comparative evaluation of the substantivity of …
TRANSCRIPT
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Soneta et al. World Journal of Pharmacy and Pharmaceutical Sciences
COMPARATIVE EVALUATION OF THE SUBSTANTIVITY OF
VARIOUS CONCENTRATION OF A PLANT BASED IRRIGANT ON
CANDIDA ALBICANS. – IN VITRO
Sanjana Pramod Soneta*, Dr. Shalini Aggarwal1, Dr. Anamika Borkar
1,
Dr. Karan Bhargava2, Dr. Anita Tandale
1, Dr. Nikhil Borse
3
*Intern, D.Y. Patil Vidyapeeth, Pune.
1Professor, D.Y. Patil Vidyapeeth, Pune.
2Lecturer, D.Y. Patil Vidyapeeth, Pune.
3Lecturer, S.M.B.T Dental College Ghoti, Nasik.
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
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 6.041
Volume 5, Issue 6, 1835-1847 Research Article ISSN 2278 – 4357
*Corresponding Author
Sanjana Pramod Soneta
Intern, D.Y. Patil
Vidyapeeth, Pune.
Article Received on
14 April 2016,
Revised on 04 May 2016,
Accepted on 25 May 2016
DOI: 10.20959/wjpps20166-6992
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Soneta et al. World Journal of Pharmacy and Pharmaceutical Sciences
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-
oxidant; it also assists in the smooth functioning of the gastrointestinal tract.[7, 8, 9]
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-L-
rhamnopyranosyl-(1→6)-β-D-glucopyranoside.)[11]
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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
drug addiction.[12, 13]
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 36o for 24hrs, 48 hrs, and 3 days.
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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).
Zone of inhibition (mm)
Group I-
Morinda
citrifolia (n=6)
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±1.08 16.83±0.75 36.18±0.49 0.83±0.41
IInd day 33.67±0.60 17.92±0.74 37.0±0.63 0.92±0.49
IIIrd day 35.08±0.37 19.08±0.66 37.80±0.30 1.08±0.37
Student’s Paired
‘t’ test value 9.49 61.25 20.89 15.31
‘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
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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
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Group IV: Negative control Saline.
Day 1 Day2 Day3
Image No-4
The results seen were statistically significant, at the end of 1st, 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
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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.
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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
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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 (1st, 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)
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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. 2014-04352)
REFERENCES
1. Deivanayagam Kandaswamy and Nagendrababu Venkateshbabu: .Root Canal Irrigants.
Journal Of Conservative Dentistry., 2010; 13(4): 256–264.
2. 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: 658-66.
3. Zehnder M. Root Canal Irrigants. J Endod., 2006; 32(5): 389-98.
4. Spangberg L, Engstrom B, Langeland K. Biologic effects of dental material. Oral Surg
Oral Med Oral Pathol., 1973; 36: 856-71.
www.wjpps.com Vol 5, Issue 6, 2016.
1846
Soneta et al. World Journal of Pharmacy and Pharmaceutical Sciences
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: 424-8.
6. McCome D, Smith DC. A preliminary scanning electron microscopic study of root canals
after endodontic procedures. J Endod., 1975; 1: 238-42.
7. 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: 394-8.
8. Peter E. Murray et al. Evaluation of Morinda citrifolia as an Endodontic Irrigant.Journal
Of Endodontics., 2008; 34: 66-70.
9. 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): 555-59.
10. 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: 394-398.
11. Shixin Deng et al. Phytochemical, Antioxidant and Toxicology Investigation of Morinda
Citrifolia L. Blossoms, ISRN Analytic Chemistry., 2012; 1-5.
12. 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: 66-67
13. 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: 29–33.
15. 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: 352–355.
16. Gomes et al. microbiological examination of infected dental root canal, oral microbial
immunol., 2004 Apr; 19(2): 71-6.
17. Elka Radeva, B. Indjov, R. Vacheva. In vitro study of the effectiveness of intracanal
irrigant on candida albicans , Journal of IMAB., 2007; 13(2): 3-7.
www.wjpps.com Vol 5, Issue 6, 2016.
1847
Soneta et al. World Journal of Pharmacy and Pharmaceutical Sciences
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: 170–5.
19. Whistler, W. A. Polynesian Herbal Medicine. Lawai, Kaua’i, Hawai’i. National Tropical
Botanical Garden., 1992.
20. Selvam et al. Studies of antiviral activity and cytotoxicity of Wrightia tinctoria and
Morinda citrifolia. Indian J Pharm Sci., 2009 Nov-Dec; 71(6): 670-672.
21. Vennila Srinivasahan and Brindha Durairaj. Antimicrobial activities of hydroethanolic
extract of Morinda citrifolia fruit. Int. J. Curr. Microbiol., 2014; 3(9): 26-33.