SCREENING OF MINIMAL INHIBITORY & FUNGICIDAL CONCENTRATION (MIC & MFC) OF PURIFIED THURUSU ( COPPER SULPHATE) ETHEL SHINY.S1, BHARATH CHRISTIAN.C.B.S2, GOMATHI.P3 1Associate Professor & HOD, Department of Gunapadam - Marunthiyal , Santhigiri Siddha Medical College, 2Assistant Professor, Department of Maruthuvam , Santhigiri Siddha Medical College, 3Assistant Professor, Department of Varmam, Pura Maruthuvam and Sirappu Maruthuvam , Santhigiri Siddha Medical College, Thiruvananthapuram, Kerala.

INTRODUCTION : A Siddha medical system is an ancient one in holistic management and it was being practiced by a large population in South India. Traditional system of medicines were used by 60% of the world's population for their health care in developing and developed countries even though where modern medicines dominate. Thus this of medicine was a boon offered by the spiritual scientist called Siddhars. There are so many Siddhars, out of them eighteen Siddhars are the most important and they all have more knowledge about the universe and its contents. Siddhars believes that, there is a connection between the celestial bodies of our universe to the living beings of earth. Any changes in the external world, brings changes to human beings. So they believe a healthy body is essential to attain eternal life. The fundamental principles of Siddha science is 96 principles, Three humors (Vadha, Pittha, Kapha),“Panchaboothas” which advocates curative and preventive measures and educates systemized life style through natural way and gives total perfection for life. According to the Siddha science of medicine, diet and lifestyle plays a major role in maintain health and curing diseases. Standardization is essential for globalization traditional medical systems. Mortality rate was increased day by day due to severity of diseases but also with the adverse effect of the synthetic drugs. Because of this people from different parts of the World preferred to choose natural products as medicines for their health care remedies. Thus it is the best time to explore siddha medicines to the World with minimum adverse effects, less expensive and easy affordable. Thus attempt was made to standardize the siddha drug through a scientific technique MIC & MFC. 2. DETERMINATION OF MINIMAL INHIBITORY CONCENTRATION 2.1.Materials required: • Nutrient Broth 13g of Nutrient Broth media (Hi Media) was dissolved in 1000ml distilled water and was autoclaved at 121°C; 15lbps for 15 minutes. • Culture of Test organisms (Growth adjusted to 1% McFarland Standard): Candida albicans (ATCC 25175) • DMSO: Dimethyl Sulphoxide (Hi Media) • 96 Well micro titer plate • ELISA plate reader (ERBA, Lisa Scan) • MBC values are calculated by using ED 50 PLUS V1.0 Software 2.2.Methods required: Minimal inhibitory concentration (MIC) was determined by using two fold serial dilution method. The growth of stock inoculum was adjusted to 1% McFarland Standard. The broth dilution assay was done in 96 well micro titer plate. Each wells in the plate were added with 100μl of the diluted (two times) conidial inoculum suspensions (final volume in each well, 200 μl). Sample was dissolved in DMSO to a final concentration of 10mg/mL and was added in increasing concentration such as 62.5µg, 125µg, 250µg, 500µg, 1000µg to the wells respectively and incubated overnight at room temperature. A control well was kept with organism alone. Growth was observed by visual inspection and by measuring the optical density (OD) at 630 nm using an ELISA plate reader. The OD was measured immediately after the visual reading. The growth inhibition for the test wells at each extract dilution was determined by the formula: Percentage of inhibition = (OD of control - OD of test)/ (OD of control) × 100 Organism: Candida albicans Concentration(µg) OD 1 OD 2 OD 3 Average % of inhibition Control 0.5967 0.5812 0.5468 0.5749 0 Sample Code- A 62.5 0.4200 0.3799 0.3633 0.3877 32.55 125 0.3539 0.3352 0.3322 0.3404 40.78 250 0.2319 0.2465 0.2257 0.2347 59.17 500 0.1587 0.1674 0.1556 0.1605 72.07 1000 0.1211 0.1257 0.1226 0.1231 78.58 Table.1. Results of Minimal Inhibitory Concentration Fig.1. Results of MIC of purified Thurusu ( Copper Sulphate ) against Candida albicans i) Control ii) Sample ( MIC 50 Value-Sample A – 243.713 µg/mL) (Calculated using ED 50 PLUS V1.0 Software) 2.4. Inference - MIC of purified Thurusu ( Copper Sulphate) : By increasing the concentration of administrated purified thurusu (Copper Sulphate)in 1000 µg, showed that the suppression of Candida albicans growth in the selected media with 78.58 % MIC value. 3. DETERMINATION OF MINIMAL FUNGICIDAL CONCENTRATION (MFC) 3.1. Materials required: • Nutrient Broth 13g Nutrient broth was suspended in 1000ml of distilled water and was autoclaved at 121°C; 15lbps for 15 minutes. • Potato dextrose Agar plates: 39g PDA medium (Hi Media) was weighed and was dissolved in 1000ml distilled water and was autoclaved at 121°C; 15lbps for 15 minutes. After autoclaving, the media (20ml) was allowed to cool to 60°C and was poured to pre sterilized Petri plates. The plates were allowed to solidify in a laminar air flow chamber. • Culture of Test organisms (Growth adjusted to 1% McFarland Standard): Candida albicans (ATCC 25175) • 96 Well micro titer plate 3.2.Methods required: The in vitro minimum fungicidal activities (MFCs) were determined for each sample against Candida albicans. The initial steps were done as in MIC protocol described earlier; (Each wells in the plate were added with 100μl of the two times diluted inoculum suspensions (final volume in each well, 200μl). Samples were added in increasing concentration such as 125, 250, 500, 1000µg to the wells respectively and incubated for 24 hours at room temperature. A control well was kept with organism alone. After 24hours of incubation, 20μl from each well (250, 1000µg) was swabbed onto Potato dextrose agar plates; the contents of the wells were not agitated prior to removal of the specified volumes. The plates were then incubated at 37˚C for 48 hours. After incubation the plates were observed for the presence of colony forming units. The MBC was the lowest drug concentration that showed either no growth or fewer than three colonies to obtain approximately 99 to 99.5% killing activity. Sample Concentration ( µg) No of colony counted CFU/mL Control Control 1358 67.9* 103 Sample A 250 1208 60.4* 103 Sample A 1000 3 0.15* 103 Table.2. Observations of Minimal Fungicidal Concentration (MFC) Figure. 2. Observations - MFC of purified Thurusu ( Copper Sulphate ) against Candida albicans i) Control ii) Sample 250µg iii) Sample 1000µg ( MIC 90 Value-Sample A – 243.713 µg/mL) (Calculated using ED 50 PLUS V1.0 Software) 3.4. Inference - MFC of purified Thurusu ( Copper Sulphate) : By increasing the administration of purified thurusu ( Copper Sulphate)in 1000µg concentration, showed that the disappearance of Candida albicans colonies in the selected media. 4. RESULTS AND DISCUSSION : The Minimal Inhibitory Concentration and Minimal fungicidal Concentration of purified thurusu against the fungus Candida albicans were significantly noted. 5.CONCLUSION : This study shows that thurusu (copper sulphate) inhibits almost all the fungal colonies of Candida albicans in the concentration of 1000µg . However, further studies are needed to assess the microbial response to thurusu (copper sulphate) in vitro and vivo. The biological variability of individual fungal cells and strains needs to be identified to optimize the thurusu (Copper sulphate) concentration required to inhibit fungal growth. The results of this study will be useful for developing a quantitative microbial risk assessment. 6.REFERENCES 1. Drugs and Cosmetics act 1940 with Drugs and Cosmetics Rules, (Ministry of Health and Family Welfare, Government of India, New Delhi) (2016) p. 317. 2. Subbarayappa BV. Chemical practices and alchemy. In: Bose DM, Sen SN, Subbarayappa BV, editors. 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