Research Article

DOI: http://dx.doi.org/10.26510/2394-0859.pbe.2017.14

Antifungal potential of improved crude extracts of Mitracarpus scaber (Zucc) against Candida guilliermondii and Candida parapsilosis

E. K. Kporou1,2*, C. Ibourahema1, A. J. Ackah1,2, S. Ouattara2

1Biochemistry and Microbiology Laboratory, Bioactives natural Substances Unit, UFR Agroforestery Faculty, Jean Lorougnon Guede University, BP 150, Daloa, Côte D'Ivoire

2Pharmacodynamics biochemical Laboratory, UFR Biosciences, Felix Houphouet Boigny University, BP 582 Abidjan, Côte D'Ivoire

*For correspondence

Dr. E. K. Kporou,

Biochemistry and Microbiology Laboratory, Bioactives Naturals Substances (BIONAS) Jean Lorougnon Guédé University (UJLoG), BP 150, Daloa, Côte D'Ivoire.

Email: elykoua@yahoo.fr

 

 

 

 

 

 

 

Received: 02 March 2017 Accepted: 16 March 2017

ABSTRACT

Objective: The present study was undertaken to evaluate antifungal potential of improved crude extracts of Mitracarpus scaber Zucc., an ivorian traditional medicinal plant against two species of clinical yeast isolates: Candida parapsilosis and Candida guilliermondii.

Methods: For evaluation of antifungal activity, double dilution method in slope tubes was used. Crude extracts of M. scaber were prepared by extracting with a blender in aqueous and hydroalcohol. In first; Aqueous (XAq) and Hydroalcoholic (X0) extracts were evaluated, and secondary the crude extract X0 was improved by partition in mixture of aqueous and hexanic solvent 50:50 v/v. Each dried phase was also examined. A phytochemical screening has been performed on most active extracts.

Results: In vitro antifungal activity showed that crude extract X0 was the most active with MIC = 6.25 mg/ml. Among the extracts obtained by partition from extract X0, extract X11 (Hexanic phase) was the better with lowest antifungal parameters MIC and MFC range between 1.562 mg/ml and 0.781 mg/ml and IC50 range between 0.73 mg/ml to 0.598 mg/ml. In addition, the most active extract contained only terpens and steroids.

Conclusions: Antifungal activity of M. scaber was improved by partition of an hydroalcoholic extract (X0) in mixture hexane-water 50:50 v/v. Hexanic phase (X11) was more active against C. parapsilosis and C. guilliermondii.

 

Keywords: Mitracarpus scaber, Antifungal, Performed extract

Introduction

Fungal infections are emerging opportunistic infections that often occur in immune-compromised people and especially those living with HIV / AIDS. In these people, attacks commonly called mycosis are both superficial (skin) and invasive (systemic). Fungi implicated in these infections are moulds and yeasts.1 Among yeasts, Candida species are the most frequently isolated samples, and the most often found species are Candida albicans, Candida tropicalis, Candida glabrata, C. guilliermondii and C. parapsilosis.2,3 Candidiasis attacks are recurrent and therapeutic management is complicated by the emergence of multi-drug resistant (MDR) fungi. This situation calls for research for new antifungal compounds with broad spectrum of activity in addition to the already existing molecules. One solution is to explore traditional medicine to identify plants with interesting therapeutic properties to extract the active ingredients. To this end, in Africa and more specifically in Côte d'Ivoire many plants are used in medicinal preparations for the treatment of skin disorders such as greenish, ringworm and athletes foot.4,5 Among these plants, Mitracarpus scaber figures prominently. This plant has been the subject of several studies including the study of antimicrobial; antifungal, antibacterial activity, anti-inflammatory, antioxidant activities and bio-tolerance.6-12 In addition, work on this plant led to isolation of secondary metabolites, some of them of therapeutic interest and even extracts of this plant have been formulated in ointments, and syrups.6,13-15 Antifungal activity has been evaluated on C. albicans, C. glabrata and C. tropicalis in 2009 and 2010.16-19 This study completes data on anticandidosic spectrum of organic extracts of this plant in comparison to ketoconazole, a standard antifungal. To achieve this general objective, the following specific objectives are defined: to prepare vegetable extracts and to assess antifungal activity of these extracts.

Materials and Methods

Plant material

Mitracarpus scaber plant was collected from District of Abidjan, Cote d'Ivoire in June 2009. Samples of this crop were sent to National Centre Floristic Felix Houphouet Boigny University to be identified specimen Chevalier 16005.

Candida strains used for the in vitro assay

For antifungal assay, clinical strains of Candida guilliermondii (ID My 0375 458 / D) and Candida parapsilosis (ID My 3826 63 / D) isolated from patients with superficial mycosis and resistant to ketoconazole were used. There were provided by Mycology unit of Pasteur Institute of Abidjan, Côte d'Ivoire.

Culture media

Sabouraud Agar (Biomerieux/ Réf: 51078: 777666501) was used for culture. The medium was prepared according to the instructions of the manufacturer's protocol.

Plant extraction

Plant was collected, washed, dried with sun's shelter at a temperature between 25°C and 27°C and was returned out of powder fine with a grinder IKA-MAG type. Aqueous extract (XAq) and hydroalcoholic extract (X0) were obtained by extracting separately hundred (100 g) grams of plant's powder in distilled water and in alcohol 70%. Extract XAq was filtered and freeze-dried. Extract X0 was concentrated with a rotary evaporator of BÜCHI at 60°C.18 To perform hydroalcoholic extract (X0), this extract was used to prepare 6 other extracts:

Three portions of 10 g extract X0 were prepared and were submitted separately to a solid /liquid extraction in 300 ml of 3 different mixtures of solvents (Hexane-Water; ethyl acetate-water and butanol-water; v/v 50:50). After decantation, the various phases were collected singly and were concentrated by evaporation for organic extracts and freeze-dried for aqueous extract. The following extracts were obtained:

hexanic phase (X11) and aqueous phase (X12) coming from partition Hexane-Water,

acetatic phase (X21) and aqueous phase (X22) coming from partition Ethyl Acetate-Water,

butanol phase (X31) and aqueous phase (X32) of the partition Butanol-Water.

All extracts obtained from X0 were tested separately on the in vitro growth of C. parapsilosis and C. guilliermondii.

Antifungal activity assay

Antifungal tests were performed on Sabouraud culture medium. Incorporating of the plant extract in agar was made by the method of double dilution in slope tubes. Our series included 11 test tubes with 9 tests tubes (containing the plant extract or ketoconazole), and 2 control tubes (one without a plant extract or ketoconazole and the second without germs and extract). Our series of tests tubes contained binary dilutions of extract from 100 to 0.390 mg/ml. All 11 tubes were sterilized by autoclaving at 121°C, 1 bar for 15 minutes and then tipped with small pellet temperature of the room to allow cooling and solidification agar. After solidification, the culture media were inoculated with the Candida strains and allowed to incubate for 48 hours. After incubation time, colonies were directly counted with a counter colonies per extract type and the growth in 9 experimental tubes was evaluated as a percentage of survival, calculated on 100% survival in the growth control tube (1).19 The processing of these data allowed to determine the values ​​of MIC (minimal inhibitory concentration), MFC (minimal fungicidal concentration) and IC50 (concentration for 50% inhibition). MFC (minimal fungicidal concentration) was determined by seeding the culture medium of experimental tube indicating MIC on new agar medium.

Method of calculating the survival S

S = Survival of fungi (percentage)

N = Number of colonies in the control tube

n = number of colonies in the test tube.

There were performed three replicates for each extract concentration against fungi

Statistical analysis

Statistical analysis was carried out using Microsoft excel version 2010. An ANOVA (Analysis of Variance) was conducted to find the significant differences of the extracts at P<0.05.

Phytochemical screening

Phytochemical analysis of aqueous (XAq) hydroalcoholic (X0), hexanic phase (X11) of M. scaber were performed using different chemical tests to check the presence of active metabolites such as sterols/terpens, alkaloids, flavonoids, saponins, anthracens derivatives, glycosids, reducing compounds, anthocyanins and tannins in the plant by standard methods.20-22

Test for sterols/terpens

Liebermann-Buchard test: Anhydrous acetic acid (1 ml) was added to 1 ml of chloroform and cooled to 0°C then a drop of concentrated sulphuric acid was added to the cooled mixture followed by the plant extract. The resultant mixture was observed for blue, green, red or orange color that changes with time.

Salkowski test: A little quantity of the plant extract was dissolved in 1 ml of chloroform, thereafter; 1 ml of concentrated sulfuric acid was added down the test tube to form two phases. Formation of red or yellow coloration was taken as an indication for the presence of sterols.

Test for flavonoids

Shinoda test: To each extract solution of the plant extract, 3 pieces of magnesium chips were added followed by a few drops of concentrated hydrochloric acid. Appearance of an orange, pink or red to purple color indicates the presence of flavonoids.

Sulphuric acid test: Plant extract (0.5 g) was dissolved in concentrated sulfuric acid and notable color change was observed (if any).

Ferric chloride test: Plant extract (0.5 g) was boiled in distilled water and filtered to 2 ml of the filtrate, two drops of freshly prepared ferric chloride solution was added; green, blue or violet coloration indicated the presence of phenolic hydroxyl group.

Sodium hydroxide test: each extract (0.5 g) was dissolve in 2 ml of 10% aqueous sodium hydroxide solution and filtered to give yellow color, a change in color from yellow to colorless on addition of dilute hydrochloric acid indicated the presence of flavonoids.

Test for alkaloids

Extract (0.5 g) was stirred with 5 ml of 1% aqueous hydrochloric acid on a water bath and filtered. 3 ml of the filtrate was divided into three. To the first 1 ml of freshly prepared Dragendoff's reagent was added and observed for formation of orange to brownish precipitate. To the second, 1 ml of Mayer's reagent was added and observed for formation of white to yellowish or cream colored precipitate. To the third 1 ml of Wagner's reagent was added to give a brown or reddish or reddish brown precipitate.

Test for tannins

Ferric chloride test: A small quantity of the extract was boiled in water and filtered. Two drops of ferric chloride was added to the filtrate, the formation of a blue-black, or green precipitate was considered for the presence of tannins.

Test for saponins

Frothing test: The plant extract (0.5 g) was shaken with water in a test tube. Frothing which persisted for 15 min indicates the presence of saponins.

Tests for anthracens derivatives

Microscopic observation: Microscopic observation of M. scaber powders supplemented with 10% NaOH. The presence of red coloration indicates presence of anthracens derivatives.

Test for coumarins

Ammonia solution test: 5 ml of extract of three extracts are evaporated to dryness. The residue thus obtained is taken up in hot water. A volume of this aqueous phase is added with an ammonia solution (NH4OH) 10% and another volume is kept as control. The appearance of fluorescence in observation under UV indicates the presence of coumarins.

Test for reducing compounds

Fehling test for reducing compounds: Their detection involves treating 1 ml of the extract with 2 ml of distilled water and 2 ml of Fehling's solution (1 ml of Fehling's solution A + 1 ml of Fehling's solution B) and then the tubes are heated in a water bath at 40°C. Positive a test is revealed by the formation of a brick-red precipitate.

Test for anthocyanins

Acid sulphuric test: A 1 ml portion of the infused; 5 ml of sulfuric acid (H2SO4) at 10% is added, then ammonium hydroxide (NH4OH) to 25 %. If the color is not acidification increases, then turns blue violet in basic medium, it can be concluded the presence of anthocyanins.

Results and Discussion

After 48 hours of incubation, the different extracts of Mitracarpus scaber inhibited in vitro growth of Candida guilliermondii and Candida parapsilosis strains with MIC values ​​ranging between 50 mg/ml and 0.781 mg/ml (Figure 1). In contrast, inhibition was observed on both the two strains at 0.781 mg/ml for the standard antifungal, ketoconazole. Also, it was noticed a progressive decrease in the number of colonies gradually as the concentrations of plant extract increased. Between the two crude extracts (XAq, X0), X0 extract inhibited the growth of each two strains at 6.25 mg / ml whereas XAq extract carried out this inhibition at 25 mg / ml on C. guilliermondii and at 50 mg / ml on C. parapsilosis. From extract X0, improved organics extracts showed MIC values ​​lower than extract X0, but aqueous equivalents extract coming from the same partition had MIC higher than extract X0 and equal to extract XAq on each Candida (Figure 1). In addition, For each sample considered and tested on a Candida, reseeding of the tube indicating MIC on new agar culture medium, showed no visible growth and all experimental tests showed MIC=MFC. IC50 values varied from 2.5 mg/ml to 9.6 mg/ml for XAq extract and aqueous phase coming from partition of X0 extract. This parameter was comprised between 0.593 mg/ml to 3.85 mg/ml for organic solvent (Table 1). IC50 was used to discriminate susceptibility between strains possessing the same FMC.

A phytochemical screening on XAq, X0 extracts of M. scaber showed presence of flavonoids, sterols/terpens, saponins, tannins, alkaloids, coumarins and reducing compounds; while X11 extract contained only sterols/terpens (Table 2).

Figure 1: Histogram of MIC values of Mitracarpus scaber extracts on the growth of Candida guilliermondii and Candida parapsilosis.

Table 1: IC50 parameters of Mitracarpus scaber whole plant extracts.

Extracts C. guilliermondii C. parapsilosis
IC50 (mg/ml) IC50 (mg/ml) 9.6 ± 0.10 1.15 ± 0.12 0.598 ± 0.05 2.687 ± 0.15 0.593 ± 0.05 2.50 ± 0.15 1.31 ± 0.12 2.625 ± 0.16 0.45± 0.05
XAq 6.75 ± 0.23
X0 2.82± 0.15
X11 0.73 ± 0.05
X12 4.75 ± 0.19
X21 2.23 ± 0.15
X22 4.85 ± 0.21
X31 3.85 ± 0,17
X32 5.10 ± 0,24
Ketoconazole 0.25± 0.05

 

Inhibition observed for each extract illustrated that M. scaber extracts have fungicidal activity as revealed by Ali and al, Ekpendu and al, Bisignano and al.9,11,13 Considering the decreasing of number of Candida in the experimental tubes while the concentrations increased, it was found that the extracts act in a dose-effect relationship. MIC values ​​(Figure 1) of crude extracts XAq and X0 indicated that X0 extract was respectively 8 and 4 times more active than XAq, but 4 and 8 times less active than X11 on C. parapsilosis and C. guilliermondii. The antifungal potency of X0 and X11 has been showed by previous works of Kporou and al in 2009 and 2010 on clinical isolates of C albicans, C. glabrata, C. tropicalis. Efficacy of extract X11 is similar to ketoconazole on C. guilliermondii, but if we consider the IC50 values, the standadrd antifungal is more active because its IC50 is lower. In addition, the clinical strain of C. guilliermondii is more sensitive to M. scaber extracts than C. parapsilosis strain because the antifungal parameters (MIC, FMC, IC50) on this strain were the lowest.

Table 2: Phytochemical constituents of crude (XAq and X0) and performed (X11) extracts of M. scaber.

Tests XAq X0 X11
Flavonoids      
Shinoda test

-

-

-

Ferric Chloride test

+

+

-

Sulphuric acid test

-

-

-

Sodium hydroxide test

-

-

-

Sterols/terpens      
Libermann-Buchard test

+

+

+

Salkowski's test

+

+

+

Saponins      
Frothing test

+

+

-

Tannins      
Ferric chloride tests

+

+

-

Alkaloids      
Mayer's reagent test

+

+

-

Dragendoff's reagent

+

+

-

Wagner's reagent

+

+

-

Coumarins      
Ammonia solution Test

+

+

-

Anthracens derivates      
Microscopic observation

-

-

-

Reducing compounds      
Fehling test

+

+

-

 

Comparing activities between aqueous crude extract and aqueous extracts (X12, X22 and X32) coming from partitions of extract X0, it appeared that IC50 values ​​of the latter were smaller, so their performance was better than extract XAq. Furthermore, it was noted that extracts from organic phases of X11, X21 and X31 sheets, have respectively better activities than those of their aqueous counterparts X12, X22 and X32. It follows from this that active ingredients of M. scaber are compounds soluble in organic solvents as demonstrated by others authors.6,13 A phytochemical screening showed that the most active extract contained only sterols/terpens and it's known that these secondary metabolits are antimicrobial, antibiotic and antifungal. Also, this result has been shown by Gbaguidi and al., 2006.14 This study revealed and confirmed that the performance of anticandidosic activity of M. scaber could be achieved by preparing an extract of alcoholic 70% base followed by a partition of this extract in a Hexane / Water mixture (V / V).

Conclusions

This study showed that Mitracarpus scaber extracts had good antifungal activity against C. guilliermondii and C. parapsilosis but the best activity was obtained with Hexanic extract (X11) obtained from the partition of hydroalcoholic extract 70% in a mixture of Hexane/Water V/V and after decanting hexanic phase was collected then dried. Also, M. scaber extracts exhibited their fungicidal activity in a dose-response relationship on the two tested strains, Candida guilliermondii was more sensitive to the tested extract.

Extract X11 containing Sterols/Terpens can be a good source of natural antifungal agent.

Acknowledgements

The authors are grateful to:

  • National Floristic Center for identifying plant species.
  • Pasteur Institute of Côte d'Ivoire for providing us different clinical isolates of Candida.

Funding: No funding sources

Conflict of interest: None declared

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