Cytotoxicity Effects of Guava Leaf Extract on Fibroblast Cells- An Invitro Study
Running Title: Guava Leaf Extract On Fibroblast Cells Vijayapriyangha Senthilkumar
Department of Conservative Dentistry and Endodontics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences,
Saveetha University, Chennai, India Sindhu Ramesh
Professor, Department of Conservative Dentistry and Endodontics Saveetha Dental College and Hospitals,
Saveetha Institute of Medical and Technical Sciences, Saveetha University,
Chennai, India Corresponding Author
SINDHU RAMESH
Professor, Department of Conservative Dentistry and Endodontics, Saveetha Dental College and Hospitals,
Saveetha Institute of Medical and Technical Sciences , Saveetha University,
162 , PH Road , Chennai 600077, TamilNadu , India Introduction
Root canal infections are polymicrobial in nature and as we know there are a number of mechanisms involved in eliminating these infectious microorganisms. From access opening to obturation,a constant used to eliminate the biofilm is endodontic irrigants. The aim of this study was to evaluate the effect of Sodium hypochlorite and Guava leaf extract on the L929 fibroblast cell line.
Materials and Method
The guava leaf powder was obtained and the extract was prepared. For cell viability assay, cells were seeded onto 96- well plates at a concentration of 5×103 cells/well, followed by the addition of test samples of various concentrations prepared in cell culture media. MTT assay was done which measures the reduction of yellow 3-(4,5dimethiazol-2-yl)- 2, 5-diphenhydramine tetrazolium bromide(MTT) by mitochondrial succinate dehydrogenase. The effect of test samples (25-100μg/ml) on cell growth inhibition will be assessed as percentile viability, where vehicle-treated cells will be taken as 100% viable.
Results
Statistical analysis was done using One-way Anova and a post hoc test. p<0.05 was considered as significant. P<0.001 as compared with negative control. Guava leaf extract at different concentrations are P<0.001 as compared with sodium hypochlorite.
Conclusion
Guava leaf extract had no or less cytotoxic effect based on its concentration. Hence it can be used as an alternative endodontic irrigant.
Keywords
Endodontic Irrigants, Fibroblast Cells, Root Canal Treatment,Guava Leaf , Natural Irrigant.
Introduction
Root canal treatment is the one which is carried out for pulpal diseases in dentistry. The main aim of root canal treatment is to remove bacteria and metabolite waste inside the root canal system, thereby preventing reinfection of the canal and healing process to take place.Irrigation is the important step of root canal treatment . Irrigation material serves as a debris solvent and makes the instrument movement easier during root canal preparation (Estrela et al., 2014;
Kandaswamy & Venkateshbabu, 2010). The ideal root canal irrigation material should have low surface tension and low toxicity as well as should be smear layer-free, inexpensive, and easy to use(Dotto, Sarkis Onofre, Bacchi, & Rocha Pereira, 2020; Jaju & Jaju, 2011; ‗Root Canal Irrigants in Horizon: A Review‘, 2016).
Chemomechanical preparation and three dimensional filling with an adequate seal are considered essential for long- term endodontic success(Martinho & Gomes, 2008; Sedgley, Lennan, & Clewell, 2004). Intracanal irrigants play an essential role in removal of smear layer and disinfection. Combined use of sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA) has been advocated as the gold standard for the above purpose(Johal, Baumgartner, & Marshall, 2007).
NaOCl (1%–5.25%) and EDTA (15%–17%) have been recommended for removal of organic and inorganic components of the smear layer, respectively. Although NaOCl is a potent antimicrobial irrigant, only higher concentrations are recommended in eradicating Enterococcus faecalis(Önçağ et al., 2003; Radcliffe et al., 2004). Currently, E. faecalis biofilm were found to be resistant to 5.25% NaOCl(Liu, Wei, Ling, Wang, & Huang, 2010). On the contrary, longer exposure times and higher concentrations are recommended for NaOCl to disinfect the canal system(Retamozo, Shabahang, Johnson, Aprecio, & Torabinejad, 2010). EDTA has limited or no antimicrobial efficacy and thus serves as an adjunct rinse only(Buck, Eleazer, & Staat, 1999; Yoshida, Shibata, Shinohara, Gomyo, & Sekine, 1995).
Most complications of the use of NaOCl appear to be the result of its accidental injection beyond the root apex which can cause violent tissue reactions characterized by pain, swelling, hemorrhage, and in some cases, development of secondary infection and paresthesia. A great deal of caution should therefore be exercised when using NaOCl during endodontic irrigation(Spencer, Ike, & Brennan, 2007; Verma, Goel, Bala, & Singh, 2012). Ehrich et al. suggested that a clinician should check, both clinically and radiographically for immature apices, root resorption, apical perforations, or any other conditions that may result in larger than normal volumes of irrigant being extruded from the root-canal system into the surrounding tissue(Ehrich, Brian, & Walker, 1993).
The most common bacteria found in root canals are Enterococcus faecalis. These bacteria are usually found in the failed root canal treatment. The prevalence of E. faecalis bacteria in root canal failure reached 38% (Rôças & Siqueira, 2012;
Zoletti et al., 2011) The irrigation material that commonly used in root canal treatment is sodium hypochlorite, which has certain weaknesses, such as becoming toxic when in contact with periradicular tissue. Sodium hypochlorite is antimicrobial to bacteria, both Gram-positive and negative, spores, fungi, and viruses (Mack, 2014; Vidana, Sullivan, Billström, Ahlquist, & Lund, 2011). However, the optimal property of NaOCl to dissolve organic tissue is non-selective that means it is able to dissolve both necrotic and vital pulp remnants indistinguishably, especially at high concentrations.The in vitro studies showed that 0.5% sodium hypochlorite has a longer incubation time than other irrigation ingredients in eradicating E. faecalis (Borzini, Condò, De Dominicis, Casaglia, & Cerroni, 2016).
Toxicity of irrigants has been evaluated on various cell lines depending on their growth potential, metabolic characteristics, and potential of DNA damage. Cell lines of target or non-target tissues have been utilized and the results obtained have been mixed; thus no definite data exists to confirm simultaneously on both cell lines(Hauman & Love, 2003; Ravinanthanan, Hegde, Shetty, & Kumari, 2018). Hence there is a need to check the potential of all endodontic irrigants for the toxic effects on the fibroblast cells.
Psidium guajava is a phytotherapic plant commonly known as Guava. It belongs to the Family Myrtaceae species(Ismail, Minhas, Khanum, & Sahana, 2012; Jang et al., 2014). The leaves of the plant P. guajava Linn are reported to possess antioxidant, hepatoprotective, anti allergy, antimicrobial, antigenotoxic, antiplasmodial, cytotoxic, antispasmodic, cardioactive, anti cough , antidiabetic, anti inflammatory and antinociceptive activities(Jaiarj et al., 2000; Jayakumari et al., 2012 (April - June); Jose et al., 2016; Nayak et al., 2019; Ukwueze, Osadebe, & Okoye, 2015).Guava plants have been reported to contain phytochemicals active against some viruses, included HIV-1(Mao et al., 2010; Sriwilaijaroen et al., 2012). The leaves of guava are rich in flavonoids, particularly quercetin as the main active substance, as well as saponins, among others(Lozoya et al., 2002). The aim of this study was to evaluate the effect of Sodium hypochlorite and Guava leaf extract on the L929 fibroblast cell line.
Materials and Methods Chemicals
The materials used for MTT test were 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 10% fetal bovine serum (FBS), 100 units/ml of fungizone, dimethyl sulfoxide (DMSO), human fibroblast cell lines (primary culture), Eagle‘s minimum essential medium (EMEM), kanamycin, and phosphate-buffered saline.
Plant Materials
The guava leaf was purchased from the local market, shade dried and made into fine powder for the extract preparation.
The plant sample was authenticated by a Botanist from Central Drug Research Institute, Tamil Nadu.
Ethanolic Extract
About 100 gm of the powdered sample was mixed with 1000 ml of ethanol and kept for 48 hrs at room temperature to ensure maximum metabolite extraction. The extract obtained was filtered and concentrated. The extraction method is based on the solubility of the constituents of the sample in ethanol. The filtrate is placed into the thimble of the Soxhlet extraction apparatus chamber. The sample was extracted for 12 hours at 4 cycles per hour. After extraction, the solvent was removed by the means of a rotary evaporator, yielding the extracted compound. The final concentration was maintained as 100mg/ml by redissolving the crude extracts in 10% dimethyl sulfoxide for bioassay analysis.
Maintenance of Cell Lines
L929 fibroblast cell lines were purchased from NCCS Pune. The L929 Cells were cultured in a humidified atmosphere at 37 °C in the cell growth DMEM medium with 10% fetal bovine serum, L–glutamine ,1% penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified CO2 (5%) chamber and 95% air. The cells were detached using 0.25% EDTA Trypsin. Neutralization of the Trypsin was achieved using DMEM containing 10% FBS and PSGF, and cells were mechanically separated using a pipette. There were 96-well plastic culture plates filled with 200µl of medium containing in each well. The plates were then incubated at 37°C in a humidified atmosphere containing 5%
CO2 and 95% air for 24 h to permit attachment of the cells to the plates.
Cytotoxicity test by MTT assay
The EMEM, Kanamycin, 1% pen strep, 10% FBS, and 100 units/ml of fungizone 100 μl was added as a control media to the 96 well microtiter plates. The microplates are filled with 100 μl of fibroblast cells with a density of 3×103 in EMEM, kanamycin, 1% pen strep, 10% FBS, and 100 units/ml of fungizone as negative control. The cells were permitted to adhere for 24 hours, and the growth medium using micropipette and the monolayer of cells was washed twice with MEM without FBS to remove dead cells and excess FBS.
1ml of medium (without FBS) containing different dilution of guava leaf extract (25-100µg/ml) were added in respective wells; 20 μl of MTT (5 mg/ml in PBS) were added to each well, and the cells incubated for a further 6‑7 hrs in 5% CO2 incubator. After removal of the medium, 1ml of DMSO was added to each well and the positive control
(Sodium hypochlorite) was tested. The supernatant was removed and 50 µl of propanol was added and the plates were gently shaken to solubilize the formed formazan. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. The plates were placed on a shaker for 15 min and the absorbance was read on an enzyme-linked immunosorbent assay (ELISA) reader at 570 nm. Each experiment was carried out in triplicate and the half maximal inhibitory concentration (IC50) of the test samples as the percentage survival of the cells was calculated.(Figure 1).
Figure 1- Microplate images showing the colour changes for different test groups. 1-L929 cells untreated negative control; 2-L929 cells treated with GE (25µg); 3- L929 cells treated with GE (50 µg) ; 4- L929 cells
treated with GE (100 µg); 5- L929 cells treated with NaOCl.
Statistical analysis
Results were expressed as mean ± SD. Statistical significance was determined by one-way analysis of variance (ANOVA) and post hoc least-significant difference test. P<0.05 was considered as significant.(Table 1).
S.No Treatment Conc (µg/ml)
Absorbance 570nm
% of cell Viability
1 L929 untreated
cells - 0.512±0.05 100
2 Guava leaf
extract 25 0.481 ±
0.08*a 93.9
3 Guava leaf
extract 50 0.415 ±
0.03*a 81.0
5 Guava leaf
extract 100 0.396 ±
0.09*a 77.3
6 NaOCl 3% 0.108±
0.08* 21.0
Table 1-Values are expressed as Mean±SD (n=3); *P<0.001, as compared with Negative control. aP<0.001, as compared with NaOCl. The LC50 of the Guava leaf extract is <100µg/ml
Results
Cytotoxicity in cell culture is typically expressed as LC50. The LC50 of the guava leaf extract is <100ug/ml. P<0.001 as compared with negative control. Guava leaf extract at different concentrations are P<0.001 as compared with sodium hypochlorite.
Discussion
The present in-vitro study evaluated the cytotoxicity of guava leaf extract on the L929 fibroblast cell line. The results showed that the guava leaf extract at 25μg/ml concentration showed about 93.9% cell viability and even at 100μg/ml showed cell viability of about 77.3 % cell viability, Which is less toxic to L929 fibroblasts cells.
Guava leaf is rich in bioactive compounds and in order to utilize these compounds in the preparation of value added products, these are extracted using ethyl acetate. Seo et al, reported the extraction of essential compounds of guava using water, ethanol and methanol, and found the highest content of phenolic compounds in water extract(Seo et al., 2014). The ethyl acetate extract was found to be rich in sesquiterpenes and fatty acids. Sesquiterpenes such as cubenol, 6S-2,3,8,8-tetramethyl tricyclo[5.2.2.0(1,6)]undec-2-ene,benzene,(1,3,3-trimethyl nonyl) and 1,6,10-dodecatrien-3-ol, 3,7,11-trimethyl-, (E) act as flavouring and fragrance agents. Apart from these, cis-à-bisabolene, an intermediate in the biosynthesis of hernandulcin which is a natural sweetener was also identified in the extract(Christianson, 2017).
Another major compound identified in the extract was fatty acid esters which are found to be fragrance ingredients.
Thus, the GC–MS analysis of guava leaf extract identified various beneficial compounds with enormous medicinal importance.
The essential oil of P. guajava is efficient in inhibiting the growth of both Gram-positive and Gram-negative bacteria at higher concentration(Nisha, Darshana, Madhu, & Bhupendra, 2011).The phenolic compound content of water extract was higher than pure ethanol and methanol extract. The phenolic compound content of hydroethanolic extracts was higher than water extracts. The antioxidant activity of those hydroethanolic extracts was higher than that of the water extracts and was significantly high in the 50% hydroethanolic extract that had the highest content of phenolic compounds(Seo et al., 2014).
Psidium guajava extract reduced the cell population in both in vitro and in vivo the extract accelerated wound healing and helps in faster healing of tissues (Porta, Fernandes, Bussadori, Marques, & Martins, 2010).The ethanolic and water extract of guava leaves possess antibacterial activity against L.acidophilus with 20% ethanolic extract being as efficacious as 0.2% chlorhexidine(Jain et al., 2014). Extracts rich in guava flavonoids have demonstrated their potential for preventing dental caries due to the growth inhibition of the oral flora(Thalikunnil, Ashok, & Sukesh, 2012).
Ethanolic and water extracts of guava leaves possess antibacterial activity against L. acidophilus with 20% ethanolic extract being as efficacious as 0.2% chlorhexidine(Jain et al., 2014).
Cytotoxicity in cell culture is typically expressed as LC50, which means that the concentration of a given agent is lethal to 50% of the cells. Moreover, the most common way to describe cytotoxicity in cell culture is LC50, the concentration of a drug that kills half of the tested cells in culture (Zhang et al., 2007).
In this research, the cytotoxic effects of the psidium guajava leaf extract at the concentrations of 25μg/ml, 50μg/ml and 100μg/ml were examined on human gingival fibroblast using MTT assay in comparison with positive and negative control. The results showed that the psidium guajava leaf extract has no cytotoxic effect. Percentage cell viability at MIC (25μg/ml) was 93.9% , MIC (50μg/ml) was 81% and MIC (100μg/ml) was 77.3%. Psidium guajava leaf extract even at 100 % concentration was not cytotoxic and also it is capable of killing E. faecalis, so it has the potential to be further investigated as a root canal irrigation material. Further studies will be designed to investigate its biomedical applications with a detailed mechanism through appropriate experimental models.
Conclusion
Guava leaf extract had no or less cytotoxic effect based on its concentration. Comparatively most of the cells showed less cytotoxic effect when compared to sodium hypochlorite at different concentrations. Hence it can be used as an alternative endodontic irrigant for better prognosis and outcome of the root canal treatment.
Acknowledgement
With sincere gratitude, we acknowledge the staff members of the department of Conservative dentistry and Endodontics and Saveetha dental college for the extended support towards the completion of the research.
Financial support and Sponsorship Nil
Conflicts of interest
There are no conflicts of interest.
References
1. Borzini, L., Condò, R., De Dominicis, P., Casaglia, A., & Cerroni, L. (2016). Root Canal Irrigation:
Chemical Agents and Plant Extracts Against Enterococcus faecalis. The Open Dentistry Journal, 10, 692–
703.
2. Neelam Tiwari, S. S. Singh, Rajesh Singh & Neeraj K. Charmkar, ―Effect of Organic-Cum-Inorganic Sources of Nutrients on Growth, Yield and Economical Gain from Guava‖, International Journal of Agricultural Science and Research (IJASR), Vol. 8, Issue 1 pp, 79-84
3. Buck, R., Eleazer, P. D., & Staat, R. H. (1999). In vitro disinfection of dentinal tubules by various endodontics irrigants. Journal of Endodontia, 25(12), 786–788.
4. Bibha Kumari & Suraj Prakash, ―Economics of Plant Prepared through Air Layers of Guava (Psidium guajava L)‖ , International Journal of Agricultural Science and Research (IJASR), Vol. 7, Issue 2, pp , 313- 318
5. Christianson, D. W. (2017). Structural and Chemical Biology of Terpenoid Cyclases. Chemical Reviews, 117(17), 11570–11648.
6. Vasanthi Padamanabhan, Manimekalai Ganapathy, Vasthi Kennedy Evanjelene, Nirmala Ayyavuv &
Jagajothi Angamuthu, ―Invitro Antioxidant Activity of Albizzia lebbeck (L) Benth‖, International Journal of General Medicine and Pharmacy (IJGMP), Vol. 3, Issue 2, pp , 9-16.
7. Dotto, L., Sarkis Onofre, R., Bacchi, A., & Rocha Pereira, G. K. (2020). Effect of Root Canal Irrigants on the Mechanical Properties of Endodontically Treated Teeth: A Scoping Review. Journal of Endodontia.
Retrieved from https://doi.org/10.1016/j.joen.2020.01.017
8. Sangita Warade & Kiran Jayade, ―Progressive Achievement of Horticultural Crops in India‖, International Journal of Applied Mathematics Statistical Sciences (IJAMSS), Vol. 7, Issue 2, pp ; 55 - 64
9. Ehrich, D. G., Brian, J. D., & Walker, W. A. (1993). Sodium hypochlorite accident: inadvertent injection into the maxillary sinus. Journal of Endodontics, 19(4), 180–182.
10. Manuj Awasthi & Manju Negi, ―Effect of Different Time and Methods of Budding on the Bud Take Success of Nectarine on Peach (Prunus Persica L.) Seedling Rootstock‖, IMPACT: International Journal of Research in Applied, Natural and Social Sciences (IMPACT: IJRANSS), Vol. 4, Issue 11, pp , 25-30 11. Estrela, C., Holland, R., Estrela, C. R. de A., Alencar, A. H. G., Sousa-Neto, M. D., & Pécora, J. D. (2014).
Characterization of successful root canal treatment. Brazilian Dental Journal, 25(1), 3–11.
12. Rupal Dhoot, Meenakshi Dhoot & Varun Kumar Badaya, ―Rootstock Breeding for Fruit Tree Crops‖, IMPACT: International Journal of Research in Applied, Natural and Social Sciences (IMPACT:
IJRANSS), Vol. 5, Issue 5, pp, 27-32
13. Hauman, C. H. J., & Love, R. M. (2003). Biocompatibility of dental materials used in contemporary endodontic therapy: a review. Part 1. Intracanal drugs and substances. International Endodontic Journal, 36(2), 75–85.
14. Ismail, M., Minhas, P. S., Khanum, F., & Sahana, V. M. (2012). Antibacterial activity of leaves extract of Guava (Psidium Guajava). International Journal of. Retrieved from https://www.cabdirect.org/globalhealth/abstract/20123216652
15. Jaiarj, P., Wongkrajang, Y., Thongpraditchote, S., Peungvicha, P., Bunyapraphatsara, N., & Opartkiattikul, N. (2000). Guava leaf extract and topical haemostasis. Phytotherapy Research: PTR, 14(5), 388–391.
16. Jain, D., Dasar, P., Nagarajappa, S., Kumar, S., Airen, B., Warhekar, S., & Others. (2014). In vitro activity
of ethanolic and water extract of guava leaves at various concentrations against Lactobacillus acidophilus.
Journal of Indian Association of Public Health Dentistry, 12(3), 232.
17. Jaju, S., & Jaju, P. P. (2011). Newer root canal irrigants in horizon: a review. International Journal of Dentistry, 2011, 851359.
18. Jang, M., Jeong, S.-W., Cho, S. K., Ahn, K. S., Lee, J. H., Yang, D. C., & Kim, J.-C. (2014). Anti- inflammatory effects of an ethanolic extract of guava (Psidium guajava L.) leaves in vitro and in vivo.
Journal of Medicinal Food, 17(6), 678–685.
19. Jayakumari, S., Anbu, J., Ravichandiran, V., Nithya, S., Anjana, A., & Sudharani, A. D. (2012 (April - June)). Evaluation Of Toothache Activity Of Methanolic Extract And Its Various Fractions From The Leaves Psidium Guajava Linn. International Journal of Pharma and Bio Sciences, 3 Issue 2. Retrieved from http://ijpbs.net/abstract.php?article=1297
20. Johal, S., Baumgartner, J. C., & Marshall, J. G. (2007). Comparison of the antimicrobial efficacy of 1.3%
NaOCl/BioPure MTAD to 5.25% NaOCl/15% EDTA for root canal irrigation. Journal of Endodontia, 33(1), 48–51.
21. Jose, J., Krishnamma, S., Peedikayil, F., Aman, S., Tomy, N., & Mariodan, J. P. (2016). Comparative Evaluation of Antimicrobial Activity of QMiX, 2.5% Sodium Hypochlorite, 2% Chlorhexidine, Guava Leaf Extract and Aloevera Extract Against Enterococcus faecalis and Candida albicans - An in-vitro Study.
Journal of Clinical and Diagnostic Research: JCDR, 10(5), ZC20–3.
22. Kandaswamy, D., & Venkateshbabu, N. (2010). Root canal irrigants. Journal of Conservative Dentistry:
JCD, 13(4), 256–264.
23. Liu, H., Wei, X., Ling, J., Wang, W., & Huang, X. (2010). Biofilm formation capability of Enterococcus faecalis cells in starvation phase and its susceptibility to sodium hypochlorite. Journal of Endodontia, 36(4), 630–635.
24. Lozoya, X., Reyes-Morales, H., Chávez-Soto, M. A., Martínez-García, M. del C., Soto-González, Y., &
Doubova, S. V. (2002). Intestinal anti-spasmodic effect of a phytodrug of Psidium guajava folia in the treatment of acute diarrheic disease. Journal of Ethnopharmacology, 83(1-2), 19–24.
25. Mack, H. L. (2014). Enterococcus Faecalis: Molecular Characteristics, Role in Nosocomial Infections and Antibacterial Effects. Nova Science Publishers, Incorporated.
26. Mao, Q. C., Zhou, Y. C., Li, R. M., Hu, Y. P., Liu, S. W., & Li, X. J. (2010). Inhibition of HIV-1 mediated cell-cell fusion by saponin fraction from Psidium guajava leaf. Zhong Yao Cai= Zhongyaocai= Journal of Chinese Medicinal Materials, 33(11), 1751–1754.
27. Martinho, F. C., & Gomes, B. P. F. A. (2008). Quantification of endotoxins and cultivable bacteria in root canal infection before and after chemomechanical preparation with 2.5% sodium hypochlorite. Journal of Endodontia, 34(3), 268–272.
28. Nayak, N., Varghese, J., Shetty, S., Bhat, V., Durgekar, T., Lobo, R., … U, V. (2019). Evaluation of a mouthrinse containing guava leaf extract as part of comprehensive oral care regimen- a randomized placebo-controlled clinical trial. BMC Complementary and Alternative Medicine, 19(1), 327.
29. Nisha, K., Darshana, M., Madhu, G., & Bhupendra, M. K. (2011). GC-MS analysis and anti-microbial activity of Psidium guajava (leaves) grown in Malva region of India. American Journal of Respiratory Medicine: Drugs, Devices, and Other Interventions, 3(4), 237–245.
30. Önçağ, Ö., Hoşgör, M., Hilmioğlu, S., Zekioğlu, O., Eronat, C., & Burhanoğlu, D. (2003). Comparison of antibacterial and toxic effects of various root canal irrigants. International Endodontic Journal, 36(6), 423–
432.
31. Porta, K., Fernandes, S., Bussadori, S. K., Marques, M. M., & Martins, M. D. (2010). Healing and cytotoxic effects of Psidium guajava (Myrtaceae) leaf extracts. Brazilian Journal of Oral Sciences, 9(4).
Retrieved 24 April 2020 from http://dx.doi.org/
32. Radcliffe, C. E., Potouridou, L., Qureshi, R., Habahbeh, N., Qualtrough, A., Worthington, H., & Drucker,
D. B. (2004). Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A. naeslundii, Candida albicans and Enterococcus faecalis.
International Endodontic Journal, 37(7), 438–446.
33. Ravinanthanan, M., Hegde, M. N., Shetty, V., & Kumari, S. (2018). Cytotoxicity effects of endodontic irrigants on permanent and primary cell lines. Biomedical and Biotechnology Research Journal (BBRJ), 2(1), 59. Retrieved 23 April 2020 from
34. Retamozo, B., Shabahang, S., Johnson, N., Aprecio, R. M., & Torabinejad, M. (2010). Minimum contact time and concentration of sodium hypochlorite required to eliminate Enterococcus faecalis. Journal of Endodontia, 36(3), 520–523.
35. Rôças, I. N., & Siqueira, J. F., Jr. (2012). Characterization of microbiota of root canal-treated teeth with posttreatment disease. Journal of Clinical Microbiology, 50(5), 1721–1724.
36. Root Canal Irrigants in Horizon: A Review. (2016). International Journal of Science and Research (IJSR), 5(3), 1240–1243.
37. Sedgley, C. M., Lennan, S. L., & Clewell, D. B. (2004). Prevalence, phenotype and genotype of oral enterococci. Oral Microbiology and Immunology, 19(2), 95–101.
38. Seo, J., Lee, S., Elam, M. L., Johnson, S. A., Kang, J., & Arjmandi, B. H. (2014). Study to find the best extraction solvent for use with guava leaves (Psidium guajava L.) for high antioxidant efficacy. Food Science & Nutrition, 2(2), 174–180.
39. Spencer, H. R., Ike, V., & Brennan, P. A. (2007). the use of sodium hypochlorite in endodontics—potential complications and their management. British Dental Journal, 202(9), 555–559.
40. Sriwilaijaroen, N., Fukumoto, S., Kumagai, K., Hiramatsu, H., Odagiri, T., Tashiro, M., & Suzuki, Y.
(2012). Antiviral effects of Psidium guajava Linn. (guava) tea on the growth of clinical isolated H1N1 viruses: its role in viral hemagglutination and neuraminidase inhibition. Antiviral Research, 94(2), 139–
146.
41. Thalikunnil, S. T., Ashok, A., & Sukesh, K. (2012). Screening of psidium gaujava for effective phytomedicines and study on its antibacterial effect against dental caries bacteria. International Journal of Pharmacy and Pharmaceutical Sciences, 4, 400–401.
42. Ukwueze, S. E., Osadebe, P. O., & Okoye, F. B. C. (2015). A new antibacterial benzophenone glycoside from Psidium guajava (Linn.) leaves. Natural Product Research, 29(18), 1728–1734.
43. Verma, S., Goel, M., Bala, S., & Singh, M. (2012). Issues Of Biocompatibility Associated With Commonly Used Endodontic Irrigants: A Review. Indian Journal of Dental Sciences, 4. Retrieved from http://search.ebscohost.com/login.aspx?direct=true&profile=ehost&scope=site&authtype=crawler&jrnl=09 764003&AN=102146331&h=fzf4fY3WC%2BhmlZsQnGMxtiXB8oB8yOP0RQOwZUqADV%2B5Uf%2 B9Y2htZPZZibGx6AQDpaFkOpYuN%2Bf%2ByExWya24VA%3D%3D&crl=c
44. Vidana, R., Sullivan, Å., Billström, H., Ahlquist, M., & Lund, B. (2011). Enterococcus faecalis infection in root canals--host-derived or exogenous source? Letters in Applied Microbiology, 52(2), 109–115.
45. Yoshida, T., Shibata, T., Shinohara, T., Gomyo, S., & Sekine, I. (1995). Clinical evaluation of the efficacy of EDTA solution as an endodontic irrigant. Journal of Endodontia, 21(12), 592–593.
46. Zhang, M., Aguilera, D., Das, C., Vasquez, H., Zage, P., Gopalakrishnan, V., & Wolff, J. (2007).
Measuring cytotoxicity: a new perspective on LC50. Anticancer Research, 27(1A), 35–38.
47. Zoletti, G. O., Pereira, E. M., Schuenck, R. P., Teixeira, L. M., Siqueira, J. F., Jr, & dos Santos, K. R. N.
(2011). Characterization of virulence factors and clonal diversity of Enterococcus faecalis isolates from treated dental root canals. Research in Microbiology, 162(2), 151–158.
