G.J. Christian* S. Elansekaran, M. Ramamurthy, V. Srinivasan, P. Shanmugapriya and M. Nijavizhi

National Institute of Siddha, Tambaram Sanatorium, Chennai – 600 047, India.

Abstract

Non healing ulcers constitute apotentially 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 nonviable tissue. The Siddha formulation “OonPoochuThailam”(OPT)is indicated for curingsloughing 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 OonPoochuThailam and to analyze the elemental constituents of the ingredients [salt of Achyranthesaspera(AA), and salt of Sesamumindicum(SI)plant] by using Inductively Coupled Plasma Mass Spectrometry (ICP-MS).The antimicrobial activity of the ingredients of OPT were studied in theconcentration of 100μg/ml against Escherichia coli, Klebsiellapneumonia, Proteus mirabilis, Staphylococcusaureus, Pseudomonasaeruginosa, Shigellasonnei, Aeromonashydrophila, 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.

Keywords: OonPoochuThailam; Sinus ; Fistula; Debridement ; Wound.

Introduction

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 necrotizingtissue take longer to close and heal, without proper clearing ofinfected and non-viable tissue. Non healing ulcers constitute apotentially 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 intodifferent typessuch assurgical, mechanical and chemical debridement.

The Siddhaformulation “OonPoochuThailam”, which is mentioned in the literature AgathiarRana Vaithyam1is indicatedfor curingsloughing ulcers, sinuses and fistulae, supposedly by acting as a chemical debriding agent; eithersolely perfecting the debriding process or complementing the surgical or parasurgicaldebridement carried out in conjunction. Sangu(Turbinellarapa, Conch shell), Salt precipitated from the filtered solution in which ash of whole plantAchyranthesaspera(Naayuruvisamoolam) is a solute andin the same way herbal salt prepared fromcapsules of Sesamumindicum fruit(EllukaiThol)areused asingredientsof which are homogenously mixed withsesame oil as a liquid vehicle to be applied externally. This homogenous physical mixture of the above ingredients with the Sesame oil is termed as OonPoochuThailam (OPT).

All the ingredients of OPTare of alkaline nature that leadsto thelysis andliquefaction of necrotic tissues. Sanguparpam(Calcinate of Conch shell) is said to have a pH range of (9.12– 9.33)2and antiulcer property3; Achyranthesaspera and Sesamumindicum herbal ash extract salts possess anti-inflammatory, anti-microbial, anti-oxidant and wound healing properties4,5; The above mentioned salt prepared of Achyranthesaspera (Kshar, pH:10.61-11.12) is used in Karanool (Kshar sutra) for treating fistula-in-ano,haemorrhoids and fissure-in-anoconditions.6

In this present study it was aimed to study the antimicrobial activities of the ingredients of OonPoochuThailam andto analyze the elemental constituents of the plant salts.

 

Materials and Methods

Preparation of Oonpoochuthailam
Ingredients:

Turbinellarapa(Conch shell, Sangu), Whole plant of Achyranthesaspera(Naayuruvisamoolam), Capsule of Sesamumindicum fruit(EllukaiThol).

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 onepart of ash, stirred with oar like spoon 3 hours once for 3 days. On the 4th day, the supernatant was put into another mud pot, boileduntil it became semisolid and the effervescence was settled. Then the semisolid end product was crystallized by drying it in the Sunlight.

Usage:

Powdered salt forms of above 3 Ingredients, were taken in the1:1:1 ratio and mixedwith sesame oil to apply on wound for healing.7

ICP-MS

The elemental analysis of salt of Achyranthesaspera(AA), and salt of Capsule of Sesamumindicum(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 is 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

Antimicrobial activity of OonPoochuThailam
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).

CultureofPathogens

The Microbial strains used in the sensitivity assay were Escherichia coli (MTCC 1687), Klebsiellapneumoniae (MTCC 432), Proteus mirabilis (MTCC 3310), Staphylococcusaureus (MTCC 737), Pseudomonasaeruginosa (MTCC 424), Shigellasonnei (MTCC 646), Aeromonashydrophila (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 Achyranthesaspera (AA), Ingredient 2- Salt of Capsule of Sesamumindicum fruit (SI), Ingredient 3- Sanguparpam (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)

Figure 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 –BTM cream (Clotrimazole-betamethasone) was used as a standard inthe 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)
Figure 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

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 1012 (parts per trillion). It is an automated, simple and unique quantitative and qualitative analysis. It measures elemental isotopes ratio. The result of elemental analysis of salt of Achyranthesaspera (Naayuruvisamoolam), and salt of Capsule of Sesamumindicum fruit (EllukaiThol) are furnished in Table 1.

Table 1: Result of  ICP-MS Analysis

Elements

 

Elemental Symbol

 

Concentration of Elements present in salt of Achyranthesaspera

 

Concentration of Elements present in

salt of Capsule of Sesamumindicum fruit

Copper Cu BDL

 

BDL

 

Arsenic

 

As BDL

 

BDL

 

Calcium

 

Ca

 

0.27 mg/L

 

0.32 mg/L

 

Iron

 

Fe 24.11 mg/L

 

4.425 mg/L

 

Mercury

 

Hg 0.91 mg/L

 

0.63 mg/L

 

Manganese

 

Mn 1.18 mg/L

 

0.415 mg/L

 

Lead

 

Pb 1.09mg/L

 

BDL

 

Sodium

 

Na 410.3 mg/L

 

BDL

 

Sulfur

 

S 14.12 mg/L

 

8.51 mg/L

 

Zinc

 

Zn 12.16 mg/L

 

6.72 mg/L

 

Phosphorous

 

P 2.82 mg/L

 

1.54 mg/L

 

Chromium

 

Cr 1.45 mg/L

 

0.21 mg/L

 

Cadmium

 

Cd 0.451 mg/L

 

0.18 mg/L

 

 

Table 2: Antimicrobial activity of OPT ingredients in terms of Zone of Inhibition.

 

S.No Test Organism Inhibition Zone Diameter in mm
Standard Control TF-1

(AA)

TF-2

(SI)

TF-3

(SP)

TF- 4 (Mixture of AA, SI &SP)
1 Escherichia coli 29-32 22-25 22-26 0 20-24
2 Pseudomonas aeruginosa 29-33 23-26 23-25 0 21-24
3 Proteus mirabilis 19-22 22-25 26-31 0 22-25
4 Staphylococcus aureus 28-31 23-27 19-22 0 21-25
5 Shigellasonnei 25-29 26-31 27-31 0 24-28
6 Aeromonashydrophila 25-27 24-28 24-27 0 20-23
7 Klebsiella pneumonia 26-30 25-28 24-29 0 23 -26
8 Salmonella typhimurium 29-33 24-27 26-31 0 24-27
9 Vibrio cholerae 30-34 24-29 25-28 0 23-27
10 Bacillus cereus 24-28 25-30 23-26 0 22-23
11 Candida albicans 14-17 24-26 26-31 0 22-26

The value represented in the table is a triplicate performed for each formulation against each organism.The zone diameter mentioned as a range of minimum to maximum values in mm;

TF- Test Formulation,  .AA- Salt of AA – Achyranthesaspera; SI-Sesamumindicum fruit; SP-Sanguparpam.

The antimicrobial activity of the ingredients of Oonpoochuthailam were studied in the

concentration of 100μg/ml against Escherichia coli (MTCC 1687), Klebsiellapneumoniae(MTCC 432), Proteus mirabilis (MTCC 3310), Staphylococcusaureus (MTCC 737), Pseudomonasaeruginosa (MTCC 424), Shigellasonnei (MTCC 646), Aeromonashydrophila (MTCC 1739), Salmonella typhimurium (MTCC 733), Vibrio cholerae  (MTCC 3906), Bacillus cereus (MTCC 430) and Candida albicans. (Diploid fungus) (MTCC 854).Antibacterial and antifungal potential of the ingredients and formulation were assessed in 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. The percentage of antimicrobial activity of each test substanceas compared to standard drug is given in Table 3.

Table 3: Percentage of Zone of inhibition compared with standard.

S.No Test Organism Corresponding % of inhibition zone with Standard
TF- 1

(AA)

TF- 2

(SI)

TF- 3

(SP)

TF-4

(Mixture of AA, SI &SP)

1 Escherichia coli 76-78 76-81 0 69-85
2 Pseudomonas aeruginosa 78-79 76-79 0 72-73
3 Proteus mirabilis 113-115 136-140 0 113-115
4 Staphylococcus aureus 82-87 68-71 0 75-81
5 Shigellasonnei 104-106 106-108 0 96-97
6 Aeromonashydrophila 96-103 96-100 0 80-85
7 Klebsiella pneumonia 93-96 92-97 0 86-88
8 Salmonella typhimurium 81-83 90-94 0 82-83
9 Vibrio cholerae 80-85 82-83 0 77-79
10 Bacillus cereus 104-107 93-96 0 82-92
11 Candida albicans 153-171 182-185 0 153-157

TF- Test Formulation; AA- Salt of Achyranthesaspera; SI-Salt of Sesamumindicum fruit; SP-Sanguparpam.

 

 

 

 

 

 

 

 

 

 

 

 

 

A-Escherichia coli,

B-Pseudomonas aeruginosa,

C-Bacillus cereus,

D-Salmonella typhimurium,

E-Vibrio cholera,

F-Shigellasonnei,

G-Proteus mirabilis,

H-Aeromonashydrophila ,

I-Klebsiella pneumonia,

J-Staphylococcus aureus,

K-Candida albicans (Diploid fungus).

 

Well marking ‘c’- Standard control; 1- Test ingredient 1( AA- Salt of Achyranthesaspera); 2- Test ingredient 2(SI- Salt of Capsule of Sesamumindicum fruit); 3- Test formulation  3(SA-Sanguparpam);  4- Test formulation 4 (Combination of Formulation 1 to 3 mixed at equal proportion).

 

Figure 3: Anti-microbial effect of Test formulations against selected Pathogens.

 

Test substance 1(Salt of Achyranthesaspera -AA) showed maximum zone of inhibition against Proteus mirabilis, Shigellasonnei, Aeromonashydrophila and Bacillus cereus which were higher than the standard drug (Ciprofloxacin).Antibacterial activity of AAwas nearly equal to standardagainstKlebsiella pneumonia, Staphylococcus aureus, Vibrio cholera andSalmonella typhimurium.

Test substance 2 (Salt of Capsule of Sesamumindicum fruit -SI) showed maximum zone of inhibition against Proteus mirabilis andShigellasonneiwhich were higher than the standard drug. Antibacterial activity of SIis nearly equal to standard againstAeromonashydrophila, Klebsiella pneumonia, Salmonella typhimurium.Vibrio cholerae andBacillus cereus.

No measurable activity was observed with respect to test substance 3 (Sanguparpam- 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 againstAeromonashydrophila, Klebsiellapneumoniae, 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 albicanswhich is significantly higher when compare to the standard clotrimazole and Beclomethasone (candid BTM).

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. With respect to bacteria, test formulations exerted promising activity against both gram positive and gram negative organisms.

 

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