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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 29  |  Issue : 2  |  Page : 90-94

Comparison of antifungal effect of Aloevera gel and Triphala: An in vitro study


1 Department of Oral Medicine and Radiology, New Horizon Dental College and Research Institute, Bilaspur, Chhattisgarh, India
2 Department of Oral Medicine and Radiology, College of Dentistry, Jazan University, Saudi Arabia
3 Department of Conservative Dentistry and Endodontics, New Horizon Dental College and Research Institute, Bilaspur, Chhattisgarh, India
4 Department of Oral Medicine and Radiology, Triveni Institute of Dental Sciences Hospital and Research Centre, Bilaspur, Chhattisgarh, India
5 Department of Oral and Maxillofacial Pathology, Daswani Dental College, Kota, Rajasthan, India

Date of Submission27-Dec-2016
Date of Acceptance24-Oct-2017
Date of Web Publication9-Nov-2017

Correspondence Address:
Supreet Jain
Department of Oral Medicine and Radiology, New Horizon Dental College and Research Institute, Sakri, Bilaspur, Chhattisgarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaomr.JIAOMR_167_16

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   Abstract 

Aim: The aim of the present study was to determine and compare the antifungal and inhibitory activities of various concentrations of Aloevera gel and Triphala against oral Candiada albicans. Materials and Methods: The fungi (C. albicans) were isolated from 10 patients with pseudomembranous candidiasis and patients with denture stomatitis and transferred to Sabouraud's broth, which was later incubated in Sabouraud's dextrose agar (SDA). Antifungal activity of the Aloevera gel and Triphala was tested by the disc diffusion method, and minimum inhibitory concentration was determined by the broth microdilution method. Results: The mean value for zone of inhibition for Aloevera was 3.35 ± 0.59 mm and 1.06 ± 0.41mm at 100% and 50%, respectively. For Triphala, it was 4.19 ± 0.57 mm and 1.79 ± 0.43 mm at 100% and 50%, respectively (P value < 0.001). Zone of inhibition of 100% Triphala showed higher value than that of Aloevera at the same concentration (P = 0.004). Similar results were obtained at 50% concentration of Triphala and Aloevera (P = 0.004). Minimum inhibitory concentration of Aloevera gel and Triphala against C. albicans was 25% and 12.5%, respectively. Conclusion: Aloevera gel and Triphala both showed antifungal property at higher concentrations and can be used as a promising adjunct for antifungal agents.

Keywords: Antifungal, culture, natural


How to cite this article:
Jain S, Mujoo S, Daga M, Kalra S, Nagi R, Laheji A. Comparison of antifungal effect of Aloevera gel and Triphala: An in vitro study. J Indian Acad Oral Med Radiol 2017;29:90-4

How to cite this URL:
Jain S, Mujoo S, Daga M, Kalra S, Nagi R, Laheji A. Comparison of antifungal effect of Aloevera gel and Triphala: An in vitro study. J Indian Acad Oral Med Radiol [serial online] 2017 [cited 2017 Nov 20];29:90-4. Available from: http://www.jiaomr.in/text.asp?2017/29/2/90/217911


   Introduction Top


Candida is a genus of yeasts and is the most common cause of oral fungal infection. Most of the species of Candida are harmless commensals or endosymbionts of hosts including humans; however, when there is compromised immune system or when mucosal barriers are disrupted, they can invade and cause disease. Candida albicans is the most commonly isolated species and is responsible for oral thrush, pseudomembranous candidiasis.[1]

The cost, side effects, and increased resistance to commercially available antifungal drugs had led the researchers to explore the natural herbal remedies against fungal infections.[2],[3] Traditionally used medicinal plants are rich source of antimicrobial agents and are readily available in rural areas. They are relatively cheaper than modern medicine. Secondary metabolite products, produced by medicinal plants, are an important source of many pharmaceutical drugs.[1]

Aloevera is a cactus like plant that belongs to the Liliacea family. Aloevera gel is a mucilaginous colorless gel, secreted by the parenchymatous cells of fresh Aloevera leaves. It contains 98–99% water and 1–2% active compounds such as aloin, aloe-emodin, aloemannan, flavonoids, aloesin, sterols, amino acids, and vitamins.[4] Triphala means three fruits, is an Indian ayurvedic formulation comprising Terminali chebula, T. Belerica, and Phyllantus embelica in 1:1:1 proportion.[5],[6] Its phytochemical constituents are tannin, gallic acid, ellagic acid, phenol, and glycosides. Both Aloevera and Triphala have antibacterial, antioxidant, antifungal, anti-inflammatory, and immunomodulatory properties.[5],[6],[7] The effects of Aloevera and Triphala extracts on oral Candida infection have been studied by very few researchers in India.[8] The aim of the study was to determine the antifungal and inhibitory activities of various concentrations of Aloevera gel and Triphala against C. albicans.


   Materials and Methods Top


The study was conducted in the Department of Microbiology, New Horizon Dental College and Research Institute, Bilaspur. Ethical clearance was taken from the institutional ethical committee. Informed consent was taken from all the patients. Candida swab was taken from the patients suffering from pseudomembranous candidiasis using denture stomatitis and transferred to Sabouraud's broth [Figure 1]. All Candida isolates were confirmed by germ-tube test and spore germination test. This isolated Candida sample was incubated in Sabouraud's dextrose agar SDA (HIMEDIA laboratories, Mumbai) at 4°C.
Figure 1: Candida growth in Sabouraud's broth

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Preparation of the Aloevera extract

Thick epidermis of fresh Aloevera leaf was removed and the solid mucilaginous gel was collected in a sterile container [Figure 2]. Ten millimeter of the gel was mixed in 100 ml of 1% dimethyl sulfoxide (DMSO) and kept at 4°C.[3] DMSO (≤1%) has insignificant effect on the growth of fungi and was therefore used as a solvent.[9]
Figure 2: Extraction of Aloevera gel from fresh leaves in sterile plate

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Preparation of the Triphala extract

One hundred gram of Triphala churna (Dabur Triphala churna) was mixed in 100 ml of water to make an aqueous solution [Figure 3]. Then this solution was boiled for 45 min. Later, it was cooled and filtered. This filtrate was used to check its antifungal effect. Antifungal property of Aloevera gel and Triphala was detected using disc diffusion method.
Figure 3: Triphala churna

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Activation of Candida albicans

Loopful fungal spores were streaked on potato dextrose agar (PDA) plate and incubated at 37°C for 24 h prior to test. All plates (n = 10) were maintained at 4°C for further use.

Disc diffusion method

Eight millimeter wells were bored in each plate (n = 10 for Candida) having activated C. albicans, with an 8 mm borer in seeded agar in which 100%, 50%, 25%, and 12.5% of Aloevera gel extract was poured. Similarly, for Triphala, 8 mm wells were bored in different plate (n = 10 for Triphala) having activated C. albicans, with 8 mm borer in seeded agar in which 100%, 50%, 25%, 12.5% of Triphala extract was poured. After it normalized to room temperature, plates were incubated at 37°C for 24 hours. The zone of inhibition was measured and recorded [Figure 4].
Figure 4: (a) Zone of inhibition at 100% concentration of Triphala. (b) Zone of inhibition at 50% concentration of Triphala. (c) Zone of inhibition at 100% concentration of Aloevera. (d) Zone of inhibition at 50% concentration of Aloevera

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Minimum inhibitory concentration

Minimum inhibitory concentration (MIC) was done using the microbroth dilution method. The highest dilution that yielded no single fungal colony was taken as the MIC. Aloevera extracts were mixed with 1% DMSO to obtain various concentrations of the stock i.e. 100%, 50%, 25%, 12.5%, and 6.25%. Similarly, various concentration of Triphala were obtained by mixing extract with distilled water. Equal volume of the various concentration of each extract and were mixed in micro-tubes (Sabouraud dextrose broth) to make up 0.5 ml of solution. The tubes were incubated at 37°C for 24 hours. Later, test dilution was subcultured on SDA and incubated for 24 h to check for fungal growth [Figure 5].
Figure 5: Minimum inhibitory concentration for Triphala and Aloevera

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Statistical analysis

The data were compiled in MS Office Excel. Statistical analysis was done using SPSS version 21 software package (SPSS Statistics for Windows, Version 21.0. Chicago: SPSS Inc.). Analysis of variance (ANOVA) test was performed for intra and intergroup comparisons. Post hoc test (Tukey HSD) was also performed to compare different concentrations of Aloevera, Triphala, and both. Results were measured as mean ± SD. P value less than <0.05 was considered to be statistically significance and <0.001 as highly significant.


   Result Top


The Candida samples collected were analyzed by Gram staining, which showed presence of clusters of pseudohyphae suggesting C. albicans species.[10] The mean value for zone of inhibition for Aloevera was 3.35 ± 0.59 mm and 1.06 ± 0.41 mm at 100% and 50%, respectively. For Triphala, it was 4.19 ± 0.57 mm and 1.79 ± 0.43 mm at 100% and 50%, respectively [Table 1]. Both Triphala and Aloevera gel showed antifungal property at higher concentrations (100% and 50% concentration, P value < 0.001).
Table 1: Mean value of zone of inhibition of Aloevera and Triphala at different concentration

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There was highly significant difference observed between 100% and 50% Aloevera (P< 0.001, t = 19.17). Similar results were observed for 100% and 50% Triphala (P< 0.001, t = 31.58) [Table 2]. When ANOVA was performed, degree of freedom between Aloevera and Triphala was 3 and within groups (100% v/s 50%) it was 36 [Table 3]. On Post hoc test, zone of inhibition of 100% Triphala showed higher value than Aloevera at the same concentration (P = 0.004). Similar results were obtained at 50% concentration of Triphala and Aloevera (P = 0.000) [Table 4]. At lower concentration (25% and 12.5%), there was no effect against Candida. MIC of Aloevera gel and Triphala against C. albicans was 25% and 12.5%, respectively.
Table 2: Student's t-test to show comparison within the group

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Table 3: ANOVA test showing degree of freedom between the groups and within the group

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Table 4: Post hoc Test (Tukey HSD) showing comparison within groups

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   Discussion Top


The emergence of multidrug resistant (MDR) bacteria and fungi and high cost of pharmaceutical drugs have necessitated the development of alternative drugs from natural and other sources. Medicinal plants, which are being used since ancient times in Ayurveda, could be one of those alternatives as they are cheap, safe, and are effective against many microbes.[11] Many researchers have checked antibacterial effect of Aloevera and Triphala.[3],[4],[7],[12] Very few studies have been conducted to check the antifungal effect of these plants against oral candidial infection.[12],[13],[14],[15],[16],[17],[18] The present study was conducted to determine the antifungal and inhibitory activities of various concentrations of Aloevera gel and Triphala against C. albicans. In the present study, both Triphala and Aloevera showed anticandidal property at 100% and 50% concentrations. No significant changes were seen at lower concentration (25% and 12.5%).

Sheta et al. used 0.5% DMSO and distilled water as solvent and mixed Triphala (suspension) to get a final concentration of 100 mg/ml. They found that zone of inhibition for Triphala against Candida was 12 mm when water was used as solvent and 20 mm when DMSO was used as solvent.[12] Authors have also checked antifungal property for DMSO and found that it has no antifungal effect. They did not boil water suspension, which could be the cause of better results than our study. Randhawa suggested that DMSO increases the permeability of cell by binding with cell membrane, thus causing synergistic effect with anticandidal drugs.[9] In the presented case, water was used as a solvent for Triphala. El-Mekkawey et al. found that T. chebula may inhibit Candida species (including clotrimazole-resistant C. albicans).[11] Malhotra et al. in theirin vitro study found that colony forming units (CFUs) of C. albicans dropped to zero after 10 min by use of 6% Triphala mouthwash.[13]

Khaing found that ethanolic extract of Aloevera has no anticandidal effect.[14] A similar study by Kaur et al. showed Aloevera to be effective against C. albicans.[15] They suggested that a positive result in their study could be because of change in procedure of gel extraction, i.e., they extracted gel at room temperature whereas a prior study extracted gel at 90°C. Hence, the active component might have been lost in the ethanolic extract if extraction was done at a higher temperature.

A study by Stanley et al. found that zone of inhibition for Aloevera against C. albicans was 4 mm and MIC was 50%, which was similar to the present study.[16] Shireen et al. found that antifungal property of Aloevera is dose dependent. They took Aloevera juice (1000, 500, 250, 100 μg) from fresh leaves and mixed it with 10 ml of ethanol. Zone of inhibition was 14 mm against C. albicans at 1000 μg concentration, which was comparable to amphotericin B.[17] Chainani et al. used ethanolic extract of Triphala and found that it showed anticandidal effect at concentration 5% and above.[18] Triphala showed better results when compared with Aloevera, with greater zone of inhibition in both 100% and 50% concentration. Moreover, Triphala inhibits Candida growth at lower concentration (12.5%) when compared to Aloevera (50%).

The antimicrobial effect of Aloevera is attributed to the natural component called anthraquinones (emodin, aloetic acid, aloin, anthracene, anthranol, barbaloin, chrysophanic acid, ethereal oil, ester of cinnamic acid, isobarbaloin, and resistannol).[17],[19] The purified aloe protein of 14 kDa from Aloevera leaf gel exhibited a potent antifungal activity against C. albicans.[19]Antifungal effect of Triphala is due to its two component [T. Chebula (Harde), T. Belerica (Baheda)].[12]

We have found that both Triphala and Aloevera have shown antifungal activity against C. albicans. However, the mechanism of action of these herbal agents on microbes is yet to be understood. Furthermore, more studies are required on various fungal loads and various concentrations of both Triphala and Aloevera to check its potency against various Candida species.


   Conclusion Top


Increase in fungal pathogens, limited therapeutic options, side effects of therapeutic drug, and emergence of MDR together put a burden on patients with compromised immunity. C. albicans is the most common opportunistic infection of oral cavity, frequently isolated from tissue samples of affected patients. Traditional medicines have used plant products since ages. Because of their ease of availability and less toxic effect, efforts have been made for developing therapeutic options using molecules from plant sources. This study is also a similar effort to check the antifungal effects of Triphala and Aloevera. In this study, Aloevera and Triphala were found to show antifungal activity.

Limitations

The present study was conducted inin vitro and involved a small sample size. In future, more trials should be carried out on larger population (in vivo) to prove the antifungal efficacy of Aloevera and Triphala against C. albicans to characterize the mechanism of action for further translation into clinical use and overcoming the challenges of fungal therapeutics.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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Malhotra A, Malhotra I, Solanki R, Khangwal M, Kumar D. Evaluation of antibacterial and antifungal properties of herbal mouthwash- A prospective study. Int J Enhanced Res Med Dent Care 2015;2:33-7.  Back to cited text no. 13
    
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Khaing TA. Evaluation of the antifungal and antioxidant activities of the leaf extract of Aloe vera (Aloe barbadensis Miller). World Acad Sci Engineer Tech 2011;5:3-23.  Back to cited text no. 14
    
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Kaur H, Goyal RR, Bhattacharya A, Gupta R, Lal NK, Arora B, et al. Antifungal activity of phyto-extracts of Piper Longum, Aloevera, and Withania somnifera against human fungal opportunistic pathogen Candida albicans. DU J Undergrad Res Innov 2015;1:107-15.  Back to cited text no. 15
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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