|Year : 2021 | Volume
| Issue : 4 | Page : 272-277
comparative evaluation of antimicrobial activity and surface tension of a combination of herbal extracts in different ratios: An in vitro study
Sayali Belsare, Alok Patel, Shweta Jajoo, Chetana Jagtap, Sneha Desai, Preetam Shah
Department of Paediatric and Preventive Dentistry, Bharati Vidyapeeth Dental College and Hospital, Pune, Maharashtra, India
|Date of Submission||22-Jun-2021|
|Date of Acceptance||13-Aug-2021|
|Date of Web Publication||20-Dec-2021|
Omkar, Anantkripa Society, Plot No. 11, Paud Road, Pune - 411 038, Maharashtra
Source of Support: None, Conflict of Interest: None
Aim: The aim of this research was to evaluate the antimicrobial activity and surface tension of a combination of herbal extracts and compare them with the gold standard in irrigating solutions that is sodium hypochlorite. Methodology: A combination of lime peel and reetha extract and ashwagandha and triphala extract was prepared in the ratio of 1:1 and 2:1, and the antimicrobial activity of all the four groups was evaluated against Enterococcus faecalis and Candida albicans. The microbial testing was done using the standard protocols for testing the zone of inhibition. The surface tensions of the prepared solutions were also evaluated using a tensiometer. The gold standard in irrigating solutions, 5.25% sodium hypochlorite, was used as the positive control. Results: Results from the study showed that all the four solutions had antimicrobial activity, however, it was significantly lower than the control group. Intergroup comparison using one-way ANOVA within the groups showed a significant difference between the zone of inhibition of the solutions against E. faecalis and C. albicans with P = 0.001. Post hoc Tukey's test was applied to the results, and the groups showed a significant difference with the positive control. Within the groups, a significant difference was seen, indicating that there were differences between the zone of inhibition due to the separate solutions. The surface tension which gives an idea of the surface wetting capacity of the irrigating solution was better in the test groups. It was highest (67.94 dynes/cm) for the positive control that is NaOCl which signifies a lower surface wetting capacity. Conclusion: It was seen that the herbal ingredients did show antimicrobial activity, however, it was not as good as the gold standard NaOCl. The test groups, however, had a lower surface tension as compared to the control group which implies a better surface wetting capacity and a better ability of the solutions to reach the uninstrumented areas.
Keywords: Antimicrobial, Citrus aurantifolia, irrigation, Sapindus mukorossi, surface tension, triphala, Withania somnifera
|How to cite this article:|
Belsare S, Patel A, Jajoo S, Jagtap C, Desai S, Shah P. comparative evaluation of antimicrobial activity and surface tension of a combination of herbal extracts in different ratios: An in vitro study. J Dent Res Rev 2021;8:272-7
|How to cite this URL:|
Belsare S, Patel A, Jajoo S, Jagtap C, Desai S, Shah P. comparative evaluation of antimicrobial activity and surface tension of a combination of herbal extracts in different ratios: An in vitro study. J Dent Res Rev [serial online] 2021 [cited 2022 May 23];8:272-7. Available from: https://www.jdrr.org/text.asp?2021/8/4/272/332912
| Introduction|| |
A successful endodontic treatment requires a combination of factors, such as an accurate diagnosis, thorough cleaning, a predictable disinfection protocol achieved with the help of various intracanal medicaments and irrigation solutions which is followed by obturation and adequate final restoration.
The primary objective of pulp therapy is to maintain the integrity and health of the teeth and their supporting tissues. Due to the bizarre anatomy of the root canal system in the primary teeth,, instrumentation followed by irrigation with a solution that promotes disinfection of these areas is important, mainly because infected primary teeth can harbor microbes within the dentinal tubules. Irrigation is presently the best method as it allows the debris and the other contaminated material to be flushed away before it is inadvertently pushed deeper into the canal and apical tissues which can compromise the periapical tissue and permanent tooth bud.
Sodium hypochlorite ranging from the concentration of 0.5%–6% has been used as an endodontic irrigant due to its properties that contribute to the effective chemomechanical debridement of a root canal system including a good antimicrobial activity against the microbes present in the infected root canal. However, it has disadvantages like its inability to penetrate well into confined areas of a root canal system. There are also a few case reports that describe life-threatening airway obstruction.,
Hence, in the recent years, the quest has been on to search for an alternative to sodium hypochlorite and the direction of research is toward herbal solutions.
It has been observed that very few individual herbal irrigants have an action comparable to that of sodium hypochlorite. Due to this reason, in this study, a combination of different herbal irrigants was evaluated for its antimicrobial property and its surface wetting capacity, with 5.25% sodium hypochlorite being used as a positive control.
The herbal powders used in the current study were as follows:
- Sapindus mukorossi (vernacular names – ritha, aritha, dodhan, and soapnut) which is known to have insecticidal, spermicidal, antiprotozoal, anti-inflammatory, cytotoxic (for cancer cells), hepatoprotective, antiplatelet aggregation, and antifungal activities
- Citrus aurantifolia (vernacular names – lime and key lime) which contains alkaloid, flavonoid, glycoside, saponin, steroid, phenols, terpenoid, and tannins and is known to have antifungal, antimicrobial, anticancer, immunomodulatory, anti-obesity, antifertility, antihelminthic, and anticholinesterase activities
- Withania somnifera (vernacular names – ashwagandha or winter cherry) which contains alkaloids (ashwagandhine, cuscohygrine, anahygrine, tropine, etc.) and steroidal compounds and is known to possess antioxidant, anxiolytic, adaptogen, memory-enhancing, antiparkinsonian, antivenom, anti-inflammatory, antitumor, immunomodulatory, and antimicrobial activities
- Triphala which is a herbal formula composed of the three fruits of Terminalia chebula, Terminalia bellirica, and Phyllanthus emblica or Emblica officinalis which possess compounds such as tannins, phenolic compounds, fatty acids, and triterpenoids and is known to have antimicrobial, free radical scavenging, antioxidant, anti-inflammatory, antipyretic, analgesic, antimicrobial, wound healing, anticariogenic, antistress, hypoglycemic, anticancer, chemoprotective, radioprotective, and chemopreventive effects. It has also been seen to have a vast number of applications in dentistry.
The antimicrobial effects of the combinations were studied against Candida albicans and Enterococcus faecalis.
Surface tension is related to the ability of the solutions to spread within the root canal and hence should be considered as an important parameter when evaluating any irrigating solution. Hence, the surface tension of the prepared solutions was also evaluated as a descriptive parameter.
The aim of the current study was to evaluate the antimicrobial activity and surface tension of a combination of herbal extracts and compare them with the gold standard in irrigating solutions that is sodium hypochlorite.
| Methodology|| |
The current study was carried out at Bharati Vidyapeeth Dental College and Hospital, Pune, and at Monera Lab, Pune. The materials were obtained from Manakarnika Aushadhalaya, Pune [Table 1].
|Table 1: Comparison of zone of inhibition for Enterococcus faecalis group in terms of mean (standard deviation) using one-way ANOVA test|
Click here to view
- Reetha powder (S. mukorossi) (Vanaspati Botanical, Pune)
- Lime peel powder (Citrus aurantifolia) (Manakarnika Aushadhalaya, Pune)
- Ashwagandha powder (Withania somnifera) (Manakarnika Aushadhalaya, Pune)
- Triphala powder (E. officinalis, T. bellirica, and T. chebula) (Bhardwaj Pharmaceutical Works, Indore)
- 5.25% sodium hypochlorite (Prime Dental, Pune).
Preparation of the materials
- Group 1 – 100 mg/ml of lime peel + 100 mg/ml of reetha extracts in distilled water (1:1 ratio)
- Group 2 – 120 mg/ml of lime peel + 60 mg/ml of reetha extract in distilled water (2:1 ratio)
- Group 3 – 80 mg/ml triphala extract + 80 mg/ml of ashwagandha extract in distilled water (1:1 ratio)
- Group 4 – 160 mg/ml of ashwagandha extract + 80 mg/ml triphala extract in distilled water (2:1 ratio)
- Group 5 – 5.25% sodium hypochlorite (Prime Dental, Pune) was used as the positive control.
The microbial inhibition assay was performed by standard norms and protocol by using the agar well diffusion method. All the instruments required were sterilized using the hot air oven or the autoclave, and the tests were carried out in the biosafety cabinet to avoid the contamination of the test materials. Adequate amount of Mueller Hinton agar (MHA) was evenly distributed over the surface of Petri dish More Details and allowed to solidify under aseptic conditions. The strains of E. faecalis and C. albicans obtained from the National Chemical Laboratory were subcultured. The turbidity of culture solution was obtained at 0.5 McFarland Standard by adding the organism. The swabs of test samples of strains of C. albicans and E. faecalis were then inoculated with a sterile spreader on the surface of solid MHA medium on different plates. Standard wells were made in the plates with the help of a cup borer. Each plate had 5 wells. The test materials were inserted in the wells of different agar plates inoculated with C. albicans and E. faecalis. Each group had 25 samples. The sample size was estimated using GPower software v22.214.171.124. The level of significance was fixed at 5%, the power of the study was kept at 80% and effect size of 0.39, and the sample size estimated was 19 per group. These wells were then incubated at 37°C ± 0.1°C for 24 h. After incubation, the plates were observed for zone of inhibition and the diameters of these zones were measured in millimeters by using Vernier calipers.
Surface tension testing
The surface tension of all the materials was checked using an Automatic Surface Tensiometer DY-300(Kyowa) which uses the Wilhelmy plate and du Nouy ring method to evaluate the surface tension. The test was carried out at Laxmi Analytical Laboratories, Mumbai. The results for the surface tension were expressed in dynes/cm.
| Results|| |
The obtained data were analyzed using SPSS v20 software, keeping the level of significance at 5%. One-way ANOVA test was used for the comparison of the zone of inhibition and post hoc Tukey's test was used for the intergroup comparison.
Against Enterococcus faecalis
The results of this study showed that within the test groups, Group 1 (1.12 mm) and Group 2 (0.34 mm) showed a lower mean zone of inhibition against E. faecalis as compared to Group 3 (1.38) and Group 4 (1.28 mm). The difference between the zone of inhibition among the groups was highly significant (P = 0.001) [Table 1] and [Graph 1].
The intergroup comparison using the Tukey's post hoc analysis [Table 2] and [Graph 2] gave the following results:
The zone of inhibition in Group 1 was significantly higher than that in Group 2 (difference: 0.78, P = 0.001), from which it can be concluded that an increase in the quantity of C. aurantifolia significantly improved the antimicrobial activity against E. faecalis. Group 3 and Group 4 were seen to be better than Group 2 with a statistically significant difference (P = 0.001). From this, it can be concluded that according to the current study, the combination of triphala and ashwagandha may have a better antimicrobial efficacy against E. faecalis as compared to a combination of C. aurantifolia and S. mukorossi. However, the difference between Group 1 and Group 3 and Group 1 and Group 4 was not statistically significant.
|Table 2: Intergroup comparison of zone of inhibition for Enterococcus faecalis (Tukey's post hoc analysis)|
Click here to view
Against Candida albicans
The comparison of the zone of inhibition among the four groups and the positive control group was seen to be statistically significant (P = 0.001) according to the ANOVA test [Table 3] and [Graph 3].
|Table 3: Comparison of zone of inhibition for Candida albicans group in terms of mean (standard deviation) using one-way ANOVA test|
Click here to view
Tukey's post hoc analysis [Table 4] and [Graph 4] gave the following results – Group 1 and Group 2 showed a better zone of inhibition against C. albicans as compared to Group 3 and Group 4. In the current study, the solutions in Group 1 and Group 2 seem to have a slightly better antimicrobial efficacy against C. albicans as compared to E. faecalis.
|Table 4: Intergroup comparison of zone of inhibition for Candida albicans using Tukey's post hoc analysis|
Click here to view
The intergroup comparison using the Tukey's post hoc analysis showed that there was no statistically significant difference between any of the test groups. However, all the test groups showed a significant difference with the positive control. The zone of inhibition for 5.25% NaOCl was seen to be significantly higher than the test groups (P = 0.001).
Hence, it can be concluded that even though the test groups did show some activity against E. faecalis and C. albicans, none of them were as good as 5.25% sodium hypochlorite.
In the current study, the surface tension of Group 2 was seen to be the lowest (36.86 dynes/cm) followed by that of Group 1 [Table 5] and [Graph 5]. This could be because of the saponin content from the S. mukorossi which is known to have an emulsification action which results in reduced surface tension. However, in contrast to the initial assumption after considering that S. mukorossi would reduce the surface tension, the results showed that the surface tension was lower in Group 2 which had a lower content of S. mukorossi.
Among the test groups, Group 4 showed the highest surface tension which could indicate that the ashwagandha (W. somnifera) resulted in an increase in the surface tension. However, the surface tension values were seen to be lower in all the test groups than the positive control that is sodium hypochlorite.
Hence, it can be seen from the results that the water-based solution of the abovementioned compounds seems to have a better surface wetting capacity and hence a better ability to reach even the uninstrumented parts of the root canal, however, the antimicrobial efficacy of the combinations against E. faecalis and C. albicans was not as good as 5.25% sodium hypochlorite.
| Discussion|| |
Triphala is one of the most evaluated herbal materials in dentistry. In a study by Prabhakar et al., triphala was shown to eradicate a 3-week-old E. faecalis biofilm. This is in accordance with the current study where the combination of triphala and ashwagandha showed a better antimicrobial efficacy against E. faecalis as compared to the other groups. Aneja et al. evaluated the antimicrobial activity of S. mukorossi and E. officinalis and the extracts of S. mukorossi exhibited antimicrobial activity against Saccharomyces cerevisiae, while extracts of E. officinalis showed inhibitory activity against Streptococcus mutans. S. mukorossi is also known to reduce the surface tension by emulsification and hence improve the surface wetting capacity. A saponin compound from the pulp of S. mukorossi has been identified, 16 μg/mL of which caused death of more than 99.9% C. albicans within 4 h. In the current study, the combination of C. aurantifolia and S. mukorossi showed a better antimicrobial efficacy against C. albicans.
C. aurantifolia has been tested for its smear layer removal capabilities. It is known to have citric acid as one of the main constituents which has a good smear layer removal efficiency. However, a study by Bolhari et al. concluded that both alcoholic and completed mixtures of C. aurantifolia extracts were not able to effectively remove smear layer compared with 17% ethylenediaminetetraacetic acid (EDTA) during root canal therapy.
The smear layer removal efficacy of solutions of S. mukorossi and C. aurantifolia was evaluated in a previous study by Chhabra et al., and they observed that the combination of the extracts in 2:1 ratio was slightly better than 1:1 ratio. This was further improved when accompanied with sonic agitation.
Similarly, ashwagandha and triphala have both been individually evaluated for their antimicrobial effects, however, the results have shown that they are not as good as NaOCl. Hence, a combination of these two was evaluated in this study. Somayaji et al. have evaluated this combination in their study for the antimicrobial effects and have concluded that there was a considerable reduction in the E. faecalis count after irrigation with these solutions. This is in accordance with the results of the current study where a reduction in the E. faecalis counts was seen.
A search of literature for the studies evaluating the surface tension of different herbal alternatives to the chemical-based irrigating solutions did not yield any results.
In a study by Pécora et al., drugs used in the pulpectomies had a high surface tension and the surface tension of Dakin's solution was similar to that of water. They also concluded that distilled water has the highest surface tension while the drugs with phenol groups had a lower surface tension. Milli-Q water, sodium hypochlorite 5.25%, and EDTA 17% had the highest surface tension, whereas those of cetrexedin and tetraclean have shown the lowest surface tension value in a study by Giardino et al. This is in accordance with the current study where sodium hypochlorite showed the highest surface tension among the tested solutions.
From the current study, it can be seen that even though sodium hypochlorite had significantly better antimicrobial activity as compared to the test groups, the surface tension was the highest. Hence, according to the current data available, that limits its penetration into irregularities of the root canal system such as fins, isthmi, and dentinal tubules and also the uninstrumented areas of the primary root canals.
The current study had the following limitations – the extracts used were water-based solutions prepared from commercially available powders obtained from an ayurvedic formulation store. An ethanolic extract may yield better results than these water-based formulation, however, the ethanol may have adverse effects on the tissues. The solubility of the powders may also not be complete in distilled water and hence the powders may be dissolved in dimethyl sulfoxide and may give better results due to the active compounds.
Furthermore, the pure extracts obtained from the store also seemed to have some contamination as seen on the plates and hence even this could have affected the results in the current study.
One more limitation of the current study was that 5.25% sodium hypochlorite was used. However, it is available in varying concentrations, and clinically, it is used in varying dilutions, especially in pediatric patients. Hence, the antimicrobial efficacy of the test groups can also be compared with lower concentrations of sodium hypochlorite ranging from 1% to 6%.
| Conclusion|| |
The use of herbal extracts in pediatric dentistry has been on the rise over the past few years. These products are known to avoid the toxic effects to the tissues and also have minimal or no systemic effects.
In the current study, both the combinations that is triphala and ashwagandha and lime peel and reetha, in both the ratios used, were seen to have an antimicrobial activity against both the organisms tested (E. faecalis and C. albicans), however, triphala and ashwagandha (W. somnifera) seemed to have a better antimicrobial efficacy against E. faecalis while the combination of lime peel (C. aurantifolia) and reetha (S. mukorossi) seemed to have a better antimicrobial efficacy against C. albicans. None of the tested groups showed an antimicrobial efficacy as good as the gold standard that is sodium hypochlorite.
The surface tension of sodium hypochlorite was the highest and it was lower in the group which had reetha (S. mukorossi) and hence it can be concluded that the surface wetting capacity was better for the test groups as compared to the gold standard. This is especially of importance in primary teeth where it is important for the irrigant to reach the uninstrumented areas of the root canal.
Further studies using the abovementioned combinations in extracted teeth and clinical trials could give us a better idea as to whether these products can be used clinically to achieve a successful endodontic treatment outcome in the primary teeth.
As the given study has no human intervention, the institute did not require an ethical clearance for the same.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
American Academy of Pediatric Dentistry. Pulp therapy for primary and immature permanent teeth. The Reference Manual of Pediatric Dentistry. Chicago, III.: American Academy of Pediatric Dentistry 2021:399-407.
Finn SB. The children's dentist, his practice and his community. In: Finn SB, editor. Clinical Pedodontics. 4th
ed. Philadelphia: WB Saunders; 1973.
Ash M, Wheeler RC, Nelson SJ. Wheeler's Dental Anatomy, Physiology and Occlusion. 8th
ed. Philadelphia: WB Saunders; 2003.
Hobson P. Pulp treatment of deciduous teeth and factors affecting diagnosis and treatment. Br Dent J 1970;128:232-8.
Kaur R, Singh R, Sethi K, Garg S, Miglani S. Irrigating solutions in pediatric dentistry: Literature review and update. J Adv Med Dent Sci 2014;2:104-15.
Pradhan MS, Gunwal M, Shenoi P, Sonarkar S, Bhattacharya S, Badole G. Evaluation of pH and chlorine content of a novel herbal sodium hypochlorite for root canal disinfection: An experimental in vitro
study. Contemp Clin Dent 2018;9:S74-8.
Senia ES, Marshall FJ, Rosen S. The solvent action of sodium hypochlorite on pulp tissue of extracted teeth. Oral Surg Oral Med Oral Pathol 1971;31:96-103.
Al-Sebaei MO, Halabi OA, El-Hakim IE. Sodium hypochlorite accident resulting in life-threatening airway obstruction during root canal treatment: A case report. Clin Cosmet Investig Dent 2015;7:41-4.
Kaushik N, Rehani U, Agarwal A, Kaushik M, Adlakha V. Antimicrobial efficacy of endodontic irrigants against Enterococcus faecalis
and Escherichia coli
: An in vitro
study. Int J Clin Pediatr Dent 2013;6:178-82.
Suhagia BN, Rathod IS, Sindhu S. Sapindus mukorossi
(Areetha): An overview. IJPSR 2011;2:1905-13.
Enejoh OS, Ogunyemi IO, Bala MS, Oruene RS, Suleiman MM, Ambali SF. Ethnomedical importance of Citrus aurantifolia
(Christm) Swingle. Pharma Innov J 2015;4:1-6.
Gupta LG, Rana AC. PHCOGMAG: Plant review. Withania somnifera
(Ashwagandgha): A review. Pharmacol Rev 2007;1:129-36.
Baliga MS, Meera S, Mathai B, Rai MP, Pawar V, Palatty Pl. Scientific validation of the ethnomedicinal properties of the ayurvedic drug triphala: A review. Chin J Integr Med 2012;18:946-54.
Naumovich DB. Surface tension and PH of drugs in root canal therapy. Oral Surg Oral Med Oral Pathol 1963;16:965-8.
Prabhakar J, Senthilkumar M, Priya MS, Mahalakshmi K, Sehgal PK, Sukumaran VG. Evaluation of antimicrobial efficacy of herbal alternatives (Triphala and green tea polyphenols), MTAD, and 5% sodium hypochlorite against Enterococcus faecalis
biofilm formed on tooth substrate: An in vitro
study. J Endod 2010;36:83-6.
Aneja KR, Joshi R, Sharma C. In vitro
antimicrobial activity of Sapindus mukorossi
and Emblica officinalis
against dental caries pathogens. Ethnobotanical Leaf 2010;14:402-12.
Balakrishnan S, Varughese S, Deshpande AP. Micellar characterization of saponin from Sapindus mukorossi
. Tenside Surf Det 2006;43:262-8.
Hu Q, Chen YY, Jiao QY, Khan A, Li F, Han DF, et al.
Triterpenoid saponins from the pulp of Sapindus mukorossi
and their antifungal activities. Phytochemistry 2018;147:1-8.
Bolhari B, Sharifian MR, Aminsobhani M, Monsef Esfehani HR, Tavakolian P. Assessing the efficacy of citrus aurantifolia extract on smear layer removal with scanning electron microscope. Iran Endod J 2012;7:88-97.
Chhabra N, Gyanani H, Kamatagi L. Smear layer removal efficacy of combination of herbal extracts in two different ratios either alone or supplemented with sonic agitation: An in vitro
scanning electron microscope study. J Conserv Dent 2015;18:374-8.
] [Full text]
Somayaji SK, Ballal ND, Shobha KL, Mohandas Rao KG. Comparison of antimicrobial efficacy of triphala, Withania somnifera
and sodium hypochlorite against Enterococcus faecalis
biofilm – An in vitro
study. Int J Pharm Pharm Sci 2014;6:808-11.
Pécora JD, Guimarães LF, Savioli RN. Surface tension of several drugs used in endodontics. Braz Dent J 1992;2:123-7.
Giardino L, Ambu E, Becce C, Rimondini L, Morra M. Surface tension comparison of four common root canal irrigants and two new irrigants containing antibiotic. J Endod 2006;32:1091-3.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]