Project Report Standardization and quality control studies of MML oil Principal Investigator Dr. Surendra Kr. Sharma Institute Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science & Technology, Hissar-125001, Haryana Supported by Department of AYUSH, Ministry of Health & Family Welfare, Govt. of India, New Delhi FINAL REPORT 1. Project Title : STANDARDIZATION AND QUALITY CONTROL STUDIES ON MML OIL 2. Principal Investigator : Prof. SURENDRA KR. SHARMA (Pharmacognosy and Phytochemistry) Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science & Technology , Hissar, Haryana 3. Co-Investigator : Nil 4. Scientific Staff : Senior Research Fellow 5. Non-Scientific Staff : Lab.-cum Animal Attendant 6. Implementing Institute : Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science & Technology , Hissar, Haryana 7. Date of Start : 17-03-2008 8. Duration : Two Years 9. Date of Completion : 31-03-2010 10. Objective as Approved : To Standardize and Evaluate the Biological activity of MML Oil 11. Deviation made from : Nil Original Objectives 12. Experimental Work : 13. Detailed Analysis of : Results 14. Conclusions Summarizing : C Achievements and Scope for Future Work 15. Procurement / Usage of : Equipment 16. Manuscript for Publication : Prof. Surendra Kr. Sharma (Principal Investigator) STANDARDIZATION AND QUALITY CONTROL STUDIES ON MML OIL Final Report Acid Value Principle Fats are readily broken down by lipase into free fatty acids and glycerol during storage, particularly when the temperature and moisture contents are high. Fat hydrolysis gets accelerated due to microbial contamination. The acid value of a fat is the number of mg of KOH required to neutralize the free acid in 1g of the oil. Regents 1. 0.1 M KOH 2. Ethanol 3. Diethyl ether 4. 1% Phenolphthalein solution Procedure: Dissolved 10g of the oil in 50 ml of a mixture of equal volumes of ethanol (95%) and ether, previously neutralized with 0.1M potassium hydroxide to phenolphthalein solution. 1ml of phenolphthalein solution was then added and titrated with 0.1M potassium hydroxide until the solution remain faintly pink after shaking for 15 seconds. The titre value in ml (n) was noted. Observation: Sr. No. Volume (ml) 1 14.4 2 14.6 3 14.4 4 14.2 5 14.4 Average 14.4 STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 1 Calculation Acid value = 5.61n/w Where, n = the number of ml of 0.1M potassium hydroxide required w = the weight, in g, of the oil Result: Acid Value was found to be 8.08 Saponification Value Principle The saponification value is the number of milligrams of potassium hydroxide necessary to neutralise the free acids and to saponify the esters present in 1 g of the substance. It is an index of mean molecular weight of the fatty acids of the glycerides. Lower saponification value indicates higher molecular weight of fatty acids and vice-versa. The oil sample is saponified by refluxing with a known excess of ethanolic KOH. The alkali required for saponification is determined by titration of the excess potassium hydroxide with standard hydrochloric acid. Reagents 1. 0.5M ethanolic Potassium hydroxide 2. Pumice powder 3. 1% phenlphthalien solution 4. 0.5M HCl Procedure Introduce 2 g of the substance being examined, accurately weighed, into a 200 ml flask of borosilicate glass fitted with a reflux condenser. Add 25.0 ml of 0.5 M ethanolic potassium hydroxide and a little pumice powder and boil under reflux on a water-bath until the solution become clear (30 minutes). While the solution still hot, add 1 ml of phenolphthalein solution and titrated immediately with 0.5 M hydrochloric acid (a ml). Repeat the operation omitting the substance being examined (b ml). STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 2 Observation Sr. No a (ml) b (ml) 1 1.1 15.0 2 1.2 15.1 3 1.2 15.1 4 1.3 15.2 5 1.2 15.1 Average 1.2 15.1 Calculation Saponification value = 28.05 (b-a)/w Where, w = weight in g, of the substance. Result: Saponification Value was found to be 194.95 Ester Value Principle The ester value is the number of milligrams of potassium hydroxide required to saponify the esters present in 1 g of the substance. Procedure The acid value and the saponification value of the oil is to be determined, as per above procedures. Calculation Ester value = Saponification value Acid value. = 194.95 8.08 = 186.87 Result: Ester value was found to be 186.87 STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 3 Hydroxyl Value: Principal The hydroxyl value is the number of milligrams of potassium hydroxide required to neutralise the acid combined by acylation in 1 g of the substance. Reagents 1. Pyridine-acetic anhydride reagent 2. Ethanol (95%) 3. 0.5M ethanolic potassium hydroxide 4. Phenolphthalein solution Procedure 2g of oil poured in a 150-ml acetylation flask fitted with a condenser and 10ml of pyridine-acetic anhydride reagent was added. It was boiled for 1 hour on a water-bath, adjusting the level of the water to maintain it 2 to 3 cm above the level of the liquid in the flask all through. Cooled, 5 ml of water through the top of the condenser added and cooled. The condenser and the walls of the flask was rinsed with 5 ml of ethanol (95 %), previously neutralised to dilute phenolphthalein solution. It was titrated with 0.5 M ethanolic potassium hydroxide using dilute phenolphthalein solution as indicator. Repeat the operation without the substance being examined Observation: Sr. No For oil For blank (ml) (ml) 1 43 41 2 43 41 3 44 42 4 43 41 5 45 40 Average 43 41 STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 4 Calculation Hydroxyl value = Acid Value + 28.05 v/w Where, v = difference in ml between the titrations; w = weight in g of the substance Result: Hydroxyl value was found to be 36.13 Iodine Value Principle The iodine value of a substance is the weight of iodine absorbed by 100 parts by weight of substance. This value indicates the degree of unsaturation of the oil. Reagents 1. Chloroform 2. Iodine monobromide solution 3. Potassium iodide solution 4. 0.1M sodium thiosulphate 5. Starch solution Procedure Hanus Method As per IP, 3 different quantities of oil that is 1, 0.5 and 0.25g weighed accurately, place it in a dry 300ml iodine flask respectively. Add 15ml of chloroform and dissolved. Add slowly from a burette 25ml of iodine monobromide solution, insert the stopper, allow to stand in the dark for 30 minutes. Add 10ml of potassium iodide solution and 100ml of water and titrate with 0.1ml sodium thiosulphate using starch solution, added towards the end of the titration, as indicator. Note the number of ml required (a). Repeat the operation omitting the substance being examined and note the number of ml required (b). STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 5 Observation: Sr. No For 1g oil For 0.5g oil For 0.25g oil Blank a (ml) a (ml) a (ml) b (ml) 1 8.1 14.5 20.6 39.2 2 8.2 14.3 20.8 39.6 3 8.1 14.2 21.5 39.2 4 8.3 14.3 21.1 39.3 5 8.5 14.1 21.0 39.5 Average 8.24 14.28 21.0 39.36 Calculation Iodine value = 1.269(b-a)/w Where w = weight in g of the oil Result: Iodine value was found to be 39.49 for 1g, 63.65 for 0.5g and 93.20 for 0.25g of oil. Peroxide Value Principle The peroxide value is the quantity of those substances in the sample, expressed in terms of milliequivalents of active oxygen per Kg, which oxidize potassium iodide under the operating conditions described. Reagents 1. Glacial acetic acid 2. Chloroform 3. Saturated potassium iodide solution 4. Activated charcoal 5. 0.01M sodium thiosulphate 6. Starch solution STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 6 Procedure The IP original method is as follows: 5g of the oil was accurately weighed, into a 250 ml glass-stoppered conical flask, 30 ml of a mixture of 3 volumes of glacial acetic acid and 2 volumes of chloroform added, swirled until dissolved and 0.5 ml of saturated potassium iodide solution was added. Allowed to stand for exactly 1 minute, with occasional shaking, added 30 ml of water and titrated gradually, with continuous and vigorous shaking, with 0.01M sodium thiosulphate until the yellow colour almost disappears. Add 0.5 ml of starch solution and continue the titration, shaking vigorously until the blue colour just disappears (a ml). Repeat the operation omitting the substance being examined (b ml). The volume of 0.01 M sodium thiosulphate in the blank determination must not exceed 0.1 ml. The method above was amended, because there was no discoloration of yellow colour by titration with 0.01M Sodium thiosulphate, since the original colour of the oil was itself yellowish orange. To overcome this problem original method was amended as follows: Warmed 10 g of oil with 500mg activated charcoal for 1 hour, left for overnight and filtered. 5g of oil was weighed in 250ml glass stopperd conical flask and add 30ml of a mixture of 3 volumes of glacial acetic acid and 2 volumes of chloroform, swirl until dissolved and add 0.5ml of saturated potassium iodide solution. Allowed standing for exactly 1 minute, with occasional shaking, adding 30 ml of water and titrated gradually, with continuous and vigorous shaking, with 0.01M sodium thiosulphate until the yellow colour almost disappears. Then added 0.5ml of starch solution and continued the titration with vigorous shaking until the blue-black colour just disappears (oil, a ml).