In Vitro Modulation of Clotrimazole, Ketoconazole, Nystatin, Amphotericin B and Griseofulvin by Acmella Caulirhiza and Senna Didymobotrya Extract Against Candida Spp
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Journal of Applied Biosciences 142: 14478 - 14508
ISSN 1997-5902
In vitro modulation of clotrimazole, Ketoconazole, Nystatin, Amphotericin B and Griseofulvin by Acmella caulirhiza and Senna didymobotrya extract against Candida spp
Olwenya Fredrick Igunza, 1Joseph Ngeranwa, 2 George Orinda, 3 Mugo Peter 4 1, 2, 3: Department of Biochemistry, Microbiology and Biotechnology; Kenyatta University, Box 43844, Nairobi- Kenya. 4: Department of Medical Laboratory Services; Kenyatta University, box 43844, Nairobi-Kenya. Corresponding Author Email: [email protected]
Original submitted in on 24th August 2019. Published online at www.m.elewa.org/journals/ on 31st October 2019 https://dx.doi.org/10.4314/jab.v142i1.4
ABSTRACT Objectives: This research aimed at determining if Acmella caulirhiza and Senna didymobotrya extracts have a modulation effect on some of the conventional antifungal agents used and to provide alternative antifungal combination treatment regimens that can delay development of resistance or prevent it; thus improving on treatment of mycoses that is currently posing a challenge. Methodology and results: Candida species used were; C. albicans; ATCC 14053, C. duabus haemulonii; ATCC 2052030, C. haemulonii; ATCC 1609496, C.auris; ATCC 2050582, C.famata; ATCC 2037476, C. orientaris; ATCC 6258 and C.krusei; ATCC 14243. Antifungal drug susceptibility of the seven Candida spp. to plant extracts and modulated conventional drugs ;mainly Clotrimazole, ketoconazole, Nystatin, amphotericin B and Griseofulvin; was determined by broth micro-dilution and disk diffusion methods using the Clinical Laboratory Standard Institute protocols; with a few adjustments. Phytochemical analysis was done on the plant extracts. Phytochemical analysis showed that the plants contained Terpenoids, Cardiac glycosides, Flavonoids, Tannins and traces of Anthraquinones. The plant extracts were found to have antifungal activity with MICs ranging from 0.125μg/ml - 8μg/ml. The effects of plant extract- conventional antifungal combination, was evaluated by calculation of the fractional inhibitory concentration index. Acmella caulirhiza extract - Clotrimazole combination showed synergy while the other combinations showed indifference and antagonism. Conclusion and application of findings: This study found that Acmella caulirhiza and Senna didymobotrya have potent antifungal phytochemicals. It also found that Acmella caulirhiza extract modulates Clotrimazole. It is thus recommended that pure active antifungal components of these plants be determined and pure active components of Acmella caulirhiza be used to develop new antifungal regimens in combination with Clotrimazole. Keywords: Modulation, Candida haemulonii, Candida famata, Acmella caulirhiza and Senna didymobotrya
14478
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
INTRODUCTION Mycoses in general has become of clinical effects of drugs such as dose limiting significance in the recent past with high mortality nephrotoxicity shown by amphotericin B, drug-drug rates reported. This increase may be associated interaction, most antifungal agents being with advances in medical practice such as use of fungistatic in nature, rise of fungal strains that are intravascular catheters and total parenteral showing multiple-drug resistance and the nutrition which provide a platform for development persistence of fungal infection in of biofilms by fungi and this is known to confer immunocompromised individuals. This resistance, use of broad spectrum antimicrobials necessitates the search for new drugs from plant that kill normal flora and change the pH leading to species in order to address the above challenges colonization by fungi. Dialysis, Solid organ and associated with mycoses treatment. The presence bone marrow transplant patients, those with of tannins, alkaloids, saponins, and terpenes acquired immune deficiency syndrome (AIDS) and among other phytochemicals gives plants inherent patients on intensive chemotherapy regimens are antifungal properties making them a potential at high risk of developing mycoses due to source for new antimicrobial agents (Bristol et al., compromised immunity (Vendettuoli et al., 2009). 2012). Several plant species from folklore have the Currently there is an increase of non-albican reputation of being able to cure fungal infections candida pathogens such as Candida glabrata, but no scientific evaluation exists for most of the Candida krusei, Candida parapsilosis and Candida plants. The anti-fungal effects of combination of tropicalis that are of clinical importance since they the plant extracts with the conventional drugs is are known to cause life threatening fungemia. also little known. This research endeavors to They show elevated MICs for most antifungal provide such information thus improving on agents; a likely indicator of resistance. The treatment of mycoses that is currently posing a treatment of mycoses is in itself facing challenges great challenge. arising from increased incidences of adverse side
MATERIALS AND METHODS Plant material: The plants were selected due to their Senna didymobotrya methanol extract, Senna wide spread use as anti-mycotic agents in western didymobotrya hexane extract, Acmella caulirhiza Kenya. Plants were obtained from the wild around methanol extract and Acmella caulirhiza hexane extract Kakamega forest and a specimen deposited at Kenya dried under shade before the extraction. Solvent National Museum for assignment of specimen voucher extraction was done sequentially with a view of number. Extracts of plants used in the study were; minimizing analogous compounds.
14479
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Plate 1: Acmella caulirhiza
Plate 2: Senna didymobotrya
Plant extract and drug concentrations: Clinical and Clotrimazole, Ketoconazole, Nystatin, Amphotericin B Laboratory Standards Institute procedure on and Griseofulvin powders were dissolved in Dimethyl preparation of concentrations of drugs and solvents Sulfoxide to give stock solutions of concentrations used as documented in M44-A were followed. 64μg/ml,32μg/ml, 16μg/ml, 8μg/ml, 4μg/ml,2μg/ml,
14480
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
1μg/ml, 0.5μg/ml, 0.25 and 0.125μg/ml; that gave disks were evenly dispensed onto the surface of the concentrations of 32 μg/ml,16 μg/ml, 8 μg/ml, 4 μg/ml, 2 inoculated agar plate and pressed down to ensure its μg/ml,1 μg/ml, 0.5 μg/ml, 0.25 μg/ml, 0.125 and 0.0625 complete contact with the agar surface. The plates μg/ml when diluted to the final concentration by media. were inverted and placed in an incubator set to 37 °C. Methanol and hexane extracts of Acmella caulirhiza The inhibition zones were read after 24 hours of and Senna didymobotrya were also weighed and incubation. The zones read were used to calculate the dissolved in Dimethyl Sulfoxide to also give stock percentage inhibition of diameter growth (PIDG) as a solutions that gave concentrations of; 32 μg/ml,16 measure of the strength of inhibition of the extract in μg/ml, 8 μg/ml, 4 μg/ml, 2 μg/ml,1 μg/ml, 0.5 μg/ml, 0.25 relation to the control drug. It is given by; μg/ml, 0.125 and 0.0625 μg/ml when dissolved in media. PIDG= Microorganisms used: The test organisms that were Diameter of sample- Diameter of positive control x 100 used are: Candida albicans; ATCC 14053, C. Diameter of positive control duabushaemulonii; ATCC 2052030 , C.haemulonii; ATCC 1609496, C.auris; ATCC 2050582, C.famata; Broth Micro-dilution Test to determine MIC values: ATCC 2037476, C.orientaris; ATCC 6258 and C.krusei; The MIC values were determined by broth micro ATCC 14243.They were sub cultured on SDA to test dilution as provided for in M27-A3 document of Clinical viability and purity before use. The test organisms were Laboratory Standards Institute with a few modifications. selected based on the fact that C. albicans has been Briefly, 100μml of the plant extract each of different known to cause diseases for a long time and is showing concentrations was put in a separate round bottomed the development of resistance or elevated MIC for well and 100μml of the inoculum added to the wells. A drugs that were effective on it. the non- albicans; C. well containing the growth media and drug alone was duabushaemulonii, C.haemulonii, C.auris, C.famata, used as a negative control while that containing the C.orientaris and C.krusei are emerging as clinically inoculum without test drug was used as appositive significant fungi with elevated MIC or showing control. They were incubated at 37oc for 24 hours resistance to most antifungal agents in use. before readings taken. For modulation, 50μml of the Inoculum preparation: Clinical Laboratory Standards drug was mixed with 50μml of the plant extract against Institute reference method M27-A3 protocol was used a concentration gradient; as the concentration of the to prepare the inoculum. Colonies from 24 hour culture drug ranged from 32 μg/ml,16 μg/ml, 8 μg/ml, 4 μg/ml, 2 plates of Candida were suspended in 0.85% saline then μg/ml,1 μg/ml, 0.5 μg/ml, 0.25 μg/ml, 0.125 to 0.0625 the turbidity adjusted to 0.5 McFarland standard, giving μg/ml, that of the plant extract ranged in the reverse a stock solution of 1 x 106 to 5 x 106 cells per ml. concentration from 0.0625 μg/ml up to 32 μg/ml. Disk preparation: Sterile blank disks of 6mm diameter 100μml of the inoculum was also added to each well had 10ml volume of each extract at different and incubated at 37oc for 24 hours. concentration were impregnated onto them. They were Phytochemical tests dried and stored at 8oc pending use. Enough disks Test for Alkaloid: 3ml of aqueous extract was added were prepared since the experiment was replicated. to 3ml of 10% HCl on a steam bath. Mayer’s reagent Sterile blank disks of 6mm diameter had 10ml volume was then added to extract. Turbidity of the resulting of DMSO impregnated on them and used as a negative yellow precipitate was evidence for the presence of control. The procedure was used for amphotericin B Alkaloid (Yadav and Munin., 2011; Lemino et al., 2013; that acted as a positive control. Pradeep et al., 2014). Inoculation of Test Plates for disk diffusion: A Test for Tannins –ferric chloride test: 2ml of the sterile cotton swab was dipped into the inoculum aqueous extract was stirred with 3ml of distilled water suspension, rotated several times and pressed firmly and few drops of FeCl3 solution were added. The against the inside wall of the tube above the fluid level formation of green colour precipitate was indication of to remove excess fluid from the swab. Dry surface of presence of Tannins (Yadav and Munin., 2011; Lemino sterile Sabouraud’s Dextrose Agar plate was inoculated et al., 2013; Pradeep et al., 2014). by evenly streaking the swab over the entire agar Test for Phlobatannins: 3ml of aqueous extract was surface and repeating the procedure two more times, added to 2ml of 10% HCl and the extract was boiled. rotating the plate approximately 60° each time to Deposition of a red precipitate was taken as an ensure an even distribution of inoculum. Antifungal evidence for the presence of Phlobatannins (Yadav and
14481
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Munin., 2011; Lemino et al., 2013; Pradeep et al., presence of steroids (Yadav and Munin. 2011; Lemino 2014). et al., 2013; Pradeep et al., 2014). Test for Flavonoids –lead acetate test: To 3ml of Test for phenols and tannins: Crude extract was methanoic extract 1ml of 10% lead acetate solution was mixed with 2ml of 2% solution of FeCl3. A blue-green or added. The formation of a yellow precipitate was taken black coloration indicated the presence of phenols and as a positive result for Flavonoids (Yadav and Munin., tannins (Yadav and Munin. 2011; Lemino et al., 2013; 2011; Lemino et al., 2013; Pradeep et al., 2014) Pradeep et al., 2014). Test for Anthraquinones: 0.5 ml of the extract was Test for cardiac glycosides -Keller-killiani test: boiled with 10 ml of sulphuric acid (H2SO4) and filtered Methanol crude extract was mixed with 2ml of glacial while hot. The filtrate was shaken with 5 ml of acetic acid in a test tube. 1-2 drops of 2% solution of chloroform. The chloroform layer was pipetted into FeCl3 was added followed by 2ml of concentrated another test tube and 1 ml of dilute ammonia was H2SO4. A brown ring at the interphase indicated the added. The resulting solution was observed for colour presence of cardiac glycosides (Yadav and Munin., changes (Yadav and Munin., 2011; Lemino et al., 2013; 2011; Lemino et al., 2013; Pradeep et al., 2014). Pradeep et al., 2014). Data management and statistical analysis: The in Test for Terpenoids (Salkowski test): 0.5ml of each vitro antifungal data obtained was tabulated on of the extract was added 2 ml of chloroform. Microsoft Excel spreadsheet program. It was then Concentrated H2S04 (3 ml) was carefully added to form transferred to IBM SPSS version 20.0 software for a layer. A reddish brown colouration of the interface statistical analysis. The data was exposed to indicates the presence of Terpenoids (Yadav and descriptive statistic and expressed as the mean ± Munin., 2011; Lemino et al., 2013; Pradeep et al., standard deviation of the mean. Wilcoxon Signed 2014). Ranks Test was done to find significant difference Test for steroid: Crude extract was mixed with 2ml of between the reference drug (Amphotericin B) and the chloroform and concentrated H2SO4 was added plant extract treatments. Friedman’s test was executed sidewise. A red colour produced in the lower chloroform to compare the means of the four different plant layer indicated the presence of steroids. Another test extracts and reference drug on each of the fungal was performed by mixing crude extract with 2ml of species. The values of p ≤ 0.05 were considered chloroform. Then 2ml of each of concentrated H2SO4 statistically significant. The analysed data was and acetic acid were poured into the mixture. The presented in tables. development of a greenish coloration indicated the
RESULTS Phytochemical analysis of plant extract: In this study concentration. On the other hand A. caulirhiza extract it was found that S. didymobotrya extract contained contained steroids, terpenoids, flavonoids, tannins, phenols, tannins, cardiac glycosides, terpenoids, anthraquinones and cardiac glycosides also in varying flavonoids and alkaloids in varying degrees of degrees of concentration (Table 1).
Table 1: Phytochemical analysis of the plant extracts Senna didymobotrya Acmella caulirhiza Alkaloids + - Tannins +++ ++ Phlobatannins - - Flavonoids ++ +++ Anthraquinones + ++ Terpenoids ++ +++ Steroids - +++ Phenols +++ - Cardiac glycosides ++ ++ KEY: + Faintly present ++ moderately present +++ highly present - Nil
14482
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Inhibition of Candida species by the plant extracts: (amphotericin B) (p˂0.05) (Appendix v and vi). The The crude extracts of S. didymobotrya and A. caulirhiza hexane extract of A. caulirhiza and methanol extract of as shown in Table 2 had antifungal activity against the S. didymobotrya when tested against C. haemulonii Candida species used in this experiment at varying and C. daubus haemuloni respectfully produced zones degrees of concentration. In all the Candida species that were not significantly different from those by the tested, the plant extracts demonstrated zones of control drug (p>0.05) (Appendix vi and vii). This inhibition at MICs ranging from 1μg/ml to 32μg/ml as indicates that the extracts are relatively less sensitive to indicated in Table 2. S. didymobotrya hexane extract the fungi when compared to amphotericin B. For the among all the extracts demonstrated the lowest efficacy control drug (amphotericin B) the zone of inhibitions at since the zones of inhibition were only observed at concentrations ranging from 0.063µg/ml to 32µg/ml concentrations of 16μg/ml to 32μg/ml (Table 2). In all were found to be 14mm to 21mm for Candida krusei, the extracts the zones of inhibition were inferior to those 13mm to 23mm for Candida orientaris, 9mm to 21mm obtained with the reference drug (Amphotericin B). The for Candida famata, 13mm to 22mm for Candida auris, levels of inhibition for methanol and hexane extracts of 8mm to 18mm for Candida haemulonii, 7mm to 14mm S. didymobotrya and A. caulirhiza, against C. albicans, for Candida daubus haemulonii and 15mm to 31mm for C. krusei, C. orientaris, C. famata and C. auri were Candida albicans (Table 2). significantly different from those by the control drug
14483
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Table 2: Zone diameters of inhibition (mm) for plant extract Organism Extract/ control Drug extract concentration in μg/ml (Log2) -5 -4 -3 -2 -1 0 1 2 3 4 Inhibition zone diameters in mm (mean± standard deviation) Candida krusei Acmella hexane - - - 10 9 14 16 15 16 23 extract ±0.31 ±0.55 ±0.8 ±0.25 ±0.14 ±0.22 ±0.7 Senna methanol - - - - 6 6 12 10 15 11 extract ±0.09 ±0.61 ±0.91 ±0.12 ±0.87 ±0.65 Acmella methanol - - - 8 8 8 10 7 18 8 extract ±0.55 ±0.77 ±0.12 ±0.91 ±0.24 ±0.44 ±0.13 Senna hexane ------6 14 extract ±0.08 ±0.13 Amphotericin B 14±0.13 14±0.04 16±0.06 17 18 20 20 21 21 21 ±0.32 ±0.19 ±0.11 ±0.02 ±0.05 ±0.62 ±0.81 Candida orientaris Acmella hexane - - - - - 10 11 11 15 17 extract ±0.09 ±0.11 ±0.31 ±0.68 ±0.47 Senna methanol ------7 10 14 extract ±0.005 ±0.12 ±0.85 Acmella methanol ------8 12 13 13 extract ±0.0 ±0.81 ±0.06 ±0.7 Senna hexane ------10 10 extract ±0.21 ±0.21 Amphotericin B 13±0.71 15± 15±0.33 17 18 20 20 23 23 23 0.51 ±0.08 ±0.10 ±0.94 ±0.23 ±0.75 ±0.13 ±0.04 Candida famata Acmella hexane - - - - - 10 6 8 8 10 extract ±0.01 ±0.07 ±0.73 ±0.98 ±0.11 Senna methanol - - - - - 10 10 10 8 10 extract ±0.36 ±0.51 ±0.8 ±0.41 ±0.05 Acmella methanol - - - - - 8 11 6 10 11 extract ±0.08 ±0.01 ±0.12 ±0.69 ±0.31 Senna hexane ------6 6 6 extract ±0.33 ±0.52 ±0.01 Amphotericin B 9± 9± 11±0.03 17± 18 16 17 19 19 21 0.69 0.05 0.47 ±0.01 ±0.07 ±0.18 ±0.43 ±0.72 ±0.33 Candida auris Acmella hexane - - - 6 8 11 11 10 16 18 extract ±0.91 ±0.42 ±0.21 ±0.01 ±0.6 ±0.55 ±0.72
14484
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Senna methanol ------7 10 9 9 extract ±0.1 ±0.09 ±0.02 ±0.63 Acmella methanol ------6 7 extract ±0.00 ±0.03 Senna hexane ------6 extract ±0.09 Continuation of Table 2
Drug /extract concentration in μg/ml (Log2) Organism Extract/control -5 -4 -3 -2 -1 0 1 2 3 4 Inhibition zone diameters in mm (mean± standard deviation)
Amphotericin B 13±0.11 14± 15±0.61 17± 17 19 20 21 23 22 0.82 0.09 ±0.03 ±0.14 ±0.22 ±0.31 ±0.17 ±0.01 Candida Acmella hexane - - - - 11 13 13 14 17 17 haemulonii extract ±0.35 ±0.21 ±0.9 ±0.73 ±0.66 ±0.1 Senna methanol ------11 11 13 extract ±0.08 ±0.061 ±0.7 Acmella methanol ------14 14 17 15 extract ±0.06 ±0.15 ±0.99 ±0.8 Senna hexane ------8 12 extract ±0.42 ±0.95 Amphotericin B 8± 7± 8± 8 10 10 11 14 15 18 0.19 0.03 0.55 ±0.71 ±0.09 ±0.42 ±0.85 ±0.16 ±0.09 ±0.03 Candida Acmella hexane ------11 11 daubushaemulonii extract ±0.41 ±0.32 Senna methanol ------10 12 12 12 extract ±0.32 ±0.28 ±0.19 ±0.71 Acmella methanol - - - - 7 10 14 14 16 16 extract ±0.01 ±0.12 ±0.19 ±0.77 ±0.62 ±0.16 Senna hexane ------6 extract ±0.42 Amphotericin B 7± 9± 9± 11 10 12 12 13 14 14
14485
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
0.52 0.12 0.51 ±0.01 ±0.42 ±0.05 ±0.52 ±0.75 ±0.55 ±0.19 C. albicans Acmella methanol 6±0.3 11 11 ±0.8 8 ±0.00 16 ±0.52 17 ±0.47 21 ±0.99 22 23 26 extract ±0.57 ±0.03 ±0.48 ±1.08 Senna methanol - - - 7 8 8 10 8 14 11 extract ±0.15 ±0.51 ±0.12 ±0.95 ±0.23 ±0.46 ±0.12 Acmella hexane - - - - - 6 6 10 11 12 extract ±0.08 ±0.13 ±0.37 ±0.25 ±0.17 Senna hexane ------6 8 10 extract ±0.69 ±0.42 ±0.82 Amphotericin B 15±0.01 16± 19±0.05 22 23 26 27 27 31 31 0.15 ±0.45 ±0.75 ±0.98 ±0.85 ±0.99 ±0.80 ±0.65 Values are expressed as mean diameter ± standard deviation of the replica.
Table 3: Zone diameters of inhibition (mm) for combined plant extract and conventional antifungal agents Organism Drug and extract concentration in μg/ml Drug(Log2) 4 3 2 1 0 -1 -2 -3 -4 -5 extract(Log2) -5 -4 -3 -2 -1 0 1 2 3 4 Drug/extract combination Inhibition zone diameters in mm (mean± standard deviation) Candida krusei Ketoconazole/Acmella - - - - - 14± 22± 22± 24± 23± hexane extract 0.05 0.75 0.82 0.69 0.19 Clotrimazole/Senna 12±0.3 ------methanol extract Clotrimazole/Acmella 18±0.51 8±0.32 ------methanol extract Clotrimazole/Senna hexane 8±0.1 ------extract Candida orienntaris Ketoconazole/Acmella - - - - - 14± 20± 22± 26± 28± hexane extract 0.04 0.66 0.15 0.78 0.96 Clotrimazole/Senna 10 14±0.24 ------methanol extract ±0.25 Clotrimazole/Acmella 18 12±0.75 ------methanol extract ±0.15 Clotrimazole/Senna hexane 10 12±0.03 ------extract ±0.88
14486
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Candida famata Clotrimazole/Senna 10 12±0.51 8 8± 14± - - - - - methanol extract ±0.25 ±0.0 0.9 0.02
AmB /Acmella hexane - - - - - 10± 10± 10± 8± - extract 0.16 0.23 0.17 0.33 Clotrimazole/Acmella - - - - - 28± 22± 16± 10± - methanol extract 0.93 0.87 0.27 0.13 Clotrimazole/Senna hexane - 14±0.086 - - - 14± 12± 12± 16± 16± extract 0.43 0.25 0.125 0.53 0.11 Continuation of Table 3 Candida auris Ketoconazole/Acmella 12 12±0.07 12 - 22 - - 28± 26± - hexane extract ±0.64 ±0.01 ±0.61 0.86 0.72 Clotrimazole/Senna hexane 12 10±0.005 8 8± ------extract ±0.78 ±0.29 0.32 Candida haemulonii Ketoconazole/Acmella - - - - 16± 22± 25± 20± 24± 28± hexane extract 0.08 0.77 0.69 0.88 0.38 0.57 Candida Ketoconazole/Acmella 14 14±0.681 - 12± 15± 11± 16± 18± 19± 20± daubushaemulonii hexane extract ±0.01 0.09 0.14 0.03 0.011 0.05 0.41 0.81 Clotrimazole/Senna hexane 10 10±0.12 8 8± 8± - - - - - extract ±0.91 ±0.67 0.02 0.031 Values are expressed as mean ±standard deviation of the replica.
14487
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Inhibition of Candida species by the plant albicans, C. krusei, C. orienntaris C. famata, C. auris, extract/conventional drug combinations: The C. haemulonii and C. duabus haemulonii (Table 4). The diameters of inhibition produced by combined extracts hexane extra had MIC’s as 4µg/ml, 2µg/ml, 0.5µg/ml, and antifungal drugs were relatively higher compared to 2µg/ml, 2µg/ml, 1µg/ml and 0.5µg/ml for Candida the extract alone implying potentiation. Wilcoxon albicans, C. krusei, C. orientaris, C. famata, C. auris, C. Signed Ranks Test indicated a significant difference haemulonii and C. duabus haemulonii respectively when the combined form was compared to extract (Table 4). With Acmella caulirhiza hexane extract the alone (P < 0.05) (Appendix V). The results indicated a MIC’s were found to be; 8µg/ml, 4µg/ml, 4µg/ml, shift from inhibition at higher extract concentrations to 2µg/ml, 1µg/ml, 4µg/ml and 2µg/ml respectively for lower extract concentrations for most of the Candida albicans, C. krusei, C. orientaris, C. famata, C. combination (Table 2 and 3). auris, C. haemulonii and C. duabus haemulonii. The Minimum Inhibitory Concentrations (MIC50) for methanol extract had the MICs as 2µg/ml, 1µg/ml, various antifungal drugs in µg/ml: Griseofulvin, 1µg/ml, 2µg/ml, 4µg/ml, 4µg/ml and 4µg/ml for Clotrimazole and Ketoconazole produced high MIC Candida albicans, C. krusei, C. orientaris, C. famata, C. values in comparison to the control drug. For nystatin auris, C. haemulonii and C. duabus haemulonii however the results were erratic with growth at low respectivel as shown in Table 4. The minimum concentration and no zone of inhibition at higher inhibitory concentrations for amphotericin B was found concentrations meaning that all the pathogens died. to be; 0.5µg/ml, 4µg/ml, 4µg/ml, 0.25µg/ml, 4µg/ml, Senna didymobotrya methanol extract had MIC’s as 0.25µg/ml and 1µg/ml for Candida albicans, C. krusei, 1µg/ml, 0.125µg/ml, 1µg/ml, 1µg/ml, 0.25µg/ml, C. orientaris, C. famata, C. auris, C. haemulonii and C. 0.5µg/ml and 0.25µg/ml respectively for Candida duabus haemulonii respectively.
Table 4: Minimum Inhibitory Concentrations (MIC50) for uncombined drugs in µg/ml ±S.D Candida spp AmB keto Griseo Nyst Clotr SME SHE AHE AME C. albicans 0.5± 0.5± 32± _ 32± 1± 4± 8± 2± 0.32 0.01 0.06 0.75 0.11 0.13 0.66 0.02
C.krusei 4± 2± 32± _ 32± 0.125± 2± 4± 1± 0.07 0.31 0.32 0.01 0.40 0.38 0.83 0.20
C.orientaris 4± 4± 0.01 32± _ 32± 1± 0.5± 4± 1± 0.15 0.01 0.07 0.99 0.00 0.01 0.71
C.famata 0.25± 0.125± 32± _ 32± 1± 2± 2± 2± 0.50 0.00 0.06 0.61 0.01 0.01 0.30 1.02
C.auris 4± 8± 0.94 32± _ 16± 0.25± 2± 1± 4± 0.22 0.013 0.06 0.04 0.02 0.01 0.03
C.haemulonii 0.25± 8± 0.17 32± _ 16± 0.5± 1± 4± 4± 0.62 0.03 0.97 0.01 0.11 0.36 0.81 C. daubushaemuonii 1± 8± 0.47 32± _ 16± 0.25± 0.5± 2± 4± 0.07 0.82 0.25 0.02 0.04 0.01 0.01 Values are expressed as mean concentration ±standard deviation of the replica. KEY: AmB = Amphotericin B, keto = Ketoconazole, Griseo = Griseofulvin, Nyst = Nystatine, Clotr = Clotrimazole, SME = Senna didymobotrya methanol extract, SHE = Senna didymobotrya hexane extract, AME = Acmella caulirhiza methanol extract and AHE =Acmella caulirhiza hexane extract. N/B Nystatin values are not indicated as it showed erratic results i.e. at concentrations ranging from 0.125 ˗ 32 µg/ml all the wells showed growth turbidity equivalent to that of the positive control while at concentrations above 32µg/ml up to 64µg/ml it showed growth turbidity equivalent to that of the negative control i.e. no growth at all.
14488
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Minimum inhibitory concentration (MIC50) for however, Amphotericin B /A. caulirhiza methanol combined drugs and plant extract in µg/ml: From extract and Ketoconazole/ S. didymobotrya hexane the plant extract/conventional drug concentration deviated from this observation where the extract gradients most of the combination MIC values were concentration was lower than the conventional drug observed at lower conventional drug concentration and concentration (Table 5). higher extract concentration. Two combinations
Table 5: Mean MIC50 for combined drugs and plant extracts in µg/ml C. albicans C.krusei C. C.famata C. auris C.haemalonii C. orientaris daubushaemalonii
EA 0.25,8 0.25,8 0.5,4 _ 0.25,8 4,0.5 0.125,16
EB 0.25,8 0.25,8 0.125,16 0.5,4 0.125,16 0.25,8 0.25,8
EC 0.25,8 1,2 2,1 8,0.25 16,0.125 _ _
ED 1,2 0.5,4 2,1 0.125,16 0.25,8 _ _
FA 0.125,16 0.0625,32 0.0625,32 0.25,8 0.125,16 0.25,8 0.25,8
FB 8,0.25 8,0.25 8,0.25 8,0.25 8,0.25 8,0.25 8,0.25
FC _ 2,1 4,0.5 _ 1,2 _ _
FD _ 0.25,8 0.5,4 1,2 0.5,4 0.25,8 0.25,8
HA 0.25,8 1,2 0.5,4 1,2 0.5,4 0.25,8 _
HB 0.25,8 0.125,16 16,0.125 0.25,8 0.25,8 1,2 1,2
HC 2,1 4,0.5 4,0.5 4,0.5 1,2 4,0.5 2,1
HD 1,2 1,2 1,2 1,2 1,2 1,2 1,2
The first MIC value recorded is that of the conventional drug followed by that of the extract at given mixture ratios of the concentration gradient. KEY: EA = Clotrimazole and Senna didymobotrya methanol extract combination, EB = Clotrimazole and Senna didymobotrya hexane extract combination, EC = Clotrimazole and Acmella caulirhiza methanol extract combination, ED = Clotrimazole and Acmella caulirhiza hexane cutting combination, FA = Ketoconazole and Senna didymobotrya methanol extract combination, FB = Ketoconazole and Senna didymobotrya hexane extract combination, FC = Ketoconazole and Acmella caulirhiza methanol extract combination, FD=Ketoconazole and Acmella caulirhiza hexane extract combination, HA = Amphotericin B and Senna didymobotrya methanol extract combination, HB = Amphotericin B and Senna didymobotrya hexane extract combination, HC = Amphotericin B and Acmella caulirhiza methanol extract combination, HD = Amphotericin B and Acmella caulirhiza hexane extract combination.
The Fractional Inhibitory Concentration Index: hexane extract combination was synergistic against C. Amphotericin B / Acmella caulirhiza hexane extract albicans, C. krusei and C. orientaris but antagonistic combination was synergistic when used against C. against the other candida species. The other krusei and C. orientaris; while with the other candida combinations were indifferent and antagonistic against species it was antagonistic. Clotrimazole / A. caulirhiza the candida species used (Table 6)
14489
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Table 6: Fractional Inhibitory Concentration Index Candida Candida Candida Candida Candida Candida C. daubus albicans krusei orientaris famata auris haemulonii haemulonii
EA 8.0 (ant) 64.0(ant) 4.0(ant) _ 32.0(ant) 1.25(ind) 64.0 ant)
EB 2.0( ind) 4.0 (ind) 32.0(ant) 2.0 (ind) 8.0(ant) 8.0(ant) 16.0(ant)
EC 4.0 (ant) 2.0 (ind) 1.1 (ind) 0.38(syn) 1.0 (syn) _ _
ED 0.28 (syn) 1.0 (syn) 0.31(syn) 8.0 (ant) 8.0 (ant) _ _
FA 16.3(ant) 256.0 ant) 32.0 (ant) _ 64.0 (ant) 16.0 (ant) 32.0 ant)
FB 16.1 (ant) 4.1 (ant) 2.5 (ant) _ 1.1 (ind) 1.25 (ind) 1.5 (ind)
FC _ 2.0 (ind) 1.5 (ind) _ 0.63 (syn) _ _
FD _ 2.1 (ant) 1.1 (ind) _ 4.1 (ant) 2.0 (ind) 4.0 (ind)
HA 8.5 (ant) 16.3 (ant) 4.1 (ant) 6.0(ant) 16.1 (ant) 17.0(ant) _
HB 2.5 (ant) 8.0 (ant) 4.3 (ant) 5.0(ant) 4.1 (ant) 5.0 (ant) 5.0 (ant)
HC 4.5 (ant) 1.5 (ind) 1.5 (ind) 16.3 (ant) 0.75 (syn) 16.1 (ant) 2.3 (ant)
HD 2.3 (ant) 0.75 (syn) 0.75 (syn) 5.0 (ant) 2.3 (ant) 4.5 (ant) 2.0 (ind) ind. = Indifferent, syn. = Synergy and ant. = Antagonistic KEY: EA=Clotrimazole and Senna didymobotrya methanol extract combination, EB=Clotrimazole and Senna didymobotrya hexane extract combination, EC=Clotrimazole and Acmella caulirhiza methanol extract combination, ED=Clotrimazole and Acmella caulirhiza hexane extract combination, FA=Ketoconazole and Senna didymobotrya methanol extract combination, FB=Ketoconazole and Senna didymobotrya hexane extract combination, FC=Ketoconazole and Acmella caulirhiza methanol extract combination, FD=Ketoconazole and Acmella caulirhiza hexane extract combination, HA=Amphotericin B and Senna didymobotrya methanol extract combination, HB=Amphotericin B and Senna didymobotrya hexane extract combination, HC=Amphotericin B and Acmella caulirhiza methanol extract combination, HD=Amphotericin B and Acmella caulirhiza hexane extract combination.
DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS Currently more attention is given to the treatment and The extract of S. didymobotrya was found to contain management of infections caused by fungi than in the phenols, tannins, cardiac glycosides, terpenoids, past though not as much as that given to bacterial and flavonoids and alkaloids as shown in Table 1.Unlike in viral infections. Conventional therapeutic drugs have this study, Bonareri et al. (2015) found no alkaloids in several limitations associated with them. From folklore both hexane and methanol extracts of this plant while certain plant species have been used in the treatment Jeruto et al. (2017) demonstrated presence of alkaloids and management of fungal infections with success from specimens collected from Nakuru County and (Bristol et al., 2012). This study was aimed at none from those collected from Siaya and Nandi determining the antifungal effects of combined crude counties. In this study also steroids and phlobatannins extract of Senna didymobotrya and Acmella caulirhiza were absent from the plant extract as was also found by hexane and methanol extracts with conventional drugs: Bonareri et al. (2015) while Jeruto et al. (2017) had griseofulvin, ketoconazole, nystatin and clotrimazole. shown that samples collected from three different
14490
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques counties (Nakuru, Nandi and Siaya) all had steroids caulirhiza showed zones of inhibition from present in the leaf extracts. Further, phenols, flavonoids concentrations of 0.5µg/ml and 0.063µg/ml and above and terpenoids were found to be present in this plant for hexane and methanol respectively. The zone of extract just as was shown by Jeruto et al. (2017). In inhibition increased with increase in concentration. contrast, Bonareri et al. (2015) had found that the Likewise the hexane extract of S. didymobotrya methanol extract of this plant lacked flavonoids. These exhibited a dose related fungal inhibition. Methanol varied observations are a clear indication that presence extract of A. caulirhiza exhibited inhibition at lower or absence of secondary metabolites is in part extract concentration than hexane probably as an dependent on geographical location where the plants indication that the active compounds were more in are growing, on genotype variation, season of methanol extract than in the hexane extract. Since collection, method of extraction and dilution (Jeruto et methanol has a higher polarity than hexane, it is most al., 2017). This information is very important for likely that the inhibitory effect was from polar herbalist from different locations when using plants that compounds. The findings of this study are in agreement are known to cure some conditions in different with a study by Thakur et al. (2015) who by using agar locations. Evaluation of efficacy of plant extracts well diffusion found inhibition of Candida albicans by therefore would best be done in the geographical Acmella caulirhiza methanol extract in a dose related location where obtained. Specific growth conditions that manner. The extract concentrations used by Thakur produce medicinal plants with high efficacy should be et al. (2015) were much higher than those used in this investigated in order to customize the plants and work study although the results were comparable. The on them at any place. In this study the extract of A. variation is likely to arise from the difference in caulirhiza lacked phenols, phlobatannins and alkaloids concentrations of the secondary metabolites in these but contained terpenoids, flavonoids, tannins, two different studies. In deed the concentration of anthraquinones and cardiac glycosides. Other secondary metabolites are dependent on geographical phytochemicals found in this plant extract by other location (Jeruto et al., 2017). Mining et al. (2014) scholars are coumarin and sterols from aqueous extract observed similar results as in this study while using by Jespher et al. (2017), alkaloids, carbohydrates, hexane extract of roots and pods of Senna steroids, saponins, tannins and phenols by Thakur et al. didymobotrya against Candida albicans meaning that (2015). As observed for S. didymobotrya these varied that the secondary metabolites obtained from the roots, reports are all attributed to different geographical leaves and pods have relatively similar activity against locations of the plants. The inhibitory effect observed on Candida albicans. The zone of inhibition for Candida the Candida pathogens can be attributed to the albicans in this study was higher for the non albicans phytochemical compounds such as; terpenoids, Candida, an indication that they require high dose of glycosides and flavonoids that have been shown to the extract for treatment. Generally the extracts showed contain antifungal activities (Tasleem et al., 2009; moderate antifungal sensitivity. It was found that the Doughari et al., 2011; Altemimi et al., 2017). extracts strength of inhibition was relatively inferior in Phytochemicals have varied mechanisms of action comparison to the positive control drug (amphotericin against fungal strains. These include and not limited to; B) with an exception of Acmella caulirhiza methanol the formation of ion channels in the microbial extract against C. duabus haemulonii that showed membrane as shown by peptides that contain superiority to the control drug. This study used crude disulphide bonds and are positively charged. Peptides extract and thus the exact concentration of the active also competitively inhibit microbial proteins adhesion to component is unknown; likewise the active component host polysaccharide receptors and this gives them the is unknown and maybe it could be superior. The inhibitory nature to microorganisms (Tasleem et al., presence of a higher concentration of flavonoids, 2009). Terpenes are lipophilic in nature thus act by terpenoids and steroids in A. caulirhiza might be membrane disruption which then increases permeability responsible for this superiority in contrast to extracts of thus loss of cellular components and impairment of the S. didymobotrya that had lower concentrations of these enzymes involve in cellular metabolism (Tasleem et al., metabolites. The MIC values for methanol extract of S. 2009). Phenolic compounds are able to inhibit fungal didymobotrya against the various Candida species; enzyme by the oxidized compounds through reaction were relatively lower compared to those of hexane with sulfhydryl groups or nonspecific protein extract. This is due to the deference in the components interactions (Tasleem et al., 2009). The extracts from A. of the crude extract that is largely affected by the type
14491
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques of solvent. It is likely that the polar components of the al., 2011). Through such complex interaction, the extract are better antifungal agents than the non-polar extract might have potentiated the efflux of the components. Mining et al (2014) found that conventional drug; they might have prevented the dichloromethane extract of Senna didymobotrya conventional drugs from combining with specific target showed moderate antifungal activity against C. molecules or prevented their conversion to active albicans. In contrast to this study, Nelofar et al. (2016) metabolites consequently contributing to the found the main component extracted from flower heads antagonistic effect observed. While working on of Acmella caulirhiza to be Spilanthol; this compound essential oils from P. graveolens, Seungwons. et al. showed MICs of hexane extract that were lower than (2003) found them to have synergistic effects when those of methanol extract against Candida albicans. combined with ketoconazole and amphotericin B Shefali et al. (2011) showed that the methanol extract against Aspergilus spp. Ulrich-Merzenich et al. (2010) of Acmella caulirhiza had activity against Penicillum showed that an extract of cannabidiol when combined chrysogenum, Rhizopus arrhigus and Rhizopus with tetrahydrocannabinol, the transport of the later stolonifer which were not considered in this study thus across the membrane is increased leading to indicating its antifungal ability against a variety of fungi. synergism. The resorption of 1-Hyoscyamin is Savitha et al. (2015) found Acmella caulirhiza extract to increased when combined with flavonol-triglycosides have high inhibition of Candida albicans in comparison (Ulrich-Merzenich et al., 2010). The synergism to calcium hydroxide. In a study done by Rex John et observed is attributed to several factors like; enhancing al. (2001), they found the MIC of amphotericin B pharmacokinetic effects like absorption and transport against Candida spp. to be less than what was across membrane, elimination of neutralization of observed in this study and less than what is given by toxically acting constituents, multitarget effect which is CLSI as the breakpoint for susceptible Candida spp. attributed to most plant extracts among other factors Working on clinical isolates, Shallu et al. (2015) found (Ulrich-Merzenich et al., 2010). The dynamics of the MIC values for amphotericin B for Candida auris, infections has changed greatly to an increase in people Candida haemulonii and Candida daubus haemulonii to with multiple infections; thus this inherent phenomena be higher than what was observed in this study. This is of plant extracts having multitarget effect should be likely an indication that clinical isolates have a higher exploited to the advantage of treatment of these chance of being resistant. In comparison to the control diseases. Clotrimazole combined with Acmella drug (amphotericin B) there was no significant caulirhiza methanol and hexane extracts largely difference in the treatments (P> 0.05). Friedman’s test produced synergistic effect. The presence of terpenoids however, indicated that the seven groups (Candida and tannins which function by membrane disruption spp.) were significantly different from each other (Fernandes et al., 2010; Shamar et al., 2010) might (p=0.007). The extracts largely were antagonistic and have potentiated the effect of clotrimazole since it indifferent. Both methanol and hexane extracts of functions by affecting membrane integrity thus efflux of Senna didymobotrya were largely antagonistic and potassium. Acmella caulirhiza extract also contained indifferent when combined with ketoconazole, anthraquinones; quinones are known to complex with clotrimazole, griseofulvin and amphotericin B. cell wall and also inactivate membrane bound enzymes Phytochemicals exert their effect by cell membrane (Fernandes et al., 2010; Shamar et al., 2010). This is disruption, binding and inhibiting specific proteins or likely to have contributed to the potentiating of adhere to and intercalate into DNA or RNA (Efferth et clotrimazole by this extract.
CONCLUSIONS From this study the following conclusions are made; ii) Senna didymobotrya extract when combined with i) Hexane and methanol extracts of Senna ketoconazole, clotrimazole, griseofulvin and didymobotrya and Acmella caulirhiza are potent amphotericin B were largely indifferent and antagonistic antifungal agents since the MIC’s ranged from 0.25 while Acmella caulirhiza extract when combined with µg/ml to 8 µg/ml depending on organism. clotrimazole was synergistic.
14492
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
RECOMMENDATIONS This study recommends that; Recommendations for future studies i) The continued use of Senna didymobotrya and i) The pure active antifungal components of these Acmella caulirhiza can combat mycoses since they are plants are determined since the crude extracts showed potent antifungal agents. antifungal potency. ii) Acmella caulirhiza can be used with clotrimazole ii) Acmella caulirhiza showed high potential of since they are synergistic while the other combinations synergism thus its pure active components to be used should be avoided due to antagonism. to develop new antifungal regimens to combat the challenges faced in treatment of mycoses.
REFERENCES Adefemi, S. A., Odeigah, L. O. and Alabi, K. M (2011). Candida albicans. East African Medical Prevalence of dermatophytosis among primary Journal, 79: 3. school children in Oke-oyi community of Blanka, H., Viktor, A., Czaika, and Markus, F. (2008). Kwara state. Nigerian Journal of Clinical Epidemiological trends in skin mycoses Practice, 14:23-28. worldwide. Mycoses, 51(4):2–15. Alastruey-Izquierdo, A., Melhem, M.S.C., Bonfietti, L.X. Bristol, S.A., Rodrigues, F.F., Campos, A.R., and da and Rodriguez-Tudela, J.L. (2015). Costa, J.G. (2012). Evaluation of the Susceptibility test for fungi: clinical and antifungal activity and modulation between laboratorial correlations in medical mycology. Cajanus cajan (L.) Mill sp. Leaves and roots Rev. Inst. Med. Trop. Sao Paulo, 57(Suppl ethanolic extracts and conventional 19): 57-64. antifungals. Pharmacognosy Magazine, 8:103- Ammar, A., Naoufal, L., Azam, B., Dennis, G. W. and 106. David, A. L. (2017) Phytochemicals: Chen, Q., Lin, M.H., Chen, M.L., Liu, Z.Y., Chai, D. and Extraction, Isolation, and Identification of Wang, R. (2012).Efficacy and safety of Bioactive Compounds from Plant Extracts. micafungin for invasive Candida infections: a Plants, 6, 42; doi: 10.3390/plants6040042. meta-analysis of randomized controlled trials. Ana, A., Marcia, S.C. M., Lucas, X. B. and Juan, L. R. Efficacy and Safety Medical Journal, 125: 345- (2015).Susceptibility test for fungi: clinical and 351. laboratorial correlations in medical mycology, Christine, C. B., Koichi, M., Shigeru, A., Haruhiko, T., Rev. Inst. Med. Trop. Sao Paulo; 57(Suppl. Olga M. M., Gutura, R., Njeri, C. W. and 19):57-64. Shigeru, K. (2007). Antifungal drug Annette, W. F., Deanna, A. S., Dora, I. M., Nathan, P. susceptibility of Cryptococcus neoformans W.(2014).Impact of New Antifungal from clinical sources in Nairobi, Kenya. Breakpoints on Antifungal Resistance in Mycoses, 50 (1):25–30. Candida Species, Journal of Clinical Cleophas, T.J. (2009).Statistics Applied to Clinical Microbiology, 14:994–997. Trials, Springer Science.175–184. Baddley, J.W., Winthrop, K.L., Nivedita, M.P., Delzell, Clinical and Laboratory Standards Institute (CLSI) E., Beokelman, T., Xie, F., Lang, C. and (2012).Reference Method for Broth Dilution Curtis, J.R. (2011).Geographic distribution of Antifungal Susceptibility Testing of Yeasts; endemic fungal infection among older person, CLSI document M27-A2 Clinical and United States. Emerging Infectious Diseases, Laboratory Standards Institute, 940 West 17:1664-1669. Valley Road, Suite 2500, Wayne, Beyda,N.D., Shen H. C., Jahangir,M. A., Dhara N. S., Pennsylvania 19087, USA. (CLSI M27-S4). Tat, M.N., Laurie, M.and Kevin W. G. (2012). Clinical and Laboratory Standards Institute Treatment of Candida famata bloodstream (CLSI).Method for Antifungal Disk Diffusion infections: case series and review of the Susceptibility Testing of Yeasts; Approved literature; Journal 0f Antimicrobial Guideline – Second Edition. CLSI document Chemotherapy, 0: 1; doi:10.1093/jac/dks388. M44-S3 (2009). Clinical and Laboratory Bii, C.C., Ouko, T.T., Amukoye, E. and Githinji, L.W. Standards Institute, 950 West Valley Road, (2002). Antifungal drug susceptibility of
14493
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Suite 500, Wayne, Pennsylvania 19087, USA. extracts of Senna didymobotrya (Fresen.). (CLSI M44-S3). Journal of Applied Biosciences, 114: 11357- Crowley, P.D., and Gallagher, H.C. (2014).Clotrimazole 11367. as a pharmaceutical: past, present and future. Kang, K., Wong, K.S., Jayampath, C.S., Journal of Applied Microbiology ISSN 1364- Samaranayake, L.P., Fong, W.P. and Tsang, 5072. P.W.K. (2009).In vitro synergistic effects of Donno, D., Beccaro, G.L. ,Mellano, M.G. , Cerutti, A.K. , metergolino and antifungal agents against Bounous, G.(2013).Medicinal plants, chemical Candida krusei. Mycoses, 53:495-499. composition and quality: may blackcurrant Lackner, M., Rezusta, A., Villuendas, M.C., Palacian, buds and blackberry sprouts be a new M.P., Meis, J.F. and Klaassen, H.C. polyphenol source for herbal preparations? (2011).Infection and colonization due to Journal of Applied Botany and Food Quality Scedosporium in Northern Spain. An in vitro 86: 79 – 89. antifungal susceptibility and molecular Efferth, T., Egon, K. (2011). Complex Interactions epidemiology study of 60 isolates. Mycoses, between Phytochemicals. The Multi-Target 54:12-21. Therapeutic Concept of Phytotherapy. Current Liao, H., Dong, W., Shi, X., Liu, H. and Yuan, K. Drug Targets, 12: 122-132. (2012).Analysis and comparison of the active Enemuor, S.C. and Amedu, A.S. (2009).Prevalence of components and antioxidant activities of superficial mycoses in primary school children extracts from Abelmoschus esculentus L. in Anyigba, Kogi state, Nigeria. African Journal Pharmacognosy Magazine, 8:156-61. of Microbiology Research, 3(2):62-65. Lydia, B. N., Moses, N., Margaret, M., Agola, E. L., Espinel-Ingroff, A., Manuel, C., Emilia, C. (2013). Johnstone, I., Albert, K.(2015)Phytochemical EUCAST and CLSI: Working Together constituents of Senna didymobotrya Towards a Harmonized Method for Antifungal fresenirwin roots used as a traditional Susceptibility Testing; Current Fungal Infection medicinal plant in Kenya International Journal Report. 7:59–67 of Education and Research 3 Feng, Y., Anatoliy, K., Gabor, B., Stephen, W. and Mailӓnder-Sa'nchez, D., Wagener, J. and Schaller, M. Elena, R. (2013) Chromosome 5 Monosomy of (2010).Potential role of probiotic bacteria in the Candida albicans Controls Susceptibility to treatment and prevention of localized Various Toxic Agents, Including Major Candidasis. Mycoses, 55:50-55. Antifungals. Antimicrobial Agents Mallappa, K. S. and Uma, R. S (2015). A Chemotherapy; 57(10): 5026-5036. Comprehensive Review on the Phytochemical Ghulam, M., Muhammad, M., Aysha, S. (2015).A Constituents and Pharmacological Activities of Review: Antifungal potentials of medicinal Pogostemon cablin Benth. An Aromatic plants, Journal of Bioresource Management2 Medicinal Plant of Industrial Importance. (2): 23-31. Molecules, 20: 8521-8547. Humberto, F. B., Sarah, D. B.C., Gilda, M. N., Manoel Mikolajewska, A., Schwartz, S. and Ruhnke, M. (2010). J. B. G., Elaine, C. F., Kelly, I., Olga, F. G. Antifungal treatment strategies in patients with (2016). Candida duobus haemulonii: an hematological diseases or cancer: from emerging rare pathogenic yeast isolated from prophylaxis to empirical, pre-emptive and recurrent vulvovaginal candidiasis in Brazil; targeted therapy. Mycoses, 55:2-16. Mem Inst Oswaldo Cruz, 111(6): 407-410. Mining, J., Lagat, Z.O., Akenga, T., Tarus, P., Imbuga, Jackson, C., Agboke, A. and Nwoke, V. (2009).In-vitro M., Tsanuo. M. K. (2014). Bioactive evaluation of antimicrobial activity of metabolites of Senna didymobotrya used as combinations of Nystatin and Euphorbia hirta biopesticide against Acanthoscelides obtectus leaf extract against Candida albicans by the in Bungoma, Kenya. Journal of Applied checker board method, Journal of Medicinal Pharmaceutical Science; 4 (09): 056-060. Plants Research, 3(9):666-669. Moses, A.G. M., Leonard, G., Erastus, G. and Henry, R. Jeruto, P., Arama, P. F., Anyango, B., Maroa, G. (2012).Phytochemical Analysis of Phenol and (2017).Phytochemical screening and Flavonoid in Eight Selected Medicinal Herbs antibacterial investigations of crude methanol Used for the Treatment of Diabetes, Malaria
14494
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
and Pneumonia in Kisii, Kenya. Academic Sanglard, D. (2016). Emerging Threats in Antifungal- Journal of Cancer Research, 5 (2): 31-39. Resistant Fungal Pathogens. Front. Med. 3:11. Nasira, S., Vilas, J, Sarita, K. and Basavraj, N. doi: 10.3389/fmed.2016.00011. (2013).Antifungal Drug Resistance in Candida Sathyaprasad, S., Jose, B., K., Chandra, H., Species. European Journal of General S.(2015).Antimicrobial and antifungal efficacy Medicine. 10(4): 254-258. of Spilanthes acmella as an intracanal Nathan, P.W. (2017).Antifungal resistance: current medicament in comparison to calcium trends and future. Infection and Drug hydroxide: An in vitro study. Indian Journal of Resistance, 10: 249–259; Dove press. Dental Research, 26:528-532. Nelofar, G. N., Tareq, A.W., Mukta, S., Abid, w., Syed, Schmeller, W. and Dzikus, A. ( 2001). Skin diseases in N. S. (2016). Spilanthes acmella an children in rural Kenya: long-term results of a endangered medicinal plant - its Traditional, dermatology project within the primary health Phytochemical and Therapeutic properties – care system. British Journal of An overview. International Journal of Dermatology.144 (1):118–124. Advanced Research, 4(1): 627 – 639. Scorzon, L., Benaducci, T., Almeida, A.M.F., Silva, Otang, W.M., Grierson, D.S. and Ndip, R.N. D.H.S., Bolzani, V.S. and Mendes-Giannini, (2012).Antifungal activity of Arctotisarctotoides M.J.S. (2007).Comparative study of disc (L.f.) O. Hoffm.and Gasteriabicolor Haw. diffusion and micro dilution for evaluation of Against opportunistic fungi associated with antifungal activity of natural compounds human immunodeficiency virus/acquired against medical yeast Candida spp. and immunodeficiency syndrome. Pharmacognosy Cryptococcus spp. Journal of Basic and Magazine, 8:135-140. Applied Pharmaceutical Sciences, 28:25-34. Pedro, M., Martina, C., Michele, T., Kelli, A. H., Chiara, Shallu, K.,Pradeep, K. S.,Cheshta, S.,Anupam, D. and Daniele, D. (2016). Phytochemical P.,Aradhana, M.,Anil, K.,Jacques, F. Profiling of Flavonoids, Phenolic Acids, M.,Anuradha, C. (2015). Multidrug-Resistant Terpenoids, and Volatile Fraction of a Candida auris Misidentified as Candida Rosemary (Rosmarinus officinalis L.) Extract. haemulonii: Characterization by Matrix- Molecules, 21(11): 1576. Assisted Laser Desorption Ionization–Time of Pfaller, M.A. and Diekema D.J. (2010).Epidemiology of Flight Mass Spectrometry and DNA Invasive Mycoses in North America. Critical Sequencing and Its Antifungal Susceptibility Reviews in Microbiology, 36:1-53. Profile Variability by Vitek 2, CLSI Broth Playford, E.G., Webster, A.C., Sorrell, T.C. and Craig, Microdilution, and Etest Method. Journal of J.C. (2006).Antifungal agents for preventing Clinical Microbiology. 53(6): 1823–1830. fungal infections in non‐neutropenic critically ill Shefali, A., Saurabh, V., Deepak, K. (2011). patients. Cochrane Database of Systematic Phytochemical and antimicrobial studies on ReviewsIssue1. Art. No.: CD004920. the leaves of Spilanthes acmella. Journal of Rossana, A. C. , Carlos, E. C. T., Raimunda, S. N. B. Chemical and Pharmaceutical Research, ,Débora, S. C. M. C., Manoel, A. N. P., João, 3(5):145-150. J. G., Daniel, T. L.,Andr é J. M., Jos é J. C. S. Shokohi, T., Badali, H., Amirrajab, N., Ataollahi, M.R., and Marcos, F. G. R. (2013).Minimum Kouhpayeh, S.A., Afsarian, M.H. (2016). In inhibitory concentrations of amphotericin B, vitro activity of five antifungal agents against azoles and caspofungin against Candida Candida albicans isolates, Sari, Iran. Current species are reduced by farnesol. Medical Medical Mycology, 2(2): 34-39. Mycology, 51: 53–59. Silva, N.C.C., Fernandes, J.A. (2010). Biological Sa'nchez, V.L.O., Eraso, E., Carrilo-Muñoz, J.A., properties of medicinal plants: a review of their Aguirre, J.M., Gaitán-cepeda, L.A. and antimicrobial activity .The Journal of Quindós, G. (2009). In vitro activity of Venomous Animals and Toxins including voriconazole against Mexican oral yeast Tropical Diseases; 16 (3): 402-413. isolates. Mycoses, 53:200-203. Talya, F., Anna, N., Shiri, Z., Edna, B., Judith, B., Ronen, B.(2016).Candida haemulonii and Candida auris: Emerging MultidrugResistant
14495
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Species With Distinct Virulence and Wayne W. LaMorte (, 2017). Mann Whitney U Test Epidemiological Characteristics. Epidemiology (Wilcoxon Rank Sum Test). Boston University and Outcomes in Mycology; Session: 43. School of Public Health. Tarcieli, P. V., Luana, R., Tatiana, B. S., Giordano, R. WHO (1998) Updated edition of Quality control T., Maria, I. A., Caroline, B. W., Janio, M. S., methods for medicinal plant materials. Sydney., Hartz, A. (2011). In vitro synergisms Xin, G. and Neil, J. S (2012). Encyclopaedia of obtained by amphotericin B and voriconazole Research Design. "Nonparametric Statistics" associated with non-antifungal agents against .SAGE Publications, Inc. City: Thousand Oaks Fusarium spp. Diagnostic Microbiology and Print ISBN: 9781412961271. Infectious Disease, 71: 126–130. Xu, R., Chen, Y., Wan, D. and Wang, J. (2012). Tasleem, A., Bhosalea, J.D., Naresh, K., Mandala, T.K., Identification of four Sedum plant medicines by Bendreb, R.S., Lavekara, G.S. and Rajesh, D. Fourier transform infrared spectra. (2009. Natural products – antifungal agents Pharmacognosy Magazine, 8:107-111. derived from plants. Journal of Asian Natural Yim, M.S., Ko, J.H., Lee, W.Y., Kim, W.H., Lee, Y.J., Products Research, 11: 621–638. Kim, N.I., Kye, Y.C., Park, K.C., Choi, J.H., Tay, T.S., Rohani, M.Y., HooSoo, T.S. and Hamimah, Lee, H.K., Kim, N.M., Kim, K.J., Ro, S.Y. and H. (2009).Epidemiology of Cryptococcosis in Ahn, K.J. (2009). Study to compare efficacy Malaysia. Mycoses, 53:509-514. and safety of fluconazole cream with Tian-yang, W., Qing, L., Kai-shun, B. (2018).Bioactive flutrimazole cream in the treatment of flavonoids in medicinal plants: Structure, superficial mycoses: a randomized, double- activity and biological fate. Asian, 13(1): 12-23. blind, phase III trial. Mycoses, 53:522-529. Vila, T.V.M., Chaturvedi, A.K., Rozental, S., Lopez- Ying, S. and Chunyang, L. (2011).Correlation between Ribot, J.L. (2015). In vitro activity of phospholipase of Candida albicans and miltefosine against Candida albicans under resistance to fluconazole. Mycoses, 55:50-55. planktonic and biofilm growth conditions and in Zulfiqar, A. and Bala, B.S. (2016). Basic statistical tools vivo efficacy in a murine model of oral in research and data analysis. Indian Journal candidiasis. Antimicrobial Agents of Anesthesia.60 (9): 662–669. doi: Chemotherapy, 59:7611–7620. 10.4103/0019-5049.190623.
ACKNOWLEDGEMENT I acknowledge and thank the Kenya National Research his technical advice and support. I also acknowledge Fund for funding this study. I acknowledge the support, the technical advice and support given to me by Mugo advice and guidance given to me by my supervisors Peter of Medical laboratory Sciences Kenyatta Prof. Ngeranwa Joseph and Dr. Orinda George. I University and any other person who helped me acknowledge Edward Karis of the department of materially or morally during my study. Pharmacy School of Medicine Kenyatta University for
14496
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
APPENDICES Appendix i
24hr culture plates showing zones of inhibition.
Appendix ii
Broth microdilution wells with inoculum and drug at different concentrations i.e. regions with intense colour are having a high concentration of the plant extract with reducing gradient to the less intense coloured regions.
14497
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Appendix iii: Percentage inhibition of diameter growth of the extracts against Candida spp.
14498
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
14499
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Appendix vi (non parametric output) i) WILCOXON=Candida krusei treated with the different extracts and treatment with Amphotericin B as the control. Basing on -ZONE DIAMETERS OF INHIBITION.
Wilcoxon Signed Ranks Test Ranks N Mean Rank Sum of Ranks Negative Ranks 1a 1.00 1.00 Positive Ranks 9b 6.00 54.00 Ampotericin B - Acmella hexane extract Ties 0c Total 10 Negative Ranks 0d .00 .00 Positive Ranks 10e 5.50 55.00 Ampotericin B - Senna methanol extract Ties 0f Total 10 Negative Ranks 0g .00 .00 Positive Ranks 10h 5.50 55.00 Ampotericin B - Acmella methanol extract Ties 0i Total 10 Negative Ranks 0j .00 .00 Positive Ranks 10k 5.50 55.00 Ampotericin B - Senna hexane extract Ties 0l Total 10 a. Ampotericin B < Acmella hexane extract, b. Ampotericin B > Acmella hexane extract c. Ampotericin B = Acmella hexane extractd. Ampotericin B < Senna methanol extract e. Ampotericin B > Senna methanol extract f. Ampotericin B = Senna methanol extract g. Ampotericin B
14500
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Friedman Test Ranks Mean Rank Acmella hexane extract 3.45 Senna methanol extract 2.20 Acmella methanol extract 2.55 Senna hexane extract 1.80 Test Statisticsa N 10 Chi-Square 13.299 df 3 Asymp. Sig. .004 a. Friedman Test
ii) WILCOXON=Candida orientaris treated with the different extracts and treatment with Amphotericin B as the control. Basing on -ZONE DIAMETERS OF INHIBITION.
Wilcoxon Signed Ranks Test Ranks N Mean Rank Sum of Ranks Negative Ranks 0a .00 .00 Positive Ranks 10b 5.50 55.00 Ampotericin B - Acmella hexane extract Ties 0c Total 10 Negative Ranks 0d .00 .00 Positive Ranks 10e 5.50 55.00 Ampotericin B - Senna methanol extract Ties 0f Total 10 Negative Ranks 0g .00 .00 Ampotericin B - Acmella methanol Positive Ranks 10h 5.50 55.00 extract Ties 0i Total 10 Negative Ranks 0j .00 .00 Positive Ranks 10k 5.50 55.00 Ampotericin B - Senna hexane extract Ties 0l Total 10 a. Ampotericin B < Acmella hexane extract g. Ampotericin B
14501
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Friedman Test Ranks Mean Rank Acmella hexane extract 3.15 Senna methanol extract 2.25 Acmella methanol extract 2.65 Senna hexane extract 1.95 Test Statisticsa
N 10 Chi-Square 11.045 df 3 Asymp. Sig. .011 a. Friedman Test
iii) WILCOXON=Candida famata treated with the different extracts and treatment with Amphotericin B as the control. Basing on -ZONE DIAMETERS OF INHIBITION.
Wilcoxon Signed Ranks Test Ranks N Mean Rank Sum of Ranks Negative Ranks 0a .00 .00 Positive Ranks 10b 5.50 55.00 Ampotericin B - Acmella hexane extract Ties 0c Total 10 Negative Ranks 0d .00 .00 Ampotericin B - Senna methanol Positive Ranks 10e 5.50 55.00 extract Ties 0f Total 10 Negative Ranks 0g .00 .00 Ampotericin B - Acmella methanol Positive Ranks 10h 5.50 55.00 extract Ties 0i Total 10 Negative Ranks 0j .00 .00 Positive Ranks 10k 5.50 55.00 Ampotericin B - Senna hexane extract Ties 0l Total 10 a. Ampotericin B < Acmella hexane extract g. Ampotericin B
Test Statisticsa Ampotericin B -Ampotericin B - Senna Ampotericin B -Ampotericin B - Senna Acmella hexane extractmethanol extract Acmella methanol hexane extract extract Z -2.842b -2.818b -2.810b -2.809b Asymp. Sig. (2-tailed) .004 .005 .005 .005 a. Wilcoxon Signed Ranks Test b. Based on negative ranks.
14502
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Friedman Test Ranks Mean Rank Acmella hexane extract 2.60 Senna methanol extract 2.80 Acmella methanol extract 2.80 Senna hexane extract 1.80
Test Statisticsa N 10 Chi-Square 8.870 df 3 Asymp. Sig. .031 a. Friedman Test
iv) WILCOXON=Candida auris treated with the different extracts and treatment with Amphotericin B as the control. Basing on -ZONE DIAMETERS OF INHIBITION.
Wilcoxon Signed Ranks Test Ranks N Mean Rank Sum of Ranks Negative Ranks 0a .00 .00 Positive Ranks 10b 5.50 55.00 Ampotericin B - Acmella hexane extract Ties 0c Total 10 Negative Ranks 0d .00 .00 Positive Ranks 10e 5.50 55.00 Ampotericin B - Senna methanol extract Ties 0f Total 10 Negative Ranks 0g .00 .00 Ampotericin B - Acmella methanol Positive Ranks 10h 5.50 55.00 extract Ties 0i Total 10 Negative Ranks 0j .00 .00 Positive Ranks 10k 5.50 55.00 Ampotericin B - Senna hexane extract Ties 0l Total 10 a. Ampotericin B < Acmella hexane extract g. Ampotericin B
Test Statisticsa Ampotericin B -Ampotericin B -Ampotericin B - Ampotericin B - Acmella hexane Senna methanol Acmella methanol Senna hexane extract extract extract extract Z -2.805b -2.814b -2.812b -2.807b Asymp. Sig. (2-tailed) .005 .005 .005 .005 a. Wilcoxon Signed Ranks Test b. Based on negative ranks.
14503
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Friedman Test Ranks Mean Rank Acmella hexane extract 3.35 Senna methanol extract 2.65 Acmella methanol extract 2.00 Senna hexane extract 2.00 Test Statisticsa N 10 Chi-Square 15.245 df 3 Asymp. Sig. .002 a. Friedman Test
v) WILCOXON=Candida haemulonii treated with the different extracts and treatment with Amphotericin B as the control. Basing on -ZONE DIAMETERS OF INHIBITION.
Wilcoxon Signed Ranks Test Ranks N Mean Rank Sum of Ranks Negative Ranks 4a 3.38 13.50 Positive Ranks 5b 6.30 31.50 Ampotericin B - Acmella hexane extract Ties 1c Total 10 Negative Ranks 0d .00 .00 Positive Ranks 10e 5.50 55.00 Ampotericin B - Senna methanol extract Ties 0f Total 10 Negative Ranks 2g 1.75 3.50 Ampotericin B - Acmella methanol Positive Ranks 7h 5.93 41.50 extract Ties 1i Total 10 Negative Ranks 0j .00 .00 Positive Ranks 10k 5.50 55.00 Ampotericin B - Senna hexane extract Ties 0l Total 10 a. Ampotericin B < Acmella hexane extract g. Ampotericin B
Test Statisticsa Ampotericin B - Ampotericin B -Ampotericin B -Ampotericin B - Acmella hexane Senna methanol Acmella methanol Senna hexane extract extract extract extract Z -1.072b -2.812b -2.263b -2.814b Asymp. Sig. (2-tailed) .284 .005 .024 .005 a. Wilcoxon Signed Ranks Test b. Based on negative ranks.
14504
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Friedman Test Ranks Mean Rank Acmella hexane extract 3.20 Senna methanol extract 2.15 Acmella methanol extract 2.80 Senna hexane extract 1.85 Test Statisticsa N 10 Chi-Square 13.776 df 3 Asymp. Sig. .003 a. Friedman Test
vi) WILCOXON=Candida daubus treated with the different extracts and treatment with Amphotericin B as the control. Basing on -ZONE DIAMETERS OF INHIBITION.
Wilcoxon Signed Ranks Test Ranks N Mean Rank Sum of Ranks Negative Ranks 0a .00 .00 Positive Ranks 10b 5.50 55.00 Ampotericin B - Acmella hexane extract Ties 0c Total 10 Negative Ranks 0d .00 .00 Positive Ranks 10e 5.50 55.00 Ampotericin B - Senna methanol extract Ties 0f Total 10 Negative Ranks 4g 2.88 11.50 Ampotericin B - Acmella methanol Positive Ranks 6h 7.25 43.50 extract Ties 0i Total 10 Negative Ranks 0j .00 .00 Positive Ranks 10k 5.50 55.00 Ampotericin B - Senna hexane extract Ties 0l Total 10 a. Ampotericin B < Acmella hexane extract g. Ampotericin B
Test Statisticsa Ampotericin B - Ampotericin B -Ampotericin B -Ampotericin B - Acmella hexane Senna methanol Acmella methanol Senna hexane extract extract extract extract Z -2.809b -2.812b -1.643b -2.807b Asymp. Sig. (2-tailed) .005 .005 .100 .005 a. Wilcoxon Signed Ranks Test b. Based on negative ranks.
14505
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Friedman Test Ranks Mean Rank Acmella hexane extract 2.10 Senna methanol extract 2.60 Acmella methanol extract 3.40 Senna hexane extract 1.90
Test Statisticsa N 10 Chi-Square 16.080 df 3 Asymp. Sig. .001 a. Friedman Test
vii) WILCOXON test for Candida albicans treated with the different extracts and treatment with Amphotericin B as the control. Basing on -ZONE DIAMETERS OF INHIBITION.
Wilcoxon Signed Ranks Test Ranks N Mean Rank Sum of Ranks Negative Ranks 0a .00 .00 Ampotericin B - Acmella hexane Positive Ranks 10b 5.50 55.00 extract Ties 0c Total 10 Negative Ranks 0d .00 .00 Ampotericin B - Senna methanolPositiv e Ranks 10e 5.50 55.00 extract Ties 0f Total 10 Negative Ranks 0g .00 .00 Ampotericin B - AcmellaPositive Ranks 10h 5.50 55.00 methanol extract Ties 0i Total 10 Negative Ranks 0j .00 .00 Ampotericin B - Senna hexane Positive Ranks 10k 5.50 55.00 extract Ties 0l Total 10 a. Ampotericin B < Acmella hexane extract g. Ampotericin B
14506
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques
Friedman Test Ranks Mean Rank Acmella hexane extract 4.00 Senna methanol extract 2.50 Acmella methanol extract 2.10 Senna hexane extract 1.40
Test Statisticsa N 10 Chi-Square 25.256 df 3 Asymp. Sig. .000 a. Friedman Test
viii) WILCOXON test for Ketoconazole, Griseofulvin, Clotrimazole, Senna methanol extract, Senna hexane extract, Acmella hexane extract, and Acmella methanol extract WITH Amphotericin Bas the control. Basing on -MINIMUM INHIBITORY CONCETRATIONS.
Wilcoxon Signed Ranks Test Ranks N Mean Rank Sum of Ranks Negative Ranks 3a 4.00 12.00 Positive Ranks 2b 1.50 3.00 Ampotericin B - Keto Ties 2c Total 7 Negative Ranks 7d 4.00 28.00 Positive Ranks 0e .00 .00 Ampotericin B - Griseo Ties 0f Total 7 Negative Ranks 7g 4.00 28.00 Positive Ranks 0h .00 .00 Ampotericin B - Clot Ties 0i Total 7 Negative Ranks 3j 2.17 6.50 Positive Ranks 4k 5.38 21.50 Ampotericin B - SME Ties 0l Total 7 Negative Ranks 3m 3.83 11.50 Positive Ranks 4n 4.13 16.50 Ampotericin B - SHE Ties 0o Total 7 Negative Ranks 4p 3.00 12.00 Positive Ranks 1q 3.00 3.00 Ampotericin B - AHE Ties 2r Total 7 Negative Ranks 4s 3.25 13.00 Positive Ranks 2t 4.00 8.00 Ampotericin B - AME Ties 1u Total 7 a. Ampotericin B < Ketoconazole f. Ampotericin B = Griseofulvin b. Ampotericin B > Ketoconazole g. Ampotericin B < Clotrimazole c. Ampotericin B = Ketoconazole h. Ampotericin B > Clotrimazole d. Ampotericin B < Griseofulvin i. Ampotericin B = Clotrimazole
14507
Olwenya et al., J. Appl. Biosci. 2019 Caractérisation de quelques variétés Algériennes de blé dur (Triticum turgidum L. var. durum) par le biais des marqueurs phénotypiques e. Ampotericin B > Griseofulvin j. Ampotericin B < Senna methanol extract p. Ampotericin B < Acmella hexane extract k. Ampotericin B > Senna methanol extract q. Ampotericin B > Acmella hexane extract l. Ampotericin B = Senna methanol extract r. Ampotericin B = Acmella hexane extract m. Ampotericin B < Senna hexane extracts. Ampotericin B
Friedman Test Ranks Mean Rank SME 1.21 SHE 2.43 AHE 3.36 AME 3.00 Test Statisticsa N 7 Chi-Square 12.141 df 3 Asymp. Sig. .007 a. Friedman Test
14508