substance seem to be an alternative to this problem. Thus the aim of this research was to investigate the acute toxicity, antioxidant activities and the in vitro antibacterial of the methanol extract of Ricinodendron heudelotii (Euphorbiaceae) against twelve pathogenic bacteria involved in infectious diseases. The major bioactive components were also screened. The antibacterial activity of the extract was investigated against 12 strains including 10 Gramand 2 Gram+ bacteria by disc diffusion method and micro dilution method, followed by another agar disc diffusion for the determination of inhibition diameters, the minimum inhibitory concentration (MICs) and the minimum bactericidal concentration (MBC), respectively. 2,2-Diphényl-1-Picrylhydrazyl (DPPH) assay was used to evaluate antiradical activity. The acute toxicity study was performed according to World Health Organization (WHO) protocol. The results of the antibacterial assays indicated that the crude extract was active on 8 of 12 strains tested, with MIC ranging from 188 to 750 μg/ml and MBC from 375 to 1500 μg/ml for the extract from barks of R. heudelotii. Overall, the results of this study indicated that the crude extract represented a potential source of antibacterial and antiradical compounds as shown in previous studies and justified their traditional use in the treatment of bacterial infections and other diseases in Cameroon.


INTRODUCTION
Ricinodendron heudelotii is a perennial native tree in the tropic and subtropic areas, reaching 40 m in height and 1.2 m of diameter, belonging to the Euphorbiaceae family and widely distributed in the Centre, South and West regions of Cameroon (Noumi and Yomi, 2001).The barks are brown or grey color and smooth.R. heudolotii morphology varies from one region to another by the color of the bark, the foliage and the fruits produced ; some species like those found in South region of Cameroun have deciduous leaves and big fruits, with 2 or 3 lobes in yellow green color and black color in maturity and this is different from those in other regions of Cameroon (Noumi and Yomi, 2001).The bark extract of this plant is used against cough, as poison antidote and for the treatment of intestinal diseases.Barks is also used to treat yellow fiver, malaria, headeach, stomac pains and some times it can help pregnant women (Momeni et al., 2005).Infusion of barks of R. heudelotii has been shown to possess diuretic and aphrodisiac effects.The leaves are used to treat dysentery and the fruits can be used as spices (Momeni et al., 2005).Plants possess secondary metabolits different from primary metabolits such as proteins, lipid and carbohydrate.Secondary metabolits are classified into many groups: phenolic compounds, terpens, essential oils and nitrogenous compounds as alcaloids.Each group contains a diversity of components with biological activities (Li et al., 2007).The stem bark of R. heudolotii contains dinorditerpenoids for example, heudelotenol, heudelotinone as well as E-ferulic acid octacosylate and some natural chemopreventive agents (Suh et al., 1995).
Several researches demonstrated that many strains of Gram-positive and Gram-negative bacteria currently developed outstanding drug resistance, making the search of new, safe, non toxic and effective antibacterial agents to become strictly a necessity.Many antibacterial agents are available in nature for the treatment of systemic infections.Plants therefore constitute good source of active agents for this purpose and many plant extracts have been reported to possess various antimicrobial activities (Nawel et al., 2005).Although R. heudelotii is widely used for the mentioned biological activities, no toxicological study of the plant has been reported previously as well as its in vitro antibacterial and antioxidant activities in Cameroon.As a contribution to the search of non toxic, novel antibacterial principle from medicinal plants of Cameroon, results of in vitro antibacterial and antioxidant investigations is being reported here.

Chemicals
Gentamicin usually used was acquired from a local pharmacy.Pure methanol, chloroform, nutrient agar and nutrient broth were purchased from Merck Company and other chemicals used were from Sigma Company.

Plant
The barks of R. heudelotti were collected in Minlamizibi, a village in the South region of Cameroon in January, 2009.Botanical identification was made in the Cameroon National Herbarium and the voucher specimen was deposited under the number 16610/SFR/CAM.Preparation of extract the collected plant was dried at room temperature (30 ± 3°C), pulverized and sieved.For the extraction, the powders of 500 g each were separately macerated in pure methanol (MeOH) solvents for 72 h.The solution was filtered using Whatman filter paper N°1 and concentrated in an air circulating oven at 54°C until total dryness.

Microorganisms
Twelve species of strains isolated from patients at "Centre Pasteur of Cameroon", a reference center for diagnostic and identified by lab technicians of Centre Pasteur were used for the evaluation of antibacterial activity.These micro-organisms were Gram-negative (Escherichia coli, Salmonella typhi, Shigella flexneri, Pseudomonas aeroginosa, Enterobacter cloacae, Klebsiella pneumoniae, Klebsiella oxytoca, Morganella morganii, Citrobacter freundi and Proteus vulgaris) and positive gram (Staphylococcus aureus and Streptococcus feacalis).

Inocula preparation
An inoculum for each micro-organism was prepared from broth cultures containing approximately 5.10 5 to 9.10 6 colony forming units per mililiter (CFU/ml).Each diluted (1:50) inoculum was applied as a lawn with a micropipette calibrated to deliver 50 μl containing around 9.10 6 CFU.The discs impregnated with extract and pure methanol only to show if there is any activity noticed using pure methanol solvent, were evaporated for 24 h at the sterile condition.The inoculated plates were incubated at 37°C for 24 h.Each assay in this experiment was repeated 3 times.

Determination of the diameters of inhibition zone
The extracts was tested in vitro for antibacterial activity by the standard disc diffusion method against the micro-organisms at a concentration of 80 mg/ml with pure methanol.Gentamycin used as standard antibiotic (positive control) was tested at a concentration of 1 μg/μl.The diameters of inhibition zones produced by these extracts and dics impregnated with pure methanol were then compared to standard antibiotic (gentamycin).

MIC determination
For MICs determination, only the most sensitive microorganisms were tested with the extract.Serial dilutions were from 93.75 to 3000 µg/ml of extract in the nutrient broth medium.100 μl of the suspension of each pathogenic bacterium (10 6 cells/ml) were added and incubated at 37°C for 24 h.Standard antibiotic (positive control) was tested in the concentration between 2.5 to 80 µg/ml.The lowest concentration which did not show any macroscopic growth of tested microorganism was identified as the MIC.

Minimum bactericidal concentration (MBC) determination
For MBC determination, 100 µl of each tube following the MIC tube was subcultured in the nutrient broth medium at 37°C for 24 h.The absence of any macroscopic growth of the nearest MIC tube was identified as the MBC (Mims et al., 1993).

Antioxidant activity
Evaluation of the curd extract of R. heudelotti is based on the reduction of 2,2-Diphényl-1-Picrylhydrazyl (DPPH) by antioxidant component (Brand-Williams et al., 1995).100 mM of concentrated methanolic solution of DPPH is prepared and 2 ml shared in tubes where 10 µl of ascorbic acid added.The content was mixed on a plate shaker at 300 rpm for 30 min and incubated at 25°C.Antioxidant activity was determined by absorbance at 517 nm using spectrophotometer UV-120-01 at different concentrations 1, 2, 3, 4, 5 and 6 mg/ml.Each test was repeated 3 times.

Experimental animals
Albino Wistar rats (100 to 200 g) of about two and half months were obtained from the animal laboratory of the Biochemistry Department of the Yaounde I University, Cameroon.All the rats were kept under environmental conditions (27 ± 2°C) and they had free access to water and food.These rats were deprived of food but notwater (16 to 18 h) prior to the administration of the extract.The principles of laboratory animal care were followed while the Department's ethical committee approved the use of the animals and the study design.

Acute toxicity
The bioassay was conducted according to the World Health Organisation guideline for the evaluation of safety and efficacy of herbal medicines (WHO, 2002).For the study, albino Wistar rats were divided into six groups of five animals each.The methanol extract of R. heudelotii was suspended in the distilled water.This extract was employed because of its moderate or high antibacterial activity against all the bacteria considered.This solution was administrated per os to rats groups in a single oral dose of 2, 4, 6, 8, and 10 g/kg body weight (bw), respectively by intra gastric gavages using a feeding needle.The control group received an equal volume of distilled water as vehicle.Observations of toxic symptoms were made and recorded systematically at 1, 2, 4, 6 and 24 h after administration of the extract.The number of rats that survived were noted after 24 h and then maintained for the further 8 days with daily observations.This visual observation included skin changes, mobility and aggressiveness, sensitivity to sound and pain, as well as respiratory movements.The toxic effects of the extract were assessed on the basis of mortality which was expressed as LD50 (Schorderet, 1992).During the experiment, the animals were weighed, food and water intake were monitored.At the end of the experiment, all surviving animals fasted overnight and were sacrificed by decapitation.The organs such as liver, lungs, heart and kidneys were excised and weighed.The pathological observations of these tissues were performed on gross.The blood samples were also collected freshly in the dry non-heparinised centrifuge tubes.This blood was allowed to coagulate before being centrifuged to separate the serum.This serum was assayed for biochemical parameters.The liver was excised, rinsed in ice-normal solution followed by cold 0.1 M Tris-HCl (pH 7.5), blotted, dried and weighed.The 20% (w/v) liver homogenates was prepared in the 0.1 M Tris-HCl buffer and the supernatant was used for biochemical analysis.Lung, kidney and heart were removed, washed in 0.9% NaCl weighed and examined as previously mentioned.Determination of biochemical parameters blood collected into non heparinised tubes was centrifuged at 3,000 rpm for 10 min to separate the serum.This serum was used to evaluate the liver enzyme function through some biochemical parameters such as aspartate aminotransferase (AST), alanine aminotransferase (Reitman and Frankel, 1957), alkaline phosphatase (Oliver et al., 1954), creatinine (Bartels et al., 1972 ), urea and total protein (Allan et al., 1949), total bilirubine and direct bilirubin (George, 1959).

Phytochemical screening
Qualitative phytochemical tests of R. heudelotti methanolic extract were carried out according to Odebiyi and Sofowora (1978) methods to identify some components as alkaloids, saponins, tannins, flavonoids, polyphenols, anthraquinones.
Test for alkaloids: 0.5 g of the sample was stirred with 5 ml of 1% aqueous HCl on a steam bath and then filtered.1 ml of the filtrate was treated with a few drops of Mayer's reagent and a second 1 ml portion was treated similarly with Dragendroff reagent.Turbidity or precipitation with either of these reagents was taken as evidence for the presence of alkaloids in the extract.
Test for saponins: The ability of saponins to produce frothing in aqueous solution and to haemolyse red blood cells was used for the screening test.0.5 g of plant extract was shaken with water in a test tube.Frothing which persisted on warming was taken as evidence for the presence of saponins.
Test for tannins: 0.5 g of dried extract was stirred with 5.0 ml of distilled water.This was filtered and ferric chloric reagent was added to the filtrate.A blue-black precipitate was taken as evidence for the presence of tannins.
Test for phenol and polyphenols: 0.5 g of plant extract was heated for 30 min in a water bath.3 ml of 5% FeCl2 was added to the mixture then followed by the addition of 1 ml of 1.00% potassium ferrocyanide.The mixture was filtered and green (phenol) and blue (polyphenol) colours were observed.
Test for anthraquinones: 0.5 g of plant extract was shaken with 5 ml of benzene, filtered and 2 ml of 10% ammonia solution was added to the filtrate.The mixture was shaken and the presence of a pink or violet colour in the ammoniacal (lower) phase indicated the presence of free hydroxy anthraquinones.
Test for flavonoids: 0.5 g of plant extract was dissolved in 5 ml of NaOH at 1 N.The change of the yellow colour obtained after adding HCl 1 N indicated the presence of flavonoïds.

Statistical analysis
The values were expressed as mean ± standard deviation (SD).Each value was a mean of five or six tests.The one-way analysis of variance (ANOVA) was used to determine the significant differences between parameters and the student-Newman Keurls test served to compare these differences at p < 0.05 using statistical package used was SPSS 10.1.

Antibacterial activity
The results of the in vitro antibacterial activity of methanol extracts determined by diameters of inhibition zones are presented in Table 1.These results indicated that the diameters of inhibition zones varied from 12 to 19.67 mm   2. The MICs ranged between 188 and 750 µg/ml and the MBC ranged between 375 and 1500 µg/ml.These results revealed that P. aeruginosa was the most sensitive with values of 188 μg/ml (Table 2).
The MIC of the extract of R. heudelotii was less active than standard antibiotic (gentamycin) ranged between 5 and 10 µg/ml.No result of MIC and MBC was determined for S. typhi, K. oxytoca, S. flexneri and P. vulgaris isolates.

Acute toxicity
For the acute toxicity studies, no death of rats was neither recorded in the control nor in the treated groups.The animals exhibited slight changes in general behaviour (slow response to stimuli, stretching and sluggishness) but did not expressed changes in their physiopatological activities.Although there was an increase in the weights of rats and their organs in the treated groups, this addition remained statistically not significant compared with control.The pathological examinations of the tissues on a gross basis indicated no detectable abnormalities at the end of the experiment.Table 4 shows the results of the blood and hepatic parameters.These results indicated that alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) of the treated rats increased significantly (p > 0.05) at the higher concentration of the extract compared to the control.We noticed also an increase of urea, total protein, creatinin (CRT) and bilirubin of the treated rats but the diference is not significant (p < 0.05).

Phytochemical screening
The phytochemical screening of the extract of R. heudelotii indicated the presence of three main classes of compounds: polyphenols, alkaloids and steroids.The polyphenols group constitutes the principal component of R. heudelotii extract with three bioactives subgroups: tannins, flavonoids and saponins.Anthraquinone was absent in the extract of R. heudelotii.Alkaloids and steroids whose antimicrobial activities have been demonstrated were also found in this extract (Table 5)

DISCUSSION
This work permitted the evaluation of some biological properties of R. heudelotii; among which the antimicrobial activity directed on some pathogens frequently encountered in infections as well as the toxicological and antioxidant studies of this extract.The diameters of inhibition zones varied from 12 to 19.67 mm and 26 to 34.67 for the extracts and gentamycin, respectively.The  (Dinan et al., 2000).The variation observed from the diameters of inhibition zone of the bacteria tested can be attributed either to the difference of the bioactive molecules present in the extract or to their mechanism of action on Gram-positive and Gramnegative bacteria.The mechanism of action of the glycosides, polyphenols, tannins and alkaloids on Grampositive and Gram-negative bacteria was demonstrated (Deeni and Sadiq, 2002;Kalemba, 2003).The MICs ranged between 188 and 750 µg/ml and the MBC ranged between 375 and 1500 µg/ml.These results revealed that P. aeruginosa was the most sensitive with values of 188 μg/ml.The MIC of the extract of R. heudelotii was less active than standard antibiotic (gentamycin) ranged between 5 and 10 µg/ml.The antibacterial property of P. aeruginosa is well documented (Kuete et al., 2007;Armelle et al., 2008).The rate MBC/MIC revealed that the extract presents a bactericidal action (MBC/MIC < 4).This action can justify the use of this plant against bacterial infections.No result of MIC and MBC was determined for S. typhi, K. oxytoca, S. flexneri and P. vulgaris whose isolates were the most resistant bacteria isolate with no diameters of inhibition zones and this can explain the result obtained.
The evaluation of trapping of DPPH radical revealed a high antiradical activity of the extract from barks of R. heudelotii with a SC 50 of 7.21 mg/ml.This antioxidant activity observed in our study can be attributed to the presence of various bio-actives components such as tannins, polyphenols, alkaloids, flavonoids and steroids found in this extract (Sokol-Letowska et al., 2007).However this trapping rate remained lower than the one of ascorbic acid (SC 50 = 3,28 mg/ml).The work done by Afolabi et al., in 2007(Afolabi et al., 2007) showed a large correlation between the percentage of phenolic components of the extract and the inhibitory activity of the extract on the DPPH.
The acute toxicity test has been investigated to establish the adverse effects of the administration of the methanol extract of R. heudelotii on some behavioural and biochemical parameters.The results indicated that up to 10 g/kg bw, no abnormal symptoms and no death of the rats was observed.According to the OCED protocol (Organisation of Economic Co-operation and Development, 2001) R. heudelotii extract can be classified as non toxic since the limited dose of an acute toxicity is generally considered to be 5.0 g/kg bw (Schorderet, 1992).If no mortality is observed at this level, a higher dosage is generally not necessary (Wallace, 1989).However, the body weight of the rats increased during the experiment.Compared to the control, the weight gained by the treated rats was higher but statistically not significant.This result showed that the extract slightly stimulates the appetite of the rats and probably not irritates directly the gastrointestinal tract.
The significant changes noted in some of the blood chemistry parameters such as ALT, AST, ALP and CRT were an indicator of some toxic effects undoubted.It should be noted that these modifications were observed at high dose (more than 6 g/kg bw).Even though the changes noted were not significant, they were statistically different compared to control.The variation of biochemical parameters indicated the malfunctioning of one or many organs.The increase of the activity of ALP after the administration of the extract may indicate the obstruction of the bile duct.However, this variation can not be attributed only to a dysfunction of the bile duct since many sources of ALP are known (liver cells; osteoblast, intestinal cells and placenta tissue).Although the variation of ALT and AST activities are associated with the hepato-cellular damage, only ALT is specific for the evaluation of liver damage.AST is highly concentrated in cardiac muscle, liver, skeletal muscle and kidneys.The significant increase of ALT and AST activities after treatment of the rats with 8 and 10 g/kg bw of extract implies an injury of the liver as well as the heart or other sources of these enzymes.The creatinine level of treated rats increased compared with the control.This last result confirms that kidneys are slightly affected at the dose of 10 g/kg body weight of the extract.The variation of other parameters such as protein and bilirubin were not statistically significant.
It has been recognized that eventual therapeutic bioactive products from plants may also contain substances which act as poisons in human (Ekramul et al., 2002).Several researches demonstrated that phenolics and polyphenols compound have antimicrobial activities.Other workers have shown that the sites and the number of hydroxyl groups on the phenol are thought to be related to their relative toxicity to microorganisms, with the evidence that an increase of hydroxylation will result to an increased toxicity (Kareem et al., 2008).The presence of three subclasses of polyphenols (tannins, flavonoids and saponins) in the extract may have significant inhibitory effect on all isolates tested.The antibacterial effects of each of these subclasses of polyphenols on Gram-positive and Gram-negative have been demonstrated as well as the microbicidal effects of alkaloids (Kalemba and Kunicka, 2003).The presence of multiple phytochemical components confers to R. heudelotii extracts in vitro antibacterial and antioxidant activities.

Conclusion
The methanol extract of R. heudelotii demonstrated effective in vitro antibacterial and antioxidant activities.However some toxic effects have been discovered after administration of high dose of this extract.Further research needs to be carried out to identify the active molecules and evaluate the in vivo antibacterial and antioxidant activities as well as sub-acute or chronic toxicities.

Table 1 .
Antibacterial activities of R. heudolotti from the disc diffusion method.

Table 2 .
MIC, MBC values (µg/ml) of R. heudolotti extract in the macro dilution assay comparable to gentamicin.

Table 3 .
Trapping percentage of the extract/ascorbic acid at different concentration.

Table 4 .
Biomedical parameters values of male rats in acute toxicity of the methanol extract from the barks of R. heudolotti.

Table 5 .
Phytochemical screening of crude methanol extract.