Indian Journal of Dental Research

: 2021  |  Volume : 32  |  Issue : 4  |  Page : 423--431

Evaluation of morinda citrifolia ethanolic extract versus morinda citrifolia fresh fruit juice for tissue response and systemic toxicity in animal model

Kavalipurapu Venkata Teja1, Sindhu Ramesh1, Karthik Ganesh2, Kaligotla Apoorva Vasundhara3,  
1 Department of Conservative Dentistry and Endodontics, Saveetha Dental College & Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamil Nadu, India
2 Department of Anatomy, Saveetha Dental College & Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamil Nadu, India
3 Department of Prosthodontics and Implantology, Private Consultant, Hyderabad, Telangana, India

Correspondence Address:
Dr. Sindhu Ramesh
Department of Conservative Dentistry and Endodontics, Saveetha Dental College & Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, 162, Poonamallee High Road, Chennai - 600 077, Tamil Nadu


Introduction: An ideal irrigant should exhibit good antimicrobial activity along with smear removal. Various plant extracts have been used for disinfection of the root canal system. The present study aims in evaluating the tissue response and systemic toxicity of ethanolic extract of Morinda citrifolia versus Morinda citrifolia fresh fruit juice in an animal model. Materials and Methods: A total 48 female adult wistar albino rats were randomly allocated into 4 groups as Group I- 3% sodium hypochlorite (n = 12), Group II- Saline (Control) (n = 12), Group III- ethanolic extract of Morinda citrifolia (n = 12), Group IV- Morinda citrifolia Fresh Fruit Juice (n = 12). A trough was made in the periapical bone, and the test sample was placed in the respective groups. The animals were euthanized for 7th day, 14th day and 30th day and the tissue specimen was prepared for histopathological evaluation, and serum analysis was done for oxidative stress markers, liver and renal function tests. Results: Serum analysis exhibited significant elevation of Alkaline Phosphatase (ALP), Glutathione Peroxidase (GPX) and Glutathione Reductase (GR) and histopathology showed a higher inflammatory response at 7th and 14th days in group II and group III (p < 0.05), as compared to the other groups. At the 30th day time interval, there was no statistically significant difference in serum analysis and inflammatory response, in all the four groups analysed (p > 0.05). Conclusion: Ethanolic extract of Morinda citrifolia was not biocompatible and more cytotoxic than a Morinda citrifolia fresh fruit juice. So, fresh fruit juice of Morinda citrifolia can be a preferred choice of natural root canal irrigant, with least possible cytotoxic effects as compared to the ethanolic extract.

How to cite this article:
Teja KV, Ramesh S, Ganesh K, Vasundhara KA. Evaluation of morinda citrifolia ethanolic extract versus morinda citrifolia fresh fruit juice for tissue response and systemic toxicity in animal model.Indian J Dent Res 2021;32:423-431

How to cite this URL:
Teja KV, Ramesh S, Ganesh K, Vasundhara KA. Evaluation of morinda citrifolia ethanolic extract versus morinda citrifolia fresh fruit juice for tissue response and systemic toxicity in animal model. Indian J Dent Res [serial online] 2021 [cited 2023 Jan 28 ];32:423-431
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The ultimate goal of endodontic therapy is to eliminate or to minimize microorganisms and its toxic products within the root canal system, as it determines the success of treatment.[1] Anatomical complexities and smear layer formation during instrumentation are the major obstacle in achieving disinfection.[2] Therefore, a combination of mechanical instrumentation with disinfection has resulted in a significant elimination of microorganisms from the root canal.[3] Mechanical instrumentation enhances the ease of access for the irrigants and intracanal medicaments into the root canal space.[4] The ideal irrigant kills the bacteria, dissolves necrotic tissue, lubricates the canal, removes the smear layer, and does not irritate healthy tissues.[4]

Sodium hypochlorite is currently being advocated in day-to-day clinical practice as a common root canal irrigating solution in a concentration ranging from 0.5 to 5.25%. Sodium hypochlorite itself can dissolve organic substances in the root canal system. However, significant issues include cytotoxicity when accidentally injected into periradicular tissues, foul smell and taste.[5]

Literature has reported various plant extracts having antimicrobial and therapeutic properties that can be used for disinfection of the root canal system.[6] Morinda citrifolia, is commonly known as Noni which belongs to the family, Rubiaceae. Its therapeutic effects include a broad range of antitumor, antihelminthic, analgesic, antihypertensive, anti-inflammatory, antibacterial and antiviral and immune-enhancing effects.[7]

Literature evidence proves the plant and its extracts to be beneficial in controlling infection and pain.[8],[9],[10],[11],[12] The wide range of therapeutic benefits of Noni include for cancer, arthritis, diabetes, hypertension, infection and pain.[13] The literature evidence proves the Morinda citrifolia extract to be beneficial in controlling infection and pain along with better antimicrobial properties and smear removal.[14],[15] So the apparent usage as a root canal irrigant and intracanal medicament of Noni extract is appropriate compared to the other herbals.

The reason for choosing the Noni as a study group is because the enormous research on natural irrigants is concentrated on Morinda citrifolia at in vitro and Ex-vivo levels.[16],[17],[18],[19],[20],[21],[22] and only two clinical reports have shown the effectiveness of Morinda citrifolia juice as a root canal irrigant.[23],[24] Till date in the endodontic literature, there were only three studies, which have reported the in vitro antimicrobial efficacy of ethanolic extract of Morinda citrifolia on Enterococcus faecalis.[19],[20],[25] Although, the specific effects of Noni on bacteria are not fully understood, the presence of l-asperuloside and alizarin may be responsible for the antibacterial and antimicrobial property of Morinda citrifolia.[15] These phytochemicals, target the bacterial cell membranes and cellular biochemical pathways,[26],[27] reacting with the lipids in bacterial cell membranes, increasing the membrane permeability and thereby disrupt the cell membranes and cause the leakage of intracellular components, facilitating the movement of antimicrobial compounds into cytoplasm, inducing cell death.[27],[28] The anti-inflammatory effect of Noni fruit is mainly attributed to the inhibition of cyclooxygenases (COX-1 and COX-2) and lipoxygenases,[29] thereby reducing the pain.

To the best of our knowledge, there are no previous reports on tissue response and systemic toxicity of ethanolic extract of Morinda citrifolia. Hence this is the first study which aimed at the evaluation of tissue response and systemic toxicity of ethanolic extract of Morinda citrifolia versus Morinda citrifolia fresh fruit juice in an animal model. The primary objective of the study was to compare and analyse serum toxicity of ethanolic extract and fresh fruit juice of Morinda citrifolia in a rat model, using the liver and renal function tests and serum oxidative stress marker levels at 7th, 14th and 30th day time intervals. The secondary objective of the study was to compare the tissue inflammatory response of ethanolic extract and fresh fruit juice of Morinda citrifolia at 7th, 14th and 30th day time intervals.

 Materials and Methods

The present study was approved by the Institutional Animal Research Ethics Committee (IAEC), approval number (BRULAC/SDCH/SIMATS/IAEC/02-2019/011), India. The present research was conducted according to Helsinki Guidelines. A total of 48 female adult Wistar albino rats were used with an average weight of 200 to 220 g. Each animal was housed in a separate cage in a temperature-controlled environment (23°c-25°c) with water and food throughout the study. The rats were randomly allocated into 4 groups as 12 per each group. Group I- 3% sodium hypochlorite (n = 12), Group II- Saline (Control) (n = 12), Group III- ethanolic extract of Morinda citrifolia (n = 12), Group IV- Morinda citrifolia fresh fruit juice (n = 12) for 7th day, 14th day and 30th day evaluations. In this study, in addition to ethanolic extract of Morinda citrifolia, 3% sodium hypochlorite (Parcan, Septodont, UK), Saline (Fresenius Kabi; Normal Saline) and Fresh fruit juice of Morinda citrifolia were used. Opaque sealed envelope method was carried out for allocation concealment of all the four agents tested.

Preparation of Morinda citrifolia fresh fruit juice

The method of extraction of juice from the Noni fruits was similar to the previous study.[30] Fresh Noni fruits were collected and were selected at maturity stage 4 (hard white).[31] The fruits were allowed to ripen for at room temperature for two days under closed condition before treatments. The weighted ripened fruits are washed using tap water and subjected to hot process technique, where on steam blanching was done at 100°c for 2, 4, 6, 8 and 10 min respectively and then cooled to room temperature for 5 min. After pre-treatment, the juice was extracted by passing in nylon net, and the fresh fruit juice was collected in a sterilised steel container. The fresh fruit juice was only subjected to blanching, and no other sterilisation process was followed, nor was the attempts done to store the agents. Because the agents used were freshly prepared before the experimentation period.

Preparation of ethanolic extract of Morinda citrifolia

Ethanolic extraction of Morinda citrifolia fruit was carried out by dissolving the commercially obtained Morinda citrifolia Fruit extract powder (Noni extract Powder; APEX BIOTECHNOL, India) in 1:5 ratio of solvent ethanol. The extraction was carried out in a shaker water bath at 40 degree Celsius for 48 hours. The extract was then filtered through Whatman NO: 1 filter paper and concentrated to dryness. This dried extract was then dissolved in the required concentration of 10% dimethyl sulfoxide (DMSO). The minimum inhibitory concentration of the different groups was determined by the tube dilution method. The extracts in the powder were dissolved at a concentration of 64 mg/ml in 10% DMSO. Only ethanolic subjection was done, and no sterilisation process was carried out and the ethanolic extract was freshly prepared before experimentation. There were no attempts made to store the extracts as the extracts were freshly prepared and used immediately.

The literature reports on the ethanolic extract of Morinda citrifolia are scarce. There were only three studies till date, which compared or assessed the antimicrobial activity of ethanolic extract of Morinda citrifolia against the endodontic pathogen, Enterococcus Faecalis.[19],[20],[25] As, the activity of ethanolic extract of Morinda citrifolia was explored recently on endodontic pathogens, the studies assessing on the cytotoxicity of this newer agent is relatively less. Especially the cytotoxic assessments were concentrated in the medical field, and none of the evaluations were done on periodontal fibroblast cell lines. Ideally, the literature on cytotoxic assessments related to novel root canal irrigants or intra canal medicaments would be considered as more reliable, when assessments were done on periapical fibroblastic cell lines. There was only one study which assessed the cytotoxicity of ethanolic extract of Noni fruit on Michigan Cancer Foundation-7 human breast cells (MCF-7), and results showed that the extract had a cytotoxic effect on MCF-7 cells, which was dose-dependent.[32]


The animals were then anesthetized with an intraperitoneal injection of 47.5 mg/kg of 10% ketamine hydrochloride (Alfasan, Woerden, The Netherlands) and 10 mg/kg of 2% xylazine hydrochloride (Alfasan, Woerden, The Netherlands). A block randomisation method was followed, and a sequence numbered opaque sealed envelope for allocation concealment. Both the operator and the evaluator were blinded. The 300 microliters of the test materials (all the four agents tested) were mixed with 10 mg of cellulose and sealed in an opaque zip lock cover before the experiment.

After anesthetised rats, the neck of the rats was shaved and disinfected with a povidone-iodine solution (Betadine, Win Medicare Pvt., Ltd.,). The incision was made in the midline, parallel to the border of the mandible and the periapical region was accessed. A trough measuring 1*1 mm was made in the periapical bone using carbide burs under continuous saline irrigation. Three hundred microliters of the test sample which was mixed priorly with 10 mg of cellulose were removed from opaque zip lock cover and placed into the trough made. The edges of the skin were stitched with surgical nylon sutures, and disinfection again is done using a povidone-iodine solution (Betadine, Win Medicare Pvt., Ltd.,) [Figure 1]. All the four test samples including 3% sodium hypochlorite, saline, ethanolic extract of Morinda citrifolia, Fresh fruit juice of Morinda citrifolia were mixed with 10 mg of cellulose before placement in the periapical bone. After the experimental stage, the animals were kept in cages according to the group and placed in isolation and were maintained on a balanced diet. Each rat was placed separately in a cage for isolation after the experimental period with labelling of the group allocated and the date.{Figure 1}

Four animals per each group were dissected at the end of experimental periods 7, 14 and 30 days. The surrounding tissue was removed from the surgical site where the test material was implanted and stored in 10% formalin.

The tissue responses were graded as slight, moderate and severe based on FDI criteria evaluation,[33] by correlating the changes to the normal specimen. The criteria for scoring the inflammatory response were as follows:

If the thickness of the reaction zone is similar or slightly wider than the normal tissue, with few or no inflammatory cells:

Grade 0- no inflammation.Grade 1- slight inflammation.

If there is an increased reaction zone in which plasma cells, macrophages or both present:

Grade 2- moderate inflammation.

If there is an increased zone of reaction with macrophages, plasma cells lymphocytes and occasional foci of neutrophil lymphocytes or both present:

Grade 3- severe inflammation.

Before the animal was sacrificed, the blood was collected from the orbital sinus. The protocol followed for the blood sample collection was based on S Parasuram et al.,[34] This method is also known as periorbital, posterior orbital and orbital venous plexus bleeding. Collection of the blood sample is under general anaesthesia. The topical anaesthetic agent is applied to the eye before bleeding. The animal was scuffed firmly with the thumb and forefinger of the non-dominant hand and the skin around the eye was pulled taut. A capillary tube was inserted into the medial canthus of the eye at 30-degree angle to the nose. Slight thumb pressure was used to puncture the tissue and enter plexus or sinus to collect the blood through the capillary tube. Once the required volume of blood is obtained, the tube is gently removed and wiped with sterile cotton. Bleeding from the site is stopped by applying gentle finger pressure at the site of puncture.

The collected blood was immediately sent for serum analysis of oxidative stress markers Glutathione (GSH), Lipid Peroxidase (LPX), Superoxide Dismutase (SOD), Glutathione Peroxidase (GPX), Glutathione Reductase (GR). Serum levels of LFT (Liver function tests) and KFT (Kidney/Renal function tests) were assessed using Serum Glutamic-Oxaloacetic Transaminase (SGOT), Serum Glutamic Pyruvic Transaminase (SGPT), Alkaline Phosphatase (ALP), (UREA).

Statistical analysis

The collected data was analysed IBM. SPSS Statistics Software for Windows Version 23.0 (Armonk, NY: IBM Corp). Descriptive statistics were assessed using mean and standard deviation. Multivariate analysis was done using the one-way ANOVA with Tukey's Post-Hoc test. For repeated measures, the repeated measures of ANOVA was used with Bonferroni correction to control the type I error on multiple comparisons. In all the above statistical tools, the probability value 0.05 was considered as a significant level.


Serum analysis of LFT and RFT

Serum analysis for liver and renal function tests, [Table 1] revealed a statistically significantly elevated levels of ALP in all the three groups except Group I at 7th day, (P < 0.05), whereas the Group II and III showed elevated levels of ALP levels even at 14th and 30th day. SGOT levels were raised in Group II and III at the 14th-day interval, with no statistically significant difference at 7th and 30th day.{Table 1}

Serum analysis of oxidative stress markers

On evaluating the oxidative stress markers, [Table 2] significantly elevated levels of GPX and GR were seen in all the three groups except for Group I at 7th and 14th day with significantly elevated levels in group II (P < 0.05). Whereas the levels of GPX and GR were not statistically significant at 30th day in all the groups, (P > 0.05), with elevated levels in Group II, that was not significant.{Table 2}

Histopathological analysis

On evaluating histopathologically, [Table 3] and [Table 4] significantly elevated levels inflammatory cells were seen in Group II and III at 7th and 14th day, (P < 0.05), whereas the levels of the inflammatory cells, were not statistically significant at 30th day in all the groups, (P > 0.05). On assessing the inflammatory levels in group II on the 7th day, severe inflammatory cells were evident, which gradually decreased on the 14th day and 30th day. Histopathological features at 40X magnification at the 30th day time interval are depicted in [Figure 2].{Table 3}{Table 4}{Figure 2}


Root canal irrigants used must be biocompatible and non-irritant to the periapical tissues. Risk of leakage through the apical foramen is of prime concern during the treatment. Therefore, the assessment of the toxicity of materials used in endodontic therapy is of utmost importance. Inadvertent damage or irritation to periapical tissues could cause degeneration of the periapical tissue and delayed wound healing.

Sodium hypochlorite has been the most commonly used endodontic irrigant because of its antimicrobial and tissue-dissolving activity.[35],[36] It acts by glucose oxidation inhibition and adenine nucelotide depletion.[37] Authors concluded that the organic tissue dissolution is related to the concentration.[38],[39] Higher the concentration, more rapid tissue dissolution and greater the cytotoxicity, but our study was not concentrated on assessing the pulpal dissolution; instead, it was focused on the cytotoxicity evaluation. It is understood that the cytotoxicity of the sodium hypochlorite is majorly dependent on concentration. Studies evaluated the cytotoxic potential of sodium hypochlorite[40],[41] As our study was aimed at comparing the natural irrigants as a comparative agents we choose an optimum and lower concentration of sodium hypochlorite as a positive control.

A recent literature review on efficiency of different concentrations of sodium hypochlorite concluded that 2.5% of sodium hypochlorite as an effective concentration used with greater effectiveness and lower cytotoxic.[42] The review also concluded that concentrations ranging from 0.5-1% was claimed to be the least cytotoxic. But, a positive control, should create the optimal and higher cytotoxic effect. As the widely used concentration among Indian endodontists ranged from 2.5 to 4%,[43] we selected optimal 3% concentration of sodium hypochlorite for our study. The other reason was commercial availability of 3% sodium hypochlorite, especially in India. So we choose this concentration as altering the higher concentrations by diluting or adjusting the concentrations, might not standardise the exact concentration of the agent used. Previous animal studies conducted by Indian authors also evaluated various root canal irrigants and used 3% concentration of sodium hypochlorite as a positive control in their studies.[44],[45] 3% concentration of sodium hypochlorite was chosen mainly based on the previous literature, which analysed the lesser concentrations of sodium hypochlorite to assess the effectiveness when compared with Morinda citrifolia.[16],[20] So, we had to choose 3% sodium hypochlorite.

In the present study, the systemic toxicity of different materials was evaluated. Sometimes there might be accidental ingestion of root canal irrigant by the patient when there is improper isolation. Reports have shown that ingestion of sodium hypochlorite caused throat irritation to severe upper airway obstruction.[46] Acute laryngotracheobronchitis, profuse drooling from the mouth and stridor was reported following the accidental ingestion of sodium hypochlorite.[47] The results of the present study showed the increased levels of ALP, Gpx and Glutathione reductase in 3% sodium hypochlorite at 7th and 14th days which indicates the increased toxic levels which have a direct impact on the systemic organs.

Even though the ethanolic extract of Morinda citrifolia showed increased inflammatory response and increased level of enzymes on toxicology screening, its levels were less when compared to 3% sodium hypochlorite and it was almost nil at the end of 30th day. Whereas the fresh fruit juice of Morinda citrifolia showed the least response in both histopathological and toxicity screening at all the experimental periods. Hence, it can be concluded from the present study that fresh fruit extracts are least cytotoxic and do not induce any reaction on accidental ingestion of the irrigant.

Among all the groups compared, the saline group elicited statistically insignificant rise in levels of the serum oxidative stress markers and LFT, RFT levels. When the histopathological response was evaluated, only moderate inflammation was seen at 7th day, and the inflammatory reaction was mild and absent at 14th and 30th day respectively. The findings were in correlation with the previous literature,[35],[44],[45] suggesting that saline resulted in favourable reaction in the connective tissues and can be considered as biocompatible.

In the present study, the blood was collected from the orbital sinus, before sacrificing the animal. The main reason for using this technique was because we required reasonable sample volumes, and repeat sampling was not needed. The blood sample was obtained only before the sacrifice of the animal. As it was a reliable and safe method, we chose this technique for blood sample collection. When the selection of rats for the study is analysed, our research was not concentrated on assessing the gender or sex-related data or the sex-specific diseases, and the experiment was not requiring the higher volumes of blood collection for serum analysis. The other factor was the more availability of female rats in the lab than males. So, we choose female rats for our study design.

The reason for making a trough in the periapical bone was mainly to create an experimental periapical reservoir simulating a periapical defect clinically. So, our aim in creating a trough was to assess the localised reactionary environment and the biocompatibility of the agents used. Ideally, the cytotoxic effect of irrigant or intracanal medicament cannot be evaluated arbitrarily. So to judge the biocompatibility of the agent in a specific periapical area, we had to make a trough of specific dimension. The other justification was cases with irrigant, intracanal medicament or sealer extrusions, beyond the confines of the root canal, into periapical regions said to have damaging effects and induce the evident bone necrosis and continued inflammation. Therefore to evaluate the cytotoxic response and the property of the agent tested, it is appropriate to create a trough over the periapical bone.

In the present study, we chose a 300 microliter of test for assessing the cytotoxicity. We did not choose the other various concentrations. The main reason was the observed dose-dependent cytotoxic effects of different concentrations of tested irrigants in our preliminary cell line study (unpublished). Our study results (unpublished) proved 300 microliters to be least cytotoxic. So we choose this concentration for the present study.

Previous studies evaluated only the cytotoxicity by assessing the subcutaneous reactions by circles made on the dorsum of the single animal.[44],[45] So, they required lesser sample sizes as all the groups were tested in a single rat. But the present study was interested in assessing the toxicity of the agents through serum analysis, along with the histopathological assessments. Hence, it was impossible to determine all the agents in a single animal. Also, our study was carried out at three different time intervals (7th, 14th, 30th day), where the animals were sacrificed respectively. Hence we included a total of 48 animals, and it was justified to obtain at least 12 animals per group, especially for these kinds of study designs.

The previous literature on animal studies assessing the endodontic irrigants was mainly limited to the assessment of cytotoxic profile by evaluating the subcutaneous tissue reaction.[44],[45] But, there are situations, where accidental ingestion of lower volumes of irrigant happens clinically in humans, which may or may not be therapeutically lethal to cause the effect. But, ideally, the endodontic animal studies should also concentrate on assessing the toxicity of the irrigating solutions used, before the clinical testing. So, to evaluate the utility of a root canal irrigant, prior research should also be carried out to assess the liver and renal function tests. These tests were done, mainly to determine the systemic toxicity of the agent before clinical application on humans. Hence, this study laid a unique pathway to evaluate the novel root canal irrigants. These in-vivo experimental models provide a scientific background for clinical practice.

Previous study investigated four major photochemical components such as iridoids, deacetylasperulosidic acid and asperulosidic acid, as well as two flavonoids, quercetin -3-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-glucopyranoside and kaempferol-3-O-α-L-rhamnopyranosyl-(1 → 6)-β-D- glucopyranoside, were quantitatively determined to be the major phytochemicals in the noni.[48] The medicinal properties of Noni are mainly due to these four photochemical components. The previous study reported that Noni has a property of scavenging reactive oxygen species, which is very appropriate for an intracanal medicament, this justifies the usage of Morinda citrifolia as an intracanal medicament.[49]

Studies have reported subcutaneous local tissue reaction at various concentrations of sodium hypochlorite (0.5%, 2.5%, and 5.25%).[50] There was no tissue reaction against different concentration of sodium hypochlorite at 24 hours but after two weeks, the higher concentration of sodium hypochlorite (2.5% and 5.25%) showed foreign body granuloma formation.[37] It was also reported that cytotoxicity of sodium hypochlorite is concentration dependant.[35] However, the results of the present study have shown that 3% sodium hypochlorite presented with severe inflammatory cells on the 7th day, which gradually reduced on 14th and 30th day. The inflammatory response induced by 3% sodium hypochlorite was in correlation with the previous literature, which stated an intense inflammatory reaction at specific periods assessed and slowly reduced after a 14th-day evaluation.[44],[45],[46],[51]

On comparing the tissue response of ethanolic extract of Morinda citrifolia and Fresh Fruit Juice, both the materials evoked mild to moderate inflammatory response when compared to sodium hypochlorite on the 7th day which gradually subsided on the 30th day. The results of this study cannot be compared with previous reports as there are no reports on the ethanolic extract of Morinda citrifolia.

The results of the present study on toxicology screening correlated with the previous studies which showed least cytotoxic and genotoxic effects of Noni.[52] Previous studies on animal models evaluated the chronic toxicity of various extracts of Morinda citrifolia.[53] One study[54] evaluated the chronic toxic effects with the aqueous extract of Morinda citrifolia, and the other two studies[55],[56] reported on the hepatotoxicity of Noni fruit juice. Fruit dried powder exhibited the subchronic oral toxicity[57] and aqueous leaf extract of Morinda citrifolia exhibited the genotoxicity, oral short term and subchronic toxicity.[58]

The ethanolic extract seemed to induce a certain degree of an inflammatory response and had a potent toxic profile at 7th and 14th days which was almost nil at the 30th day. The present study was first to report the ethanolic extract of Morinda citrifolia in animal models, and none of the studies till date have reported on systemic toxicity of the root canal irrigants used. There were no previous studies, which evaluated the serum toxicity levels of various irrigants investigated in the present study. So, the results of the serum analysis cannot be compared with the previous literature. So this study laid a novel pathway for evaluating the toxicological screening of the irrigant before passing into the clinical trials.

The other naturally available root canal irrigants investigated include[59] Aloe Vera, Propolis, Burdock (Articum Lappa), Cinnamon (Cinnamomum zeylancium), Ferula Gummosa, German Chamomile (Marticaria Recutta), Grapefruit (Citrus Paradisi), Green tea (Camellia Sinensis), Key lime (Citrus Aurantifolia), Mango (Mangifera Indica), Neem (Azadirachta Indica), Passion Fruit (Passiflora Edulis), Triphala, Tulsi (Ocimum Sanctum), Turmeric (Curcuma Longa). Although many naturally available products and extracts are available, the dearth of studies comparing root canal irrigants are concentrated on assessing the antimicrobial efficacy only at in-vitro and ex-vivo studies. Present study results cannot be compared with any of these extracts as none of the animal experimentation was carried out before the in vivo assessments. One study conducted the cytotoxicity of Triphala and liquorice extract and proved that herbal extracts were least cytotoxic as compared to 3% sodium hypochlorite.[60] There was no animal experimentation to date on the usage of these herbal products to assess the cytotoxicity, especially as root canal irrigant.

When the limitations of the present study are considered, acute subcutaneous tissue reactions at 1 hr, 2 hr and 1 day time intervals were not evaluated. So ideally, biocompatibilty studies should include acute time intervals when evaluating the inflammatory responses. The other factor is the protocol of assessment. Most of the previous studies assessed the reactions by injecting the irrigant directly into subcutaneous tissue, but the present study was planned to determine the specific response in the periapical area by creating an experimental trough. So, the validity of the current protocol still needs to be justified with more future studies. The major limitation was the extracts used. Every time the extracts were prepared freshly before experimentation and were not stored. So, ideally, in a clinical scenario, it is cumbersome to prepare the extracts daily. Thus, the clinical reliability and usage of these extracts are questionable.

The other major limitation of the study was the need for fresh preparation of extract, which is cumbersome in a clinical situation. Although the present study was standardised, ideally, these results cannot be generalised to clinical situations, as the human environment is different from an animal model and laboratory conditions. So, more standardised protocols are encouraged before proceeding to the testing in humans.


Within the limitations of the present study it can be concluded that the ethanolic extract of Morinda citrifolia showed almost a similar inflammatory response and systemic toxicity as 3% sodium hypochlorite at 7th and 14th days. So, fresh fruit juice of Morinda citrifolia can be a preferred choice of natural root canal irrigant, with least possible cytotoxic effects as compared to the ethanolic extract.

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Conflicts of interest

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