IJCRR

IJCRR - 9(2), January, 2017

Pages: 09-13

Date of Publication: 20-Jan-2017

A Comparative Evaluation of the Efficacy of Andrographis Paniculata, 2% Chlorhexidine and 5.25% Sodium Hypochlorite Against Enterococcus Faecalis- An Invitro Study

Author: Ravi Varman C.1, Thillainayagam S.2, Dinesh D.S.3, Jaisenthil A.4, Bharath N.B.5, Karthikeyan S.6

Category: Healthcare

Abstract:Aim: To evaluate and compare the antimicrobial efficacy of 0.5% phytochemical extracts of Andrographis Paniculata (AP), 2% Chlorhexidine (CHX) and 5.25% Sodium hypochlorite (NaOCl) as an endodontic irrigant against Enterococcus faecalis.
Methodology: Forty freshly extracted human single rooted mandibular premolars were selected, decoronated and standardized to 12mm root length. Cleaning and shaping was done using ProTaper\? (Dentsply) up to size F3 and irrigated with 3% NaOCl. Contamination of the specimens was carried out for 21 days at 37ºC. The specimens were then divided into four groups (n=10) for irrigation with 0.5% AP, 2% CHX, 5.25% NaOCl and distilled water as control. Before and after irrigation, microbiological samples were collected using sterilized paper points, and efficacy was expressed as reduction in microbial load using Colony Forming Units. Statistical analysis was performed using One-way Anova/Kruskal-Wallis test with Tukey's Post Hoc test.
Result: In the root canals, the 0.5% herbal extract of AP was found to be equally effective to 2% CHX and highly effective than 5.25% NaOCl against Enterococcus faecalis with a marked decrease in the number of CFU at the 48th hr. from 2.6 x10³ to 0.9 x10³. The values were statistically significant with p< 0.5.
Conclusion: Being a herbal extract with antimicrobial efficacy similar to 2% CHX, 0.5% AP seems to be a better alternative. However further tests and randomised clinical trials are required on AP to use it as an endodontic irrigant in vivo

Keywords: Enterococcus faecalis, Endodontic irrigant, Leaf extracts, Andrographis paniculata, Colony forming unit

Full Text:

Introduction:

Microorganisms and their toxic metabolic products are responsible for the development and persistence of apical periodontitis of endodontic origin. Enterococcus faecalis, facultative anaerobic gram-positive cocci is the most commonly isolated species in persistent root canal infections¹.

Sodium hypochlorite, the most commonly used irrigant has some undesirable characteristics like tissue toxicity, allergic potential and disagreeable smell and taste. Chlorhexidine used as an irrigant is active against E.faecalis but has less tissue dissolving property².

The constant increase in antibiotic resistant strains and side effects caused by synthetic drugs has prompted researchers to look for herbal alternatives. Various natural plant extracts has antimicrobial and therapeutic effects suggesting its potential to be used as endodontic irrigants. Pharmacological studies have acknowledged the value of medicinal plants as potential source of bioactive compounds³.

Andrographis Paniculataor kalmeghis available abundantly in India, Pakistan and Srilanka and also one of the most widely used plants in Ayurvedic formulations. A.paniculata  was  recommended  in Charaka Samhita  dating  to  175  BC  for  treatment  of jaundice  along  with  other  plants  in  multi  plant preparations⁴. Andrographis  has demonstrated significant  activity  in  fighting  common cold,  flu  and  upper  respiratory  infections. The pharmacological studies suggest its anti-inflammatory, antipyretic, anti-viral, immunostimulatory, potential cancer therapeutic agent, anti-hyperglycaemic and antioxidant properties^5-11. Researchers have found that the acetone and  methanol  extracts  of  leaves  and  stems  of A.paniculata  showed  greater  inhibitory  effect (30.33±0.88mm)  on  the  growth  of  Enterococcus feacalis⁴.Hence the purpose of this study was to evaluate the antimicrobial efficiency of the leaf extracts of A.paniculata against Enterococcus faecalis  as an endodontic irrigant.

Materials and methods:

Phytochemical extract: Shade dried powder of leaves of A.paniculata were obtained (Aravindh herbals, Chennai). The coarse powder of the sample was extracted with 2.5 litres of 95% ethanol by maceration process until the extraction was completed. Then the extract was filtered and the solvent was removed by distillation under reduced pressure.

Specimen preparation:Forty extracted human, permanent straight single-rooted mandibular premolar teeth with no caries, apical fractures and resorption were selected and stored in saline. The teeth samples were decoronated using rotary diamond disc with a standardized length 12 mm below the CEJ. In order to standardize the samples, each canal was prepared up to size 30 with ProTaper rotary nickel-titanium instruments (Dentsply Tulsa Dental, Johnson City, TN) according to manufacturer's instructions. The root apices were coated with nail varnish to seal the apical foramen. The canals were irrigated with 10ml of 5.25% NaOCl and 10ml of 17% EDTA. The specimens to be tested were sterilized at 121°C for 15 minutes at 26 psi and stored in 100% humidity at 30°C until use.

Isolation of micro-organisms: Purestrain of Enterococcus faecalis from American Type Culture Collection (ATCC #29212) was used. Respectively cultures were grown overnight at 37°C in brain heart infusion (BHI) broth on a rotary shaker 150 rpm and microbial growth were checked by changes in turbidity at 24 hours.

Antimicrobial activity test:As a preliminary test, the minimum inhibitory concentration(MIC) of A.paniculata against E.faecalis was checked by well diffusion method. Test pathogens were spread on the test plates- Mueller Hinton Agar (MHA). Sterile well of 6mm diameter was made and 5, 10, 20 µl of the medicament was loaded in the well. The test plates were incubated for 24hr. the zone of inhibition (mm in diameter) were read and taken as activity against the test pathogen. Chlorhexidine was loaded as the positive control and the MIC of A.paniculata against E.faecalis was found to be 0.5%.

Contamination of the specimens: Contamination of specimens werecarried out for 21 days at 37°C with E.faecalis adjusted to a degree of turbidity 1 according to McFarland scale, which corresponds to a microbial load of 3 × 10³ cells/ml. Under laminar flow, the samples were recontaminated every second day with fresh broth containing the microorganism.

Irrigation procedure: The specimens were irrigated with 25-G needle tip was placed to a depth of 1mm short of WL, respectively.

Group 1: Irrigation was done with 2% CHX

Group 2: Irrigation was done with 5.25% NaOCl

Group 3: Irrigation was done with extracts of A.Paniculata

Group 4: Irrigation was done with distilled water.

CFU procedure: After incubation, the tooth canal was drilled and the invaded bacteria was collected and placed in nutrient broth. Then 100µl of the broth was plated on the sterile nutrient agar medium and spread evenly with L-rod and incubated overnight. CFU was evaluated at 0^thhr and 48^thhr using digital counter.

Results:

In the root canals , the 0.5% herbal extract of AP was found to be equally effective to 2%CHX and highly effective than 5.25%NaOcl against Enterococcus faecalis with a marked decrease in the number of CFU at the 48^thhr from 2.6x10³ to 0.9x10³. The mean values of No.of bacterial colonies before and after irrigation are listed in Table [1].

Table [1]: Mean values of No.of bacterial colonies before and after irrigation 

Statistical analysiswas performed using One-way Anova/Kruskal-Wallis test with Tukey's post-hoc test.The values were statistically significant with p<0.5

Discussion:

 The success of endodontic treatment lies in the complete debridement of root canal system with the help of chemical irrigants and mechanical instruments¹. Owing to the rising incidence of antimicrobial resistance against various chemotherapeutic and antimicrobial agents, the treatment of bacterial infection requires special consideration which may otherwise lead to grave prognosis. Simultaneously, the evolution of multi drug resistant (MDR) bacterial strains has further aggravated the present situation¹².

In this scenario, herbs have been a valuable source of medication worldwide due to their important antimicrobial principles, phytoconstituents and wider therapeutic potentials. In the present study, the herbal extracts of A.paniculata were used due to its wide range of pharmacological effects. Diterpenoids and flavonoids are the main phytochemical constituents which are believed to be responsible for the most biological activities of this plant¹³.Andrographolide, a diterpenoid found in leaf extracts is said to possess antimicrobial property and the flavanoids are nature’s biological response modifiers. Phenolic compounds are the largest group of phytochemicals that accounts for most of the antioxidant   and antimicrobial properties. At low  concentration  tannins  can  inhibit  the  growth  of microorganisms  and  act  as  an  antifungal  agent  at higher concentration by coagulating the protoplasm of the microorganism⁴.

A study conducted by Radha R et al., concluded, extracts  of  A.paniculata  showed  greater  inhibitory  effect (30.33±0.88mm)  on  the  growth  of  Enterococcus faecalis. The herbal extracts can also be used as an endodontic irrigant against E.faecalis, the most common pathogen found in persistent root canal infection.

In the present study, the minimum inhibitory concentration (MIC) of the crude extracts of A.paniculata against E.faecalis was determined by agar dilution method and it was found to be 0.5%. The root canal specimens were contaminated and incubated for 3 weeks which has been shown to produce a dense infection reaching 200 μm to 400 μm into the dentinal tubules³. The antimicrobial activity was evaluated by the number of colony forming units at 0^th hour and 48^th hour.

The results of the study showed that, 0.5% leaf extracts of A.paniculata was highly efficient to 5.25% NaOCl and equally efficient to 2% CHX. A.paniculata is biocompatible and severe tissue injuries and adverse effects were less when compared to NaOCl and CHX.Bitter taste associated with this plant can be altered by different formulations due to addition of sweeteners and flavours to increase the patient's compliance and acceptability.

Conclusion:

Being an herbal extract with several pharmacological activities including its antioxidant property, A.paniculata seems to be a better alternative. However further clinical trials and randomised control studies are required to use A.paniculata as an endodontic irrigant in vivo.

References:

1. Ghonmode WN, Balsaraf OD, Tambe VH, Saujanya KP, Patil AK, Kakde DD. Comparison of the antibacterial efficiency of neem leaf extracts, grape seed extracts and 3% sodium hypochlorite against E. feacalis–An in vitro study. Journal of international oral health: JIOH. 2013 Dec;5(6):61.
2. Dutta A, Kundabala M. Comparative anti-microbial efficacy of Azadirachta indica irrigant with standard endodontic irrigants: A preliminary study. Journal of conservative dentistry: JCD. 2014 Mar;17(2):133.
3. Vinothkumar TS, Rubin MI, Balaji L, Kandaswamy D. In vitro evaluation of five different herbal extracts as an antimicrobial endodontic irrigant using real time quantitative polymerase chain reaction. Journal of Conservative Dentistry. 2013 Mar 1;16(2):167.
4. R. Radha, M. Sermakkani ,V. Thangapandian. Evaluation of phytochemical and antimicrobial activity of Andrographis paniculatanees (Acanthaceae) aerial parts. Int. J. of Pharm. and Life Sci. (IJPLS). 2011 Feb;2( 2): 562-7
5. Shen YC, Chen CF, Chiou WF. Andrographolide prevents oxygen radical production by human neutrophils: possible mechanism (s) involved in its anti?inflammatory effect. British journal of pharmacology. 2002 Jan 1;135(2):399-406.
6. Madav S, Tripathi HC, Mishra SK. Analgesic, antipyretic and antiulcerogenic effects of andrographolide. Indian Journal of Pharmaceutical Sciences. 1995 May 1;57:121-.
7. Chang RS, Ding L, Gai-Qing C, Qi-Choa P, Ze-Lin Z, Smith KM. Dehydroandrographolide succinic acid monoester as an inhibitor against the human immunodeficiency virus. Experimental Biology and Medicine. 1991 May 1;197(1):59-66.
8. Puri A, Saxena R, Saxena RP, Saxena KC, Srivastava V, Tandon JS. Immunostimulant agents from Andrographis paniculata. Journal of Natural products. 1993 Jul;56(7):995-9.
9. Rajagopal S, Kumar RA, Deevi DS, Satyanarayana C, Rajagopalan R. Andrographolide, a potential cancer therapeutic agent isolated from Andrographis paniculata. Journal of Experimental therapeutics and Oncology. 2003 May 1;3(3):147-58.
10. Yu BC, Chen WC, Cheng JT. Antihyperglycemic effect of andrographolide in streptozotocin-induced diabetic rats. Plantamedica. 2003 Dec;69(12):1075-9.
11. Zhang XF, Tan BK. Antihyperglycaemic And Anti-Oxidant Properties Of Andrographis Paniculata In Normal And Diabetic Rats. Clinical and Experimental Pharmacology and Physiology. 2000 May 1;27(5-6):358-63.
12. Ewam UP. Evidence based antibacterial potentials of medicinal plants and herbs countering bacterial pathogens especially in the era of emerging drug resistance: An integrated update. International Journal of Pharmacology. 2014;10(1):1-43.
13. Sule A, Ahmed QU, Samah OA, Omar MN. Screening for antibacterial activity of Andrographis paniculata used in Malaysian folkloric medicine: A possible alternative for the treatment of skin infections. Ethnobotanical leaflets. 2010;1(4):8-10.