Extraction of the plant
The extraction was performed according to Ogbiko et al. [18]. The powdered leaves of 200 grams were weighed on an analytical balance (Mettler Toledo, Switzerland) and macerated in 1000 ml of absolute methanol and acetone in Erlenmeyer flask at the ratio of 1:5 after three days extract was collected and marc was re-macerated. Collection of the extract was carried out at the interval of three days, so leaves were macerated totally for 9 days with the intermittent shaking on the rotary-shaker (VWR DS-500; The Lab World Group, Boston, MA, USA). The pool of collected extracts was first filtered through sieve mesh then followed by filtration via Whatman no.1 by using filtration apparatus or unit. A filtrate of extracts was concentrated in a rotary evaporator (Buchii model R-200, Switzerland) at 40oC temperature and 40 revolutions per minute (RPM) until solvents were completely removed and solid extracts were formed.
Crude Extract Solvent Fractionation
The crude methanol extract was subjected to further solvent fractionation by increasing polarity including n-hexane, chloroform, ethyl acetate, and aqueous. Voukeng et al. [19] method was used for solvent fractionation with modification on the concentration of extract residue between fractionation intervals. The methanol crude extract was not completely dissolved in water, therefore, 90% methanol solvent was used as diluent. The methanol crude extract 60 grams were weighed on an analytical balance and subjected to dissolve completely in 100 ml of 90% methanol (10 ml water and 90 ml methanol) in the beaker. The completely dissolved 100 ml methanol crude extract was mixed with 100 ml n-hexane for solvent partitioning in separatory funnel having a capacity of 250 ml. The mixture in separatory funnel was fixed to the standing stage pole and waited until a clear and separated layer formed between two solvents. Once a clear layer formed, the methanol part was taken first carefully to beaker and n-hexane partition to another container. This procedure was repeated three times, the n-hexane partition was collected together for future concentration. While the remaining crude methanol extract solution was subjected to evaporation in a rotary evaporator at 40o C and 40 rpm to remove methanol solvent. Then, 90 ml of water was added to the concentrated crude methanol extract to form a 100 ml aqueous solution. The 100 ml aqueous solution of crude methanol extract was mixed with 100 ml of chloroform in the separatory funnel. The separatory funnel was fixed on the standing stage pole and waited until a clear layer formed between the aqueous solution of crude methanol extract and chloroform. The chloroform portion was held lower layer and collected first in the container and aqueous portion in another container. It was replicated three times and the chloroform portion pooled in the container for later concentration. The remnant aqueous portion of crude methanol extract was concentrated on a rotary evaporator to remove the remaining chloroform. The concentrated 100 ml aqueous portion of crude methanol extract was mixed with 100 ml ethyl acetate in the separatory funnel. The separatory funnel was fixed on the standing stage pole and waited till a clear layer appeared between aqueous fraction and ethyl acetate fraction. It was repeated three times, and aqueous fraction and ethyl acetate fraction were collected in different containers. The aqueous fraction was lyophilized by lyophilizer (Operon, Korea vacuum limited, Korea), but n-hexane, chloroform, and ethyl acetate fractions were concentrated in a rotary evaporator.
Preliminary Phytochemical Screening
The extracts were screened for phytochemical constituents such as alkaloids, flavonoids, terpenoids, phenols, tannins, steroids, saponins, anthraquinones, and cardiac glycosides using different chemicals and reagents for the detection of secondary metabolites in extracts and solvent fractions [20, 21, 22, 23].
Test Organisms
Microorganisms selected for the experiment were standard strains including Staphylococcus aureus (ATCC 25923), Streptococcus agalactiae (ATCC 12386), Streptococcus pyogenes (ATCC 19615), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Klebsiella pneumoniae (ATCC 700603) brought from Ethiopian Public Health Institute, and clinical isolates Staphylococcus aureus and Escherichia coli obtained from Animal Products, Veterinary Drug and Animal Feed Quality Assessment Centre of Veterinary Drug and Animal Feed Administration and Control Authority, and Candida albicans (ATCC10231) brought from Ethiopian Biodiversity Institute. The gram staining, selective media, haemolysin, and catalase test were conducted to confirm test microorganisms according to [24, 25].
Standard Drugs
Gentamicin 10 µg disc was used as positive control against bacteria and brought from Animal Products, Veterinary Drug, and Animal Feed Quality Assessment Centre of Ethiopian Veterinary Drug and Animal Feed Administration and Control Authority. Amphotericin-B 20 µg/ was used as positive control against fungus and obtained from the Ethiopia Food and Drug Administration and Control Authority.
Antibacterial Activity
The brain heart infusion (BHI) broth was prepared for streptococcal species and nutrient broth for other test bacteria. Overnight cultured 3–5 distinct colonies of bacteria based on their colony size were inoculated into 4 ml broth media and incubated at 37oC overnight. The nutrient or BHI broth was added to the overnight incubated bacterial suspension, and vortexed on a vortex mixer (Fisher Scientific. Ltd., England) for one minute to attained uniformly distribution. The vortexed bacterial suspension was adjusted to 0.5McFarland standard (Remel, Lenexa Kansas 66215, USA) (equivalent to 1–2 × 108 CFU/ml) through contrasting against white paper black line striped and was used for experiment within 15 minutes [26].
The 100 µl of adjusted bacterial suspension was pipetted using a micropipette and applied on the surface of Mueller Hinton agar, and was swabbed at 60o rotation to uniformly distribute bacteria throughout media surface using a cotton swab. The swabbed Mueller Hinton agar was allowed to stand for 15 minutes to provide time for the attachment of bacteria on the media. Then, the sterilized cork borer of 6 mm diameter was perforated with the swabbed media to create 6 mm diameter wells. At the time of punching media for different test bacteria, cork borer was sterilized by immersing in alcohol and burning with bunsen burner flames [27, 28]. The concentration of extracts for the experiment was determined based on a previous study on the plant [16]. The created wells were filled with 50 µl extracts or fractions at a concentration of 400 mg/ml, 200 mg/ml and 100 mg/ml, and negative control, but positive control disc (gentamicin) was placed on the media surface. After all wells on petri dishes were filled, and positive control was placed on petri dishes, then petri dishes were placed in the refrigerator at 4oC for 2 h to facilitate diffusion of extracts or fractions in the media. Subsequently, petri dishes were incubated at 37oC for 24 h in the incubator (BioTechnics India). The inhibition zone diameter after 24 h incubation was measured by a ruler in millimetre and recorded [15, 16, 29]. The experiment was done in triplicate.
Antifungal Activity
Agar well diffusion assay
The assay determined inhibition zone diameter for crude extracts, solvent fractions, and positive control, but not for negative control. The aqueous fraction exhibited the highest inhibition zone diameter of 21 mm, but no inhibition zone diameter observed for n-hexane and chloroform fractions against C. albicans (Table 6). Table 6: Mean inhibition zone diameter (mm) of Ricinus communis Linn leaf crude extracts and solvent fractions of methanol extract against fungi.
Test extract and
fraction
|
Candida albicans
|
Mean ± SEM
|
R2
|
Methanol extract
|
100 mg/ml
|
7.33 ± 0.333a3d2
|
0.735
|
200 mg/ml
|
8.67 ± 0.333a3
|
400 mg/ml
|
10.00 ± 0.577a3b2
|
Control
|
20 µg/ml
|
23.33 ± 0.333
|
|
Acetone extract
|
100 mg/ml
|
7.33 ± 0.333a3d2
|
0.746
|
200 mg/ml
|
8.67 ± 0.333a3
|
400 mg/ml
|
9.67 ± 0.333a3b2
|
Control
|
20 µg/ml
|
22.67 ± 0.333
|
|
n-hexane fraction
|
100 mg/ml
|
--
|
|
200 mg/ml
|
--
|
400 mg/ml
|
--
|
Control
|
20 µg/ml
|
23.33 ± 0.333
|
|
Chloroform fraction
|
100 mg/ml
|
--
|
|
200 mg/ml
|
--
|
400 mg/ml
|
--
|
Control
|
20 µg/ml
|
23.33 ± 0.333
|
|
Ethyl acetate fraction
|
100 mg/ml
|
11.33 ± 0.333a3b1d3
|
0.890
|
200 mg/ml
|
13.33 ± 0.333a3b1d1
|
400 mg/ml
|
15.33 ± 0.333a3b3c1
|
Control
|
20 µg/ml
|
22.67 ± 0.333
|
|
Aqueous fraction
|
100 mg/ml
|
14.67 ± 0.333a3c1d3
|
0.928
|
200 mg/ml
|
17.00 ± 0.577a3b1d2
|
400 mg/ml
|
21.00 ± 0.577a1b3c2
|
Control
|
20 µg/ml
|
23.33 ± 0.333
|
|
Values expressed as Mean ± SEM for n = 3. The mean comparisons for different extracts, crude methanol extract’s fractions, and control (Amphotericin-B 20 µg/ml) were performed by one-way ANOVA followed by Tukey HSD Post Hoc multiple comparison test. Where, compared to apositive control, b100mg/ml, c200mg/ml, and d400mg/ml. 1P<0.05, 2P<0.01, 3P<0.001. No activity = -- |
Determination of minimum inhibitory concentration of crude extracts and solvent fractions of methanol extract against pathogenic bacteria.
The minimum inhibitory concentration of methanol crude extract ranging from 6.25 mg/ml (S. aureus) to 25 mg/ml (E. coli, K. pneumoniae, P. aeruginosa) and acetone crude extract ranging from 8.33 mg/ml (S. pyogenes) to 100 mg/ml (K. pneumoniae). Also, minimum inhibitory concentration ethyl acetate fraction ranging from 1.5625 mg/ml (S. aureus) to 12.5 mg/ml (P. aeruginosa) and for aqueous fraction ranging from 6.25 mg/ml (S. aureus and S. pyogenes) to 66.67 mg/ml (K. pneumoniae) (Tables 7 and 8). The minimum inhibitory concentration for clinical isolate bacteria ranging from 3.125 mg/ml of ethyl acetate fraction (S. aureus) to 100 mg/ml of n-hexane and chloroform fractions (S. aureus and E. coli) (Table 9).
Determination of minimum bactericidal concentration of crude extracts and solvent fractions of methanol extract.
The MBC of methanol extract ranging from 100 mg/ml (gram-positive bacteria) to 200 mg/ml (P. aeruginosa) and was not detected in E. coli and K. pneumoniae. The minimum bactericidal concentration of acetone extract was 200 mg/ml in gram-positive bacteria, but not detected in gram-negative bacteria. The minimum bactericidal concentration of n-hexane and chloroform fractions were not detected. However, minimum bactericidal concentration ethyl acetate fraction ranging from 25 mg/ml (S. aureus and S. agalactiae) to 200 mg/ml (E. coli and K. pneumoniae), and in clinical isolate bacteria ranging from 88.33 mg/ml (S. aureus) to 200 mg/ml (E. coli) (Tables 7, 8 and 9).
Table 7
MIC and MBC of crude extracts and solvent fractions against gram-positive bacteria.
Test extract and fraction
|
Activities
|
S. aureus
|
S. agalactiae
|
S. pyogenes
|
Mean ± SEM (mg/ml)
|
Mean ± SEM (mg/ml)
|
Mean ± SEM (mg/ml)
|
Methanol extract
|
MIC
|
6.25 ± 0.000
|
12.500 ± 0.000
|
6.25 ± 3.1250
|
MBC
|
100.00 ± 0.000
|
100.00 ± 0.000
|
100.00 ± 0.000
|
Acetone extract
|
MIC
|
18.75 ± 6.25
|
16.67 ± 4.167
|
8.33 ± 2.083
|
MBC
|
200.00 ± 0.000
|
200.00 ± 0.000
|
200.00 ± 0.000
|
n-hexane fraction
|
MIC
|
100.00 ± 0.00
|
100.00 ± 0.00
|
100.00 ± 0.00
|
MBC
|
ND
|
ND
|
ND
|
Chloroform fraction
|
MIC
|
83.33 ± 16.667
|
16.67 ± 4.167
|
16.67 ± 4.167
|
MBC
|
ND
|
ND
|
ND
|
Ethyl acetate fraction
|
MIC
|
1.5625 ± 0.00
|
4.17 ± 1.0417
|
3.125 ± 0.000
|
MBC
|
25.00 ± 0.000
|
25.00 ± 0.000
|
50.00 ± 0.000
|
Aqueous fraction
|
MIC
|
6.25 ± 0.00
|
12.50 ± 0.000
|
6.250 ± 0.000
|
MBC
|
200.00 ± 0.000
|
200.00 ± .000
|
200.00 ± .000
|
Mean value expressed as Mean ± SEM (n = 3), ND = not detected |
Table 8
MIC and MBC of crude extracts and solvent fractions against gram-negative bacteria.
Test extract and fraction
|
Activities
|
E. coli
|
K. pneumoniae
|
P. aeruginosa
|
Mean ± SEM (mg/ml)
|
Mean ± SEM (mg/ml)
|
Mean ± SEM (mg/ml)
|
Methanol extract
|
MIC
|
25.00 ± 0.000
|
25.00 ± 0.000
|
25.00 ± 0.000
|
MBC
|
ND
|
ND
|
200.00 ± 0.000
|
Acetone extract
|
MIC
|
66.67 ± 16.667
|
100.00 ± 0.000
|
66.67 ± 16.667
|
MBC
|
ND
|
ND
|
ND
|
n-hexane fraction
|
MIC
|
100.00 ± 0.000
|
100.00 ± 0.000
|
NT
|
MBC
|
ND
|
ND
|
NT
|
Chloroform fraction
|
MIC
|
83.33 ± 16.667
|
83.33 ± 16.667
|
50.00 ± 0.000
|
MBC
|
ND
|
ND
|
ND
|
Ethyl acetate fraction
|
MIC
|
4.17 ± 1.0417
|
6.250 ± 0.000
|
12.50 ± 0.000
|
MBC
|
200.00 ± 0.000
|
200.00 ± 0.000
|
50.00 ± 0.000
|
Aqueous fraction
|
MIC
|
50.00 ± 0.000
|
66.67 ± 16.667
|
25.00 ± 0.000
|
MBC
|
ND
|
ND
|
200.00 ± 0.000
|
Mean value expressed as Mean ± SEM (n = 3) ND = not detected, NT = not tested |
Table 9
MIC and MBC of crude extracts and solvent fractions against clinical isolate bacteria.
Test extract and fraction
|
Activities
|
Clinical E. coli isolate
|
Clinical S. aureus isolate
|
Mean ± SEM (mg/ml)
|
Mean ± SEM (mg/ml)
|
Methanol extract
|
MIC
|
25.00 ± 0.000
|
12.50 ± 0.000
|
MBC
|
ND
|
ND
|
Acetone extract
|
MIC
|
25.00 ± 0.000
|
12.50 ± 0.000
|
MBC
|
ND
|
ND
|
n-hexane fraction
|
MIC
|
100.00 ± 0.000
|
100.00 ± 0.000
|
MBC
|
ND
|
ND
|
Chloroform fraction
|
MIC
|
100.00 ± 0.000
|
100.00 ± 0.000
|
MBC
|
ND
|
ND
|
Ethyl acetate fraction
|
MIC
|
6.250 ± 0.000
|
3.125 ± 0.000
|
MBC
|
200.00 ± 0.000
|
83.33 ± 16.667
|
Aqueous fraction
|
MIC
|
66.67 ± 16.667
|
25.00 ± 0.000
|
MBC
|
ND
|
ND
|
Mean value expressed as Mean ± SEM (n = 3), ND = not detected |
Determination of minimum inhibitory and fungicidal concentration of crude extracts and solvent fractions of methanol extract against Candida albicans.
The minimum fungistatic concentration of crude extracts and solvent fractions of methanol extract ranging from 3.125 mg/ml of the aqueous fraction to 66.67 mg/ml of methanol crude extract against C. albicans. The minimum fungicidal concentration of solvent fractions of methanol extract ranging from 50 mg/ml of the aqueous fraction to 200 mg/ml of ethyl acetate fraction. However, the minimum bactericidal concentration of methanol and acetone crude extracts were not detected against C. albicans (Table 10).
Table 10
MIC and MFC of crude extracts and solvent fractions against fungi.
Test extract and fraction
|
Activities
|
Candida albicans
|
Mean ± SEM (mg/ml)
|
Methanol extract
|
MIC
|
66.67 ± 16.667
|
MFC
|
ND
|
Acetone extract
|
MIC
|
41.67 ± 8.333
|
MFC
|
ND
|
n-hexane fraction
|
MIC
|
ND
|
MFC
|
ND
|
Chloroform fraction
|
MIC
|
ND
|
MFC
|
ND
|
Ethyl acetate fraction
|
MIC
|
16.67 ± 4.167
|
MFC
|
200.00 ± 0.000
|
Aqueous fraction
|
MIC
|
3.125 ± 0.000
|
MFC
|
50.00 ± 0.000
|
Mean value expressed as Mean ± SEM (n = 3), ND, = not detected |
Preliminary screening of phytochemical constituents of Ricinus communis Linn leaf
The results of preliminary phytochemical investigations of Ricinus communis Linn leaf crude extracts and solvent fractions is indicated on Table11. The phytochemical screening indicated alkaloids, flavonoids, terpenoids, tannins, cardiac glycosides, steroids, anthraquinones, saponins, and phenols in crude methanol extract and ethyl acetate fraction of Ricinus communis Linn leaf (Tables 11 and 12).
Table 11
Phytochemical constituents of crude extracts and solvent fractions of methanol extract of Ricinus communis Linn leaf.
Secondary metabolites
|
Crude extract
|
Solvent fractions
|
Methanol
|
Acetone
|
n-hexane
|
Chloroform
|
Ethyl acetate
|
Aqueous
|
Flavonoids
|
+
|
+
|
‒
|
+
|
+
|
+
|
Alkaloids
|
+
|
+
|
+
|
+
|
+
|
+
|
Saponins
|
+
|
‒
|
‒
|
+
|
+
|
+
|
Cardiac glycosides
|
+
|
+
|
‒
|
‒
|
+
|
+
|
Terpenoids
|
+
|
+
|
‒
|
‒
|
+
|
+
|
Tannins
|
+
|
+
|
+
|
+
|
+
|
+
|
Steroids
|
+
|
‒
|
‒
|
‒
|
+
|
+
|
Phenols
|
+
|
+
|
+
|
+
|
+
|
+
|
Anthraquinones
|
+
|
‒
|
‒
|
‒
|
+
|
‒
|
-, absence, +, presence |
Determination Of Minimum Inhibitory Concentration (mic) For Pathogenic Bacteria
The minimum inhibitory concentrations were determined using the broth microdilution technique for extracts or solvent fractions as their inhibition zones equal to or greater than 7 mm in agar well diffusion techniques [31]. The overnight cultured 3–5 distinct bacterial colonies were inoculated into 4 ml Mueller Hinton broth and incubated at 37oC overnight. Overnight incubated bacterial suspension had been adjusted (0.5McFarland standard) was diluted at a ratio of 1:20 with Mueller Hinton broth (0.5 ml bacterial suspension was added to 9.5 ml broth) and vortexed to have uniformly distributed bacterial suspension (5 × 106 CFU/ml). The UV radiated sterile microtiter plate (Greiner Bio-One, Germany) wells were filled with 100 µl Mueller Hinton broth which commenced from well one to twelve. The serial double dilution technique was employed for extracts and fractions in broth filled wells. The serial double dilution was performed as 100 µl extracts or fractions were added to the first well and thoroughly mixed for five times by rinsing using micropipette and 100 µl of the mixture was transferred to the second well using a new micropipette tip and thoroughly mixed as above. A 100 µl of the second well mixture was pipetted using a new micropipette tip and transferred to third well then was thoroughly mixed. The process was continued until the tenth well and 100 µl mixture of the tenth well was pipetted and discarded to have an equal volume of fluid in wells [26]. The twofold serially diluted concentrations of extract for the experiment were determined from a previous study on the plant. The serially diluted concentrations used in the experiment were (200 mg/ml, 100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, 3.125 mg/ml, 1.5625 mg/ml, 0.78125 mg/ml, and 0.3906 mg/ml) [16]. The 100 µl broth filled eleventh and twentieth wells were used as growth and sterility control, respectively. The 10 µl diluted bacterial suspension (10% of 100 µl well volume) was pipetted to wells from eleventh to first wells to reduce contamination to sterility control and attained final concentration of 5 × 105CFU/ml bacteria in each well, but 10 µl broth was pipetted to twelfth well. Finally, microtiter plates were sealed using parafilm and incubated at 37oC for 24 h [26]. The incubated microtiter plate wells were filled with 0.01% resazurin sodium salt indicator from twelfth to first well and incubated for 2 h at 37oC. The resazurin sodium salt reaction with actively growing microorganisms produces colour changes which are important to determine the MIC of extracts or fractions based on colour changes. The blue or purple colour appears if the growth of microorganisms is inhibited, while pink or colourless change is observed for those actively growing cells which reduced resazurin sodium salt to resorufin. Resazurin sodium salt solution was prepared by dissolving 0.01gram in 100 ml sterile distilled water and filtered through 0.2 µ pore size filter paper and stored in a dark container at 4oC refrigerator until use [29, 32]. The experiment was performed in triplicate.
Determination Of Minimum Inhibitory Concentration For Pathogenic Fungi
Overnight cultured three colonies of yeast were inoculated into sabouraud dextrose broth and incubated at 37oC overnight. Overnight incubated yeast suspension had been adjusted (0.5McFarland standard) was diluted at a ratio of 1:20 with sabouraud dextrose broth (0.5 ml yeast suspension was added to 9.5 ml broth) and vortexed to have uniformly distributed yeast suspension (0.5–2.5 × 105 CFU/ml). The sterile microtiter plate wells were filled with 100 µl sabouraud dextrose broth started from well one to twelve. The serial double dilution technique was employed for extracts and fractions in broth filled wells commenced from first to tenth wells. The serial double dilution was performed as 100 µl extracts or fractions were added to the first well and thoroughly mixed five times by rinsing using micropipette and 100 µl of the mixture was transferred to the second well using a new micropipette tip and thoroughly mixed as above. A 100 µl of the second well mixture was pipetted using a new micropipette tip and transferred to third well then was thoroughly mixed as above. The process was continued until the tenth well and 100 µl mixture of the tenth well was pipetted and discarded to have an equal volume of fluid in wells [34]. The twofold serially diluted concentrations of extract for the experiment were determined from a previous study on the plant. The serial double dilution concentrations used in the experiment were (200 mg/ml, 100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, 3.125 mg/ml, 1.5625 mg/ml, 0.78125 mg/ml, and 0.3906 mg/ml) [15]. The 100 µl broth filled eleventh and twentieth wells were used as growth and sterility control, respectively. The 10 µl diluted yeast suspension (10% of 100 µl broth volume) was pipetted to wells from eleventh to first wells to reduce contamination on sterility control and attained final concentration of yeast suspension (2.5 × 104CFU/ml) in each well, but 10 µl broth was pipetted to twelfth well. The filled microtiter plate wells were sealed by parafilm and incubated at 37oC for 24 h [26, 34]. The incubated microtiter plate wells were filled with 0.01% resazurin sodium salt indicator from twelfth to first well and incubated for 2 h at 37oC. The MIC of extracts and fractions were determined as blue or purple resazurin colour changed to pink or colourless [29, 32]. The experiment was done in triplicate.
Determination Of Minimum Bactericidal Concentration (mbc)
The minimum bactericidal concentration was determined through sub-culturing of 10 µl content of microtiter plate well which is greater or equal to the lowest minimum inhibitory concentration on the Mueller Hinton agar and incubated for 24 h. After 24 h incubation, the petri dish was assessed for the presence of growth, and the minimum concentration of extracts or fractions with no visible growth was taken as minimum bactericidal concentration [33]. The experiment was done in triplicate.
Determination Of Minimum Fungicidal Concentration (mfc)
The minimum fungicidal concentration was determined through sub-culturing of 10 µl content of microtiter plate well which is greater or equal to the lowest minimum inhibitory concentration on the sabouraud dextrose agar and incubated for 24 h. After 24 h incubation, the Petri dish was assessed for the presence of growth, and the minimum concentration of extracts or fractions with no visible growth was taken as minimum fungicidal concentration [33]. The experiment was done in triplicate.
Data Analysis
The data were entered into an excel spreadsheet for statistical analysis using Statistical Package for Social Science (SPSS) version 20. The descriptive statistics, one-way ANOVA, Tukey Post Hoc test, and linear regression R2 were utilized for statistical analysis and inference. The descriptive statistics were employed for calculation of group mean of inhibition zone diameter as Mean ± SEM. The one-way ANOVA was performed to determine the significant difference among group means. Whereas, Tukey Post Hoc test was followed by one-way ANOVA to determine the significant difference between each group mean. The linear regression R2 was calculated to determine the concentration dependence of crude extracts and solvent fractions on antimicrobial activities against test microorganisms. Statistically significant differences were declared at a p-value of less than 0.05.