Abstract: The purpose of this investigation deals with the antibacterial, antiradical activities and the toxicity (LD50 and haematological parameters) of Alafia multiflora stem barks. A. multiflora is a medicinal plant known for its curative effects on ulcerous wounds. Antibacterial activities of crude extracts were evaluated against E. coli, S. aureus, E. agglomerans, P. aeruginosa, P. vulgaris, K. Pneumoniae and the inhibitory zones as well as the Minimum Inhibition Concentrations (MIC) determined. Antiradical activity and total phenolic compounds of the extracts were evaluated. Methanol, methylene-chloride/methanol and aqueous extracts inhibited the growth of all the test microorganisms with MIC values ranged from 2.5 to 40 mg mL-1. The best antiradical activity was obtained at 0.25 mg mL-1. These results suggest that A. multiflora possess antibacterial and antiradical constituents. Aqueous and the methanol extracts administered in single oral doses of 0, 2.5, 5, 7.5 and 10 g kg-1 to groups of 10 rats (5 males, 5 females) yielded no mortality after 7 days observation. The LD50 values of the tested extracts were found to be above 5000 mg kg-1, indicating that A. multiflora is rather poorly toxic. A significant increase of platelet count was observed in animals treated with aqueous and methanol extracts at 5 to 10 g kg-1.
INTRODUCTION
Alafia multiflora Stapf is a large liana of Apocynaceae family. It is a multiple usage medicinal plant distributed in West and Central Africa. In Ghana, the latex diluted with water is taken orally to cure stubborn wounds (Tsala and Dimo, 2006; Abbiw, 1990). The latex mixed with bark scrapings is applied to wounds and leg ulcers and to ulcers caused by syphilis. The seeds are also used as an ingredient of arrow poison in the Democratic Republic of Congo (Tsala and Dimo, 2006; Neuwinger, 2000). In Cameroon the fresh latex, either alone or mixed with Oncinotis glabrata (Baill.) Stapf ex Hiern, is also applied to treat yaws. The stem bark or fruits decoction are taken to relieve abdominal pain. According to Farrell et al. (1991) latex and resin from A. multiflora may protect itself from pathogens by sanitizing and sealing wounds. Balansard et al. (1980) found that vanillic acid isolated from the latex possesses antibacterial properties, while Pais et al. (1971) isolated alafine (a pyrrolizidine alkaloid) from the seeds as an ester of syringic acid. There is a considerable evidence indicating association between reactive oxygen species and the physiopathology of chronic diseases such as diabetes, hypertension and cancer, aging, burns and chronic wounds (Beswick et al., 2001; Tomasz, 2002; MacKay and Miller, 2003; Telgenhoff and Shroot, 2005). In order to protect themselves against free radicals, living organisms are endowed with defence systems implicating enzymes (catalase, superoxide dismutase, glutathione peroxidase/reductase) and exogenous bio-molecules (Vitamin C and E, β-carotene) and metabolite by-products (bilirubine, uric acid) (Llesuy et al., 2001; Javanmardi et al., 2003). The antioxidant potential of plants extracts is mainly related to the phenolic components, such as flavonoids, phenolic acids and phenolic diterpenes (Javanmardi et al., 2003). Antioxidants inhibit or delay membrane peroxidation and can be expected to prevent cellular senescence in wound healing and chronic diseases (Telgenhoff and Shroot, 2005; Mahmood et al., 2005; Shivananda et al., 2006). Giving that A. multiflora is used for wound healing and demonstrated protective effect against CCl4-induced oxidative stress in rats (Dimo et al., 2006). The aim of this study was to investigate the antibacterial activity, free radical scavenging effect and LD50 of various extracts of A. multiflora.
MATERIALS AND METHODS
Plant materials and preparation of extracts: Fresh stem barks were collected in May, in the village Nkolntara, near Yaoundé city (Central Province of Cameroon) and authenticated at the National Herbarium-Yaoundé, where the voucher specimen was conserved under the reference number 43196/HNC. The above plant parts were dried at room temperature, ground into a powder 5.5 kg was macerated in a mixture of methylene-chloride/methanol/ (1:1) (CM) for 48 h. The supernatant was evaporated at 80°C in a vacuum desicator to obtain 50 g of brown extract. For methylene-chloride (C) and Methanol (M) extracts, 15 g of CM extract was further fractionated in methanol to obtain 5.5 g methanol extract and 9.23 g methylene chloride extract. The hexane extract was prepared as follows: 5 g of the C were dissolved in hexane. This was mixed and filtered. The filtrate was evaporated and the Hexane (H) extract obtained (0.9 g) was used. The aqueous (W) extract was obtained by adding 5 L of distilled water to 1 kg of the fine powder. The mixture was allowed to stand and macerated at 50°C. The resulting infusion was filtered after 2 h and lyophilised to obtain 21, 17 g of a brown extract.
Antibacterial activity
Test organisms: Six species of microorganisms namely Staphylococcus
aureus, Proteus vulgaris (Gram positive bacteria), Escherichia
coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus
agglomerans (Gram negative bacteria) were used in this study. They were
all clinical isolates obtained from Centre Pasteur du Cameroun (Yaoundé).
The identity of the strains was confirmed using the standard biochemical methods
(Cheesbrough, 2000). The test organisms were maintained on agar slant at 4°C
and were subcultured on the fresh appropriate agar plate 24 h prior to any antimicrobial
test.
Culture media: Muller Hinton Agar (MHA) was used for the diffusion assays and Nutrient broth containing 0.05% phenol red and supplemented with 10% glucose (NBGP) was used for MIC determination.
The agar-well diffusion test: The antimicrobial diffusion test was carried out as described by Candan et al. (2003) using a cell suspension of about 1.5 106 cfu mL-1 obtained from a McFarland turbidity standard N° 0.5. The suspension was standardised by adjusting the optical density to 0.1 at 600 nm (SHIMADZU UV-120-01 spectrophotometer) (Tereschuck et al., 1997). This was used to inoculate by flooding the surface of MHA plates (90 mm ø). Excess liquid was air-dried under a sterile hood. Wells of 6 mm were bored into the agar plate using the bold end of a pipette tip and then 75 μL of extract of desire concentration was dropped into the wells. A well prepared with only the corresponding volume of nutrient broth was used as negative control. The plates were incubated at 37°C for 24 h. Antimicrobial activity was evaluated by measuring the diameter of the Inhibition Zone (IZ), around the well. Extracts with IZ > 7 mm was considered.
Tube dilution test: MICs of test samples found active by the diffusion test as well as the MICs of the reference antibiotic (gentamycin) were determined as follows: extract containing NBGP prepared at the concentration of 160 mg mL-1 was serially diluted two-fold to obtain concentrations range of 1.25 to 160 mg mL-1 in a total volume of 5 mL. Gentamycin containing NBGP (156.25 μg mL-1) was also diluted the same way to obtain concentrations range of 0.0625 to 8 mg mL-1. Each tube, as well as the negative control (extract-free) was inoculated with 50 μL of standard inoculum of test organism (approximately 5x106 cells) (Camporese et al., 2003) and incubated at 37°C for 24 h. Microbial growth was determined by observing the change of colour in the tubes which change to yellow when there is growth. The lowest concentration showing no colour change was considered as the MIC. The results recorded for each bioassay was the average of a simultaneous duplicated test.
Free radical-scavenging activity determination: The free radical scavenging activity of the extracts was measured by DPPH, according to the previously described technique (Brand-Williams et al., 1995). The calculation of the DPPH inhibition was done by Qian and Nohinbere (2004) method.
Total soluble phenolic compounds: The total soluble phenolic compounds present in the different plant extracts was determined according to the method described by Singleton et al. (1999). The results recorded for each bioassay was the average of simultaneous triplicate test.
LD50 investigation and haematological function: Toxicological assessment. The test was carried out following the methods described by WHO (1992). Five groups of 10 inbred Wistar rats weighing between 70 and 120 g (5 males and 5 females), were constituted based on the administered doses, i.e. 0, 2.5, 5, 7.5 and 10 g kg-1. The animals were maintained on a 12 h light/dark cycle with water ad libitum and fed with laboratory baked food made of maize (50%), soybeans (25%) and wheat flour (25%), supplemented with table salt, palm oil, fish and bone powder. They were deprived of food 18 h prior to administration of the extracts. Prior authorization for the use of laboratory animals was obtained from the Cameroon National Ethics Committee (Reg. N° FWA-IRB00001954). The test animal received intragastrically single oral doses of aqueous and methanol extracts of A. multiflora and were under close observation for 72 h after dosing. The weight, food and water intake was also recorded for 7 days. At the end of this period, animals were sacrificed by decapitation and the blood was collected for the determination of haematological parameters. The relative weights of the liver, the kidneys, the heart and the lung were also determined.
Haematological analysis: Haematological parameters were determined using an HUMACOUNT HUMAN GmbH haematological analyser. They included haemoglobin concentration, red blood cells and white blood cells count, differential white blood cells count, haematocrit, mean cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and platelet count.
Statistical analysis: The one way Analysis of Variance (ANOVA) and Student-Neuman-Keuls multiple comparison tests of the SPSS program was used to determine statistical differences between treatments. p<0.05 was considered to be statistically significant. Data are expressed as mean±SEM.
RESULTS
Antibacterial activity: Out of six plant extracts screened, three (CM, M and W extracts) inhibited the growth of test organisms in the Petri dishes (agar-well diffusion test) the (Table 1). The diameter of the IZ ranged from 10 to 23 mm. M extract showed a greater inhibiting activity compared to CM extract. The lowest IZ was obtained with W extract on S. aureus, while the highest IZ value was obtained with the M extract on K. pneumoniae. The C extract was found to be not active against S. aureus, P. vulgaris, P. aeruginosa, while the hexane extract was not inhibitory to any of the test organisms (Table 1). Three extracts (CM, M and W) inhibited like gentamycin, the growth of all the six tested bacteria. The results indicated a stronger activity of the M extract against all the bacteria tested, with Minimum Inhibitory Concentrations (MICs) ranging from 2.5 to 10 mg mL-1 (Table 2).
Antiradical activity and total soluble phenolic compounds: The results obtained showed that DPPH solution was bleached with all the samples tested. However, differences could be observed between the different antiradicals used depending to their concentrations. As indicated on Table 3, DPPH inhibition was greater for the gallic acid compared to that of A. multiflora extracts.
| Table 1: | Inhibition zones of various extracts of Alafia multiflora |
| W = Aqueous extract; C-M = Methylene-chloride-methanolic extract; C = Methylene-chloride extract; M = Methanolic extract; H = Hexane extract; G = Gentamycin; na = Not active a. Diameter of inhibition zone including diameter of well. The number is the concentration at which the extracts have been tested (mg mL-1) | |
| Table 2: | Minimum inhibitory concentration of various extracts of Alafia multiflora |
| W = Aqueous extract; C-M = Methylene-chloride-methanolic extract tested; C = Methylene-chloride extract; M = Methanolic extract; H = Hexane extract tested at 20 mg; G = Gentamycin (na = Not activea) | |
| Table 3: | Antiradical activity of gallic acid and Alafia multiflora extracts (% inhibition of DPPH) and total soluble phenols, using gallic acid as standard) |
| W = Aqueous extract; C-M = Methylene-chloride-methanolic extract; C = Methylene-chloride extract; M = Methanolic extract; H = Hexane extract | |
| Table 4: | Mean organs relative weight (mg g-1 body weight) of rats treated with various acute doses (2.5-10 g kg-1) of aqueous extract of Alafia multiflora |
| a: Indicates a statistically significant difference (p = 0.05) as compared to dose 0 g kg-1 (n = 5). d: Indicates a statistically significant difference (p = 0.05) as compared to dose 7.5 g kg (n = 5) | |
| Table 5 : | Mean organs relative weight (mg g-1 body weight) of rats treated with various acute doses (2.5-10 g kg-1) of methanol extract of Alafia multiflora |
| a: Indicates a statistically significant difference (p = 0.05) as compared to dose 0 g kg-1; b: Indicates a statistically significant difference (p = 0.05) as compared to dose 0 and 2.5 g kg-1; c: Indicates a statistically significant difference (p = 0.05) as compared to dose and 10 g kg-1 (n = 5) | |
All the extractives exhibited a range of antiradical activity above 60% for W, CM, C and M extracts, except the H extract. Total phenolic content values were evaluated in term of gallic acid equivalent mg-1 (GAE mg-1) of extract used. The total amount of phenolic groups was higher in the M extract (69.83) and the C extract (60.67), the CM extract (54.17), the W extract (47,14) and the H extract (22.47) (Table 3).
Toxicological assessment: Oral administration of 0, 2.5, 5, 7.5 and 10 g kg-1 of W and M extracts did not result in animal mortality or important morbidity. Therefore the approximate LD50 values estimated using Yamanaka et al. (1990) method should be above 5 g kg-1.
Animal weight was not reduced by single dose administration of the extracts within a period of 7 days observation. No significant food and water intake were observed between the treated groups and the control.
Above 2.5 g kg-1, W extract led to a significant (p<0.05) decrease in liver and kidney relative weight in females and had no effect on the same organs in males (Table 4). M extract increased liver relative weight of males treated only at the dose of 10 g kg-1. A significant (p<0.05) decrease of kidney relative weight was obtained with animals treated with 5, 7.5 and 10 g kg-1 of the M extract (Table 5). There was no effect on heart relative weight of rats treated with both extracts. Rats treated with 10 g kg-1 of M extract had a lung relative weight higher than those of the other treated groups (including the control group). Animal treated with W extract at 7.5 and 10 g kg-1 showed a significantly increased (p<0.05) testis relative weight. This was also observed with rats treated with 10 g kg-1 of M extract.
Haematological parameters recorded in this study are shown in Table 6 (for W extract) and Table 7 (for M extract). There was no significant change in the white blood cells and red blood cells count, haemoglobin concentration and haematocrit. However, both W and M extract induced a relative low eosinophils count, an increase lymphocytes and platelets count.
| Table 6: | Effect of acute oral doses of Alafia multiflora aqueous extract on haematological parameters in rats |
| Values are mean±SEM (n = 4). WBC = White Blood Cells; LY = Lymphocytes; MI = Monocytes; GR = Granulocytes; RBC = Red Blood Cells; HGB = Haemoglobin; HCT = Haematocrit; MCH = Mean Corpuscular Haemoglobin; MCHC = Mean Corpuscular Haemoglobin Concentration; PLT = Platelet; MPV = Mean Platelet Volume; PCT = Thrombocrit | |
| Table 7: | Effect of acute oral doses of Alafia multiflora methanol extract on haematological parameters in rats |
| Values are mean±SEM (n = 4). WBC = White Blood Cells; LY = Lymphocytes; MI = Monocytes; GR = Granulocytes; RBC = Red Blood Cells; HGB = Haemoglobin; HCT = Haematocrit; MCH = Mean Corpuscular Haemoglobin; MCHC = Mean Corpuscular Haemoglobin Concentration; RDW = Red Blood Cells Distribution Width; PLT = Platelet; MPV = Mean Platelet Volume; PCT = Thrombocrit. a: p<0.05 as compare to dose 0 g kg-1; b: p<0.05 as compare to dose 2.5 g kg-1, c: p<0.05 as compare to dose 5 g kg-1, d: p<0.05 as compare to dose 7.5 g kg-1; e: p<0.05 as compare to dose 10 g kg-1 (n = 4) | |
Moreover, all the animals treated with methanol M extract showed a significantly increase of platelet count. The mean platelet volume was significantly (p<0.05) increased with animals treated at 7.5 and 10 g kg-1.
DISCUSSION
CM, M and W extracts inhibited the growth of all the pathogens tested. Based on the values of the antimicrobial parameters (IZ, MICs), one could speculate that the stem bark extracts from Alafia multiflora would be a useful source for treating ailments caused by the bacteria tested. Since these fractions are not pure, it would be difficult to speculate about the mechanisms of the antimicrobial activities observed. The Present results confirmed the evidence of previous studies reporting that methanol is a better solvent for more consistent extraction of antimicrobial substances from medicinal plants compared to other solvents, such as water, ethanol and hexane (Karaman et al., 2003).
It is known that phenolic compounds exercise their antiradical propertied by providing a neutralising OH group that bind to the free radical compound. Therefore the higher the OH group of a given phenolic compound the higher its scavenging potential (Brand-Williams et al., 1995). The antiradical activity of extracts from A. multiflora was not strictly correlated with the phenol content indicating that other factors playing major roles as antiradicals are involved as suggest by many authors (Llesuy et al., 2001; Ilhami et al., 2004). In fact the typical phenolics that possess antioxidant activity are known to be mainly phenolics acid (gallic acid, vanillic acid, syringic acid, ascorbic acid, ferrulic acid, coumaric acid, etc.) and flavonoids (Brand-Williams et al., 1995; Qian and Nihorimbere, 2004). Phytochemical screening of the extracts used showed the presence of phenols, tannins, flavonoids, anthraquinones and alkaloids in all extracts, except for the lipid rich H extract (unpublished data).
The difficulty of healing observed with ulcerous wound is not only due to the presence of pathogenic bacteria resistant to the host defence mechanisms, but also to excess production of free radicals by the host defence cells which promote cell death and thus the persistence of the wound (Cheesbrought, 2000; Telgenhoff and Shroot, 2005). Gallic acid is known to possess both antibacterial and antiradical properties (Fernandez et al., 1996). Furthermore, vanillic acid (Balansard et al., 1980) and an ester of syringic acid (Pais et al., 1971) were isolated from stem and seeds of A. multiflora respectively. Balansard et al. (1980) found that the isolated vanillic acid was also highly antibacterial. This justify the more the healing success obtained with the sap and the bark of this plant in the treatment of ulcerous wounds and skin problems.
Giving that the toxicity assays revealed no remarkably behaviour change, no gross pathological change in animals treated with the aqueous and the methanol extracts at doses up to 10 g kg-1 and considering the relative low toxicity (LD50 > 5 g kg-1, OCED, 2000) the use of these extracts at the therapeutic doses will be safe. A chemical with such low toxicity does not need further testing for lethal effects (Yamanaka et al., 1990). The extracts led to a decreased liver and kidney relative weight at higher doses, the methanol extract significantly increased (p<0.05) platelets count at doses 7.5 and 10 g kg-1. The mechanism of action of these observed abnormalities was not established. We did not determine if renal injury may develop independently of hepatic events. However, agranulocytosis and hepatorenal toxicity of the Alafia multiflora extracts at higher dose represent some adverse effects usually attributed to topical medicines when given orally (Harms and Saurat, 1992). Despite the liver and kidney hypotrophy, no mortality was observed after 7 days in animals treated with up to 10 g kg-1. It is therefore not excluded that these abnormal observations were due solely to the quantity of extract that was given to the animals, since below 5 g kg-1 no such abnormalities were obtained. Moreover, according to OCED (2000), LD50 of plant extract greater or equal to 5000 mg kg-1 is indicative of poor toxicity. This goes further to confirm our assertion that the security margin of A. multiflora (toxicity wise) is quite large.
The fact that at 2.5 g kg-1 of the aqueous extract generated decrease of toxicity signs (liver and kidney relative weight) in female animals but not in males suggest that the female animals are more sensitive to the eventual toxic principle present in this extract. Further studies are needed to elucidate this observed sex-related side effect. With methanol extract the same abnormal signs were observed in both male and female animals. This suggest that this same toxic agent may be more concentrated in the methanol extract and thus affect both males and females, or it may be here a question of a different principle acting on the same organs.
CONCLUSIONS
The known antimicrobial mechanisms associated to each group of chemical to which the isolated compounds belong and/or others may explain the antibacterial properties of the extracts used in this study. The total phenolic content was higher in the methanol extract which confirms its free radical-scavenging activity. The results of the present study provide an important basis for the use of methanol extract from the stem bark of Alafia multiflora for the treatment of infections associated to the microorganisms tested and the management of oxidative stress. Aqueous and methanol extracts of A. multiflora showed a wide margin of safety at acute dosage. The induction of liver and kidneys relative weight abnormalities in male and females substantiates an apparent sex difference in tolerance of A. multiflora extracts. However, further pharmacological and toxicological studies currently going on in our laboratory may confirm this hypothesis. More data are also needed to determine the active antibacterial and antiradical components of the extracts.
ACKNOWLEDGMENTS
Authors acknowledge the Chinese Cooperation, the Cameroon National Herbarium, The Centre Pasteur du Cameroun for their collaboration and special thanks to Pr David Dussourd (University of Maryland, USA) for sending part of the literature exploited for this project.