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 Table of Contents  
ORIGINAL ARTICLE
Year : 2023  |  Volume : 16  |  Issue : 1  |  Page : 54-59

Comparative evaluation of antifungal efficacy and potency of soft liners incorporated with powder extracts of Lawsonia inermis and Withania somnifera on the growth of Candida albicans – An in vitro study


1 Department of Prosthodontics, Kaher's KLE VK Institute of Dental Sciences, Belagavi, Karnataka, India
2 Basic Science Research Center, Belagavi, Karnataka, India

Date of Submission04-Jan-2022
Date of Acceptance23-Mar-2022
Date of Web Publication21-Jan-2023

Correspondence Address:
Dr. Rahul Suresh Jaiswal
Nehru Nagar, Belagavi, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/kleuhsj.kleuhsj_36_22

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  Abstract 


CONTEXT: Denture relining materials are soft and resilient materials used in the treatment of patients with denture soreness. Studies have concluded that rugged surface of the liners allows the sticking of the Candida albicans and later gives rise to a condition called denture-induced stomatitis.
AIMS: The aim of this study is to evaluate and compare the antifungal efficacy and potency of denture soft liners incorporated with powder extract of Lawsonia inermis and Withania somnifera on the growth of C. albicans.
MATERIALS AND METHODS: A total of 105 samples were taken in this in vitro study and was divided into three groups: To check for the antifungal activity with 35 samples each. L. inermis and W. somnifera powder extract were incorporated in soft liner at 1%, 3%, 5%, and 7% concentration and the antifungal efficacy against C. albicans was tested by calculating the diameter of inhibition zone for 1, 7, and 14 days.
Statistical analysis used: Subgroup analysis was carried out using one-way analysis of variance (ANOVA) test. Pair-wise comparisons were carried out through Tukey's multiple post hoc procedures.
RESULTS: When comparison of four subgroups (1%, 3%, 5%, and 7%) was done with the mean zone of antifungal efficacy at 1st day, 7th day, and 14th day time points in both the groups by one-way ANOVA, statistically significant results were obtained with P of 0.0001.
CONCLUSIONS: L. inermis and W. somnifera natural herb extracts have shown to have antifungal activity against C. albicans and can be used as an alternative for the synthetic antifungal drugs.

Keywords: Antifungal agent, Candida albicans, soft liner


How to cite this article:
Jaiswal RS, Kanathila H, Uppin RC. Comparative evaluation of antifungal efficacy and potency of soft liners incorporated with powder extracts of Lawsonia inermis and Withania somnifera on the growth of Candida albicans – An in vitro study. Indian J Health Sci Biomed Res 2023;16:54-9

How to cite this URL:
Jaiswal RS, Kanathila H, Uppin RC. Comparative evaluation of antifungal efficacy and potency of soft liners incorporated with powder extracts of Lawsonia inermis and Withania somnifera on the growth of Candida albicans – An in vitro study. Indian J Health Sci Biomed Res [serial online] 2023 [cited 2023 Jan 28];16:54-9. Available from: https://www.ijournalhs.org/text.asp?2023/16/1/54/368320




  Introduction Top


The edentulous patient wearing complete denture may need refitting of complete denture after a few years of service. For relining procedure, denture relining material is frequently used in dental clinics. Denture relining materials are soft and resilient materials used in the treatment of patients with denture soreness and also use in the field of “Maxillofacial prosthetics.”[1] Acrylic resin materials are considered a gold standard for the fabrication of dental prosthesis and temporary denture soft liners provide tissue conditioning which will enhance the fit of dentures. However, problem with their inherent rough surface and long-term use of these materials will lead to more plaque accumulation and subsequent microbial adhesion and colonization.[2] These microorganisms can be removed with the help of chemical, mechanical, or using ultraviolet light. However, these methods may cause surface degradation and deformation of soft liner.[3]

Studies have concluded that rugged surface of the liners allow the sticking of the microorganisms and favor fungal growth. Kang et al. found that “acrylic-based denture soft liner” showed a greater adhesion to Candida albicans when compared with silicon-based denture liners. C. albicans, primarily adhere to the intaglio surface of the liner and later give rise to a condition called denture-induced stomatitis.[4] A rise in fungal infection has led to a greater need for antifungal drugs. Antifungal drugs used can be categorized into two groups: Natural and synthetic origins.[5] Synthetic drugs are nystatin, ketoconazole, amphotericin B, miconazole, fluconazole, chlorhexidine, clotrimazole, and itraconazole. Natural agents such as origanum oil, Thai herbs, Clinacanthus nutans, Caesalpinia sappan Linn, lemongrass oil, and seed oils such as Ocimum sanctum Linn and Linum usitatissimum. Many inorganic antifungal agents are also used such as silver zeolite, photocatalyst, silver nanoparticles, and magnesium oxide. However, the use of synthetic drugs over the time has resulted to have many harmful side effects which in need led to the application of herbal medicines in various fields.[6] Hence, there is a need to bring about an alteration in the relining material with herbal antifungal agent. Keeping this background in mind a study is planned to evaluate antifungal property of acrylic-based soft relining material by incorporating herbal extracts which will be beneficial with less adverse effects.


  Materials and Methods Top


Ethical Clearance was obtained from ethical committee of KLE VK Institute of Dental Sciences, Belagavi with Ref no MDC 2019/1328 dated 08/07/2019. A total of 108 samples were taken to assess the antifungal efficacy. These samples were divided into three groups as:
  • Group A: Control group (soft liner powder and liquid)
  • Group B: Lawsonia inermis leaf powder incorporated in soft liner.
  • Group C: Withania somnifera root powder incorporated in soft liner.


Each of these groups was further divided into four subgroups of concentration 1%, 3%, 5%, and 7% concentration with 9 samples each. Extract authenticated from indian council of medical research, Belagavi, with powder fineness <180 μm was included in the study. Powder fineness more than 180 μm were excluded from the study.

Procedure

Preparation of powder extract of henna and Ashwagandha

  • For the preparation of powder of L. inermis (Henna) and W. somnifera (Ashwagandha), the plants were collected from Ayurvedic College and were authenticated by the botanist from the “National Institute of Traditional Medicine, Belagavi.” Then, the leaves and roots were cleaned and dried until there is no moisture content
  • Dried leaves and roots were then ground into fine powder of size 180 μm using No. 180 sieve having 0.180 mm size of the aperture. Powder was sieved to achieve even powder size [Figure 1].
Figure 1: Powder extract of henna and Ashwagandha

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Preparation of composite mixture

GC soft liner polymer was taken as control in this study (Group A). The powder extracts of L. inermis and W. somnifera (Group B and Group C, respectively) were mixed with soft liner polymer at 1wt%, 3wt%, 5wt%, and 7wt% concentration. Then, Group B (powder extract of L. inermis leaves + soft liner polymer) and Group C (powder extract of W. somnifera root + soft liner polymer) mixture bottle were shook for 10 min to achieve homogeneous mix.

Susceptibility test procedure

C. albicans was obtained from KLE's “Dr. Prabhakar kore Basic Science Research Center, Belagavi” [Figure 2]. Using Sabouraud dextrose agar on 90 mm diameter Petri plates, antifungal susceptibility was assessed using the well diffusion technique. The inoculum was then smeared onto the culture plates. A sterile cork borer was used to punch a 6 mm diameter and 5 mm depth well once the inoculum has dried. Then, Group B and Group C mixtures were mixed separately with monomer of soft liner according to manufacturer instructions and placed into the punched wells on Sabouraud dextrose agar. Group A (Control Group) was mixed the same as that for Group B and Group C. Then, agar plates were incubated at 37°C ± 1°C for 14 days. The diameter of the inhibition zone (DIZ) will be measured with metallic scale after 1st, 7th, and 14th day.
Figure 2: ATCC strain of Candida albicans

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  Results Top


Data from the study were entered into a Microsoft Excel spreadsheet, and the statistical analysis was performed using IBM SPSS version 20 software International Business Machines Corporation (IBM) Armonk, New York. To explain the data's underlying structure, descriptive statistics were used.

Independent t-test was used for comparison of two groups with mean zone of inhibition in millimeter (mm) at different time intervals and pair-wise comparison of four subgroups (1%, 3%, 5%, and 7%) with mean zone of inhibition scores at different time points by Tukey's multiple post hoc procedures. Dependent t-test was used for the comparison of 1st day, 7th day and 14th day time points with mean zone of inhibition scores in two study groups. Comparison of different times (1st, 7th, and 14th day) in each subgroup of Group B and Group C were done using paired t-test. Control Group A had complete growth of Candida and therefore it was not considered in the analyses [Figure 3].
Figure 3: Control group after 14 days

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The mean value (DIZ) for the L. inermis groups at 1% concentration for the 1st day, 7th day, and 14th day were 1.78, 0.89, and 0.17 (0 means complete Candida growth), respectively. The mean value (DIZ) for the W. somnifera group at 1% concentration for the 1st day, 7th day, and 14th day were 4.11, 3.17, and 1.67, respectively. The mean value (DIZ) for the L. inermis groups at 3% concentration for the 1st day, 7th day, and 14th day were 3.44, 2.33, and 1.22, respectively. The mean value (DIZ) for the W. somnifera group at 3% the concentration for 1st day, 7th day, and 14th day were 6.22, 5.06, and 3.28, respectively. The mean value (DIZ) for the L. inermis groups at 5% concentration for the 1st day, 7th day, and 14th day were 7.39, 5.44, and 3.22, respectively. The mean value (DIZ) for the W. somnifera group at 5% concentration for the 1st day, 7th day, and 14th day were 8.39, 6.89, and 4.83, respectively. The mean value (DIZ) for the L. inermis groups at 7% concentration for the 1st day, 7th day, and 14th day were 10.61, 9.22, and 7.00, respectively [Figure 4]. The mean value (DIZ) for the W. somnifera group at 7% concentration for the 1st day, 7th day, and 14th day were 11.94, 10.28, and 7.61, respectively [Figure 5]. Thus, the W. somnifera Group C exhibited more antifungal activity as compared to the L. inermis Group B.
Figure 4: Diameter zone of inhibition of Group B (Softliner + Henna) after 14 days

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Figure 5: Diameter zone of inhibition of Group B (Softliner + Ashwagandha) after 14 days

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When comparison of four subgroups (1%, 3%, 5%, and 7%) was done with the mean zone of antifungal efficacy at 1st day, 7th day, and 14th day time points in both Group B and Group C by one-way ANOVA, statistically significant results were obtained with P of 0.0001 [Table 1] and [Table 2]., With the increase in the concentration of L. inermis and W. somnifera, antifungal activity increases, but with the increase in day point time, antifungal activity decreased in each group. When pair-wise comparison was done for four subgroups (1%, 3%, 5%, and 7%) with mean zone of antifungal efficacy at 1st day, 7th day, and 14th day time points in Group B and Group C by Tukey's multiple post hoc procedures, significant results (P of 0.0001) were obtained [Figure 6] and [Figure 7]. Higher the concentration of test sample, higher the zone of antifungal efficacy was noted.
Table 1: Comparison of four subgroups (1%, 3%, 5%, and 7%) with mean zone of antifungal efficacy at 1st day, 7th day, and 14th day time points in Group B by one-way ANOVA

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Table 2: Comparison of four subgroups (1%, 3%, 5%, and 7%) with mean zone of antifungal efficacy at 1st day, 7th day, and 14th day time points in Group C by one-way ANOVA

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Figure 6: Comparison of Group B and Group C in four subgroups (1%, 3%, 5%, and 7%) with mean zone of antifungal efficacy at 1st day, 7th day, and 14th day time points

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Figure 7: Comparison of four subgroups (1%, 3%, 5%, and 7%) with mean zone of antifungal efficacy at 1st day, 7th day, and 14th day time points in Group B and Group C

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Antifungal activity of both Group B and C was indirectly proportional to the time period [Figure 8]. With the progressing time period, the antifungal activity was found to be less effective when compared with the last time period. Although antifungal efficacy was noted in both the groups at progressive time interval, higher concentrations of these herbal antifungal agents showed more efficacies. Furthermore, the efficacy noted on the 1st day was statistically significant when compared with the 7th and 14th days.
Figure 8: Comparison of different treatment times in each subgroup of Group B and Group C

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  Discussion Top


The treatment of Candida infections is currently challenging owing to the inadequate number of available drugs, increased resistance to these drugs, high costs, and toxicity. Denture-induced candidiasis has been the most repeatedly occurring oral mucosal infection which is mostly concerned with the use of a removable dental prosthesis and is seen in almost 11%–70% of all the denture wearers.[7]

Denture soft liners are generally used for conditioning the denture-supporting mucosa which is inflamed by ill-fitting dentures. They lower the stress generated by denture and act as a cushion.[8] However, in return, they provide hub for intraoral microorganism because of increased surface roughness when compared with denture surface.[9] It is found that denture soft liners do not exhibit any antifungal activity. Conventional treatment option for denture stomatitis includes topical application of commercially available antifungal agents.[10] These commercially available drugs have side effects such as rise in the appearance of resistance. Many studies have incorporated herbal extract into denture soft liner and concluded improved efficacy against the fungal growth with no side effects such as resistance development.[7] Therefore, incorporation of antifungal medicament into the denture soft liners is done to treat denture-induced candidiasis.

In this study, herbal medicines such as L. inermis (Henna) leaf and W. somnifera (Ashwagandha) root powder extract have been incorporated into the denture soft liner and were tested against the C. albicans growth for its antifungal efficacy to 14 days. Incorporation of these powders into the resilient soft lining material resulted to have an antifungal effect and thus rejecting the null hypothesis of the study.

”Henna possesses analgesic, hypoglycemic, hepatoprotective, immunostimulant, anti-inflammatory, antibacterial, antimicrobial, antiviral, antiparasitic, antidermatophytic, antioxidant, antifertility, tuberculostatic, and anticancer effects, according to a phytopharmacological study published in 2010.”[11] Ashwagandha (W. somnifera Dunal) is widely utilized in Indian herbal medicines and nutraceuticals. It's an annual herb that grows wild in dry, arid soil and is well-known for immunomodulation and antiaging in Ayurveda, according to the ancient Indian school of plant medicine. In addition, W. somnifera has anti-inflammatory, antitumor, anti-radiosensitizing, and analgesic properties.[12]

The anticandidal activities of L. inermis (henna) in paste form were assessed using the agar diffusion technique in a research study. Henna demonstrated the most potent antifungal action (20 mm inhibition zone) against 68 (35.4%) clinical candida isolates, whereas 73 (38.0%) isolates had moderate activity (5–15 mm inhibition zone). Henna paste resistance was found in 51 (26.5%) isolates (no inhibition zone).[13]

The main goal of the study was to evaluate and check the antifungal efficacy of henna and Ashwagandha for longer duration. Leachable antifungals such as essential oils or water-soluble synthetic antifungal drugs might not last for longer time, but these herbal powder extracts will serve the purpose as these get embedded within the polymeric matrix of soft liner, thus contributing to a longer duration of antifungal activity. Furthermore, the finer particle size of these powder extracts will not impact the surface properties of the soft liner coating.[14]

In this study, henna with a concentration of more than 5% and Ashwagandha with a concentration of more than 3% showed considerable antifungal efficacy to the 14th day. Both of these natural antifungal medicines can be used to treat fungal infections caused by C. albicans for short-term requirements.


  Conclusions Top


  • Inhibition of C. albicans varies significantly by varying the concentration of herbal extracts. Increasing the concentration of these herbal extracts increased the zone of inhibition
  • Antifungal activity decreases with the time at all concentrations. 1% concentration of these extracts has minimal efficacy than 7% against C. albicans
  • Ashwagandha root powder extract was superior to henna leaf extract at all concentrations along with the time mentioned in the study.


Incorporation of these powder extracts into the denture soft liner could prove to be beneficial to improve the oral health status of geriatric patients with cognitive disturbances, medically compromised conditions, and reduced manual dexterity thus minimizing the severity of oral candidiasis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Shay K. Denture hygiene: A review and update. J Contemp Dent Pract 2000;1:28-41.  Back to cited text no. 1
    
2.
Webb B, Thomas C, Wilcox M, Harty D, Knox K. Candida associated denture stomatitis etiology and management: A review. Part 2. Factors influencing distribution of Candida species in the oral cavity. Aust Dent J 1998;43:45-50.  Back to cited text no. 2
    
3.
Muttagi S, Subramanya JK. Effect of incorporating seed oils on the antifungal property, surface roughness, wettability, weight change, and glucose sorption of a soft liner. J Prosthet Dent 2017;117:178-85.  Back to cited text no. 3
    
4.
Sivakumar I, Arunachalam KS, Sajjan S, Ramaraju AV, Rao B, Kamaraj B. Incorporation of antimicrobial macromolecules in acrylic denture base resins: A research composition and update. J Prosthodont 2014;23:284-90.  Back to cited text no. 4
    
5.
Skupien JA, Valentini F, Boscato N, Pereira-Cenci T. Prevention and treatment of Candida colonization on denture liners: A systematic review. J Prosthet Dent 2013;110:356-62.  Back to cited text no. 5
    
6.
Rogers TR. Antifungal drug resistance: Does it matter? Int J Infect Dis 2002;6 Suppl 1:S47-53.  Back to cited text no. 6
    
7.
Emami E, Kabawat M, Rompre PH, Feine JS. Linking evidence to treatment for denture stomatitis: A meta-analysis of randomized controlled trials. J Dent 2014;42:99-106.  Back to cited text no. 7
    
8.
Graham BS, Jones DW, Burke J, Thompson JP. In vivo fungal presence and growth on two resilient denture liners. J Prosthet Dent 1991;65:528-32.  Back to cited text no. 8
    
9.
Iqbal Z, Zafar MS. Role of antifungal medicaments added to tissue conditioners: A systematic review. J Prosthodont Res 2016;60:231-9.  Back to cited text no. 9
    
10.
Lee HL, Wang RS, Hsu YC, Chuang CC, Chan HR, Chiu HC, et al. Antifungal effect of tissue conditioners containing poly (acryloyloxyethyltrimethyl ammonium chloride)-grafted chitosan on Candida albicans growth in vitro. J Dent Sci 2018;13:160-6.  Back to cited text no. 10
    
11.
Kamal M. Pharmacological activities of Lawsonia inermis Linn: A review. Molecules 2010;15:2139-51.  Back to cited text no. 11
    
12.
Alam N, Hossain M, Mottalib MA, Sulaiman SA, Gan SH, Khalil MI. Methanolic extracts of Withania somnifera leaves, fruits and roots possess antioxidant properties and antibacterial activities. BMC Complement Altern Med 2012;12:175.  Back to cited text no. 12
    
13.
Yiğit D. Antifungal activity of Lawsonia inermis L. (Henna) against clinical Candida isolates. Erzincan Universitesi Fen Bilimleri Ensitusu Dergisi 2017;10:196-202.  Back to cited text no. 13
    
14.
Webb BC, Thomas CJ, Willcox MD, Harty DW, Knox KW. Candida-associated denture stomatitis. Aetiology and management: A review. Part 1. Factors influencing distribution of Candida species in the oral cavity. Aust Dent J 1998;43:45-50.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2]



 

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