In vitro modulation of clotrimazole, Ketoconazole, Nystatin, Amphotericin B and Griseofulvin by Acmella caulirhiza and Senna didymobotrya extract against Candida spp

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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

14478

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: igunzafred@gmail.com

Original submitted in on 24^th August 2019. Published online at www.m.elewa.org/journals/ on 31^stOctober 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

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14479 INTRODUCTION

Mycoses in general has become of clinical significance in the recent past with high mortality rates reported. This increase may be associated with advances in medical practice such as use of intravascular catheters and total parenteral nutrition which provide a platform for development of biofilms by fungi and this is known to confer resistance, use of broad spectrum antimicrobials that kill normal flora and change the pH leading to colonization by fungi. Dialysis, Solid organ and bone marrow transplant patients, those with acquired immune deficiency syndrome (AIDS) and patients on intensive chemotherapy regimens are at high risk of developing mycoses due to compromised immunity (Vendettuoli et al., 2009).

Currently there is an increase of non-albican candida pathogens such as Candida glabrata, Candida krusei, Candida parapsilosis and Candida tropicalis that are of clinical importance since they are known to cause life threatening fungemia.

They show elevated MICs for most antifungal agents; a likely indicator of resistance. The treatment of mycoses is in itself facing challenges arising from increased incidences of adverse side

effects of drugs such as dose limiting nephrotoxicity shown by amphotericin B, drug-drug interaction, most antifungal agents being fungistatic in nature, rise of fungal strains that are showing multiple-drug resistance and the persistence of fungal infection in immunocompromised individuals. This necessitates the search for new drugs from plant species in order to address the above challenges associated with mycoses treatment. The presence of tannins, alkaloids, saponins, and terpenes among other phytochemicals gives plants inherent antifungal properties making them a potential source for new antimicrobial agents (Bristol et al., 2012). Several plant species from folklore have the reputation of being able to cure fungal infections but no scientific evaluation exists for most of the plants. The anti-fungal effects of combination of the plant extracts with the conventional drugs is also little known. This research endeavors to provide such information thus improving on treatment of mycoses that is currently posing a great challenge.

MATERIALS AND METHODS

Plant material: The plants were selected due to their wide spread use as anti-mycotic agents in western Kenya. Plants were obtained from the wild around Kakamega forest and a specimen deposited at Kenya National Museum for assignment of specimen voucher number. Extracts of plants used in the study were;

Senna didymobotrya methanol extract, Senna didymobotrya hexane extract, Acmella caulirhiza methanol extract and Acmella caulirhiza hexane extract dried under shade before the extraction. Solvent extraction was done sequentially with a view of minimizing analogous compounds.

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14480 Plate 1: Acmella caulirhiza

Plate 2: Senna didymobotrya

Plant extract and drug concentrations: Clinical and Laboratory Standards Institute procedure on preparation of concentrations of drugs and solvents used as documented in M44-A were followed.

Clotrimazole, Ketoconazole, Nystatin, Amphotericin B and Griseofulvin powders were dissolved in Dimethyl Sulfoxide to give stock solutions of concentrations 64μg/ml,32μg/ml, 16μg/ml, 8μg/ml, 4μg/ml,2μg/ml,

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14481 1μg/ml, 0.5μg/ml, 0.25 and 0.125μg/ml; that gave concentrations of 32 μg/ml,16 μg/ml, 8 μg/ml, 4 μg/ml, 2 μg/ml,1 μg/ml, 0.5 μg/ml, 0.25 μg/ml, 0.125 and 0.0625 μg/ml when diluted to the final concentration by media.

Methanol and hexane extracts of Acmella caulirhiza and Senna didymobotrya were also weighed and dissolved in Dimethyl Sulfoxide to also give stock solutions that gave concentrations of; 32 μg/ml,16 μg/ml, 8 μg/ml, 4 μg/ml, 2 μg/ml,1 μg/ml, 0.5 μg/ml, 0.25 μg/ml, 0.125 and 0.0625 μg/ml when dissolved in media.

Microorganisms used: The test organisms that were used are: Candida albicans; ATCC 14053, C.

duabushaemulonii; ATCC 2052030 , C.haemulonii;

ATCC 1609496, C.auris; ATCC 2050582, C.famata;

ATCC 2037476, C.orientaris; ATCC 6258 and C.krusei;

ATCC 14243.They were sub cultured on SDA to test viability and purity before use. The test organisms were selected based on the fact that C. albicans has been known to cause diseases for a long time and is showing the development of resistance or elevated MIC for drugs that were effective on it. the non- albicans; C.

duabushaemulonii, C.haemulonii, C.auris, C.famata, C.orientaris and C.krusei are emerging as clinically significant fungi with elevated MIC or showing resistance to most antifungal agents in use.

Inoculum preparation: Clinical Laboratory Standards Institute reference method M27-A3 protocol was used to prepare the inoculum. Colonies from 24 hour culture plates of Candida were suspended in 0.85% saline then the turbidity adjusted to 0.5 McFarland standard, giving a stock solution of 1 x 10⁶ to 5 x 10⁶cells per ml.

Disk preparation: Sterile blank disks of 6mm diameter had 10ml volume of each extract at different concentration were impregnated onto them. They were dried and stored at 8^oc pending use. Enough disks were prepared since the experiment was replicated.

Sterile blank disks of 6mm diameter had 10ml volume of DMSO impregnated on them and used as a negative control. The procedure was used for amphotericin B that acted as a positive control.

Inoculation of Test Plates for disk diffusion: A sterile cotton swab was dipped into the inoculum suspension, rotated several times and pressed firmly against the inside wall of the tube above the fluid level to remove excess fluid from the swab. Dry surface of sterile Sabouraud’s Dextrose Agar plate was inoculated by evenly streaking the swab over the entire agar surface and repeating the procedure two more times, rotating the plate approximately 60° each time to ensure an even distribution of inoculum. Antifungal

disks were evenly dispensed onto the surface of the inoculated agar plate and pressed down to ensure its complete contact with the agar surface. The plates were inverted and placed in an incubator set to 37 °C.

The inhibition zones were read after 24 hours of incubation. The zones read were used to calculate the percentage inhibition of diameter growth (PIDG) as a measure of the strength of inhibition of the extract in relation to the control drug. It is given by;

PIDG=

Diameter of sample- Diameter of positive control x 100 Diameter of positive control

Broth Micro-dilution Test to determine MIC values:

The MIC values were determined by broth micro dilution as provided for in M27-A3 document of Clinical Laboratory Standards Institute with a few modifications.

Briefly, 100μml of the plant extract each of different concentrations was put in a separate round bottomed well and 100μml of the inoculum added to the wells. A well containing the growth media and drug alone was used as a negative control while that containing the inoculum without test drug was used as appositive control. They were incubated at 37^oc for 24 hours before readings taken. For modulation, 50μml of the drug was mixed with 50μml of the plant extract against a concentration gradient; as the concentration of the drug ranged from 32 μg/ml,16 μg/ml, 8 μg/ml, 4 μg/ml, 2 μg/ml,1 μg/ml, 0.5 μg/ml, 0.25 μg/ml, 0.125 to 0.0625 μg/ml, that of the plant extract ranged in the reverse concentration from 0.0625 μg/ml up to 32 μg/ml.

100μml of the inoculum was also added to each well and incubated at 37^oc for 24 hours.

Phytochemical tests

Test for Alkaloid: 3ml of aqueous extract was added to 3ml of 10% HCl on a steam bath. Mayer’s reagent was then added to extract. Turbidity of the resulting yellow precipitate was evidence for the presence of Alkaloid (Yadav and Munin., 2011; Lemino et al., 2013;

Pradeep et al., 2014).

Test for Tannins –ferric chloride test: 2ml of the aqueous extract was stirred with 3ml of distilled water and few drops of FeCl3 solution were added. The formation of green colour precipitate was indication of presence of Tannins (Yadav and Munin., 2011; Lemino et al., 2013; Pradeep et al., 2014).

Test for Phlobatannins: 3ml of aqueous extract was added to 2ml of 10% HCl and the extract was boiled.

Deposition of a red precipitate was taken as an evidence for the presence of Phlobatannins (Yadav and

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14482 Munin., 2011; Lemino et al., 2013; Pradeep et al., 2014).

Test for Flavonoids –lead acetate test: To 3ml of methanoic extract 1ml of 10% lead acetate solution was added. The formation of a yellow precipitate was taken as a positive result for Flavonoids (Yadav and Munin., 2011; Lemino et al., 2013; Pradeep et al., 2014) Test for Anthraquinones: 0.5 ml of the extract was boiled with 10 ml of sulphuric acid (H2SO4) and filtered while hot. The filtrate was shaken with 5 ml of chloroform. The chloroform layer was pipetted into another test tube and 1 ml of dilute ammonia was added. The resulting solution was observed for colour changes (Yadav and Munin., 2011; Lemino et al., 2013;

Test for Terpenoids (Salkowski test): 0.5ml of each of the extract was added 2 ml of chloroform.

Concentrated H2S04 (3 ml) was carefully added to form a layer. A reddish brown colouration of the interface indicates the presence of Terpenoids (Yadav and Munin., 2011; Lemino et al., 2013; Pradeep et al., 2014).

Test for steroid: Crude extract was mixed with 2ml of chloroform and concentrated H2SO4 was added sidewise. A red colour produced in the lower chloroform layer indicated the presence of steroids. Another test was performed by mixing crude extract with 2ml of chloroform. Then 2ml of each of concentrated H2SO4

and acetic acid were poured into the mixture. The development of a greenish coloration indicated the

presence of steroids (Yadav and Munin. 2011; Lemino et al., 2013; Pradeep et al., 2014).

Test for phenols and tannins: Crude extract was mixed with 2ml of 2% solution of FeCl3. A blue-green or black coloration indicated the presence of phenols and tannins (Yadav and Munin. 2011; Lemino et al., 2013;

Test for cardiac glycosides -Keller-killiani test:

Methanol crude extract was mixed with 2ml of glacial acetic acid in a test tube. 1-2 drops of 2% solution of FeCl3 was added followed by 2ml of concentrated H2SO4. A brown ring at the interphase indicated the presence of cardiac glycosides (Yadav and Munin., 2011; Lemino et al., 2013; Pradeep et al., 2014).

Data management and statistical analysis: The in vitro antifungal data obtained was tabulated on Microsoft Excel spreadsheet program. It was then transferred to IBM SPSS version 20.0 software for statistical analysis. The data was exposed to descriptive statistic and expressed as the mean ± standard deviation of the mean. Wilcoxon Signed Ranks Test was done to find significant difference between the reference drug (Amphotericin B) and the plant extract treatments. Friedman’s test was executed to compare the means of the four different plant extracts and reference drug on each of the fungal species. The values of p ≤ 0.05 were considered statistically significant. The analysed data was presented in tables.

RESULTS

Phytochemical analysis of plant extract: In this study it was found that S. didymobotrya extract contained phenols, tannins, cardiac glycosides, terpenoids, flavonoids and alkaloids in varying degrees of

concentration. On the other hand A. caulirhiza extract contained steroids, terpenoids, flavonoids, tannins, anthraquinones and cardiac glycosides also in varying 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

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14483 Inhibition of Candida species by the plant extracts:

The crude extracts of S. didymobotrya and A. caulirhiza as shown in Table 2 had antifungal activity against the Candida species used in this experiment at varying degrees of concentration. In all the Candida species tested, the plant extracts demonstrated zones of inhibition at MICs ranging from 1μg/ml to 32μg/ml as indicated in Table 2. S. didymobotrya hexane extract among all the extracts demonstrated the lowest efficacy since the zones of inhibition were only observed at concentrations of 16μg/ml to 32μg/ml (Table 2). In all the extracts the zones of inhibition were inferior to those obtained with the reference drug (Amphotericin B). The levels of inhibition for methanol and hexane extracts of S. didymobotrya and A. caulirhiza, against C. albicans, C. krusei, C. orientaris, C. famata and C. auri were significantly different from those by the control drug

(amphotericin B) (p˂0.05) (Appendix v and vi). The hexane extract of A. caulirhiza and methanol extract of S. didymobotrya when tested against C. haemulonii and C. daubus haemuloni respectfully produced zones that were not significantly different from those by the control drug (p>0.05) (Appendix vi and vii). This indicates that the extracts are relatively less sensitive to the fungi when compared to amphotericin B. For the control drug (amphotericin B) the zone of inhibitions at concentrations ranging from 0.063µg/ml to 32µg/ml were found to be 14mm to 21mm for Candida krusei, 13mm to 23mm for Candida orientaris, 9mm to 21mm for Candida famata, 13mm to 22mm for Candida auris, 8mm to 18mm for Candida haemulonii, 7mm to 14mm for Candida daubus haemulonii and 15mm to 31mm for Candida albicans (Table 2).

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14484 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

extract - - - 10

±0.31 9

±0.55 14

±0.8 16

±0.25 15

±0.14 16

±0.22 23

±0.7 Senna methanol

extract

- - - - 6

±0.09 6

±0.61

12

±0.91

10

±0.12

15

±0.87

11

±0.65 Acmella methanol

extract - - - 8

±0.55 8

±0.77 8

±0.12 10

±0.91 7

±0.24 18

±0.44 8

±0.13 Senna hexane

extract - - - 6

±0.08 14

±0.13 Amphotericin B 14±0.13 14±0.04 16±0.06 17

±0.32 18

±0.19 20

±0.11 20

±0.02 21

±0.05 21

±0.62 21

±0.81 Candida orientaris Acmella hexane

extract - - - 10

±0.09 11

±0.11 11

±0.31 15

±0.68 17

extract - - -- - - 7

±0.005 10

±0.12 14

extract - - - -- - 8

±0.0 12

±0.81 13

±0.06 13

±0.7 Senna hexane

extract - - - 10

±0.21 10

±0.21 Amphotericin B 13±0.71 15±

0.51 15±0.33 17

±0.08 18

±0.10 20

±0.94 20

±0.23 23

±0.75 23

±0.13 23

±0.04 Candida famata Acmella hexane

extract - - - 10

±0.01 6

±0.07 8

±0.73 8

±0.98 10

extract - - - 10

±0.36 10

±0.51 10

±0.8 8

±0.41 10

extract - - - 8

±0.08 11

±0.01 6

±0.12 10

±0.69 11

±0.31 Senna hexane

extract - - - 6

±0.33 6

±0.52 6

±0.01 Amphotericin B 9±

0.69 9±

0.05 11±0.03 17±

0.47 18

±0.01 16

±0.07 17

±0.18 19

±0.43 19

±0.72 21

±0.33 Candida auris Acmella hexane

extract - - - 6

±0.91 8

±0.42 11

±0.21 11

±0.01 10

±0.6 16

±0.55 18

±0.72

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14485 Senna methanol

extract - - - 7

±0.1 10

±0.09 9

±0.02 9

extract - - - 6

±0.00 7

±0.03 Senna hexane

extract - - - 6

±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±

0.82 15±0.61 17±

0.09 17

±0.03 19

±0.14 20

±0.22 21

±0.31 23

±0.17 22

±0.01 Candida

haemulonii Acmella hexane

extract - - - - 11

±0.35 13

±0.21 13

±0.9 14

±0.73 17

±0.66 17

extract - - - 11

±0.08 11

±0.061 13

extract - - - 14

±0.06 14

±0.15 17

±0.99 15

±0.8 Senna hexane

extract -- - - 8

±0.42 12

±0.95 Amphotericin B 8±

0.19 7±

0.03 8±

0.55 8

±0.71 10

±0.09 10

±0.42 11

±0.85 14

±0.16 15

±0.09 18

±0.03 Candida

daubushaemulonii Acmella hexane

extract - - - 11

±0.41 11

extract - - - 10

±0.32 12

±0.28 12

±0.19 12

extract - - - - 7

±0.01 10

±0.12 14

±0.19 14

±0.77 16

±0.62 16

±0.16 Senna hexane

extract - - - 6

±0.42

Amphotericin B 7± 9± 9± 11 10 12 12 13 14 14

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14486 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

hexane extract - - - 14±

0.05 22±

0.75 22±

0.82 24±

0.69 23±

0.19 Clotrimazole/Senna

methanol extract

12±0.3 - - - -

Clotrimazole/Acmella

methanol extract 18±0.51 8±0.32 - - - -

Clotrimazole/Senna hexane

extract 8±0.1 - - - -

Candida orienntaris Ketoconazole/Acmella

hexane extract - - - 14±

0.04 20±

0.66 22±

0.15 26±

0.78 28±

0.96 Clotrimazole/Senna

methanol extract 10

±0.25 14±0.24 - - - -

Clotrimazole/Acmella

methanol extract 18

±0.15 12±0.75 - - - -

Clotrimazole/Senna hexane

extract 10

±0.88 12±0.03 - - - -

0.52 0.12 0.51 ±0.01 ±0.42 ±0.05 ±0.52 ±0.75 ±0.55 ±0.19

C. albicans Acmella methanol

extract 6±0.3 11

±0.57 11 ±0.8 8 ±0.00 16 ±0.52 17 ±0.47 21 ±0.99 22

±0.03 23

±0.48 26

extract - - - 7

±0.15 8

±0.51 8

±0.12 10

±0.95 8

±0.23 14

±0.46 11

±0.12 Acmella hexane

extract - - - 6

±0.08 6

±0.13 10

±0.37 11

±0.25 12

±0.17 Senna hexane

extract - - - - -- - - 6

±0.69 8

±0.42 10

±0.82 Amphotericin B 15±0.01 16±

0.15 19±0.05 22

±0.45 23

±0.75 26

±0.98 27

±0.85 27

±0.99 31

±0.80 31

±0.65

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14487 Candida famata Clotrimazole/Senna

methanol extract 10

±0.25 12±0.51 8

±0.0 8±

0.9 14±

0.02 - - - - -

AmB /Acmella hexane

extract - - - 10±

0.16 10±

0.23 10±

0.17 8±

0.33 - Clotrimazole/Acmella

methanol extract - - - 28±

0.93 22±

0.87 16±

0.27 10±

0.13 - Clotrimazole/Senna hexane

extract - 14±0.086 - - - 14±

0.43 12±

0.25 12±

0.125 16±

0.53 16±

0.11 Continuation of Table 3

Candida auris Ketoconazole/Acmella

hexane extract 12

±0.64 12±0.07 12

±0.01 - 22

±0.61 - - 28±

0.86 26±

0.72 - Clotrimazole/Senna hexane

extract 12

±0.78 10±0.005 8

±0.29 8±

0.32 - - - -

Candida haemulonii Ketoconazole/Acmella

hexane extract - - - - 16±

0.08 22±

0.77 25±

0.69 20±

0.88 24±

0.38 28±

0.57 Candida

daubushaemulonii Ketoconazole/Acmella

hexane extract 14

±0.01 14±0.681 - 12±

0.09 15±

0.14 11±

0.03 16±

0.011 18±

0.05 19±

0.41 20±

0.81 Clotrimazole/Senna hexane

extract 10

±0.91 10±0.12 8

±0.67 8±

0.02 8±

0.031 - - - - -

Values are expressed as mean ±standard deviation of the replica.

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14488 Inhibition of Candida species by the plant extract/conventional drug combinations: The diameters of inhibition produced by combined extracts and antifungal drugs were relatively higher compared to the extract alone implying potentiation. Wilcoxon Signed Ranks Test indicated a significant difference when the combined form was compared to extract alone (P < 0.05) (Appendix V). The results indicated a shift from inhibition at higher extract concentrations to lower extract concentrations for most of the combination (Table 2 and 3).

Minimum Inhibitory Concentrations (MIC50) for various antifungal drugs in µg/ml: Griseofulvin, Clotrimazole and Ketoconazole produced high MIC values in comparison to the control drug. For nystatin however the results were erratic with growth at low concentration and no zone of inhibition at higher concentrations meaning that all the pathogens died.

Senna didymobotrya methanol extract had MIC’s as 1µg/ml, 0.125µg/ml, 1µg/ml, 1µg/ml, 0.25µg/ml, 0.5µg/ml and 0.25µg/ml respectively for Candida

albicans, C. krusei, C. orienntaris C. famata, C. auris, C. haemulonii and C. duabus haemulonii (Table 4). The hexane extra had MIC’s as 4µg/ml, 2µg/ml, 0.5µg/ml, 2µg/ml, 2µg/ml, 1µg/ml and 0.5µg/ml for Candida albicans, C. krusei, C. orientaris, C. famata, C. auris, C.

haemulonii and C. duabus haemulonii respectively (Table 4). With Acmella caulirhiza hexane extract the MIC’s were found to be; 8µg/ml, 4µg/ml, 4µg/ml, 2µg/ml, 1µg/ml, 4µg/ml and 2µg/ml respectively for Candida albicans, C. krusei, C. orientaris, C. famata, C.

auris, C. haemulonii and C. duabus haemulonii. The methanol extract had the MICs as 2µg/ml, 1µg/ml, 1µg/ml, 2µg/ml, 4µg/ml, 4µg/ml and 4µg/ml for Candida albicans, C. krusei, C. orientaris, C. famata, C.

auris, C. haemulonii and C. duabus haemulonii respectivel as shown in Table 4. The minimum inhibitory concentrations for amphotericin B was found to be; 0.5µg/ml, 4µg/ml, 4µg/ml, 0.25µg/ml, 4µg/ml, 0.25µg/ml and 1µg/ml for Candida albicans, C. krusei, C. orientaris, C. famata, C. auris, C. haemulonii and C.

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±

0.01 0.5±

0.06 32±

0.75 _ 32±

0.11 1±

0.13 4±

0.66 8±

0.02 2± 0.32

C.krusei 4±

0.31

2±

0.32

32±

0.01

_ 32±

0.40

0.125±

0.38

2±

0.83 4±

0.20

1± 0.07

C.orientaris 4±

0.01 4± 0.01 32±

0.07 _ 32±

0.99 1±

0.00 0.5±

0.01 4±

0.71 1± 0.15

C.famata 0.25±

0.00 0.125±

0.06 32±

0.61 _ 32±

0.01 1±

0.01 2±

0.30 2±

1.02 2± 0.50

C.auris 4±

0.013 8± 0.94 32±

0.06 _ 16±

0.04 0.25±

0.02 2±

0.01 1±

0.03 4± 0.22 C.haemulonii 0.25±

0.03 8± 0.17 32±

0.97 _ 16±

0.01 0.5±

0.11 1±

0.36 4±

0.81 4± 0.62 C. daubushaemuonii 1±

0.07 8± 0.47 32±

0.82 _ 16±

0.25 0.25±

0.02 0.5±

0.04 2±

0.01 4±

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.

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14489 Minimum inhibitory concentration (MIC50) for combined drugs and plant extract in µg/ml: From the plant extract/conventional drug concentration gradients most of the combination MIC values were observed at lower conventional drug concentration and higher extract concentration. Two combinations

however, Amphotericin B /A. caulirhiza methanol extract and Ketoconazole/ S. didymobotrya hexane deviated from this observation where the extract concentration was lower than the conventional drug concentration (Table 5).

Table 5: Mean MIC50 for combined drugs and plant extracts in µg/ml C. albicans C.krusei C.

orientaris C.famata C. auris C.haemalonii C.

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:

Amphotericin B / Acmella caulirhiza hexane extract combination was synergistic when used against C.

krusei and C. orientaris; while with the other candida species it was antagonistic. Clotrimazole / A. caulirhiza

hexane extract combination was synergistic against C.

albicans, C. krusei and C. orientaris but antagonistic against the other candida species. The other combinations were indifferent and antagonistic against the candida species used (Table 6)

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14490 Table 6: Fractional Inhibitory Concentration Index

Candida albicans

Candida krusei

Candida orientaris

Candida famata

Candida auris

Candida haemulonii

C. daubus 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 management of infections caused by fungi than in the past though not as much as that given to bacterial and viral infections. Conventional therapeutic drugs have several limitations associated with them. From folklore certain plant species have been used in the treatment and management of fungal infections with success (Bristol et al., 2012). This study was aimed at determining the antifungal effects of combined crude extract of Senna didymobotrya and Acmella caulirhiza hexane and methanol extracts with conventional drugs:

griseofulvin, ketoconazole, nystatin and clotrimazole.

The extract of S. didymobotrya was found to contain phenols, tannins, cardiac glycosides, terpenoids, flavonoids and alkaloids as shown in Table 1.Unlike in this study, Bonareri et al. (2015) found no alkaloids in both hexane and methanol extracts of this plant while Jeruto et al. (2017) demonstrated presence of alkaloids from specimens collected from Nakuru County and none from those collected from Siaya and Nandi counties. In this study also steroids and phlobatannins were absent from the plant extract as was also found by Bonareri et al. (2015) while Jeruto et al. (2017) had shown that samples collected from three different

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14491 counties (Nakuru, Nandi and Siaya) all had steroids present in the leaf extracts. Further, phenols, flavonoids and terpenoids were found to be present in this plant extract just as was shown by Jeruto et al. (2017). In contrast, Bonareri et al. (2015) had found that the methanol extract of this plant lacked flavonoids. These varied observations are a clear indication that presence or absence of secondary metabolites is in part dependent on geographical location where the plants are growing, on genotype variation, season of collection, method of extraction and dilution (Jeruto et al., 2017). This information is very important for herbalist from different locations when using plants that are known to cure some conditions in different locations. Evaluation of efficacy of plant extracts therefore would best be done in the geographical location where obtained. Specific growth conditions that produce medicinal plants with high efficacy should be investigated in order to customize the plants and work on them at any place. In this study the extract of A.

caulirhiza lacked phenols, phlobatannins and alkaloids but contained terpenoids, flavonoids, tannins, anthraquinones and cardiac glycosides. Other phytochemicals found in this plant extract by other scholars are coumarin and sterols from aqueous extract by Jespher et al. (2017), alkaloids, carbohydrates, steroids, saponins, tannins and phenols by Thakur et al.

(2015). As observed for S. didymobotrya these varied reports are all attributed to different geographical locations of the plants. The inhibitory effect observed on the Candida pathogens can be attributed to the phytochemical compounds such as; terpenoids, glycosides and flavonoids that have been shown to contain antifungal activities (Tasleem et al., 2009;

Doughari et al., 2011; Altemimi et al., 2017).

Phytochemicals have varied mechanisms of action against fungal strains. These include and not limited to;

the formation of ion channels in the microbial membrane as shown by peptides that contain disulphide bonds and are positively charged. Peptides also competitively inhibit microbial proteins adhesion to host polysaccharide receptors and this gives them the inhibitory nature to microorganisms (Tasleem et al., 2009). Terpenes are lipophilic in nature thus act by membrane disruption which then increases permeability thus loss of cellular components and impairment of the enzymes involve in cellular metabolism (Tasleem et al., 2009). Phenolic compounds are able to inhibit fungal enzyme by the oxidized compounds through reaction with sulfhydryl groups or nonspeciﬁc protein interactions (Tasleem et al., 2009). The extracts from A.

caulirhiza showed zones of inhibition from concentrations of 0.5µg/ml and 0.063µg/ml and above for hexane and methanol respectively. The zone of inhibition increased with increase in concentration.

Likewise the hexane extract of S. didymobotrya exhibited a dose related fungal inhibition. Methanol extract of A. caulirhiza exhibited inhibition at lower extract concentration than hexane probably as an indication that the active compounds were more in methanol extract than in the hexane extract. Since methanol has a higher polarity than hexane, it is most likely that the inhibitory effect was from polar compounds. The findings of this study are in agreement with a study by Thakur et al. (2015) who by using agar well diffusion found inhibition of Candida albicans by Acmella caulirhiza methanol extract in a dose related manner. The extract concentrations used by Thakur et al. (2015) were much higher than those used in this study although the results were comparable. The variation is likely to arise from the difference in concentrations of the secondary metabolites in these two different studies. In deed the concentration of secondary metabolites are dependent on geographical location (Jeruto et al., 2017). Mining et al. (2014) observed similar results as in this study while using hexane extract of roots and pods of Senna didymobotrya against Candida albicans meaning that that the secondary metabolites obtained from the roots, leaves and pods have relatively similar activity against Candida albicans. The zone of inhibition for Candida albicans in this study was higher for the non albicans Candida, an indication that they require high dose of the extract for treatment. Generally the extracts showed moderate antifungal sensitivity. It was found that the extracts strength of inhibition was relatively inferior in comparison to the positive control drug (amphotericin B) with an exception of Acmella caulirhiza methanol extract against C. duabus haemulonii that showed superiority to the control drug. This study used crude extract and thus the exact concentration of the active component is unknown; likewise the active component is unknown and maybe it could be superior. The presence of a higher concentration of flavonoids, terpenoids and steroids in A. caulirhiza might be responsible for this superiority in contrast to extracts of S. didymobotrya that had lower concentrations of these metabolites. The MIC values for methanol extract of S.

didymobotrya against the various Candida species;

were relatively lower compared to those of hexane extract. This is due to the deference in the components of the crude extract that is largely affected by the type

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14492 of solvent. It is likely that the polar components of the extract are better antifungal agents than the non-polar components. Mining et al (2014) found that dichloromethane extract of Senna didymobotrya showed moderate antifungal activity against C.

albicans. In contrast to this study, Nelofar et al. (2016) found the main component extracted from flower heads of Acmella caulirhiza to be Spilanthol; this compound showed MICs of hexane extract that were lower than those of methanol extract against Candida albicans.

Shefali et al. (2011) showed that the methanol extract of Acmella caulirhiza had activity against Penicillum chrysogenum, Rhizopus arrhigus and Rhizopus stolonifer which were not considered in this study thus indicating its antifungal ability against a variety of fungi.

Savitha et al. (2015) found Acmella caulirhiza extract to have high inhibition of Candida albicans in comparison to calcium hydroxide. In a study done by Rex John et al. (2001), they found the MIC of amphotericin B against Candida spp. to be less than what was observed in this study and less than what is given by CLSI as the breakpoint for susceptible Candida spp.

Working on clinical isolates, Shallu et al. (2015) found the MIC values for amphotericin B for Candida auris, Candida haemulonii and Candida daubus haemulonii to be higher than what was observed in this study. This is likely an indication that clinical isolates have a higher chance of being resistant. In comparison to the control drug (amphotericin B) there was no significant difference in the treatments (P> 0.05). Friedman’s test however, indicated that the seven groups (Candida spp.) were significantly different from each other (p=0.007). The extracts largely were antagonistic and indifferent. Both methanol and hexane extracts of Senna didymobotrya were largely antagonistic and indifferent when combined with ketoconazole, clotrimazole, griseofulvin and amphotericin B.

Phytochemicals exert their effect by cell membrane disruption, binding and inhibiting specific proteins or adhere to and intercalate into DNA or RNA (Efferth et

al., 2011). Through such complex interaction, the extract might have potentiated the efflux of the conventional drug; they might have prevented the conventional drugs from combining with specific target molecules or prevented their conversion to active metabolites consequently contributing to the antagonistic effect observed. While working on essential oils from P. graveolens, Seungwons. et al.

(2003) found them to have synergistic effects when combined with ketoconazole and amphotericin B against Aspergilus spp. Ulrich-Merzenich et al. (2010) showed that an extract of cannabidiol when combined with tetrahydrocannabinol, the transport of the later across the membrane is increased leading to synergism. The resorption of 1-Hyoscyamin is increased when combined with flavonol-triglycosides (Ulrich-Merzenich et al., 2010). The synergism observed is attributed to several factors like; enhancing pharmacokinetic effects like absorption and transport across membrane, elimination of neutralization of toxically acting constituents, multitarget effect which is attributed to most plant extracts among other factors (Ulrich-Merzenich et al., 2010). The dynamics of infections has changed greatly to an increase in people with multiple infections; thus this inherent phenomena of plant extracts having multitarget effect should be exploited to the advantage of treatment of these diseases. Clotrimazole combined with Acmella caulirhiza methanol and hexane extracts largely produced synergistic effect. The presence of terpenoids and tannins which function by membrane disruption (Fernandes et al., 2010; Shamar et al., 2010) might have potentiated the effect of clotrimazole since it functions by affecting membrane integrity thus efflux of potassium. Acmella caulirhiza extract also contained anthraquinones; quinones are known to complex with cell wall and also inactivate membrane bound enzymes (Fernandes et al., 2010; Shamar et al., 2010). This is likely to have contributed to the potentiating of clotrimazole by this extract.

CONCLUSIONS

From this study the following conclusions are made;

i) Hexane and methanol extracts of Senna didymobotrya and Acmella caulirhiza are potent antifungal agents since the MIC’s ranged from 0.25 µg/ml to 8 µg/ml depending on organism.

ii) Senna didymobotrya extract when combined with ketoconazole, clotrimazole, griseofulvin and amphotericin B were largely indifferent and antagonistic while Acmella caulirhiza extract when combined with clotrimazole was synergistic.

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14493 RECOMMENDATIONS

This study recommends that;

i) The continued use of Senna didymobotrya and Acmella caulirhiza can combat mycoses since they are potent antifungal agents.

ii) Acmella caulirhiza can be used with clotrimazole since they are synergistic while the other combinations should be avoided due to antagonism.

Recommendations for future studies

i) The pure active antifungal components of these plants are determined since the crude extracts showed antifungal potency.

ii) Acmella caulirhiza showed high potential of synergism thus its pure active components to be used to develop new antifungal regimens to combat the challenges faced in treatment of mycoses.

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ACKNOWLEDGEMENT

I acknowledge and thank the Kenya National Research Fund for funding this study. I acknowledge the support, advice and guidance given to me by my supervisors Prof. Ngeranwa Joseph and Dr. Orinda George. I acknowledge Edward Karis of the department of Pharmacy School of Medicine Kenyatta University for

his technical advice and support. I also acknowledge the technical advice and support given to me by Mugo Peter of Medical laboratory Sciences Kenyatta University and any other person who helped me materially or morally during my study.

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14497 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.

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14498

Appendix iii: Percentage inhibition of diameter growth of the extracts against Candida spp.

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