Antifungal efficacy of different extracts of neem and turmeric on candida species – An in vitro analysis

Nallan C. S K. Chaitanya; Diksha Chikte; Kandari Ramya; Sushmitha Yadav; C Pravallika; Sasi Vardhini; Bhargavi Priya; Baradi Keerthana; Amal Fathima

doi:10.4103/ijds.ijds_52_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 14 | Issue : 2 | Page : 53-57

Antifungal efficacy of different extracts of neem and turmeric on candida species – An in vitro analysis

Nallan C. S K. Chaitanya¹, Diksha Chikte¹, Kandari Ramya¹, Sushmitha Yadav¹, C Pravallika², Sasi Vardhini², Bhargavi Priya², Baradi Keerthana², Amal Fathima²
¹ Department of Oral Medicine and Radiology, Panineeya Mahavidyalaya Institute of Dental Sciences and Research Centre, Hyderabad, Telangana, India
² Department of Oral Medicine and Radiology, Panineeya Institute of Dental Sciences, Hyderabad, Telangana, India

Date of Submission	03-May-2021
Date of Decision	30-Sep-2021
Date of Acceptance	08-Oct-2021
Date of Web Publication	26-Apr-2022

Correspondence Address:
Nallan C. S K. Chaitanya
Assistant Professor, Department of Oral Radiology and Medicine, RAK College of Dental Sciences, Ras al Khaimah
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ijds.ijds_52_21

Abstract

Background: Curcumin is an age-old spice, which is known for its anti-inflammatory activity and antibacterial property, and neem is proven to have many medicinal properties, especially known for its antimicrobial properties. Aim: The study aimed at assessing the antifungal activity of different preparations of turmeric and neem over Candida species. Materials and Methods: Using Sabouraud's agar as the medium, Candida species colonies were transferred to the agar plates after preparation of five wells. 75 μl, 50 μl, 25 μl, 10 μl, and 5 μl of neem leaf oil, neem oil, turmeric oil, turmeric extract, and neem seed oil were added and incubated at 37°C for 18–24 h. The diameter of inhibition zone around the wells where the growth was inhibited corresponded to the antifungal activity. Results: Neem bark extract and turmeric extract showed the highest inhibitory range against Candida albicans. Neem leaf extract showed the highest range of inhibition at 75% against Candida krusei and Candida tropicalis. Conclusion: Extracts of turmeric and neem have shown definitive inhibition against different strains of candida in vitro, and hence, these can be used for treating candidiasis as an alternative to regular triazoles.

Keywords: Agar plates, Candida, curcumin, disc diffusion, neem

How to cite this article:
K. Chaitanya NC, Chikte D, Ramya K, Yadav S, Pravallika C, Vardhini S, Priya B, Keerthana B, Fathima A. Antifungal efficacy of different extracts of neem and turmeric on candida species – An in vitro analysis. Indian J Dent Sci 2022;14:53-7

How to cite this URL:
K. Chaitanya NC, Chikte D, Ramya K, Yadav S, Pravallika C, Vardhini S, Priya B, Keerthana B, Fathima A. Antifungal efficacy of different extracts of neem and turmeric on candida species – An in vitro analysis. Indian J Dent Sci [serial online] 2022 [cited 2022 May 16];14:53-7. Available from: http://www.ijds.in/text.asp?2022/14/2/53/344072

Introduction

The most common opportunistic infection of the oral cavity is oral candidiasis caused by various Candida species, and the most common of them being Candida albicans. Angular cheilitis, acute candidiasis, and chronic candidiasis are the three broad groups.^[1] Yeasts are the fourth most common organisms recovered from blood culture in hospitals with nosocomial infections.^[2],[3] The most common predisposing factors include long-term use of antibiotics, immune suppressor drugs, and steroids and chronic diseases such as diabetes, acquired immunodeficiency syndrome, and malignancy.^[4],[5],[6] The treatment modalities include topical and systemic antifungal agents, with triazoles being the first line of treatment effective against most Candida species.^{[7],[8],[9],[10],[11]} However, due to the widespread use of such agents, increase in antifungal resistance after prolonged use was encountered. Hence, there is always an increased demand for a new and effective antifungal therapeutic agent with less adverse effects and incidence of resistance.

Curcumin is derived from a plant source. Curcuma longa L. (Zingiberaceae family) is a rhizome, which is available with the common name turmeric.^[12] C. longa rhizome is being used as antimicrobial agent for a long time, with curcumin being the active ingredient.^[13] Turmeric has been used in the treatment of jaundice, liver diseases, dental pain, and as a food colorant. Its anti-inflammatory, hypoglycemic, and antimicrobial activities have been evaluated and validated.^[14] The anti-inflammatory action of curcumin is due to the inhibition of activation of nuclear factor-κB. The antifungal effect of curcumin is due to downregulation of desaturase (ERG3) which results in a reduction in ergosterol of fungal cells. Accumulation of precursors of ergosterol results in the generation of reactive oxygen species which is responsible for cell death.^[15] Neem (Azadirachta indica) is an evergreen tree, called “arista” in Sanskrit which means complete or perfect. It is endemic to the Indian subcontinent and cultivated throughout Southeast Asia, Australia, East and sub-Saharan Africa, Fiji, Mauritius, and many countries in Latin America.^[16] Talwar et al. in 1997 demonstrated that different parts of the neem tree (leaf, bark, and seed oil) exhibit wide pharmacological activities including antioxidant, antimalarial, antimutagenic, anticarcinogenic, anti-inflammatory, antihyperglycemic, anti-ulcer, and antidiabetic properties.^[17] It also has various biological activities due to the presence of phytochemical constituents such as nimbidin, nimbin, nimbolide, azadirachtin, gallic acid, epicatechin, catechin, and margolone.^[18] These are extracted from the fruits and seeds of the tree. The neem tree is particularly rich in triterpenoids. As many as 100 compounds were derived and isolated from many parts of the neem tree.^[19] According to Polaquini et al., terpenoids are known to be responsible for its fungicidal or fungistatic activity on several pathogenic fungi.^[20] Aqueous neem leaf extracts elucidated an increase in superficial hydrophobicity on cells of C. albicans.^[21] This study was carried out with a primary goal of evaluating the antifungal activity of different forms such as turmeric powder, neem leaf oil, neem bark extract, neem seed oil, and turmeric oil against C. albicans, Candida glabrata, Candida tropicalis, and Candida krusei biofilms in vitro in different concentrations. The formulations, if found effective, could be used as an alternative therapeutic agent against Candida infections.

Materials and Methods

The present in vitro study was done in Panineeya Institute of Dental sciences in collaboration with Maratha Mandal Dental College, Belgaum. It was carried out between June 2018 and December 2019.

Armamentarium and source of data

To evaluate the efficacy of different formulations of turmeric extracts and neem extracts, the following products were procured after high-performance liquid chromatography, from Minal pharmacy, Mumbai:

Turmeric powder extract – Dry powder of 100 grams
Turmeric oil formulation - 100 ml
Neem seed oil – 100 ml
Neem bark oil - 100 ml
Neem leaf oil – 100 ml.

Active cultures of Candida glabrata microbial type culture collection code

Three thousand and nineteen, C. tropicalis microbial type culture collection (MTCC) Code: 184, C. krusei MTCC Code: 9215, all in aerobic forms were procured from Institute of Microbial Technology laboratories, Chandigarh, in freeze-dried state. C. albicans was procured separately by culturing it from saliva samples.

Methodology

Sabouraud's agar was used as the medium for Candida growth. Initially, the agar plates were brought to room temperature. Within 15 min of inoculum preparation, a sterile cotton swab was dipped into inoculum and rotated against the wall of the tube to remove excess inoculum. Using the inoculating loop, the procured Candida species colonies were transferred separately to the agar plates and visually adjusted the turbidity with broth equal to that of 0.5 McFarland turbidity. Alternately, the material was adjusted to the suspension with a photometric device. The entire surface of the agar plate was swabbed three times, rotating plates approximately 60° between streaking to make sure that there is a uniform distribution. The plates were allowed to stand for at least 3 min but no longer than 15 min before applying them to agar disc

Addition of compound into the plate

Using a sterile hollow tube of 5 mm, five wells were prepared on the inoculated agar plate. All five items, such as neem leaf oil, neem bark oil, neem seed oil, turmeric oil, and turmeric powder extract, were applied to the already formed wells on the inoculated agar plates at concentrations of 75 l, 50 l, 25 l, 10 l, and 5 l of each. The plates were then incubated at 37°C for 18–24 h. The antifungal activity was assessed by measuring the diameter of inhibition zone around the wells where the growth was inhibited. The abovementioned protocol was followed for all the Candida species meant for study.

Results and Observations

Candida albicans

With 1: 2 water dilutions, neem bark extract and turmeric extract had shown the highest range of inhibition followed by neem leaf oil and turmeric oil, but their efficacy decreased as the concentration decreased. Neem leaf oil had demonstrated the least inhibition compared to all the other testing samples, whereas the neem leaf extract had failed to inhibit C. albicans at all concentrations [Figure 1] and [Table 1] and [Table 2].

Figure 1: Inhibition of candida albicans on the agar plate with different concentrations of turmeric oil

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Table 1: Candida albicans minimum inhibitory concentration with various testing agents and dilutions

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Table 2: Different strains of Candida with percentage inhibition by disc diffusion methods

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Candida krusei

At 75% concentration, the neem leaf extract plate had shown the highest inhibitory range. The neem leaf extract had shown a slightly lesser range of inhibition and neem leaf as well as the turmeric oil has failed to inhibit C. krusei. At 50% concentration, turmeric oil had shown maximum inhibition, whereas neem leaf extract, neem bark extract, and turmeric oil had demonstrated an equal range of inhibition. C. krusei was resistant to neem leaf oil and pure neem oil. At 25% and lower concentration, C. krusei was resistant to all the preparations.

Candida glabrata

At 75% concentration, turmeric oil demonstrated the highest range of inhibition. At 50% concentration, turmeric extract also had elicited the highest range of inhibition for the growth of Candida Glabrata. None of the other testing ingredients had showed any effect on the growth of the species at 50% concentration and at 25% and lower concentration, all the preparations were inefficient in inhibiting C. glabrata [Figure 2].

Figure 2: Inhibition of Candida Glabrata using agar plate with different concentrations of turmeric extract

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Candida tropicalis

After incubation of C. tropicalis in the appropriate medium, at 75% concentration, neem leaf extract and neem bark extract had shown the highest range of inhibition, whereas neem oil and neem leaf oil and all forms of turmeric oils tested were inefficient in inhibiting C. tropicalis. Neem bark extract had shown good inhibition at 75% concentration, but it had failed to show any inhibition at 50%. For those concentrations at 50%, neem leaf extract plate had exhibited inhibition for the growth of this type of Candida. At 25% and at lower concentrations, all the preparations have failed to show any inhibition [Figure 3].

Figure 3: The inhibition of Candida Tropicalis with different concentrations of Neem on the agar plate

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Discussion

The common spices, which we use in kitchen in our daily life, and natural herbal extracts effect the fungal growth adversely and hence should be considered to control fungal diseases. The bioactive compounds extracted from plants must be examined and can be used as drugs against fungal pathogens without the development of drug resistance in pathogens and without any side effects. C. albicans is the frequently associated species in denture stomatitis. C. glabrata and C. tropicalis are the emerging species next to it. Existing antifungal agents such as nystatin and fluconazole have been found to be toxic on long-term use develop resistance. De Rezende Ramos et al.^[14] established the effect of neem extracts on cell surface hydrophobicity and biofilm formation, which affected the colonization by C. albicans. The presence of several active antimicrobial ingredients in leaves of neem such as desactylimbin, quercetin, and sitosterol might be responsible for the fungicidal and bactericidal properties of extracts from neem leaves in both in vitro or in vivo trials.^[22] In the current study, neem leaf oil showed the highest antifungal activity against C. glabrata when compared to neem bark extract, whereas neem bark extract showed the highest range of inhibition against other strains of Candida. The presence of concentrations of azadirachtin, quercetin, and β-sitosterol in Azadirachta indica leaves might be responsible for the strong antibacterial and antifungal activity compared to bark and seed.^[23] Margolone, margolonone, and isomargolonone are tricyclic diterpenoids isolated from stem bark had shown to exhibit antibacterial activity.^[24] Antimicrobial effectiveness could be attributed to different concentrations of the active ingredient in different parts of the plant. In a study conducted, the antioxidant activity of leaves, fruits, flowers, and stem bark extracts were evaluated and results showed leaf aqueous extract and flower and stem bark ethanol extracts showed higher antioxidant properties.^[23]

In 2012, a study by Garcia-Gomes et al. found that curcumin has a high ability to decrease fluconazole resistance in a C. albicans isolate.^[25] In a study conducted by Lee and Lee, they evaluated the efficacy of curcumin on the cell membrane of C. albicans by measuring potassium efflux and they found that curcumin induced changes in the intracellular components, especially potassium.^[26] Curcumin, according to Sharma et al., can influence ergosterol-related gene regulation and membrane lipid homeostasis. Despite its efficacy against human ailments, curcumin's therapeutic efficacy was restricted due to low absorption, fast metabolism, and rapid systemic elimination.^[27] Thus, numerous efforts were made to improve curcumin's bioavailability by altering these features. Some adjuvants were used to increase the bioavailability of curcumin by blocking its metabolic pathway. Curcumin administration enhanced salivary radical scavenging capacity as well as plasma catalase, myeloperoxidase, and nitric oxide generation in these subjects.^[27] Overall, our findings showed that lipidated curcumin has health-promoting properties in healthy middle-aged persons. When compared to other turmeric extracts, turmeric oil showed a wider range of inhibition in our investigation. As a result, neem and turmeric extracts can be utilized to treat a variety of Candida strains.

Dikshita used tissue conditioners to compare the efficacy of neem leaf extract and three different antibacterial agents. They observed that neem leaf extract was efficient against both C. albicans and Streptococcus.^[28] Similarly, Chen et al. evaluated the antifungal efficacy of different chemical components of C. longa and found that the extracts from these leaves have shown higher antifungal activity when compared to the Curcuma rhizome extracts.^[29] It was in accordance with our study that different extracts have shown variation in antifungal efficacy.

Conclusion

The data provided here demonstrated that curcumin was effective against the different strains of Candida and can be used as adjuvant therapy along with regular triazoles for the treatment of candidiasis, but their effect is more pronounced only in higher concentrations and their effectiveness decreases as the concentration decreased. Neem leaf extracts and neem oil had shown satisfactory inhibitory range against all the strains of Candida, but they were effective only in the higher concentration. Hence, extracts of neem leaf, neem bark, and turmeric could be used as adjunctive treatment along with regular antifungal drugs.

Ethical clearance

The study was in vitro and did not require ethical clearance certification.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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