COMPARATIVE EVALUATION OF THE SUBSTANTIVITY OF VARIOUS CONCENTRATION OF A PLANT BASED IRRIGANT ON CANDIDA ALBICANS. IN VITRO

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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Soneta et al. SJIF Impact Factor 6.

1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Soneta et al. SJIF Impact Factor Volume 5, Issue 6, Research Article ISSN COMPARATIVE EVALUATION OF THE SUBSTANTIVITY OF VARIOUS CONCENTRATION OF A PLANT BASED IRRIGANT ON CANDIDA ALBICANS. IN VITRO Sanjana Pramod Soneta*, Dr. Shalini Aggarwal 1, Dr. Anamika Borkar 1, Dr. Karan Bhargava 2, Dr. Anita Tandale 1, Dr. Nikhil Borse 3 *Intern, D.Y. Patil Vidyapeeth, Pune. 1 Professor, D.Y. Patil Vidyapeeth, Pune. 2 Lecturer, D.Y. Patil Vidyapeeth, Pune. 3 Lecturer, S.M.B.T Dental College Ghoti, Nasik. Article Received on 14 April 2016, Revised on 04 May 2016, Accepted on 25 May 2016 DOI: /wjpps *Corresponding Author Sanjana Pramod Soneta Intern, D.Y. Patil Vidyapeeth, Pune. ABSTRACT Aim: Evaluation of the substantivity of a plant based irrigant with and without Chitosan on Candida albicans was evaluated. Material and Methods: Twenty four teeth were selected after radiological evaluation, decoronated at cementoenamel junction and were distributed equally into four groups. Root canal procedure was done and proper irrigation protocol was followed by using 5ml of 3% Sodium Hypochlorite after every change of instrument, subsequently 17% EDTA was also used during cleaning and shaping. Physiologic Saline was used using endodontic needle to irrigate the canal after each file change. Last irrigant used was 6% Morinda citrifolia for 30min in the canal. Dentinal discs were prepared. Results: The results showed that the zone of inhibition was best in Group III which includes Sodium Hypochlorite followed by Group I (Morinda Citrifolia) and then Group II (Morinda Citrifolia & Chitosan). KEYWORDS: Morinda Citrifolia, Chitosan, Candida albicans, root canal disinfectant. INTRODUCTION Successful root canal therapy is an outcome of proper instrumentation, irrigation, and obturation of the root canal. Of all the procedures done during root canal therapy, irrigation of the root canal is most important. The effectiveness of the irrigation protocol is evaluated Vol 5, Issue 6,

2 according to the healing of the periapical tissues. Thus the goal of the primary endodontic treatment must be to optimize root canal disinfection and to prevent reinfection. [1] An ideal root canal irrigant [2, 3] has the following properties. Helps effectively disinfect and clean the root canal system, be able to disinfect and penetrate the dentin and its tubules, have long term antibacterial effect (substantivity), remove smear layer, have no adverse effect on dentin or the sealing ability of filling materials, be inexpensive, conventional to apply, and cause no tooth discoloration, be able to dissolve pulp tissue and inactivate endotoxins. Most commonly used irrigants these days are Sodium hypochlorite and Chlorhexidine bigluconate, Dequalinium acetate, Ethylenediamine tetraacetic acid, Citric acid etc. Sodium Hypochlorite has remained a popular root canal irrigant because of its ability to dissolve organic matter. [4] The disadvantages of this irrigant are burning of the oral mucosa, unpleasant taste, high toxicity, corrosion of instrument [5], inability to remove the smear layer [6], periapical irritation if extruded, and its incapability of imparting any long term sustained effect (Substantivity) to the radicular dentin. Lots of studies and research has been done on herbal irrigants that have been explored as potential endodontic irrigants, These are: Neem, Triphala, Green Tea, Polyphenols, Turmeric and Morinda citrifolia. Morinda citrifolia is a fruit with proven antifungal properties, removes smear layer, is antibacterial and has anti-inflammatory properties, owing to the fact that it is a powerful anti- [7, 8, 9] oxidant; it also assists in the smooth functioning of the gastrointestinal tract. It is routinely used as an Over the Counter drug formulation, as a generic well being drug which requires no prescription. Morinda was introduced as an irrigant for the root canal system in 2008 by Aree Jainkittivong. [10] The chemical formula of Noni is (1-deacetylasperulosidic acid; 2-asperulosidic acid; 3- quercetin-3-o-α-l-rhamnopyranosyl-(1 6)-β-D-glucopyranoside; 4-kaempferol-3-O-α-Lrhamnopyranosyl-(1 6)-β-D-glucopyranoside.) [11] Vol 5, Issue 6,

3 Morinda Citrifolia is used in patients with diseases such as, diabetes, muscle aches, heart diseases, cancer, gastric ulcers, arthritis, blood vessel problems menstrual difficulties, and [12, 13] drug addiction. Chitosan is a non toxic biopolymer derived by the deacetylation of chitin. Chitin is a naturally occurring polymer in the exoskeleton of crustaceans. It is a bioadhesive material that readily binds to negatively charged surfaces and has admirable antimicrobial and antifungal activities. [14] In this study we have checked the substantivity of Morinda citrifolia with and without the addition of Chitosan on dentin. MATERIALS AND METHODS METHODOLOGY Tooth Preparation Twenty- four extracted human permanent teeth were selected for this study. Each tooth was digitally radiographed in both mesiodistal and buccolingual direction to confirm absence of fracture. The teeth were stored in Saline until use. Teeth were decoronated at the cementoenamel junction and pulpal remnants were extirpated using barbed broaches. Each root canal was enlarged using the protaper nickel titanium rotary system up to size 30 at 1mm from apical foramen. Proper irrigation protocol was followed by using 5ml of 3% Sodium Hypochlorite after every change of instrument, subsequently 17% EDTA was used during cleaning and shaping. Physiologic Saline was used to perform irrigation using an endodontic needle after each file change. Last irrigant used was 6% Morinda citrifolia, for 30min in the root canal. (12, 13, 15) The canals were then dried with paper points. Dentinal discs were prepared from the root portion of the tooth. CULTIVATION AND INOCULATION OF BACTERIA Reference strains of Candida albicans was obtained from National Chemical Laboratory (ATCC 10231). Dentinal discs were placed in Sabouraud Dextrose Agar cultivated with Candida albicans. The plates were incubated at 36 o for 24hrs, 48 hrs, and 3 days. Vol 5, Issue 6,

The antifungal activity was checked at the end of 24 hrs, 48 hrs and on the 3rd day. The antifungal activity of the test material was checked by measuring the diameters of Zones of Inhibition.

4 The antifungal activity was checked at the end of 24 hrs, 48 hrs and on the 3rd day. The antifungal activity of the test material was checked by measuring the diameters of Zones of Inhibition. RESULTS STATISTICAL ANALYSIS Table No.1: Comparison of mean and SD values of Zone of inhibition (mm) in Group Morinda citrifolia, Group- Morinda Citrifolia +Chitosan, Group-NaOCl and Group- Negative Control (n=6). Group I- Morinda citrifolia (n=6) Zone of inhibition (mm) Group II- Morinda Citrifolia +Chitosan (n=6) Group III- NaOCl (n=6) Group IV- Negative Control (n=6) Mean ± SD Mean ± SD Mean ± SD Mean ± SD Ist day 32.33± ± ± ±0.41 IInd day 33.67± ± ± ±0.49 IIIrd day 35.08± ± ± ±0.37 Student s Paired t test value p value and significance p<0.01, highly significant p<0.01, highly significant p<0.01, highly significant p<0.01, highly significant Graph No: 1 Vol 5, Issue 6,

5 Group I: Morinda citrifolia. Day1 Day2 Day3 Image No-1 Group II: Morinda citrifolia and Chitosan. Day1 Day2 Day3 Image No-2 Group III: Positive control Sodium hypochlorite. Day1 Day 2 Day3 Image No-3 Vol 5, Issue 6,

day Group III (Sodium Hypochlorite) showed maximum Zone of Inhibition followed by Group I (Morinda

6 Group IV: Negative control Saline. Day 1 Day2 Day3 Image No-4 The results seen were statistically significant, at the end of 1 st, 2 nd & 3 rd day Group III (Sodium Hypochlorite) showed maximum Zone of Inhibition followed by Group I (Morinda citrifolia), Group II (Morinda citrifolia and Chitosan) and Group IV (Saline) showed the least Zone of Inhibition. (Graph no:1) Other Supporting Matter Vol 5, Issue 6,

7 Vol 5, Issue 6,

DISCUSSION Endodontic infections are subsume of all types of microbes including Bacteria, Fungi and Viruses. The root canal space facilitates the growth of many micro-organisms.

8 DISCUSSION Endodontic infections are subsume of all types of microbes including Bacteria, Fungi and Viruses. The root canal space facilitates the growth of many micro-organisms. Prevotella species such as P.nigrescens and P.intermedia (16.7%) are often found in primary endodontic cases followed by Porphyromonas gingivalis(6.7%), Porphyromonas endodontalis(5%), Fusobacterium nucleatum(11.7%), and other species. [16] In secondary infected root canal the microflora most commonly found are Candida albicans (65%), Enterococcus Faecalis (24%-70%), Lactobacilli (25%), Actinomyces species (26.5%), etc. Vol 5, Issue 6,

9 There are several irrigants which are used for root canal treatment such as Sodium Hypochlorite (1%-5.25%), Chlorhexidine (0.2%-2%), Ethylenediamine Tetraacetic Acid (10%-17%), Citric acid (10%-50%). [3] Amongst them sodium hypochlorite is considered as a gold standard. Sodium Hypochlorite has many disadvantages as discussed in introduction. The major disadvantage is that it is not effective on the micro flora present in retreatment cases (i.e. Candida albicans). [17] Candida albicans and its spores are the most common fungi isolated from failed endodontic cases. [16] The constant rise in antibiotic resistant species and adverse effects that are a part of the usage of synthetic drugs has necessitated research for herbal alternatives such as Propolis, Morinda citrifolia and Azadirachta indica (Neem) which are less damaging to the surrounding tissue. In secondarily infected root canals, there are some microorganisms (Candida albicans, E. Faecalis) that show more resistance to chemical and mechanical procedures and this must be taken into account when using various endodontic irrigant and intracanal medicaments. With the reduction of concentration, the effectiveness of NaOCl against Candida albicans falls significantly. [17] The use of Sodium Hypochlorite in root canal treatment was promoted by a series of researchers, (A. Bystrom and G. Suvqvist 1985) documenting the antimicrobial efficacy of Sodium Hypochlorite in human root canals. [18] Some researchers suggest that the antimicrobial effectiveness of Sodium Hypochlorite is based upon its high ph (hydroxyl ions action) which is alike the mechanism of action of Calcium Hydroxide. The high ph of Sodium Hypochlorite interferes with the integrity of the cytoplasmic membrane with an irreversible enzymatic inhibition. Biosynthetic adaptation in cellular metabolism and phospho-lipid degradation observed in lipidic peroxidation and hence it should ideally occupy the root canal space with maximum density and depth to the working length in order to permit its biological effects to act in closest proximity to the appropriate tissues. [17] Recently, the ability of Sodium hydroxide irrigation to eradicate fungal species completely from the root canal has been questioned. For example, ex vivo studies have shown those Vol 5, Issue 6,

10 candidal strains are resistant to NaOCl (C.E.Radcliffe et. al 2004), reduction in concentration of NaOCl, and the efficacy of NaOCl against Candida albicans is reduced significantly. Substantivity is termed as the property of continuing the therapeutic action despite removal of vehicle. The substantivity of Sodium Hypochlorite is less therefore there is a need for newer root canal irrigant which has better substantivity and which can overcome the disadvantages of Sodium Hypochlorite. There are many researches being carried out to get a better root canal irrigant. Morinda citrifolia is assumed to show a better result and therefore it is also being studied as a root canal irrigant. Morinda citrifolia is an important medicinal plant for many centuries throughout the South Pacific and was used in remedies by Polynesians over 2000 years (Whistler, 1992) [19] and extracts of fruit, leaf and stem demonstrate remarkable antibacterial activity (Selvam et al., 2009). [20] It is reported to have anti-bacterial, antiviral, antifungal, antihelmintic, analgesic, anti-inflammatory hypotensive and immune enhancing effects. The fruit juice is used in treatment of different kinds of illnesses such as arthritis, diabetes, blood vessel problems, heart diseases, cancers, menstrual difficulties, gastric ulcers, muscle aches and drug addiction. M. citrifolia has been classified as an ayurvedic herb due to its therapeutic properties. Different parts of the stems, plant, leaves, including roots and fruit have been consumed purely on the basis of the supposition that it possesses healing properties. In a study, M. citrifolia (fruit, leaf and stem) antibacterial property has been evaluated and the results displayed the effect on the growth of most of the tested bacteria and MIC values auspiciously govern that the plant parts of M. citrifolia can be utilized in the treatment of infectious disease. The minimum inhibitory concentration (MIC) of Morinda Citrifolia is 50µg/ml, which means it has a good inhibitory effect. [21] In each group the best Zone of Inhibition was found on day 3 and the worst Zone of Inhibition was found on day 1 and these were statistically significant. This shows the substantivity of Morinda citrifolia. At the end of each day (1 st, 2 nd &3 rd day) Zones of Inhibition demonstrated by Group III were the best when compared to other groups and the worst Zones of Inhibition was given by group IV at the end of each day. All these results were statistically significant.(graph No 1) Vol 5, Issue 6,

11 Chitosan did not do well with Morinda citrifolia. This may be due to antagonistic action happening between Morinda citrifolia and Chitosan, and study in this direction needs further investigation. CONCLUSION The results showed that the Zone of Inhibition was best in Group III which includes Sodium Hypochlorite at the end of 24 hours, 48 hrs & 72 hrs. Group I which includes Morinda citrifolia showed good Zone of Inhibition at the end of 24 hours, 48 hours and 72 hours. Group II Morinda citrifolia + Chitosan showed a moderate Zone of Inhibition which was lower than expected. At the end of 72 hours Group I and group III almost showed equal substantivity. Group IV Saline did not show any Zone of Inhibition against Candida albicans. According to the result the Zone of Inhibition was best in group III Sodium hypochlorite but Group I Morinda citrifolia showed almost equal Zone of Inhibition. Morinda citrifolia can be used as an irrigant due to its ability to inhibit Candida albicans and it has few disadvantages as compared to Sodium hypochlorite. Morinda citrifolia needs to be subjected to future microbiological study. The reinfection of root canal occurs after the period of 3-6 months, the infection could spread between a period of 1 to 10 years before the symptoms appear. So we should check the effect of Morinda citrifolia in retreatment cases and further studies are mandatory to access the substantivity for a longer span of time. ACKNOWLEDGEMENTS I would like to thank ICMR for selecting and funding my research (Ref. No ) REFERENCES 1. Deivanayagam Kandaswamy and Nagendrababu Venkateshbabu:.Root Canal Irrigants. Journal Of Conservative Dentistry., 2010; 13(4): Torabinejad M, Handysides R, Khademi A, Bakland LK. Clinical implications of the smear layer in Endodontics: A review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod., 2002; 94: Zehnder M. Root Canal Irrigants. J Endod., 2006; 32(5): Spangberg L, Engstrom B, Langeland K. Biologic effects of dental material. Oral Surg Oral Med Oral Pathol., 1973; 36: Vol 5, Issue 6,

12 5. Gomes BP, Ferraz CCR, Vianna ME, Berber VB, Teixeira FB, de Souza-Filho FJ. In vitro Antimicrobial activity of several concentrations of Sodium Hypochlorite and Chlorhexidine Gluconate in the elimination of Enterococcus faecalis. Int Endod J., 2001; 34: McCome D, Smith DC. A preliminary scanning electron microscopic study of root canals after endodontic procedures. J Endod., 1975; 1: Jainkittivong A, Butsarakamuha T, Langlais RP. Antifungal activity of Morinda Citrifolia fruit extract against Candida albicans. oral surgery oral medicine oral pathology oral radiology endodontology., 2009; 108: Peter E. Murray et al. Evaluation of Morinda citrifolia as an Endodontic Irrigant.Journal Of Endodontics., 2008; 34: Marcio Carneiro Valera et al. Antimicrobial Activity of Sodium Hypochlorite Associated with Intracanal Medication for Candida albicans and Enterococcus Faecalis Innoculated in root canals, J Appl Oral Sci., 2009; 17(6): Aree Jainkittiong et al. Antifungal activity of Morinda Citrifolia fruit extract against Candida albicans. Oral Surgery Oral Medicine Oral Pathology Oral Radiology Endodontology., 2008 april; 3: Shixin Deng et al. Phytochemical, Antioxidant and Toxicology Investigation of Morinda Citrifolia L. Blossoms, ISRN Analytic Chemistry., 2012; Peter E. Murray, Romi M. Farber, Kenneth N.Namerow and Franklin Garica-Gooy. Evaluation of Morinda Citrifolia as an Endodontic Irrigant. JOE., 2008 Nov; 34: Hanna Rosaline, D Kandaswamy, D Gogulnath and MI Rubin. Influence of various herbal irrigants as a final rinse on adherence of E.faecalis by florescence Confocal laser scanning microscope. 14. N. V. Ballah et al. Susceptibility of Candida albicans and Enterococcus Faecalis to Chitosan, Chlorhexidine gluconate and their combination-in vitro, Aust Endod J., 2009; 35: Hannah Rosaline, D Kandaswamy, D Gogulnath and MI Rubin. Influence of various herbal irrigant as a final rinse. J. Conservative Dentistry., 2013 Jul-Aug; 16: Gomes et al. microbiological examination of infected dental root canal, oral microbial immunol., 2004 Apr; 19(2): Elka Radeva, B. Indjov, R. Vacheva. In vitro study of the effectiveness of intracanal irrigant on candida albicans, Journal of IMAB., 2007; 13(2): Vol 5, Issue 6,

13 18. Bystrom A, Claesson R, Sundqvist G. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Endod Dent Traumatol., 1985; 1: Whistler, W. A. Polynesian Herbal Medicine. Lawai, Kaua i, Hawai i. National Tropical Botanical Garden., Selvam et al. Studies of antiviral activity and cytotoxicity of Wrightia tinctoria and Morinda citrifolia. Indian J Pharm Sci., 2009 Nov-Dec; 71(6): Vennila Srinivasahan and Brindha Durairaj. Antimicrobial activities of hydroethanolic extract of Morinda citrifolia fruit. Int. J. Curr. Microbiol., 2014; 3(9): Vol 5, Issue 6,

Int.J.Curr.Microbiol.App.Sci (2018) 7(12):

Int.J.Curr.Microbiol.App.Sci (2018) 7(12): International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 12 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.712.044