Original Article
In vitro Control of Oral Thrush Causal Organisms Using Medicinal Plants Extracts
Abosede M. EBABHI
1*, Adedotun A. ADEKUNLE
2, Yoma I. OGHENERHABOKE
21University of Lagos, Distance Learning Institute, Akoka, Lagos, Nigeria; [email protected] (*corresponding author)
2University of Lagos, Faculty of Science, Department of Botany, Akoka, Lagos, Nigeria; [email protected]; [email protected]
Abstract
Oral hygiene is important to the generality of the human healthcare system. For this, the antifungal activities of the aqueous and ethanol extracts of four medicinal plants of Jatropha curcas (stem), Eucaluptus golbulus (leaves), Vernonia amygdalina (stem) and Zanthoxylum zanthoxyloides (root) were carried out in vitro against three species of Candida associated with oral thrush namely C. albicans, C. glabrata, C. tropicalis using the disc diffusion agar assay. The zones of inhibition varied with the test organisms as well as the extracts. The ethanolic extract of Jatropha curcas showed the highest zone of inhibition of 10.88 ± 0.22 mm against C. albicans while the least zone of inhibition (6.13 ± 0.13 mm) was exhibited by the ethanol extract of Z. zanthoxyloides on C. glabrata. The preliminary phytochemical screening showed the presence of tannin, saponin, alkaloids, flavonoids and reducing sugar in all plant samples. This study can be further used as a foundation for the screening of phytochemical constituents by pharmaceuticals for the control and eradication of oral thrush.
Keywords: Candida spp.; medicinal plants; mouth swab; oral thrush; phytochemicals
AcademicPres
Available online: www.notulaebiologicae.ro
Notulae Scientia Biologicae Print ISSN 2067-3205; Electronic 2067-3264
Not Sci Biol, 2019, 11(3):376-382. DOI: 10.15835/nsb11310443
Introduction
Thrush is a fungal infection (mycosis) of any species from the genus Candida. This is commonly referred to as yeast infection and technically as candidiasis, moniliasis and oidiomycosis (William et al., 2006). Candidiasis encompasses infection that range from superficial, such as oral thrush and vaginitis, to systemic and potentially life- threatening disease. It is often confined to severely immune- compromised persons such as cancer, transplant and AIDS
patients, as well as nontrauma emergency surgery patients (Koukpupetis et al., 2010). Candida abicans is the most common agent of thrush in humans (Walsh and Dixon, 1996).
Medicinal plants have been part of human medicine since the dawn of civilization. These plants have made backbones of traditional medicinal systems in Nigeria (Akerele, 1988) and other parts of the world like India (Nayak et al., 2011). The use of plant extracts to treat microbial infections is also reported in ancient Ayurvedic compendium ‘Charack Samhita’ and ‘Sushrat Samhita’
(Kelmanson et al., 2000). The search for new therapeutic treatments for various disease conditions is expanding due to increased prevalence of drug resistant microorganisms (Fidel et al., 1999; Samuel et al., 2014). In many developing countries, plants have been looked at as a very promising source of new compounds for drug discovery and development (Kong et al., 2003). Plant extracts and their products are clinically safer than antibiotics (Srinivasan et al., 2001). The aim of this study is to control oral Candidiasis in vitro using medicinal plants which will serve as a reference point for further research on controlling oral infections.
Received: 13 Feb 2019. Received in revised form: 27 Aug 2019. Accepted: 13 Sep 2019. Published online: 30 Sep 2019.
and well spread on the agar. The plates were incubated at 27
°C based on the method described by Okigbo et al. (2009).
The plates were examined for possible growth of contaminants, the absence of which confirmed the purity of the test extracts.
Antifungal assay of the plant extracts
The antifungal assay was carried out using the disc diffusion agar method of Adekunle and Ikumapayi (2006) and Valle et al. (2016). The disc preparation involved the use of filter papers (Whatman No. 1 filter paper) perforated into discs, placed in aluminium foil, wrapped and sterilized in an autoclave. The discs were dried in an oven at 60 °C for 30 minutes. Each plant extract was reconstituted with 1 ml of distilled water respectively at concentrations of 100 mg/ml and 200 mg/ml, the discs were later soaked in the prepared extracts for 24 hrs prior to use. Spore suspension of 105-107 cells/ml, counted with haemocytometer was made (Lee et al., 1981). A micropipette was used to introduce 0.1 ml of the spore suspension on sterile plates of Saboraud dextrose agar (SDA) before spreading with a glass rod under sterile conditions to ensure even distribution of the spores.
These were then dried at room temperature for 15 mins prior to the application of the discs. The discs (4) which had been soaked in plant extract were placed at the four edges of the surface of the agar plate with the aid of a sterilized forceps. These were done in triplicate. The inoculated plates were later placed in an incubator at 28 °C and observed after 48 hours. The extent of inhibition was determined by measuring the diameter of the inhibition zone using a transparent metre rule (Booths et al., 1971). The mean zone of inhibition of the three replicated tests of the plants extracts on the test organisms were expressed in millimetres.
The discs were also impregnated in equivalent volume of distilled water as negative control and griseofulvin (anti- fungal drug) served as the positive control measure.
Statistical analysis
The data were expressed as mean ± S.E.M. and were statistically analysed using one-way analysis of variance (ANOVA). Means were separated by the Duncan new multiple range test. Values were considered significant at p˂0.05 (Kim, 2015).
Results
Isolation and identification of microorganisms
The results on the study of biological control of oral candidiasis using medicinal plants revealed that three fungi isolates were obtained in the course of this study. The isolated fungi were identified as Candida albicans, Candida glabrata and Candida tropicalis. All isolated organisms showed resistance to chloramphenicol which was indicated by the growth in the presence of the discs. The antibiotic is usually added to fungi growth media to inhibit the growth of Gram positive and Gram negative bacteria. In the biochemical test, the Candida spp were able to break the urea at 72 hour of incubation which was indicated by a colour change from pale pinkish-red to pale orange colour.
The set up experiment was observed for 4 days. The lack of Materials and Methods
Isolation and identification of microorganisms
Mouth swab was collected from postgraduate students at the hall of residence in a tertiary institution in Southwest, Nigeria by using sterile swab sticks to scrap the surface of the tongues before early morning wash of the mouth as described by Ochei and Kolhatkar (2000). The swab sticks were aseptically placed in separate seal up plastic bags for transport to the lab. A total of 42 students were routinely sampled for three weeks on a weekly basis. All samples were routinely grown on Sabouraud dextrose agar (SDA) (Oxoid, Basingstoke, England) for 72-96 hours at 30 °C.
Growth medium was prepared in accordance to manufacturer’s specification. Pure cultures of isolated organisms were certified as fungi using their cultural, morphological characteristics as well as comparing them with confirmed representatives of different species in relevant texts like Ellis et al. (2007). Further biochemical tests were performed by Gram staining according to the method described by Beveridge (2001). Sugar fermentation test was carried out using a modified method of Olutiola et al. (2000). Urea hydrolysis test and sensitivity to chloramphenicol were carried out using the methods of Seeliger (1956) and Kirby et al. (1996) respectively.
Photomicrographs of the isolates were taken with Motic Camera 2000.
Collection, identification and preparation of plant extracts Plant parts of Jatropha curcas (stem), Vernonia amygdalina (stem) and Eucalyptus globules (leaves) were obtained from the Botanical and Zoological garden of the University of Lagos. The roots of Zanthoxylum zanthoxyloides were purchased from Oyingbo herbal market, Mainland Local Government Area of Lagos state.
The plants were all identified at the Lagos University herbarium, University of Lagos. The plant parts were cut into bits of 2.0 cm and shade dried for about two weeks.
The plant materials were all ground into fine powder using commercial blender. Two hundred grams of each sample was soaked in conical flasks containing different solvent of aqueous and ethanol at 1:2 w/v and observed on a shaker for 72 hours. The extracts were filtered through a Whatman No 1 filter papers and Muslin cloth severally into separate beakers and concentrated to dryness with the aid of a rotary evaporator. The stocks were kept at 4 °C in a refrigerator until further use (Silva et al., 2014).
Qualitative preliminary phytochemical screening of the plant samples was carried out using standard procedures as described by Edeoga et al. (2005), Sofowora (2006), Trease and Evans (2009) and Ashafa and Umebese (2012). The phytochemicals analyzed include tannins, phenols, saponins, alkaloids, flavonoids, steroids, cardiac glycosides and reducing sugars. The extracts were later subjected to quantitative analysis based on the methods of Edeoga et al.
(2005) and Prohp and Onoagbe (2012).
Test for purity of the organic extracts
Each extract obtained was tested to ensure its purity via dropping on a sterile plate containing potato dextrose agar
Ebabhi AM et al / Not Sci Biol, 2019, 11(3):376-382 378
colour change is an indication of a negative result. The isolated organisms were confirmed as yeast via the fermentation analysis. Result is has depicted in Table 1.
Qualitative and quantitative analysis of plant extracts The voucher number of the authenticated plants are LUH 6992 (Jatropha curcas), LUH 7045 (Vernonia amygdalina). LUH 4689 (Eucalyptus globules) and LUH 6317 (Zanthoxylum zanthoxyloides). The qualitative analysis shows that tannin, saponin, alkaloid and reducing sugar are present in all plant extracts. Phenol was absent in Jatropha curcas. Steroid and cardiac glycoside were equally absent in Eucalyptus globules while in Vernonia amygdalina, steroid was the only phytochemical absent. Result is as presented in Table 2.
For the quantitative analysis alkaloids had the highest quantity of 33.64 mg/g while steroids had the least quantity of 13.21 mg/g in J. curcas. The ethanolic extract of Eucalyptus globulus showed that steroids and cardiac glycosides were absent. Like in Jatropha curcas, alkaloids had the highest quantity of 41.98 mg/g while saponin had the least quantity of 19.38 mg/g in E. globulus. The ethanolic extract of Vernonia amygdalina revealed that alkaloid recorded the highest quantity of 46.37 mg/g while cardiac glycoside had the least quantity of 15.32 mg/g.
Zanthoxylum zanthoxyloides ethanolic extract showed that alkaloid similarly recorded the highest quantity of 32.15 mg/g with steroids being the least of 11.61 mg/g as depicted in Table 3.
Table 1. Phenotypic characteristics of the isolates
Biochemical characteristics Fungal isolates
C. albicans C. glabrata C. tropicalis
Cell morphology Ovoid Globose Round
Gram reaction + + +
Sucrose - - V
Glucose + + +
Starch - - -
Lactose + - +
Galactose - + +
Maltose - + +
Fructose + + +
Dextrose + + +
Urease + + +
Resistance to chloramphenical + + +
+ = Present - = Absent V = Variable
Table 2. Qualitative analysis of phytochemicals present in plant extracts Phytochemical
Plan extract Tannin Phenol Saponin Alkaloid Flavonoid Steroid Cardiac glycoside
Reducing sugar
J. curcas (E) + - + + + + + +
E. globulus (E) + + + + + - - +
V. amygdalina (E) + + + + + - + +
Z. zanthoxyloides (E) + + + + + + + +
+: Present, -: Absent E: Ethanol
Table 3. Quantitative analysis of phytochemicals in plant extracts
Phytochemicals (mg/ g) Plant samples
J. carcas E. globulus V. amygdalina Z. zanthoxyloides
Tannin 22.05 29.06 23.33 30.76
Phenol 0.00 19.38 15.69 21.66
Saponin 31.50 29.25 33.81 32.15
Alkaloid 33.64 41.98 46.37 39.04
Flavonoid 32.24 22.50 18.36 19.12
Steroid 13.21 0.00 0.00 11.61
Cardiac glycoside 13.99 0.00 15.32 14.26
Reducing sugar 15.77 23.76 20.09 20.56
Antifungal assay
The test for purity of the extracts showed no microbial growth after incubation. The results on Figs. 1 and 2 show the zones of inhibition of the different plant extracts on the fungal isolates. The analysis of variance revealed that the rate of inhibition differed significantly (p < 0.05) among the extracts. The ethanol extracts of the plant samples showed higher level of inhibition on the test organisms as compared with the antifungal drug used (positive control). For the extracts at 200 mg/ml, the highest rate of inhibition was observed on C. albicans (10.88±0.22 mm) using the ethanol extract from Jatropha curcas while the least zone of inhibition was observed in C. glabrata at 6.13±0.13 mm from both extracts of Zanthoxylum zanthoxyloides.
However, both aqueous and ethanolic extracts of Jatropha curcas showed a significant (p< 0.05) level of inhibition (9.88±0.32 mm; 10.38±0.24 mm respectively) on the growth of C. tropicalis while they had no significant inhibitory effects on the growth of C. glabrata. The results showed that the ethanolic extract of Eucalyptus globulus had a significant (p< 0.05) rate of inhibition (10.13±0.32 mm) than the aqueous extract (9.00±0.24 mm) on the growth of C. albicans. Both aqueous and ethanolic extracts produced significant level of inhibition (9.62±0.43 mm) on the growth of C. tropicalis while the inhibitory effect (6.25±0.13 mm) on the growth of C. glabrata was
insignificant. However, at 100 mg/ml the ethanol extract of E. globulus showed significant level (9.50±0.20 mm) of inhibition on C. tropicalis while no significant difference was observed on the other extracts though they all showed fungistatic activities on all test isolates.
Discussion
Oral candidiasis (mouth thrush) is a yeast infection which develops inside the mouth and on the tongue of human. This condition is commonly found in children, the aged and the immunosuppressed (Umeh and Okolocha, 2005; Silva et al., 2012). Oral candidiasis is a Candida disorder that can be treated medically and traditionally using herbal remedies. This study examined the effects of both aqueous and ethanolic extracts of four medicinal plants on oral candidiasis causal organisms isolated from postgraduate students of a tertiary institution. A total number of 42 students were sampled. The identified isolates were Candida albicans, Candida tropicalis and Candida glabrata. C. albicans is a well-known causal agent of candidiasis in human (Calderone, 2002) while C. glabrata has been implicated as a prevalent pathogenic yeast by Rodrigues and Henriques (2017) in their study on oral mucositis.
Fig. 1. The zone of inhibition induced by the medicinal plant extracts at 200 mg/ml on isolated Candida spp from oral thrush patients (AE=Aqueous extract, EE=Ethanol extract)
Fig. 2. The zone of inhibition induced by the medicinal plant extracts at 100 mg/ml on isolated Candida spp from oral thrush patients (AE=Aqueous extract, EE=Ethanol extract)
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According to Fidel et al. (1999), C. glabrata is ranked the second or third causative agent of superficial (oral, esophageal, vaginal, or urinary) or systemic candida infections, which are often nosocomial. Its infections have been noted to have high mortality rate in compromised, at- risk hospitalized patients. The isolation of Candida spp in our studies also confirms with the work of Redding et al.
(2002) who reported that a frequent combination of mixed species infection by Candida species is C. glabrata and C.
albicans, which has been found in approximately 70% of patients with oral candidosis. Flevari et al. (2013) also stated that Candida species remain the most important cause of opportunistic infections worldwide, affecting predominantly patients over 65 years old, while they are considered to be the fourth most common cause of nosocomial bloodstream infections.
The issue of pathogenic organisms mutating against various synthetic compounds and known therapeutic drugs has led scientists to constantly be on the search for new and effective compounds which can eradicate the menace of diseases in the human race. The phytochemicals found in most plants have been shown to be most effective in the treatment of diseases and they provide an important source of all the world’s pharmaceuticals. The results from the preliminary phytochemical screening in our present study showed that the sampled plants of Jatropha curcas, Eucalyptus globulus, Vernonia amygdalina and Zanthoxylum zanthoxyloides possess most of these bioactive compounds like tannin, cardiac glycosides, steroids, terpenoids, flavanoids, and alkaloids which were found in reasonable amount in the ethanol extracts of the plants’ parts. Alkaloid, tannin and saponin were present in all samples. The presence of these compounds have been confirmed in J.
curcas by Sharma et al. (2012); in V. amygdalina by Alara et al. (2018); in E. globulus by Takahashi et al. (2004) and Egwaikhide et al. (2009) while the phytochemical screening of Z. zanthoxyloides by Kosh-Komba et al. (2017) also revealed the presences of these bioactive compounds. The antifungal activities of all sampled plants can be linked to the presence of tannin. Tannin has been reported to inhibit the cell protein synthesis of microorganisms as it forms irreversible complexes with proline rich protein (Shimada, 2006).
The presence of phytochemical compounds in the plants extracts indicates that some of the phytochemical compounds might be responsible for the potential antifungal activities. The results of this study showed that the aqueous and ethanolic extracts of Vernonia amygdalina had insignificant inhibition on the growth of C. glabrata but had significant inhibition on the growth of Candida albicans at 200 mg/ml. In the same vain, Akinpelu (1999) had also stated that the methanolic extract at 60% had no effect on C. albicans. The aqueous and ethanolic extracts of Z. zanthoxyloides exhibited a significant inhibition on the growth of C. tropicalis while the inhibition on the growth of Candida albicans was considerable. This result is supported by Adebiyi et al. (2009) who observed in their study that the use of ethanolic extracts of the root of Zanthoxylum sp gave a considerable inhibition on the growth of Candida sp.
Thus, reducing the level of oral infection in some selected natives who use this root as chewing-stick in the western part of Nigeria. In Central African Republic, Kosh-Komba
et al. (2017) also showed that the water-alcohol (50/50, alcohol 95°) extract of Z. zanthoxyloides root is very effective against C. albicans with high concentration (4 mg/disc). The results of our study illustrated that the ethanolic extracts of Jatropha curcas had significant inhibitory effects on the growth of C. albicans and C.
tropicalis than the aqueous extract. This result is supported by Agbogidi et al. (2013) who reported that the sap from the stem of Jatropha curcas inhibited the growth of microbes on the tongues of selected people and it gave relief to toothaches. This result is also in accordance with the work of Osemene et al. (2013) that showed the antimicrobial properties of Jatropha curcas on some oral disorders.
However, the aqueous and ethanolic extracts of J. curcas had no significant inhibition on the growth of C. glabrata.
Ethanolic extracts of Eucalyptus globulus showed significant inhibition on the growth of C. albicans in comparison to the aqueous extract. The work of Hardel and Sahoo (2011) lay credence to this as they revealed that a 50% ethanolic extract of E. globulus leaves had antifungal activity against oral pathogenic microorganisms with minimum inhibition concentration (MIC) values ranging from 0.20-6.25 mg/ml.
The antifungal activities of these plants parts can be attributed to the presence of some bioactive components in them. Many authors for instance have suggested that the presence of saponins confer many plants with antifungal activities. Saponins are surface active agents which interfere with or alter the permeability of the cell wall of microorganisms. Sule et al. (2010) have reported saponins as a major antifungal secondary metabolite. It is also believed that the interaction with steroids of the fungal membrane is the mechanism of antifungal activity of saponins as reported by Arif et al. (2009) and Damke et al. (2011). The results of this study showed that the leave, stem and root extracts of E.
globulus, J. curcas and Z. zanthoxyloides respectively had significant antifungal activities on C. albicans and C.
tropicalis as compared to the antifungal drug used as control.
These extracts had no significant effects on the growth of C.
glabrata a causal agent of oesophageal thrush. However, the ethanolic extract of V. amygdalina stem showed a significant fungistatic effect on the growth of C. glabrata. C. glabrata has been severally reported (Krogh-Madsen et al., 2006;
Lass-Flörl, 2009; Rodrigues and Henriques, 2017) to be resistant to many antifungal drugs especially the azoles containing drugs.
Conclusions
The issue of oral thrush and mouth odour has become alarming in our environment especially among the youths.
Many people find it difficult to seek medical assistance as a result of economic status and possible public embarrassment. The use of various types of plants such as Jatropha curcas, Eucalyptus globulus, Vernonia amygdalina and Zanthoxylum zanthoxyloides as chewing-stick to clean the mouth is a common practice. This study therefore established the efficacy of some medicinal plants in eradicating the causal organisms of oral thrush.
These plants can be exploited for their potential in mouthwash and in economically produced pastries for the treatment of oral thrush as they are eco-friendly and economically available.
Conflict of Interest
The authors declare that there are no conflicts of interest related to this article.
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