A. Preparation of Oon poochu thailam
Ingredients
Turbinella rapa (Conch shell, Sangu), Whole plant of Achyranthes aspera (Naayuruvi samoolam), Capsule of Sesamum indicum fruit (Ellukai Thol).

Preparation of salts
Each ingredient was prepared as salt form as follows. First, the ingredients were charred each separately to convert into ash, and the ash of each ingredient was put into separate mud pot. Four parts of water were added for one part of ash, stirred with oar like spoon 3 hours once for 3 days. On the 4^th day, the supernatant was put into another mud pot, boiled until it became semisolid and the effervescence was settled. Then the semisolid end product was crystallized by drying it in the Sun light.

Usage
Powdered salt forms of above 3 Ingredients, were taken in the 1:1:1 ratio and mixed with sesame oil to apply on wound for healing.⁷

B. ICP-MS
The elemental analysis of salt of Achyranthes aspera (AA), and salt of Capsule of Sesamum indicum (SI) fruit were done by using Inductively Coupled Plasma Mass Spectrometry (ICP-MS).

The study was conducted at Centre for Laboratory Animal Technology and Research, Sathyabama University, Chennai, Tamil Nadu, India.

Machine Model: Agilent 7700 ICPMS
Samples are decomposed to neutral elements in high temperature argon plasma and analyzed based on their mass to charge ratios. Digestion of sample was carried out by transforming 0.5gm of the sample into a closed beaker and 5 ml of concentrated HNO3 was added and digested to near dryness. 16 M nitric acid was further added each time to the sample and digested until the clear solution was obtained. 5ml of 12 M Hydrochloric acid was added to ensure complete digestion.The digested solution was cooled to room temperature and made to the final volume of 100 ml with deionized water. Sample solutions were then filtered through membrane (0.45micron) filter. Finally, the digested samples were used for metal analysis using inductively coupled plasma Mass Spectrometry. Each sample was digested in triplicate. A blank solution was also prepared in a similar manner.⁸

C. Antimicrobial activity of Oon Poochu Thailam
Test Procedure
The antimicrobial study was conducted at Centre for Laboratory Animal Technology and Research, Sathyabama University, Chennai, Tamil Nadu, India.

Cleaning and Sterilization
The glass-wares used in the present study were cleaned with cleaning solution and sterilized in hot air oven to 180⁰C for 3 hours. All nutrient media were sterilized by autoclave (121⁰C, 15psi for 15-20 minutes).

Culture of Pathogens
The Microbial strains used in the sensitivity assay were Escherichia coli (MTCC 1687), Klebsiella pneumoniae (MTCC 432), Proteus mirabilis (MTCC 3310), Staphylococcus aureus (MTCC 737), Pseudomonas aeruginosa (MTCC 424), Shigella sonnei (MTCC 646), Aeromonas hydrophila (MTCC 1739), Salmonella typhimurium (MTCC 733), Vibrio cholera (MTCC 3906), Bacillus cereus (MTCC 430) and Candida albicans (Diploid fungus) (MTCC 854) were purchased from MTCC, Chandigarh , India and they were sub cultured as per the guideline and standard protocol laid down by National committee for clinical Laboratory standards. Microbial Stock cultures were maintained at 4°C on slopes of nutrient agar (Hi Media, Mumbai).

Preparation of Test samples
Test samples (Ingredient 1- Salt of Achyranthes aspera (AA), Ingredient 2- Salt of Capsule of Sesamum indicum fruit (SI), Ingredient 3- Sangu  parpam (SA), OPT formulation 4- mixture of formulations 1,2 and 3) provided for evaluation were powder in nature and in order to make the sample less viscous and suitable for handling through pipette about 2 ml of sterile distilled water was added to make up to the volume and triturated for nearly 30 minutes to make it a homogenous liquid (Fig 1).

Sample Well Details
Well 1: Test formulation 1(AA- Salt of Achyranthesaspera)
Well 2: Test formulation 2 (SI-Sesamumindicum fruit)
Well 3: Test formulation 3 (SA-Sanguparpam)
Well 4: Test formulation 4 (Combination of Formulation 1 to 3 mixed at equal proportions)
Well 6: Standard Ciprofloxacin (20 mug/ml)
Fig. 1. Sample well

Standard control
Standard drug was used as control in this study. Ciprofloxacin was used as a standard for anti-bacterial study and the marketed formulation Candid^TM cream (Clotrimazole) was used as a standard in the anti-fungal screening.

Sterility Test for Test formulations
The test substances were subjected to the preliminary sterility evaluation by disc plate method. Freshly prepared nutrient agar medium was loaded on the sterile disc and the same was used for enumeration of sterility of the test substances. As the test substances were colloidal in nature, streaking or swabbing was not possible. Diluted form of test substances at the concentration of 100µl and 200µl were loaded on to the top of the disc and incubated for the period of 48 hours with timely observations in between. Incubated plate was observed for 12, 24 and 48 hours after incubation and no growth of organism either as an isolated or as a colony was found in the incubated test substances. (Fig. 2).

Disc marking
Well Marking 1: Test ingredient 1 (AA- Salt of Achyranthesaspera)
Well Marking 2: Test ingredient 2 (SI- Salt of Capsule of Sesamumindicum fruit)
Well marking 3: Test ingredient 3 (SP-Sanguparpam)
Well Marking 4: Test formulation 4 (Combination of Formulation 1 to 3 mixed at equal proportion)
Well Making C: Standard Ciprofloxacin (20 mug/ml) – For Anti-bacterial activity;
Standard Clotrimazole- For Anti-fungal activity
Formulation 1 to 4 – Evaluated at two different concentrations (100µl and 200 µl)
Fig. 2. Disc plates of Sterility test

Preparation of inoculums
Active cultures for experiments were prepared by transferring a loopful of cells from the stock cultures to test tubes of Mueller-Hinton broth (MHB) for bacteria and Sabouraud dextrose broth (SDB) for fungi that were incubated without agitation for 24 hrs at 37°C and 25°C respectively. The cultures were diluted with fresh Mueller-Hinton and Sabouraud dextrose broth to achieve optical densities corresponding to 2.0·10⁶ colony forming units (CFU/ml) for bacteria and 2.0·10⁵ spore/ml for fungal strains.⁹

Hole-plate diffusion method
Equidistant holes of 6mm were made in the agar using sterile cork borers. A 100µL volume of sample solution was added to the holes using a pipette or (Eppendorf). The compound was allowed to diffuse for 5 minutes and the plates were kept for incubation at 37°C for 24 hrs. At the end of incubation, inhibition zones formed around the disc were measured with transparent ruler in millimeter. The same procedure was followed for the fungus also. These studies were performed in triplicate.^10,11,12