Paul Keilah Lunga, Jean De Dieu Tamokou, Gerald Ngo Teke, Donatien Gatsing and Jules Roger Kuiate*
Laboratory of Microbiology and Antimicrobial Substances, Faculty of Science, University of Dschang, P.O. Box 67 Dschang, Cameroon.
* Author to whom correspondence should be addressed. E-mail: jrkuiate@yahoo.com, Tel: +237 99 67 91 35, Fax: + 237 33 45 17 35
Abstract: The antibacterial activities, therapeutic efficacy and the antioxidant potentials of the crude methanol extracts of the stem barks of two medicinal plants, Paullinia pinnata (Sapindaceae) and Ficus thonningii (Moraceae), were studied. The agar diffusion and the broth microdilution assays were used for the evaluation of the in vitro antimicrobial activities. The therapeutic study was carried out in a Shigella-induced diarrhoeal model in rats. The DPPH● scavenging assay was used for evaluating the antioxidant potentials of the plant extracts. The in vitro antibacterial assays showed that MICs values ranged from 0.2-3.13 mg/mL and 0.39-1.56 mg/mL for P. pinnata and Ficus thonningii extracts respectively. The dose of 0.45 g/Kg body weight for both extracts completely eradicated shigellosis induced in rats after 4 and 5 days of treatment. The highest DPPH● scavenging activity was found in the methanol extract of P. pinnata (76.0 - 90.0%) compared to that of F. thonningii (17.0 - 41.0%) for concentrations of extracts ranging from 10 to 1000 µg/mL. Therefore, the extracts of P. pinnata and F. thonningii could be used in the treatment of bacterial infections and the prevention of oxidative damages due to free radicals.
Keywords: Paullinia pinnata, Ficus thonningii, antibacterial, antioxidant, therapeutic effect.
INTRODUCTION
Bacterial infections occupy the first position in the world of microbial infections. They constitute a serious universal public health hazard that has ravaged the entire world today. The high frequency of bacterial infections is attributed to poor sanitary measures in the lower income countries and lack of adequate financial means to follow treatment using antibiotics (Huang and Zhou, 2007). Also, the exploitation of chemical substances in therapeutics has registered a lot of failures in microbial therapy over the years (Boyce et al., 1992) due to the frequent emergence of resistant strains to frequently used antibiotics (Fluit et al., 2001). There is then a need for a constant search of new efficient antimicrobial substances. The plant kingdom is endowed with a large variety of bioactive substances.
Paullinia pinnata (Sapindaceae) and Ficus thonningii (Moraceae) are respectively a liana and a tree used in West Region of Cameroon for the treatment of bacterial infections like typhoid, syphilis, gonorrhoea, stomach-ache, waist pain, diarrhoea. The antioxidant properties of the methanol extract of the leaves and antibacterial activity of fatty acids of the roots of P. pinnata have been investigated (Jimoh et al., 2007; Annan et al., 2009). To the best of our knowledge, no antibacterial and antioxidant studies have been carried out on F. thonningii. The present study was undertaken on the basis of the traditional use of these plants. In east Africa, the leaves of P. pinnata are used in the treatment of gonorrhoea, for wounds and microbial infections (Chabra et al., 1991). The bark of F. thonningii is used in treating colds, sore throat, dysentery, wounds, constipation, nose bleeding and to stimulate lactation (ICRAF, 1992). This work was aimed at evaluating the in vitro antibacterial and radical scavenging activities as well as in vivo antibacterial activity in a Shigella-induced diarrhoea model in rats, of the methanol extracts of the stem barks of these plants.
Plant materials
P. pinnata and F. thonningii stem barks were collected from the locality of Bafang, West Region, Cameroon in December 2008. The identification of plant specimens was done at the National Herbarium in Yaounde, Cameroon, where voucher specimens were deposited under the reference Code Numbers 10702/SRFCam (for P. pinnata) and 13021/HNC (for F. thonningii).
Preparation of plant extracts
The stem bark of P. pinnata and F. thonningii were air-dried at room temperature (25 ± 2 °C), chopped and ground to obtain fine powders. Plant extracts were prepared by maceration using the method described by Okogun et al. (2004). Briefly, each powder was completely submerged in methanol in a mass to volume ratio of 50 g : 200 mL. Extraction was allowed to proceed for 48 h. The mixtures were then filtered using Whatman paper N° 3 and the filtrate concentrated by evaporating the solvent at 50 °C under reduced pressure, using a rotatory evaporator (Buchi R-200), to obtain the extracts. The process was repeated twice on the residue obtained above in order to maximize yield. The dried extracts were stored in bottles at +4 °C in the refrigerator till usage.
Antimicrobial assays
Microorganisms
Eight bacterial strains and isolates known to be responsible for human bacterial infections were used in the work. These included two Gram positive bacteria (Staphylococcus aureus ATCC25922 and Enterococcus faecalis ATCC10541) and six Gram negative bacteria (Escherichia coli ATCC11775, Klebsiella pneumoniae ATCC13883, Pseudomonas aeruginosa ATCC27853, Salmonella typhi ATCC6539, Proteus mirabilis and Shigella flexneri). The last two bacteria were clinical isolates obtained from “Centre Pasteur” in Yaounde, Cameroon, while the rest were reference strains obtained from the American Type Culture Collection (USA, Rockeville).
The bacterial cell suspensions were prepared at 1.5 ´ 108 colony-forming units/mL (CFU/mL) following McFarland turbidity standard N° 0.5. For this purpose, 18 hrs old overnight bacterial cultures were prepared in nutrient agar. A few colonies of bacteria were collected aseptically with a sterile loop and introduced into 10 mL of sterile 0.9 % saline distilled water solution. The concentration of the suspension was then standardised by adjusting the optical density to 0.10 at 600 nm wavelength (Tereshuck et al., 1997).
Diameters of inhibition zones were determined using Mueller Hinton agar (MHA) by the well diffusion method as described by Berghe and Wietinck (1991) with slight modifications. Bottles containing 19.80 mL of sterile molten MHA were maintained in a water bath at 40 °C to prevent solidification of the medium, and were aseptically inoculated with 200 µL of each bacterial suspension. Bacterial inocula and medium were well mixed and dispensed into sterile 90 mm diameter plastic Petri dishes and allowed to solidify at room temperature in a sterile cupboard. After solidification, 6 mm diameter wells were bored into the gel (5 wells/dish) using a sterile borer. Fifty microlitres of plant extract, prepared at 200, 100, 50, 25 and 12.50 mg/mL in 5% DMSO were introduced into each well (one concentration into each of the 5 wells per Petri dish for each bacterial strain or isolate) and allowed to pre-diffuse for one hour at 20 °C. This gave a well charge of 10, 5, 2.5, 1.25 and 0.62 mg respectively for the concentrations. Control dishes were made along side that received 50 µL of 5% DMSO solution and 50 µg/well of ciprofloxacin (reference antibiotic). The dishes were then incubated at 37 °C for 18 hrs. Antimicrobial activity was evaluated by measuring the diameter of the zone of growth inhibition around each well with a millimetre rule. The assay was done in triplicates at three different occasions and the mean diameters recorded as inhibition zones.
Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The broth micro-dilution method was used for susceptibility testing of bacteria species. The two extracts were tested against the eight bacteria species listed above. The tests were carried out in 96-micro well sterile plates as described by Newton et al. (2002). For this, the crude extracts, dissolved in 5% dimethylsulfoxide (DMSO) solution and diluted with Mueller Hinton broth to obtain a stock concentration of 50 mg/mL for each sample was used. Serial two-fold dilutions of each extract were made with Mueller Hinton broth to yield a volume of 100 µL/well. This gave a concentration range of 25.0 to 0.049 mg/mL. One hundred microlitres of each bacterial suspension (containing about 1.5×108 CFU/mL) was added to respective wells containing the test samples and mixed thoroughly to give final concentrations ranging from 12.50 to 0.024 mg/mL. The solvent control, 5% DMSO, did not show inhibitory effects on the growth of the bacteria. Ciprofloxacin at a concentration range of 12.50 to 0.024 µg/mL was used as a standard reference. Tests were done in duplicates. The cultured microtitre plates were covered and incubated at 37 °C for 24 hrs. Inhibitory concentrations of the extracts were detected after addition of 50 µl of 0.2 mg/mL p-iodonitrotetrazolium violet (INT) (Sigma–Aldrich, South Africa) and incubated at 37 °C for 30 min (Mativandlela et al., 2006). Viable bacteria change the yellow dye of INT to a pink color. All concentrations at which no visible colour changes were observed were considered as inhibitory concentrations and the lowest of these concentrations was considered as the MIC. The bactericidal concentrations were determined by adding 50 µL aliquots of the preparations (without INT), which did not show any visible colour change after incubation during MIC assays, into 150 µL of extract-free Mueller Hinton broth. These preparations were further incubated at 37 °C for 48 hrs and bacterial growth was revealed by the addition of INT as above. All extract concentrations at which no colour changes were observed were considered as bactericidal concentrations. The smallest of these concentrations was considered as the MBC. The tests were performed in duplicates at two different occasions and the results recorded as mean ± SD.
Phytochemical screening
The major classes of phytochemicals: alkaloids, flavonoids, polyphenols, triterpenes, steroids, saponins, tannins, anthocyanins and coumarins were screened for in the crude extract as described by Bruneton (1999).
In vivo therapeutic test
This test was carried out in a Shigella-induced diarrhoea in rat. Prior to the test, animals were housed under the test conditions for a period of one week. All animal experiments were conducted in strict accordance to the rules and regulations of the ethical committee of laboratory standards for experimental animals.
A Shigella flexneri suspension was prepared at 4 Mc Farland turbidity scale. For this, bacterial suspension was prepared from an 18 hrs old overnight Shigella culture on nutrient agar (NA). One millilitre (1 mL) of this solution containing about 12 x 108 colonies was orally administered to each animal (Kamgang et al., 2006). Only infected animals were selected and used.
Grouping of animals
Animals were arranged into eight groups. Each group contained four animals (two males and two females) selected from the infected stock. Treatment was done by administering the extracts orally, every morning for five days. The animals were treated as follows:
- Group one were not treated and served as negative control.
- Group two received Immodium (2.50 mg/Kg bw) and served as positive control.
- Group three, four and five received the P. pinnata extract at concentrations of 0.45, 0.22 and 0.11 g/Kg bw, corresponding to 2MIC, MIC and 0.5MIC respectively.
- Group six, seven and eight received the F. thonningii extract at the same concentrations.
Food and water were given to the animals before and during the treatment ad libitum. Each day, the faecal matter was collected just before administration of plant extract or Immodium or nothing for the negative control.
Assessment of stool bacterial density
The extent to which the animals complied with treatment was studied by counting the amount of bacterial colonies in the fecal samples using the following protocol.
- 0.1 g of fecal matter was completely dissolved in 5 mL of autoclaved distilled water.
- 50 µL of the resulting solution was spread on the surface of solidified 0.9% saline SS agar in the 35 mm type Petri dishes.
- After incubation for 18 hrs at 37 °C, the number of colonies following growth of Shigella flexneri in each Petri dish was determined and recorded.
- The results were converted into the number of colonies per gram of fecal matter per animal.
The time course for the bacteria treatment was assessed from the number of colonies obtained for each animal with time and this gave us an idea on how the animals were complying with treatment using the two extracts and thus the duration of treatment using the optimum dose regimens.
The free radical scavenging activities of the crude extracts were evaluated using the DPPH assay method as described by Mensor et al. (2001). Briefly, the test samples, dissolved in methanol were mixed with a 0.3 mM 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) methanol solution, to give final concentrations of 10, 50, 100, 500 and 1000 µg of extract per milliliter (mL) of DPPH solution. After 30 min in the dark at room temperature, the optical densities were measured at 517 nm and converted into scavenging activities (SCa). Ascorbic acid was used as a standard control. Each assay was repeated trice and results were recorded as the mean ± SD.
SCa = [(Absorbance of control−Absorbance of test sample)/Absorbance of control] ×100.
All data are presented as the mean ± SD. Results were analyzed by the one way ANOVA and where differences exist, means were compared using the Student-Newman-Keuls’ test at 0.05 probability level.
RESULTS AND DISCUSSION
P. pinnata gave an output of 24.0% while F. thonningii gave a 14.3% yield. All were presented as solid powders with P. pinnata extract having a dark brown colour whereas F. thonningii extract had a characteristic reddish colour and was very stiff. The phytochemical screening of the two extracts revealed the presence of six and three chemical groups of constituents respectively for P. pinnata and Ficus thonningii extracts (Table 1). Alkaloids, saponins and triterpene were present in both extracts. In addition to these classes of phytochemicals, P. pinnata extract had three others: phenols, tanins and sterols, not found in F. thonningii extract. The presence of these classes of compounds and their differences in composition between the two extracts may explain the presence of activity as well as the differences in activity between the two extracts. However, none of them had flavonoids, anthocyanins and coumarins.
Just like with most pharmaco-active plants of the mangrove forest, P. pinnata extract contained high amounts of polar compounds as opposed to that of F. thonningii in this context. These polar compounds, mainly phenolics, are readily extracted by maceration using MeOH (Uddin et al., 2005). This probably explains why F. thonningii with no phenols gave a relatively low yield (14.3 %) compared to the 24.0% yield of P. pinnata.
In vitro antibacterial activities of extracts
F. thonningii and P. pinnata extract exerted a concentration-dependent antibacterial activity on all the eight bacterial strains and isolates (Tables 2 and 3). This activity was also dependent on the microorganism. P. mirabilis and S. aureus appeared to be the most susceptible strains while E. faecalis and K. pneumoniae appeared to be the most resistant. P. pinnata exhibited a bactericidal activity on E. faecalis, P. aeroginosa, S. typhi and S. aureus (MIC/MBC ≤ 4). The most sensitive bacteria were P. mirabilis (MIC = 0.2 mg/mL) and Shigella flexneri (MIC = 0.39 mg/mL). The reference drug was still the most active, exerting bactericidal effects on four of the tested microbes. F. thonnigii extract was more active than that of. P. pinnata. This may be due to the phytochamical compositional difference as well as the mechanism of action of the active principle(s) (Takeo et al., 2004).
Many studies have been made pertaining to the antimicrobial activities of other members of the genus Ficus. The results of the present study corroborate those of Aswar et al. (2008) and Kuete et al. (2008) who reported on the antimicrobial properties of some species of the Ficus genus.
S. aureus, S. flexneri and S. typhi were susceptible to the crude methanol extracts of P. pinnata and F. thonningii. The MICs obtained in this study could be compared to those recorded by Lutterodt et al. (1999) on the susceptibility of S. aureus, S. flexneri and Vibrio cholerae to Psidium guajava extract. Since S. aureus was relatively more sensitive to F. thonningii and is commonly implicated in pus-causing wounds (Lutterodt et al., 1999), we could suggest that this extract could be effective as an alternative treatment for wounds infected with this bacterium.
In general, drugs with lowest MICs for a given bacterial isolate are the best indicated for treatment of an infection due to that isolate (James and Mary, 1998). It follows therefore that the crude methanol extract of Ficus thonningii can be considered as a better product candidate for treating diseases caused by Enterococcus faecalis, Pseudomonas aeroginosa, Salmonella typhi and Staphylococcus aureus. Except for Proteus mirabilis which was more sensitive to P. pinnata MeOH extract, the two extracts have comparable actiovities on E. coli, K. pneumonia and S. flexneri (Table 3). Interestingly, is the high activity of F. thonningii extract despite a low number of phytochemical classes detected. This may suggest that constituents of this extract are more active or are present in higher concentrations.
In vivo therapeutic study
In infected animals, stools were soft with mucus or liquid or were moulded and smooth but mucus coated. Sometimes, the presence of blood and mucus made the stool to appear dark and shinny. These justified the instalment of infection, further confirmed by high bacterial charge in faeces. The animals were weak and less active. The treatment improved the general condition of animals. The number of bacteria colonies/gram of faecal matter with time, dropped significantly in a dose-dependent manner during treatment (Figure 1). Animals stopped manifesting diarrhoea after four days of treatment with the 0.45 g/Kg, five days with 0.22 g/Kg body weight dose and persisted even after day five with minimum dose (0.11 g/Kg). In the negative control (the untreated group), the stool bacterial charge started reducing gradually after the first day, but remained relatively high compared to the treated groups throughout the test period. Immodium stopped diarrhoea in 3 days. This drug acts as antisecretory as well as anti-inflammatory and antibacterial agent. This may suggest that the two plant extracts possess these three biological properties.
In the case of the negative control, colony count per gram of faecal matter remained very high after 5 days. However, it was observed that from day one, the colony count started dropping gradually (Figures 1). This can be attributed to the instalment of an immune response against the foreign microbe.
Tannins, alkaloids, saponins, steroids and terpenoids indentified in these extracts may be responsible for the antimicrobial and antidiarrhoeal activities (Otshudi et al., 2000; Havagiray et al., 2004). Tannins and tannic acid present in antidiarrhoeal plants denature proteins in the intestinal mucosa by forming protein tannates which make the intestinal mucosa more resistant to chemical alteration and reduces secretion (Tripathi, 1994). The antisecretory potentials of tannins could contribute to the observed antidiarrhoeal activity of P. pinnata. The antidiarrhoeal activity may be associated with the antimicrobial activity of these extracts (Venkatesan et al., 2005).
The DPPH assay was used and the results are presented in Table 4. Both extracts possessed antioxidant properties which varied significantly between the two extracts and L-ascorbic acid. At concentrations of 1000 µg/mL and 500 µg/mL, radical scavenging activities of each sample did not vary significantly. The activity of P. pinnata extract surpassed that of F. thonningii at corresponding concentrations and was comparable to that of L-ascorbic acid at concentrations of 1000 µg/mL and 500 µg/mL. Phenolic and nitrogenous compounds are known to be potential antioxidants because of their ability to scavenge free radicals and reactive oxygen species such as singlet oxygen, superoxide anion radical and hydroxide radicals (Hall and Cuppett, 1997; Pietta et al., 1998). Like vitamins E and C, P. pinnata crude MeOH extract revealed great antioxidant potentials, doubling, tripling and even quadrupling those of F. thonningii MeOH extract as corresponding concentrations decrease (Table 4). This exceptionally high radical scavenging activity of P. pinnata extract compared with that of F. thonningii can be attributed to the presence of phenols in the former and their absence in the latter (Bjelakovic et al., 2007); the antioxidant activity of a polyphenol is directly proportional to the number of the –OH groups it contains. In human health these compounds, are thought to be instrumental in combating oxidative stress, a syndrome causative of some neurodegenerative diseases and some cardiovascular diseases (Herrera and Barbas, 2001). P. pinnata extract could therefore be recommended as an alternative to the use of vitamin C (L-ascorbic acid) as an antioxidant. This assertion comes to confirm the finding of Jimoh et al. (2007) who demonstrated potential antioxidant activity in the methanol extract of the leaves of P. pinnata.
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Table 1: Phytochemical classes identified in the crude methanolic extract of Paullinia pinnata and Ficus thonningii.
| No | Phytochemical class | Paulinia pinnata | Ficus thoningii | |
|
| 1 | Flavonoids | - | - | |
| 2 | Phenols | + | - | |
| 3 | Tanins | + | - | |
| 4 | Alkaloids | + | + | |
| 5 | Saponins | + | + | |
| 6 | Anthocyanins | - | - | |
| 7 | Triterpene | + | + | |
| 8 | Sterols | + | - | |
| 9 | Coumarins | - | - | |
+: present; - : absent
Table 2: Diameters of inhibition zones (mm) of the extracts of the stem barks of P. pinnata and F. thonningii as a function of well charge and specie of microorganism.
| Extract | well Charge (mg) | Bacteria strains |
|
| E. coli | E. faecalis | K. pneumoniae | P. aeruginosa | P. mirabilis | S. aureus | S. typhi | S. flexneri |
|
| Inhibition diameters ± SD (mm) |
|
| Pp | 10 | 18,1±1.1a | 13,3±0.5d | 14,4±0.7cd | 17,2±0.8ab | 19,4±1.0a | 19,5±0.7a | 18,1±1.3a | 19,1±1.2a |
| 5 | 15,4±1.8abc | 11,6±0.5e | 11,7±0.4e | 16,0±1.2 ab | 19,1±0.9a | 17,8±0.9ab | 16,3±1.4ab | 17,0±1.3ab |
| 2,5 | 13,6±1.8cde | 10,4±0.6ef | 10,8±0.3ef | 14,4±2.1bcde | 17,5±0.5a | 16,3±1.0ab | 14,7±0.8bcd | 15,5±0.9bc |
| 1,25 | 12,5±1.0de | 00±0.0f | 10,1±0.6f | 11,5±0.5e | 16,0±0.7bc | 14,3±0.5cd | 13,0±0.5d | 14,2±0.4cd |
| 0,625 | 10,7±1.4ef | 00±0.0f | 8,8±0.6f | 10,1±0.6f | 14,3±0.5cd | 14,0±0.0d | 11,2±0.6ef | 12,8±0.3d |
| Ft | 10 | 16,8±0.4ab | 14,2±0.8cd | 13,1±0.6d | 15,0±0.0c | 18,5±1.1a | 19,1±0.6a | 15,0±0.0c | 18,5±0.5a |
| 5 | 15,2±0.4bc | 11,5±0.5e | 10,7±0.4ef | 13,0±0.0d | 16,5±0.5ab | 17,2±0.6ab | 12,0±0.0e | 16,5±0.5ab |
| 2,5 | 12,4±0.5de | 10,4±0.7ef | 9,4±0.7f | 11,5±0.5e | 14,3±1.7bcd | 15,0±0.9bc | 11,5±0.5e | 14,3±0.5cd |
| 1,25 | 10,7±1.1ef | 9,4±0.7f | 8,6±0.9f | 10,3±0.9 ef | 13,1±0.3d | 13,5±0.5d | 10,3±0.9ef | 13,5±0.5d |
| 0,625 | 9,8±0.8 f | 8,5±0.5f | 8,0±0.5f | 9,0±0.0 f | 11,6±0.5e | 12,0±0.0d | 9,0±0.0f | 12,0±0.0d |
| Cp | 0.05 | 34.0±0.1 | 34.0±0.1 | 34.0±0.2 | 34.0±0.1 | 34.0±0.1 | 35.0±0.2 | 36.0±0.1 | 36.0±0.2 |
| | | | | | | | | | | | | | | | | | | |
Pp = Paullinia pinnata; Ft = Ficus thonningii; Cp = ciprofloxacin. The results are the mean ± SD of triplicate tests measured in two directions after 18 h of incubation at 37 °C. For the same line and column, values carrying the same letter in superscript are not significantly different at p ≤ 0.05 (Student-Newman-Keuls test)
Table 3: Minimum inhibitory concentrations (MIC) and bactericidal concentrations (MIC) of P. pinnata, F. thonningii extracts and ciprofloxacin (mg/mL).
| Bacterium | Paulinia pinnata | Ficus thoningii | Ciprofloxacin |
| MIC | MBC | MBC/MIC | MIC | MBC | MBC/MIC | MIC x 10-3 | MBC x 10-3 | MBC/MIC |
| E. coli | 0.39 | 6.25 | 16 | 0.39 | 12.5 | 32 | 0,09 | 1,56 | 17 |
| E. faecalis | 1.56 | 6.25 | 4 | 0.39 | 6.25 | 16 | 0,09 | 1,56 | 17 |
| K. pneumoniae | 0.78 | 12.5 | 16 | 0.78 | 6.25 | 8 | 0,09 | 1,56 | 17 |
| P. aeruginosa | 1.56 | 6.25 | 4 | 0.78 | 6.25 | 8 | 0,04 | 0,19 | 4 |
| P. mirabilis | 0.20 | 12.5 | 64 | 0.39 | 6.25 | 16 | 0,09 | 1,56 | 17 |
| S. aureus | 3.13 | 6.25 | 2 | 1.56 | 6.25 | 4 | 0,04 | 0,19 | 4 |
| S. typhi | 1.56 | 6.25 | 4 | 0.78 | 6.25 | 8 | 0,19 | 0,78 | 4 |
| S. flexneri | 0.39 | 6.25 | 16 | 0.39 | 6.25 | 16 | 0,19 | 0,78 | 4 |
The values are the mean of four different values. The results obtained did not show any standard variation. Incubation at 37 °C for 24 h.
Table 4: Antioxidant potential (% radical scavenging activity) of P. pinnata, F. thonningii extracts and L- ascorbic acid as a function of the concentration.
| Concentration (µg/mL) | P. pinnata | F. thonningii | L- Ascorbic acid |
| 1000 | 90,0 ± 0.0 a | 41,0 ± 1.4 fg | 94,0 ± 0.0 a |
| 500 | 90,0 ± 1.0 ab | 33,0 ± 2.0 g | 94,0 ± 0.0 a |
| 100 | 88,0 ± 0.0 b | 25,0 ± 0.0 h | 93,7 ± 0.6 a |
| 50 | 84,7 ± 0.6 b | 20,0 ± 2.0 i | 93,7 ± 0.6 a |
| 10 | 76,0 ± 1.0 c | 17,0 ± 0.0 j | 93,7 ± 0.6 a |
The differences observed on figures with the same superscripts along each column and across the lines are not statistically significant at p≤ 0.05. (Student-Newman-Keuls test)
Figure 1: Evolution of the treatment of infected animals with different doses of P. pinnata (1a) and F. thonningii (1b) extracts.
