Ethanolic Extract of Sapindus Mukorrossi as Final Endodontic Irrigant.

Preparation of ethanolic extract of S.mukorossi:

The desiccated fruit of Sapindus mukorossi will be used for the formulation of an experimental root canal irrigant. The dry pericarps of s. mukorossi (≈ 1.0 kg) which is present around the seed nut, will be separated with a sharp blade. These pericarps will be blended to produce fine particles in a sterilized home blender (Enviro 3 in 1 juicer blender ENJ301). The resultant 10gram powder will be soaked in 100ml of absolute ethanol (99%) for 24hours at normal room temperature. This solvent will be filtered and removed with help of a rotary evaporator, and extract will be kept in cleaned screw-capped vials (Premium Vials B4702-12 Glass with Screw Cap) at -20º C until use. To form an operational concentration of 5 mg/ml, the extract will be re-dissolved in distilled water.

All samples were prepared by a single operator using the following protocol. After extraction tooth was stored in 0.1% thymol. Until further use. Specimens were randomly divided into 3 groups through computer-generated software. There will be 30 teeth in each group.

Chamber opening was done with # 4 diamond cutting round bur, a #10 K-type file (Maillefer/Dentsply) was introduced into each root canal till it could be seen outside the apical foramen. The working length will be decided by decreasing this length by 1mm. A single operator using K-type files (Maillefer/Dentsply) initially than with the ProTaper Universal System (Dentsply-Maillefer) up to F2 was performed. Irrigation was performed at each change of file with 2.5mL of 3% NaOCl (canasol). After instrumentation the sample Group 1 would be irrigated with 3ml of 17% EDTA and left in the canal for 1 minute. Group 2 will be irrigated with Sapindus mukorossi finally all groups will be flushed with 5ml of 0.9% normal saline. The control group's final irrigation was done with 0.9% saline.

Afterward, the specimen was dried with F2 sterile absorbent paper points and an F2 gutta-percha cone was selected as the master cone. Sealapex (Sybron-Endo, Glendora, CA, USA) sealer was mixed according to the manufacturer's instructions and placed in the canal via the help of a 25# k file.

• Mastercone was inserted up to the working length and tug back was assessed.
• Sealer application was performed in the manner described above.
• The GP was inserted up to the working length after the apical 2mm of the master cone was evenly coated with the sealer.
• The finger spreaders #25 and #20 (MANI, Japan) were placed into the canal to make room for the accessory GP.
• The process was repeated until the #20 spreader was unable to enter the canal's coronal one-third.
• Excess GP was seared off using a hot condenser, and coronal GP was vertically compacted to produce a good coronal seal.
• The access cavity was sealed with GIC restorative type(3M, ESPE) All samples were stored in an incubator (binder GmbH, Germany) at 37oc under 100% humidity for 1 week, to allow for the sealer to set completely.

3.10.1 Preparation of the sample for stereomicroscopic analysis of dye penetration: After the samples were retrieved from the incubator they were allowed to air dry for 10 minutes. All samples in the experimental group were coated with two layers of colored nail varnish, except the apical 2mm of the root apex.

After complete drying of nail varnish, the roots were then vertically placed in 1% methylene blue dye (WellcoSol, TM) for 72hours.

After 3 days, the samples were washed for 5 minutes under running tap water and left to air dry fully. To avoid dye introduction onto the inner root surface during sectioning, nail polish and dye were scraped off the samples with a scalpel and surgical blade #15 (Bisturi Ucu, Turkey). The tooth was cut in half longitudinally, parallel to its long axis. To ensure that the sectioning was performed through the center of the root and to prevent damage to the obturated canal space, 2mm deep longitudinal grooves were placed in the center of the buccal and lingual root surface using a diamond disc in a slow-speed handpiece (Nakanishi Inc (NSK), Japan), with caution not to invade the canal space. The teeth were then fractured longitudinally with a double tapered chisel and a mallet, preserving the entire obturation space.

The samples were then viewed under Stereiomicroscope (Olympus VM-ILA-2) in the bioinformatics and Molecular Medicine Laboratory (DRIBBS, DUHS) under 30x magnification. The images were captured and uploaded to IMAGE J software (National Institute of Health, USA). The samples were examined by two blinded observers (endodontists). The investigator determined apical microleakage by measuring the linear depth of dye penetration, which is defined as the maximum dye penetration distance from the root apex to the coronal extent. In the performa, all measures were measured in millimeters (mm).

For dentinal tubule penetrance:

Another half of the tooth was left to desiccate for 24 hours in 40-100% ethyl alcohol. Then specimen will be mounted on metallic stubs and gold-sputtered. Under an electron microscope, a photomicrograph will be taken. Samples were studied for dentinal tubule penetration of sealers at two levels - 2 and 5mm from the root apex by two independent observer's reliability will be checked. Photomicrographs showing the maximum penetration at each level were taken. The distance of the points was measured from the sealer-dentin interface and the depth of penetration was calculated.

For interaction with sodium hypochlorite:

For the assessment of precipitate formation, solutions of 17% EDTA, S.mukorossi, and 3% NaOCl will be used. Each polystyrene round-bottom tube (Falcon; Thermo Fischer Scientific, Waltham, MA) will be filled with 2mL extract of S. mukorossi and17% EDTA respectively. Tube 3 will be filled with a mixture of 1 mL of S.mukorossi and 1 mL 3% NaOCl. Tube 4 was filled with 17% EDTA with NaoCl. Left on 95% humidity at 37ºC for 1 week. An unbiased observer was observing every tube for precipitate formation after every 15minutes for first 2 hours and at 24-hour intervals for 1 week. To avoid any error this procedure repeated 3 times