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ORIGINAL ARTICLE
Year : 2021  |  Volume : 13  |  Issue : 1  |  Page : 67-72

Antifungal Efficacy of Wrightia tinctoria (Roxb.) R.Br on Candida Species Isolated from the Oral Cavity: an Invitro Study


1 Postgraduate Student, Department of Oral and Maxillofacial Pathology, Adhiparasakthi Dental College and Hospital, Melmaruvathur, Tamilnadu, India
2 Associate Professor, Department of Microbiology, Melmaruvathur Adhiparasakthi Institute of Medical Sciences and Research, India
3 Professor and Head of the Department, Department of Oral and Maxillofacial Pathology, Adhiparasakthi Dental College and Hospital, Melmaruvathur, Tamilnadu, India

Date of Submission27-Nov-2020
Date of Acceptance19-Mar-2021
Date of Web Publication06-Aug-2021

Correspondence Address:
Dr. K. V. Devika
Department of Oral and Maxillofacial Pathology, Adhiparasakthi Dental College and Hospital, Melmaruvathur, Tamilnadu-603319
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jofs.jofs_284_20

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  Abstract 


Introduction: Nature is a valuable source of active ingredients that needs to be explored, especially its utilization in the medical field. Owing to the limited availability of antifungal drugs and also considering their side effects, there is always a constant need for a safe and competent alternative. Wrightia tinctoria, a medicinal tree, has been reported to possess potent antifungal activity against commercially available candida strains [American type culture collection (ATCC) and microbial type culture collection (MTCC)]. This could be beneficial clinically only if its antifungal activity could be proved against candida species isolated from clinical samples as the commercially available candida strains might have lost its pathophysiological characteristics on repeated subcultures. Hence, with this background, we performed this study to determine the antifungal efficacy of the extracts obtained from the leaves of W. tinctoria against the candida species isolated from the oral cavity. The aim of this study was to determine the antifungal efficacy of W. tinctoria on candida species isolated from the oral cavity. Materials and Methods: To determine the antifungal activity, acetone, ethyl acetate, and chloroform extracts of leaves of W. tinctoria were used. The study population consisted of five healthy volunteers above 18 years of age without any harmful habits and five patients at increased risk of candida infection. Saliva samples were collected by oral rinse technique using phosphate buffered saline. Fluconazole was used as a positive control and the antifungal efficacy was determined using disk diffusion method. Kruskal-Wallis test was used to determine the significant difference between the extracts. Results: The mean zone of inhibition of acetone, ethyl acetate, and chloroform extracts of leaves of W. tinctoria was 10.8667, 11.0000, 10.1333 mm, respectively. Conclusion: Acetone, ethyl acetate, and chloroform extracts of W. tinctoria possess antifungal activity against candida species isolated from oral cavity.

Keywords: W. tinctoria, Candida albicans, antifungal activity


How to cite this article:
Devika KV, Sabarinathan T, Shamala S. Antifungal Efficacy of Wrightia tinctoria (Roxb.) R.Br on Candida Species Isolated from the Oral Cavity: an Invitro Study. J Orofac Sci 2021;13:67-72

How to cite this URL:
Devika KV, Sabarinathan T, Shamala S. Antifungal Efficacy of Wrightia tinctoria (Roxb.) R.Br on Candida Species Isolated from the Oral Cavity: an Invitro Study. J Orofac Sci [serial online] 2021 [cited 2021 Sep 29];13:67-72. Available from: https://www.jofs.in/text.asp?2021/13/1/67/323354




  Introduction Top


Candida belonging to the kingdom Fungi causes superficial and deep/systemic opportunistic mycotic infections by expressing adhesins and invasins on its cell surface and invades the host cell by endocytosis or by elaborating enzymes.[1],[2],[3] More than 150 species of candida are detected and among which C. albicans is the most common species associated with human infections.[4] Candida infections are usually treated with antifungal drugs, but drug resistance is a major setback. W. tinctoria belonging to the family Apocynaceae has been used as medicines in Siddha, Unani, and Ayurveda since it possess antimicrobial, anti-inflammatory, anticancer, anthelmintic, and astringent properties.[5],[6],[7],[8] Numerous studies have reported its antifungal activity on candida strains obtained from the culture centers such as microbial type culture collection (MTCC) and American type culture collection (ATCC).[5],[9],[10] However, our study demonstrates the antifungal activity of W. tinctoria on candida species isolated from the oral cavity as it is a common opportunistic fungal infection encountered in day-to-day practice in dental clinics.


  Materials and Methods Top


To prepare the plant extract, first the fresh leaves of W. tinctoria were collected from Kollimalai, Namakkal district, Tamil Nadu and was authenticated by a botanist [Figure 1]. The leaves were washed with running water, shade dried for 10 days, and powdered using a mechanical grinder. The solvents used were ethyl acetate, chloroform, and acetone as the extract medium. Hundred grams of the finely powdered leaves was mixed with 300 mL of each solvent. It was kept in an orbital shaking incubator for 48 hours and filtered using Whatman Filter Paper No. 1 [Figure 2]. The filtrate was dried and the dry yield was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 mg/mL DMSO [Figure 3]. DMSO was chosen because of its amphipathic nature. The culture media was prepared by dissolving 6.5 mg of Sabouraud Dextrose Agar (SDA, Himedia- MO63) in 100 mL of water and autoclaved. Hicrome differential agar media (Himedia-M1297A) was prepared by dissolving 2 mg of Hicrome agar powder in 100 mL of water (not autoclaved) and 2 g of agar agar was added for solidification.
Figure 1 Fresh leaves of Wrightia tinctoria

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Figure 2 Filtration of extracts using Whatman’s filter paper No.1

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Figure 3 Ethyl acetate, Chloroform, acetone extracts of Wrightia tinctoria

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The study was approved (protocol no. 2019/IRB-JUN-OP 01/APDCH) by Institutional Review Board, Ethics and Research committee, Adiparashakthi Dental college and Hospitals, Melmarvathur on 1st june 2019. Five healthy volunteers above 18 years of age without any harmful habits and five patients who were prone to candida infection were initially selected for the study by the purposive and convenient method of sampling. Patients on antifungal medications (topical and systemic) were excluded from the study. After obtaining the informed consent, saliva samples were collected using oral rinse technique. Participants rinsed their mouth with phosphate buffered saline (PBS 0.1 M, pH 7.2) for 60 seconds and spitted into a sterile container. The obtained samples were centrifuged at 1700 rpm for 10 minutes. The supernatant was discarded and the centrifuged deposits were inoculated in SDA agar and incubated at 37°C [Figure 4].[11] After 48 hours, cream smooth pasty convex colonies were observed [Figure 5]. Gram staining of the cultured candida showed the presence of bacteria intermixed with candida. Hence, repeated subculturing in SDA agar was carried out until pure gram-positive candida was observed in gram staining [Figure 6].
Figure 4 Inoculation of saliva sample on SDA agar

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Figure 5 Creamy smooth pasty convex colonies in SDA agar

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Figure 6 Gram positive microorganism (yeast and hyphae)

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For species identification, these pure candida colonies obtained in SDA culture were inoculated in Hicrome Candida Differential Agar and incubated at 37°C. After 48 hours, four green colored colonies were formed, which were identified as C. albicans, and one purple colored colony formed was identified as C. tropicalis according to the manufacturer’s instruction (Himedia-M1297A) [Figure 7]. Further, the green colored colonies formed in Hicrome agar were added to 1 mL of serum and incubated at 37°C. After 2 hours, formation of the incipient hyphae (germ tube) confirmed the candida species as albicans [Figure 8]. Candida was spread on the SDA agar by lawn culture to determine the antifungal activity using the disk diffusion method. To obtain a standard concentration of candida, 0.5 McFarland standard was used as a reference. One milliliter of Brain Heart Infusion Broth was taken in a test tube and the candida was inoculated into it and mixed well. It was incubated at 37°C and checked every 1 hour till it obtained the turbidity equal to that of 0.5 McFarland standard (1.5 × 108 CFU/mL).
Figure 7 Light green coloured colonies- CANDIDA ALBICANS, purple colour - CANDIDA TROPICALIS

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Figure 8 Germ tube formation

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For disk diffusion method, 6 mm sterile antibiotic plain disks (Hi Media) were impregnated with a standard concentration (50 μL) of the plant extracts. The impregnated disks were placed at an equal distance in a  Petri dish More Details containing SDA agar, inoculated with Candida, and incubated at 37°C for 24 hours. Fluconazole was used as a positive control. After 24 hours, the zones of inhibitions were measured using a transparent ruler. The disk diffusion method was done in triplicates to obtain a reliable result and to minimize the error. The disks were infused with DMSO and each of the solvents without the plant extracts was also placed separately on the SDA agar lawn cultured with Candida, which was then incubated at 37°C for 24 hours and the zone of inhibition was measured using a ruler.


  Results Top


Statistical analysis was performed using SPSS software version 17. A positive zone of inhibition measuring 35 mm was observed with fluconazole [Figure 9]. As the zone of inhibition was observed to be within the same range in C. albicans and C. tropicalis, the average was calculated together. The zone of inhibition ranged from 8 to 14 mm for all the plant extracts. The mean zone of inhibition of acetone, ethyl acetate, and chloroform extracts of leaves of W. tinctoria was 10.8667, 11.0000, 10.1333 mm, respectively [Figure 10]. The means were compared using Kruskal-Wallis test and it was found that there was no significant difference (P = 0.47) between the extracts [Table 1]. This shows that all the three extracts possess antifungal activity and are equally effective. No zone of inhibition was observed in the negative controls − acetone, ethyl acetate, chloroform, and DMSO [Figure 11]. This proved that the anticandidal effect is credited solely to the biologically active compounds in the W. tinctoria and not to the solvents.
Figure 9 Positive zone of inhibition observed in Fluconozole (F) Negative zone of inhibition observed in Dimethyl Sulphoxide (DMSO)

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Figure 10 Zones of inhibition of acetone (A), ethylacetate (EA) and chloroform (C) extracts of leaves of Wrightia tinctoria on Candida species

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Table 1 Mean zone of inhibition of acetone (A), ethyl acetate (EA) and chloroform (C) extracts of leaves of W. tinctoria

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Figure 11 No zone of inhibition were observed in negative controls Acetone (A), Ethylacetate (E), Chloroform (C)

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  Discussion Top


A tremendous increase in fungal infections, particularly by candida species, is seen affecting human health in recent times. Phagocytes such as neutrophils and macrophages are the first line of defense against the fungal pathogens.[12] The size, shape, and composition of the fungal cell play a crucial role in the evasion of host immune response.[12] The phagocytic cells (10–15 μm) can ingest the cells larger than its size, but the fungal hyphae can grow up to 18.8 μm/hour in vitro or even more in vivo, thereby making phagocytosis difficult.[12]

The cell wall of fungi is a highly regulated and organized structure that can interfere with the functions of host immune cells by producing inhibitors of chemotaxis or phagocytosis.[12],[13] It is composed 90% of carbohydrates and 10% of proteins. The components of the fungal cell wall are polysaccharides, mannans (O-linked and N-linked), β-glucans (β-1,3-glucans and β-1,6-glucans), and chitin or chitosan (deacylated form).[13] β-1,3 Glucan is a robust pro-inflammatory mediator compared to mannans and can recruit neutrophils.[10] The organization of candida cell wall differs with the morphological forms.[12] β-1,3 Glucans and chitins are present in the inner cell wall of the yeast at the bud scars, while in hyphae these two components are masked by the mannans, thus forming a moving target for immune surveillance due to which yeast triggers the immune recognition and phagocytosis, but the hyphae evades the host immune response and invades.[12]

Most antifungals act on the fungal cell wall either directly by targeting the ergosterol of the fungal cell wall or by inhibiting the enzymes involved in the synthesis of ergosterol, such as squalene epoxidase, 14-α lanosterol demethylase, reductase, and isomerase.[14] Depletion of ergosterol affects the growth and proliferation of fungal cell.[14] Azoles are routinely employed antifungal agents and are classified as imidazoles (miconazoles, oxiconazoles, ketoconazoles, clotrimazoles) and triazoles (fluconazoles and itraconazoles) based on the nitrogen atoms.[14] Azoles are fungistatic and acts on 14-α lanosterol demethylase (ERG 11 gene), thereby interfering with the conversion of lanosterol to ergosterol resulting in fungal cell death.[14] Polyenes (amphotericin) directly bind to ergosterol and result in pore formation leading to cell death.[14] Pyrimidine analogues (5-fluorocytosine) get converted into 5-fluorouracil and get incorporated into the nucleic acids where they blocks the protein synthesis and DNA replication leading to fungal cell death.[14] The new antifungals echinocandins (caspofungin) act by targeting β-1,3 glucan synthase and thus they prevent the formation of β-1,3 glucan of the fungal cell wall.[14]

The development of resistance to the available antifungal drugs is a major hindrance in the effective treatment of fungal infections.[14] One of the commonest mechanisms of multiple drug resistance is the modification of the target protein Erg11p by chromosomal mutations that prevents binding of fluconozole to the target protein.[14] Another mechanism identified was the overexpression of the genes encoding the efflux proteins, ATP-binding cassette proteins and major facilitator superfamily proteins, which leads to an increased efflux of drugs that causes its decreased cytoplasmic concentration and hence the antifungal resistance.[14]

W. tinctoria seems to be a prime focus of research in recent times. It is commonly called “Sweet Indrajao”, “Pala Indigo Plant”, and “Dyer’s Oleander”.[5] This plant was named after a Scottish physician and botanist William Wright (1740–1827).[5] The word “tinctoria” means “tinge of dye” and hence the name “pala indigo”.[5] Traditionally, this plant was known to cure jaundice and hence called “jaundice curative tree”.[6] Antifungal activity of W. tinctoria was proven by the studies conducted by Moorthy et al. and Vedhanarayanan et al.[5],[10] Various extracts of the leaves of W. tinctoria such as ethanol, methanol, hexane, chloroform, ethyl acetate, acetone, aqueous, and petroleum ether have been shown to elicit potent antifungal activity.[6],[15],[16] In a study by Maddila and Hemalatha,[9] the crude hexane, ethyl acetate, and aqueous extracts of the leaves of W. tinctoria were proven to possess antifungal activity. The chloroform extracts of the leaves are reported to be very effective against dermatophytes and nondermatophytes and are used in the treatment of dermatophytosis.[8] Hence, we chose the leaves of W. tinctoria as specimen of our study and observed that the acetone, ethyl acetate, and chloroform extracts of the leaves of W. tinctoria exhibited inhibitory property on candida species isolated from the oral cavity. When comparing the extracts, all the three extracts showed almost the same antifungal efficacy.It was evident from numerous literature references that the possible antifungal mechanism of W. tinctoria on candida species could be attributed to its bioactive compounds.[15],[16] The phytochemical screening of the leaves of W. tinctoria confirmed the presence of alkaloids, cardiac glycosides, phenols, tannins, triterpenoids, triacontanol, myristic acid, palmitoleic acid, palmitic acid, stearic acid, arachidic acid, α- and β-amyrin, lupeol along with flavonoids such as indigotin, indirubin, tryptanthrin, rutin and isatin.[6],[15] Indirubin, a major constituent in chloroform extract, was reported to be seen only in dried leaves of W. tinctoria.[6],[7]

Phenols alter the cell surface hydrophobicity, causing leakage of cytoplasmic contents of the fungal cell.[15] It also has an immunoregulatory effect on monocyte.[17] A study by Yang et al.[18] on saponins revealed that it inhibits the yeast-to-hyphal transition, decreases phospholipase production, and increases the membrane permeability of the candida cell wall. It can cause pore formation in the cell membrane by binding to the ergosterol of fungal cell wall and causes leakage of cellular contents.[18],[19] Saponins also inhibit the growth of fluconazole and echinocandin-resistant C. albicans isolates.[19] Ponnusamy et al.[20] in their study found indirubin, a bioactive compound of W. tinctoria, to be very effective against fungal species. Hence, the zone of inhibition obtained in our study could also be credited to the phytochemicals such as phenols, saponins, and indirubin compounds of W. tinctoria.

To the best of our knowledge, no sufficient literature was available regarding the studies about antifungal efficacy of W. tinctoria on candida species isolated from the oral cavity. Since the study was first of its kind, the results obtained in the present study could not be compared with other studies. As this is a preliminary study, observations in the present investigation can be a basis for future research on a wider range. The results of our study indicate that the researches on medicinal plants having traditional claims might warrant fruitful results. These plants could be a potential source for developing new antifungal agents in the future, especially in case of drug resistance.

Limitations

This study was performed with an aim to obtain candida from immune altered and healthy patients. Isolation of candida from oral cavity was a laborious procedure and we were able to obtain only five Candida out of 20 immune altered patients. Among healthy volunteers, isolating the candida was a complex and a difficult procedure. We tried to revive C. albicans from MTCC 183 strain using culture media, but we were unable to procure candida growth.


  Conclusion Top


It can be concluded based on our study that acetone, ethyl acetate, and chloroform extracts of W. tinctoria do possess antifungal activity against the candida species isolated from oral cavity. However, this study has to be further proceeded with different concentrations of the plant extracts of W. tinctoria to identify minimum inhibitory concentration in order to acquire more clarity regarding the antifungal activity.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Rao B, Rajeswari D, Devarakonda R, Battu H. Phytochemical and pharmacological studies on Wrightia tinctoria. World J Pharm Pharm Sci 2019.  Back to cited text no. 15
    
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Srivastava R. A review on phytochemical, pharmacological, and pharmacognostical profile of Wrightia tinctoria: adulterant of kurchi. Pharmacogn Rev 2014;8(15):36.  Back to cited text no. 16
    
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