Anti-inflammatory efficacy of petasin-incorporated zinc oxide eugenol sealer - An in vivo zebrafish study.

Background: The eugenol from zinc oxide eugenol (ZnOE) sealer tends to diffuse to the periapical region resulting in inflammation. Several modifications of ZnOE sealer have been formulated to minimize the inflammatory potential of the ZnOE sealer. Petasites hybridus contains petasin which possesses anti-inflammatory property used in treatment of migraine and allergic rhinitis. Aim: The aim of the study was to evaluate the anti-inflammatory property of petasin-incorporated ZnOE sealer on zebrafish. Materials and
Methods: The study has been reviewed and approved by the Institutional Review Board (SRMU/MandHS/SRMDC/2018/S/025) and by the in-house Institutional Animal Ethics Committee (IAEC) of Pentagrit Research Lab and conducted with compliance to ICH harmonization and principles for animal housing and handling (IAEC Study No: 215/Go06/IAEC). The samples were implanted in the caudal portion of the zebrafish. The samples (n = 50) were divided into five groups (n = 10) - Group 1: incision only (negative control), Group 2: zinc oxide (ZnO) (positive control), Group 3: ZnO + eugenol and petasin in ratio of 10:1, Group 4: ZnO + eugenol and petasin in ratio of 5:1, and Group 5: ZnO + eugenol and petasin in ratio of 1:1. The experimental groups were further subdivided into two subgroups based on time intervals at 24 h and 48 h. The tissue samples were assessed using smear pathology test, and the percentage of inflammation was evaluated. The data were statistically analyzed using SPSS software version 22.0 with a significance level fixed as 5% (α = 0.05).
Results: The presence of inflammatory cells and congestion of arterioles were taken as the criteria to assess inflammatory action. It was maximum in ZnOE sealer followed by ZnOE sealer modified with the addition of petasin to eugenol in the ratio of 10:1, 5:1, and 1:1, respectively.
Conclusion: The addition of petasin extract to ZnOE reduces the inflammation potential of ZnOE sealers. ZnOE sealer containing eugenol and petasin in the ratio of 1:1 showed a maximal reduction in inflammation. Copyright:

INTRODUCTION

The main objectives of root canal therapy are complete debridement and shaping followed by fluid-tight obturation of the root canal system. Root canal sealers play an important role in providing this seal when coated on the gutta-percha points during obturation. The sealers are in contact with the periapical tissue in their freshly mixed state and post setting for a very long time. When the root canal sealers extrude beyond the apex, it could irritate the periapical tissues and might interfere with the healing process due to its potentially toxic ingredients.[1]

The most commonly used ZnOE sealer has several limitations that include hydrolysis, disintegration, and release of toxic substances in the periapical tissue, leading to inflammation and necrosis.[2] ZnOE sealers have shown to be cytotoxic in cell culture studies.[3] Short- and long-term subcutaneous implantation of these sealers induces large influx of macrophages and is found to cause moderate-to-severe inflammatory response with time.[3] This is attributed to the solubility of the sealers and availability of tissue fluids. These factors are the prime determinants for the release of free eugenol, thereby leading to toxicity.

Several attempts to improve the properties of ZnOE sealer have been made such as antibacterial activity (paraformaldehyde), anti-inflammatory (steroids), radiopacity (silver), consistency (Canada balsam), setting time (sodium borate anhydrate), and microleakage (zinc oxide nanoparticles).[45678]

Steroids were incorporated to reduce the inflammatory effect of freshly mixed ZnOE-based sealers. Endomethasone (Septodont, Cedex, France), a commercially available ZnOE-based sealer, has been shown to have sedating and antimicrobial property which is effective over a large spectrum and persistent over time.[9] The steroid present in the sealer is found to cause postoperative pain and discomfort after 6–8 weeks of insertion.[5] The steroids mask any inflammatory reaction until it is removed from the area and induces inflammatory reaction and postoperative pain.[5] It also significantly inhibited cell metabolism. Some authors showed that when pachymic acid, a naturally derived product with anti-inflammatory property, was added to resin-based sealers, it improved the physical properties of flow, setting time, film thickness, solubility, and radiopacity[10] and did not have cytotoxicity.[11] Similarly, an in vitro study done by Vinola et al. showed that petasin, a herbal medicine, extracted from the shrub of Petasites hybridus (common name: butterbur) added to ZnOE sealer significantly reduced the inflammation caused by the sealer.[12] It is being widely used for treating allergic rhinitis, tonsillitis, migraine, and asthma.[13]

However, there are no in vivo studies to validate these findings. Hence, the purpose of this in vivo histological study was to investigate the inflammatory effect of zinc oxide eugenol (ZnOE) sealer with and without incorporation of petasin extract in zebrafish model. Danio rerio (zebrafish) member of the Cyprinidae family is a freshwater fish, native to the Himalayan region. Zebrafish has been used as a model organism for biological research since 1930.[14] Being a vertebrate fish, it is an important model for vertebrate development, genomics, physiology, behavior, drug toxicology, and disease.[15] There are studies in literature stating the use of zebrafish for checking the anti-inflammatory property of herbal extracts by inserting implants.[16]

The null hypothesis proposed was that the addition of petasin in varying ratios to the ZnOE sealer will not affect the inflammation caused by the sealer.

MATERIALS AND METHODS

Zebrafish husbandry

Fifty adult zebrafishes (Pentagrit Research Lab, Chennai, India.) were segregated from the stock and acclimatized for 10 days to laboratory conditions, temperature (27 ± 2°C), and pH (7.5–7.8). The fishes in groups of 10 were housed in 5 housing units for a light-dark cycle of 14/10 h at 27 ± 1°C. The study has been reviewed and approved by the Institutional Review Board (SRMU/MandHS/SRMDC/2018/S/025) and by the in-house Institutional Animal Ethics Committee (IAEC) of Pentagrit Research Lab and conducted with compliance to ICH harmonization and principles for animal housing and handling (IAEC Study No: 215/Go06/IAEC). Good animal practice as per IAEC in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals, India, was followed in adherence to the established protocols.

The study consisted of five groups (n = 10), with Group 1 consisting of an incision alone (negative control) and Group 2: conventional ZnOE (Deepak Enterprises, Mumbai, India) – positive control. The liquid for the experimental groups (Groups 3–5) was made by mixing eugenol and 15% petasin in ratios of 10:1, 5:1, and 1:1. Petasin extract (15%) from the Petadolex capsules (Enzymatic Therapy, Germany, batch no. 20085617) was used in this study. This liquid was then mixed with zinc oxide powder to obtain the required sealer consistency.

Methods

Anesthesia

To prepare the fish for implant, the fish from the clutches was isolated and anesthetized using gradual cooling of the water. The anesthesia tank was set with two adjacent water baths 17°C and 12°C. The fish was gently transferred from the clutches to the first anesthesia tank maintained at 17°C and was observed for gradual decrease in the operculum movement, after which it was transferred to 12°C tank, until the fish was ready to handle with no response to caudal fin touch. The anesthetized fish was then placed on the observation stage with minimal oxygen support and was kept hydrated.

Implantation

Sealers of each group were mixed according to the grouping, and allowed to dry at 37°C for 24 h to form a soft chalk consistency. A small incision was placed at the caudal region of the zebrafish using a number 11 scalpel; 0.5–1 mm (1 mg by weight) of each sample was then inserted into the incision site except the control group [Figure 1]. Incisions were made in the control group also to differentiate between the incision injury and the implant material toxicity. The fishes were transferred to housing water unit immediately after implantation. At 24 h and 48 h, five fishes from each group were euthanized for histopathological evaluation.

Figure 1

(a) Incision made with a no. 11 scalpel at the caudal region of the anesthetized zebrafish. (b) Zebrafish with an incision on the caudal region. (c) The experimental material implanted in the incision site

Dissection

The fishes were euthanized at 10°C and placed on a dissection stage. The external hard scales were removed gently with the help of forceps and dissection needle exposing the internal soft skeletal muscles. A small cut section of approximately 0.5 mg of the localized muscle near the implantation site was removed and sectioned to 7-μm samples and fixed by squash cytology. The slides for each respective group were prepared for smearing and staining with hematoxylin and eosin.

Smear pathology

Smear pathology and screening for percentage of mortality were performed at 24 h and 48 h.

Imaging and quantification of cells

Smeared pathology slides were observed under bright-field microscope (Labomed LX 400). The images were analyzed and the numbers of damaged and normal cells were counted using Future Win Joe software (Hangzhou Future Optics Sci. and Tech. Co., Ltd. China). The samples were evaluated for inflammatory infiltrate, cellularity, vascularization, and macrophagic activity. The percentage of inflammation was calculated by comparing with the control and statistically analyzed using one-way ANOVA followed by post hoc test for intergroup comparison and paired t-test for intragroup comparison. SPSS software version 22.0 (Statistical Product and Service Solutions, SPSS, Chicago, USA) was used for statistical analysis with a significance level fixed as 5% (α = 0.05).

RESULTS

Percentage of mortality

There was no mortality in Groups 1 and 5 at 24 and 48 h [Figure 2]. Group 2 showed maximum mortality of 90% and 100% at 24 h and 48 h, respectively. The addition of petasin to the ZnOE sealer proportionately decreased the mortality.

Figure 2

Bar diagram – percentage of mortality at 24 h and 48 h

Histopathological findings

Histopathological analysis revealed mild inflammatory response in Group 1 at 24 h and 48 h [Figures 3 and 4]. Group 2 showed a severe inflammatory response at 24 h. Among the experimental groups, as the amount of petasin increased, there was a statistically significant reduction in the inflammation at 24 h and further reduction of inflammation at 48 h, with Group 5 showing minimal or no inflammatory changes [Figures 5 and 6].

Figure 3

Histological images of localized muscle post implant at 24 h. (a) Group 1 showed an inflamed area with mononuclear inflammatory infiltrate (×400 magnification) (circle area); inflammatory grade, moderate. (b) Group 2 showed severe infiltration of inflammatory cells (PMNs, macrophages, and lymphocytes) (×400 magnification) (circle area). Note intense mononuclear infiltrate and congested blood vessels; inflammatory grade, severe. (c) Group 3 showed severe infiltration of inflammatory cells (PMNs, macrophages, and lymphocytes) (×400 magnification) (circle area). (d) Group 4 showed slight infiltration of inflammatory cells (macrophages and lymphocytes) (circle area); inflammatory grade, moderate. (e) Group 5 showed mild infiltration of PMNs, macrophages, and lymphocytes (×200 magnification) (circle area); inflammatory grade, moderate

Figure 4

Histological images of localized muscle post implant at 48 h. (a) Group 1 showed numerous fibrous tissues and the formation of new blood vessels (×400 magnification); inflammatory grade, absent (circle area). (b) Group 3 showed severe infiltration of inflammatory cells (PMNs, macrophages, and lymphocytes) (×400 magnification) (circle area). (c) Group 4 showed moderate infiltration of inflammatory cells (macrophages and lymphocytes) (circle area); inflammatory grade, moderate. (d) Group 5 showed mild infiltration of PMNs, macrophages, and lymphocytes (×200 magnification), with formation of new blood vessels (circle area); inflammatory grade, moderate

Figure 5

Bar diagram – percentage of inflammation with implant injury at 24 h and 48 h

Figure 6

Bar diagram – percentage of inflammation without implant injury at 24 h and 48 h

DISCUSSION

The inflammatory, neurotoxic effect and cytotoxicity of ZnOE sealer have been well evidenced in the literature.[17] Free eugenol, a tissue irritant, leaches out from the sealer into the periapical region and initiates the release of inflammatory cells such as neutrophils, macrophages with induction of pro-inflammatory cytokines such as interleukin-1 (IL-1) β, tumor necrosis factor-α (TNF-α), leukotrienes (LTs), prostaglandins, interferon-γ, and transforming growth factor-β leading to infection, inflammation, and bone resorption at the periapical region.[18] The cytokines produced predominantly by activated macrophages, play a vital role in the upregulation of inflammatory reactions inducing pain.[19]

Petadolex® is a standard proprietary extract manufactured since 1988 by a patented procedure in a capsule form containing 15% petasin which is free of pyrrolizidine alkaloids. It has been accepted by the American Association of Neurologists.[13] Petasin derived from butterbur (P. hybridus) has been reported to have a range of anti-inflammatory, antispasmodic, and analgesic properties.[13] It consists of essential oils (0.4%), sesquiterpenes (petasine, isopetasine, and neopetasin), sesquiterpene lactones (bakkenolides and eremopetasitenins, and pyrrolizidine alkaloids like senecionine).[20] The inflammatory effects of the sesquiterpenes are deployed by the mechanism of inhibiting LT synthesis.[21] Zebrafish (Danio rerio) was used in this study since the genomes, molecular mechanisms of development, and signaling pathways involved in the immune response of zebrafish are highly similar to those of mammals.[2223] The genes are 75% homologous to human genes, and more than 80% of human disease-associated genes are present in them.[23]

The anti-inflammatory effect of petasin was found to be concentration dependent since 1:1 ratio showed the least inflammation. The decrease in inflammation by addition of petasin can be attributed to the presence of sesquiterpenes which possess anti-inflammatory property. LTs and other inflammatory mediators play a vital role in the inflammatory cascade affiliated with periapical inflammation. The expression of many pro-inflammatory stimulants, such as cytokines, LTs, infectious agents, or oxidative stress, activates NF-κB which thereby activates the expression of many inflammatory mediators such as enzymes, chemokines, and adhesion molecules.[2324] The inflammatory mediators promote monocyte–macrophage differentiation, macrophage relocalization, at the site of infection leading to chronic inflammation. LT is an effective chemoattractant mediator of inflammation due to the presence of seven transmembrane-spanning G protein receptors.[25] Therefore, inhibition of LTs through inhibition of 5-, 12-, 15-LOXs will lead to the reduction in chronic inflammatory conditions along with the inhibition in the production of pro-inflammatory cytokines including TNF-α and IL-1β.[24] Petasin extract possesses anti-inflammatory effect by inhibiting LT synthesis.[19] Petasin has proved to suppress the LT synthesis in human eosinophils, neutrophils, and macrophages, thereby reducing the inflammatory process.[20] The anti-inflammatory activity of petasin justifies the usage of this herbal extract in the ratio of 1:1 along with ZnOE sealer.

CONCLUSION

The addition of petasin to ZnOE sealer in the ratio of 1:1 exhibited good anti-inflammatory action compared to the conventional ZnOE sealers. Thus, null hypothesis was rejected. Within the limitations of this study, the modified ZnOE with petasin seems to be a promising formulation as a root canal sealer. Further exploratory in vivo studies will aid as supplementary investigations before introducing it in clinical practice.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.