Anti-Microbial Screening and Elemental Analysis of the Siddha Medicinal Oil ‘Oon Poochu Thailam’-A Debriding Agent used in Sloughing Ulcers and Fistulae

Non healing ulcers constitute a potentially serious medical and as well as surgical illness. Wounds that contain necrotizing and less perfused (low oxygen content) tissue take longer to close and heal without proper clearing of dead or non viable tissue. The Siddha formulation “Oon Poochu Thailam” (OPT) is indicated for curing sloughing ulcers, sinuses and fistulae, supposedly by acting as a chemical debriding agent (like topical debriding agent). In this present study it was aimed for screening the antimicrobial activities of the ingredients of Oon Poochu Thailam and to analyze the elemental constituents of the ingredients [salt of Achyranthes aspera (AA), and salt of Sesamum indicum (SI) plant] by using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The antimicrobial activity of the ingredients of OPT were studied in the concentration of 100mg/ml against Escherichia coli, Klebsiella pneumonia, Proteus mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa, Shigella sonnei, Aeromonas hydrophila, Salmonella typhimurium, Vibrio cholera, Bacillus cereus and Candida albicans. Antibacterial and antifungal potential of the OPT ingredients were assessed both individually and together in terms of zone of inhibition of bacterial and fungal growths. The test items 1(AA), 2(SI) and 4(Mixture of AA, SI & SA) have shown a broad spectrum of anti-microbial activity against bacteria and fungi.

Debridement is the medical terminology for removal of dead (necrotic), damaged or infected tissue which is least viable so as to improve the healing potential of the remaining healthy tissue, thereby facilitating the wound healing process. Thus wounds that contain less perfused (low oxygen content), infected and hence necrotizing tissue take longer to close and heal, without proper clearing of infected and non-viable tissue. Non healing ulcers constitute a potentially serious medical and surgical illness.
Sometimes, debridement is executed naturally on the body's own ability to shed off dead tissue termed as Apoptosis. However, more often, debridement warrants a medical procedure which unfolds into different types such as surgical, mechanical and chemical debridement.
The Siddha formulation "Oon Poochu Thailam", which is mentioned in the literature Agathiar Rana Vaithyam 1 is indicated for curing sloughing ulcers, sinuses and fistulae, supposedly by acting as a chemical debriding agent; either solely perfecting the debriding process or complementing the surgical or parasurgical debridement carried out in conjunction. Sangu (Turbinella rapa, Conch shell), Salt precipitated from the filtered solution in which ash of whole plant Achyranthes aspera (Naayuruvi samoolam) is a solute and in the same way herbal salt prepared from capsules of Sesamum indicum fruit (Ellukai Thol) are used as ingredients of which are homogenously mixed with sesame oil as a liquid vehicle to be applied externally. This homogenous physical mixture of the above ingredients with the Sesame oil is termed as Oon Poochu Thailam (OPT).
All the ingredients of OPT are of alkaline nature that leads to the lysis and liquefaction of necrotic tissues. Sangu parpam (Calcinate of Conch shell) is said to have a pH range of (9.12-9.33) 2 and antiulcer property 3 ; Achyranthes aspera and Sesamum indicum herbal ash extract salts possess anti-inflammatory, anti-microbial, anti-oxidant and wound healing properties 4,5 ; The above mentioned salt prepared of Achyranthes aspera (Kshar, pH:10.61-11.12) is used in Karanool (Kshar sutra) for treating fistula-in-ano, haemorrhoids and fissure-in-ano conditions. 6 In this present study it was aimed to study the antimicrobial activities of the ingredients of Oon Poochu Thailam and to analyze the elemental constituents of the plant salts.

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 180 0 C for 3 hours. All nutrient media were sterilized by autoclave (121 0 C, 15psi for 15-20 minutes).

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 4mixture 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).

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).

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 2.0·10 6 colony forming units (CFU/ml) for bacteria and 2.0·10 5 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

RESULTS AND DISCUSSION
Inductively Coupled Plasma Mass Spectrometry (ICP-MS): ICP-MS is a type of mass spectrometry that is highly sensitive and capable of the determination of a range of metals and several non-metals at concentration below one part in 10 12  terms of zone of inhibition of bacterial and fungal growths (Fig 3). The results of the antibacterial and antifungal activities are presented in Table  2. Percentages of antimicrobial activity of each test substance as compared to standard drugs Ciprofloxacin and Clotrimazole are given in Table 3.
Test substance 1 (Salt of Achyranthes aspera -AA) showed maximum zone of inhibition against Proteus mirabilis, Shigella sonnei, Aeromonas hydrophila and Bacillus cereus which were higher than the standard drug (Ciprofloxacin). Antibacterial activity of AA was nearly equal to standard against Klebsiella pneumonia, Staphylococcus aureus, Vibrio cholera and Salmonella typhimurium.
Test substance 2 (Salt of Capsule of Sesamum indicum fruit -SI) showed maximum zone of inhibition against Proteus mirabilis and Shigella sonnei which were higher than the standard drug. Antibacterial activity of SI is nearly equal to standard against Aeromonas hydrophila, Klebsiella pneumonia, Salmonella typhimurium. Vibrio cholerae and Bacillus cereus.
No measurable activity was observed with respect to test substance 3 (Sangu parpam-SA) against test pathogens.
Test substance 4 (Mixture of AA, SI & SA-OPT formulation) showed maximum zone of inhibition against Proteus mirabilis which was higher than that of the standard drug. Antibacterial activity of Test substance 4 was nearly equal to standard against Aeromonas hydrophila, Klebsiella pneumoniae, Salmonella typhimurium, Bacillus cereus and Vibrio cholera.
The test formulations 1(AA), 2(SI) and 4(Mixture of AA, SI & SA) have shown a broad spectrum of anti-fungal activity against Candida albicans which is significantly higher when compared to the standard clotrimazole (Candid TM ).
The data obtained from the present study clearly indicates that the test formulations 1(AA), 2(SI) and 4(Mixture of AA, SI & SA) have shown a broad spectrum of anti-microbial activity against bacteria and fungi. Regarding antibacterial assay, test formulations exerted promising activity against both gram positive and gram negative organisms.