This is an invitro, experimental study that was approved by the Institutional Ethical Committee (MGV/IEC/940/2021-2022). Fresh Thymus vulgaris leaves were obtained from a nursery in the city. The extract was prepared by the Soxhlet extraction method. Thyme leaves were washed first with tap water,dried in shade (approximately 4-5days) at room temperature and coarsely powdered to a suitable size. The crushed plant material was loaded into the thimble, which was placed inside the Soxhlet extractor. Following this, the solvent (alcohol) was added to a round bottom flask, which was attached to a condenser, and heated to the boiling point of the solvent. Extraction was carried out until the drug was extracted exhaustively. The extract was cooled, and the macerate was subjected to distillation to eliminate the residual solvent. The obtained semisolid extract was then transferred to a China dish and heated at a low heat to eliminate the solvent completely, and the total amount of extract was weighed.
The MIC and MBC of the thyme plant extract were determined against standard strains of P. gingivalis (ATCC 33277), A. actinomycetemcomitans (ATCC 43718) and F. nucleatum (ATCC 25586). Two repeats of the sample and control groups were performed to obtain significant results.
A) MIC TEST
Nine dilutions of each drug were taken with thioglycollate broth for MIC. In the initial tube, 20 microliters of drug was added to 380 microliters of thioglycollate broth. For dilutions, 200 microliters of thioglycollate broth was added into the next 9 tubes separately. Then, from the initial tube, 200 microliters was transferred to the first tube containing 200 microliters of thioglycollate broth. This was considered a 10-1 dilution. From the 10-1 diluted tube , 200 microliters was transferred to a second tube to make a 10-2 dilution. The serial dilution was repeated up to a 10-9 dilution for each drug. From the maintained stock cultures of required organisms,5 microliters was taken and added to 2 ml of thioglycollate broth. In each serially diluted tube, 200 microliters of the above culture suspension was added. The tubes were incubated for 48-72 hours in an anaerobic jar at 37°C and observed for turbidity13.
B) MBC TEST
From the MIC dilutions, 5 tubes were plated (which were sensitive in MIC) and incubated for 24 - 48 hrs. The next day, the colony count was taken. MBC was performed to determine whether there was a bacteriostatic or bactericidal effect of the extract against the organism. If there is no growth then, it has a bactericidal effect, and if there is growth then, it has a bacteriostatic effect 13.
C) ANTIOXIDANT ACTIVITY: DPPH (2,2-diphenyl-picryl-hydrazyl) assay
The DPPH radical scavenging activity assay is widely used for a relatively rapid evaluation of antioxidant activitieson the principle of reduction of a stable radical. Reducing DPPH by an antioxidant is accompanied by the transition from purple to yellow color of the solution, which is measured by spectrophotometer.
DPPH (0.2 mM) and 0.1 M Tris HCl (pH 7.4) was used as the reagent, and the extract was prepared by weighing it to 10 mg and dissolving it in 1 ml of DMSO. Three test tubes were taken and labelled blank, control (Trolox as DPPH-scavenging compound) and test. In a blank test tube, 600 µl ethanol with 400 µl Tris HCl was added. In the control, 100 µl ethanol with 400 µl Tris HCl and 500 µl DPPH solution was added, and in the test, 100 µl sample with 400 µl Tris HCl and 500 µl DPPH solution was added. All the tubes were mixed and kept in the dark for 30 min, and the absorbance was read at 490 nm.14
D) ANTI- INFLAMMATORY ASSAY
The electrophoresis apparatus was cleaned,plates were set up (large and small plates with spacers in between them), clamped, and screws were tightened. Agarose gel was poured between the two glass plates to seal the bottom surface and left for 5-10 minutes.An appropriate resolving gel mixture was pipetted between the glass plates to avoid bubbles. Plates were filled up to 80%, leaving space for the stacking gel and comb. A small amount of water was laid to achieve a completely flat interface between the resolving gel and stacking gel and was allowed to set for 45 minutes. After the removal of excess water from the space, the stacking gel was poured onto the resolving gel. A comb was inserted and allowed to set for 30 min. MMP samples were collected, and the extracts were incubated for 1 hr at room temp. Electrode gels were assembled, and electrophoresis was performed. The zymogram renaturing buffer was decanted, and the gel was incubated in zymogram incubation buffer at 37⁰C overnight. Staining was performed using Coomassie Brilliant Blue R-250 for one hour. Gel images were captured by gel documentation system and exported to Total Lab software (UK) for quantification of bands based on intensity.15,16
