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). Repeat the operation omitting the oil (blank, b ml).
Observation
Sr. No. For oil (ml) For blank (ml) 1 1.2 0.1 2 1.2 0.1 3 1.3 0.1 4 1.1 0.1 5 1.2 0.1 Average 1.2 0.1
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 7 Calculation
Peroxide value = 10(a-b)/w
Where
w = weight in g, of the oil.
Result: Peroxide value of oil was found to be 2.2
Weight per Milliliter (Wt. per ml):
The weight per milliliter of a liquid is the weight, in g, of 1 ml of a liquid when weighed in air at 250C.
Apparatus
1. Pycnometer
Procedure
The pycnometer was caliberated by filling it with recently boiled and cooled water at 250C and weighed the contents. Assuming that the weight of 1 ml of water at 250C when weighed in air of density 0.0012 g per ml is 0.99602 g, calculate the capacity of the of the pycnometer. Adjust the temperature of the substance being examined, to about 200C and filled the pycnometer with it. Adjust the temperature of the filled pycnometer to 250C, remove any excess of the substance and weigh. Subtract the tare weight of the pycnometer from the filled weight of the pycnometer.
Determined the weight per millilitre by dividing the weight in air, in g, of the quantity of liquid, which fills the pycnometer at the specified temperature, by the capacity expressed in ml, of the pycnometer at the same temperature (i.e. density = mass/volume).
Observation:
Sr. No Weight of Water (g) Weight of Oil (g) 1 23.712 21.585 2 23.651 21.602 3 23.682 21.577 4 23.703 21.602 5 23.685 21.572 Average 23.687 21.588
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 8 Calculation :
For water:
Density = mass or weight/volume
0.99602 = 23.687/volume
Volume = 23.687/0.99602 = 23.78
Now, wt/ml of oil is:
Density = mass or weight/volume
= 21.588/23.78
= 0.9078
Result: Weight per milliliter of oil 250C was found to be 0.9078
Relative Density:
The relative density of a substance is the ratio of the mass of a given volume of the substance to the mass of an equal volume of water, both weighed at 250C, unless otherwise specified.
Apparatus
1. Pycnometer
Procedure
Proceed as described under Weight per milliliter. Divided the weight of the substance in the pycnometer by the weight of water contained, both determined at 250C.
Calculation :
Relative density = weight of oil/weight of water
= 21.588/ 23.687
= 0.9114
Result: Relative Density of oil at 250C was found to be 0.9114 pH
Apparatus
1. Digital pH Meter
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 9 Procedure
Instrument was calibrated by standard buffer solution of 4, 7 and 9.2 pH. Immerse the electrodes in the solution being examined and measure the pH.
Observation:
Sr. No pH
1 4.234
2 4.230 Counts : 10
3 4.231 Average : 4.2323
4 4.232 Std. Dev. : 0.0013
5 4.234 Max. : 4.234
6 4.234 Min. : 4.231
7 4.232
8 4.233
9 4.232
10 4.231
Result: pH of oil was found to be 4.2323
Refractive Index:
It is the ratio of the velocity of the light in vacuum to its velocity in the substance. It may also be defined as the ratio of the sine of the angle of incidence to the sine of the angle of refraction. It generally varies with temperature and wavelength of light. It increases with the increase in unsaturation and also chain length of fatty acids in oils.
Apparatus
1. Abbe Refractometer :
Refractive index vary between 1.4220 to 1.4859 and can be read from the instrument to the fourth decimal place accurately.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 10 Calibration
The instrument is calibrated with a glass prism of known refractive index (an optical contact with the prism being made by a drop of bromonaphthalene)
Procedure
Clean refractometer with alcohol and ether. A drop of oil was placed on the prism. The prism was closed by the ground glass-half of the instrument. The dispersion screw was adjusted so that no colour line appears between the dark and illuminated halves. The dark line was adjusted exactly on the cross wires and the refractive index was read on the scale.
Observation
Sr. N0 Refractive index
1 1.492 Counts : 10 2 1.491 Average : 1.491-0.012 (correction factor) 3 1.492 Std. Dev. : 0.001 4 1.492 Max. : 1.492 -0.012 (correction factor)
5 1.492 Min. : 1.489-0.012 (correction factor)
6 1.489
7 1.491
8 1.492
9 1.490
10 1.489
Result: Refractive index of oil was found to be 1.479
Optical Rotation:
Optical rotation of the substance is the angle through which the plane of polarization is rotated when polarized light passes through the solution of substance. The optical;
250C
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 11 Apparatus
1. Polarimeter with sodium lamp
Procedure
Apparatus was calibrated by using a solution of sucrose of different concentration and measuring the optical rotation in a 2-dm tube. After calibration optical rotation was determined for oil.
Observation
Sr. No Opt. Rot
1 0.371
2 0.371 Counts : 10
3 0.370 Average : 0.3721
4 0.370 Std. Dev. : 0.0037
5 0.379 Max. : 0.379
6 0.379 Min. : 0.369
7 0.372 Temp. : 29
8 0.370
9 0.369
10 0.370
Result: Optical rotation of oil was found to be 0.3721
Viscosity:
The determination of viscosity of oil is carried out by means of a capillary viscometer. For measurement of viscosity, the temperature of the substance being measured must be accurately controlled, since small temperature changes may lead to marked changes in viscosity.
Apparatus
Ostwald Viscometer
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 12 Procedure
Fill viscometer, previously washed and completely dried with the distilled water and note the time taken by it lower meniscus to fall from one point to another point as marked on viscometer. Now repeat the same procedure with the oil.
Observation Sr. No. Time for oil Time for water (seconds) (seconds)
1 403 10
2 395 10
3 400 10
4 397 10 5 405 10
Average 400 10
Calculation: Kinematic Viscosity (v) = K.t Where, K= Constant of the instrument determined on a liquid of known viscosity t = time in seconds for the meniscus to fall from one point to other point.
The Kinematic viscosity is measured in square meter per seconds.
For water: Kinematic Viscosity (v) = K.t 0.801 x 10-6 = K x 10 K = 0.801 x 10-7 Now, Kinematic Viscosity (v) of oil is: Kinematic Viscosity (v) = K.t = 0.801 x 10-7 x 400 = 32.04 x 10-6 or 32.04 centistokes
Result: Kinematic viscosity of oil was found to be 32.04 x 10-6 m2/sec or 32.04 centistokes.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 13 Determination of microbial count:
Sample Preparation
Homogenize 10ml of material with 5g of polysorbate 80R. Add 85 ml of lactose broth , heated to not more than 400C . Maintain this temperature for the shortest time necessary until an emulsion was formed and, in any case, for not more than 30 minutes. If necessary, adjust the pH of the emulsion to about 7.
Total Viable Aerobic Bacterial Count:
(A) Culture media
(i) Soybean casein digest agar medium (SCDAM)
Ingredients Quantity (g)
Pancreatic digest of casein 15
Papain digest of soybean meal 05
Sodium chloride 05
Agar 15
Water q.s. 1000 ml
Mixed all the contents and sterilized it by autoclaving at 121 C. Adjusted the pH to 7.3 0.2.
(ii) Soybean casein digest medium (SCDM)
Ingredients Quantity (g)
Pancreatic digest of casein 17
Papaic digest of soybean meal 03
Sodium chloride 05
Dibasic potassium phosphate 2.5
Dextrose 2.5
Water q.s. 1000 ml
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 14 All the ingredients were dissolved in distilled water and warmed slightly. After cooling it to room temperature, the pH was adjusted to 7.1 0.2. It was sterilized by autoclaving at 121 C for 30 minutes.
(B) Method
10ml of pretreated sample was transferred to 100 ml of SCDM and mixed well in an incubator shaker at 125 rpm for 1-4 hours, for revivification of microorganisms. 1 ml of sample was pipetted out from SCDM broth medium into pre-sterilized petri-plates (180ºC for 2 hours) and 15-20 ml of soybean casein SCDAM was added. The contents were mixed properly for uniform distribution and the SCDAM plates were incubated in a bacteriological incubator at 35ºC for 48-96 hours. After incubation total number of bacterial colonies was counted using Colony Counter and Colony Forming Units per ml (CFU/ml) was calculated using the following formula:
Total Fungal Count:
(A) Culture media
(i) Soyabean casein digest agar medium
(ii) Soyabean casein digest medium
(B) Method
The culture media were prepared as above and 10 ml of each sample was transferred to 100 ml of SCDM and mixed well for 1-4 in incubator shaker at 125 rpm for revivification of microorganism. 1 ml of sample was pipetted out from SCDM broth medium into pre- sterilized petri-plates (180ºC for 2 hours) and 15-20 ml of SCDAM was added. The contents were mixed properly for uniform distribution and the SCDA plates were incubated in BOD incubator at 25ºC for 5-7 days. After incubation total number of fungal colonies was counted with the help of colony counter and CFU/ml was calculated using formula:
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 15
Determination for E. coli
(A) Culture media
(i) Ingredients Quantity (g) Pancreatic digest of gelatin 17 Peptone 03 Lactose 10 Sodium chloride 05 Bile salts 1.5 Agar 13.5 Neutral red 30 Crystal violet 01 Water to q.s. 1000ml
The ingredients were boiled in water for 1 minute to affect solution and after adjustment of pH to 7.1 ± 0.2, sterilization was done.
(ii)
Ingredients Quantity (g)
Pancreatic digest of gelatin 20
Lactose 10
Dehydrated ox bile 05
Bromocresol purple 10
Water q.s 1000 ml
The adjustment of pH to 7.3 ± 0.2 was done and it was sterilized.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 16 (B) Method
Aseptically 10 ml of sample was transferred to 100 ml lactose broth/soybean casein digest broth medium and the media was incubated at 37ºC for 24 hours. The flask was examined for growth and the contents were mixed by gentle shaking. 1 ml of the enriched
35ºC for 26 hours. Concomitant was done using a loopful of enriched culture and the plates were incubated at 37ºC for 24 hours.
Determination for Salmonella typhi
(A) Culture media
(i) Selenite F broth
Ingredients Quantity (g)
Peptone 05
Lactose 02
Disodium hydrogen phosphate 10
Sodium hydrogen selenite 04
Water q.s. 1000 ml
The ingredients were boiled in water for 1 minute to affect solution and after adjustment of pH to 7.1 ± 0.2, sterilization was done.
(B) Method
1.0 ml of the enriched culture was added to the tubes containing 10 ml of selenite F broth. The tubes were incubated at 35ºC for 48 hours and observed for the presence of turbidity.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 17 Determination for Pseudomonas aerginosa
(A) Culture media
(i) Cetrimide agar medium Ingredients Quantity (g) Pancreatic digest of gelatin 20 Magnesium chloride 1.4 Potassium sulphate 10 Cetrimide 0.3 Agar 13.6 Glycerin 10 Water q.s. 1000 ml The ingredients were boiled in water for 1 minute to affect solution and after adjustment of pH to 7.1 ± 0.2, sterilization was done.
(B) Method
1.0 ml of the enriched culture was added to the plates containing cetrimide agar media, mixed and incubated at 35ºC to 37ºC for 24 to 48 hours and observations for microbial growth were made.
Determination for Staphylococcus aureus
(A) Culture media
(i) Vogel Johnson agar medium (VJA)
Ingredient Quantity (g) Pancreatic digest of casein 10 Yeast extract 05 Mannitol 10 Dibasic potassium phosphate 05 Lithium chloride 05 Glycine 10 Agar 16 Phenol red 25 Water q.s. 1000 ml
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 18 All ingredients were made in to solution by heating and cooled to ~45ºC and 20 ml of a 1% w/v solution of potassium tellurite was added to it. The pH was adjusted to 7.4 ± 0.2 and the contents were sterilized.
(B) Method
Enriched culture was streaked on the surface of VJA media and incubated at 35ºC for 24 hours and observed for the presence of growth.
Table : Microbiological Count determination results
Microorganisms Count
Total bacterial count Nil
Total fungal count Nil
Escherichia coli Nil
Salmonella typhi Nil
Staphylococcus aureus Nil
Pseudomonas aeruginosa Nil
TOXICOLOGICAL STUDIES (OECD 434)
Sighting study:
The purpose of the sighting study is to allow selection of the appropriate starting dose for the main study. The test substance is administered to single animals in a sequential manner. The sighting study is completed when a decision on the starting dose for the main study can be made (or if a death is seen at the lowest fixed dose).The starting dose for the sighting study is selected from the fixed dose levels of 50, 200, 1000 and 2000 mg/kg as a dose expected to produce evident toxicity based, when possible, on evidence from existing data on the same chemical and for structurally related chemicals. In the absence of such information, the starting dose will be 1000 mg/kg.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 19 A period of at least 24 hours will be allowed between the testing of each animal. All animals should normally be observed for at least 14 days.
No morbidity and toxic signs were observed in group treating with 1000mg/kg.
Now, MML being an oil of unknown composition, and anticipating its direct relevance for protecting the human or animal health, further Sighting studies were conducted at 2000 mg/kg and 5000 mg/kg.
No morbidity and toxic signs were observed in group treating with 2000mg/kg and 5000 mg/kg.
Skin irritation test:
The degree of dermal irritation of oil was determined in rabbits using the occluded dermal irritation test method as described elsewhere [Auletta, 1995]. Six rabbits were used for this test and each animal served as its own control. On day 0 of the test period, hair was clipped from the back (about 10% of the total body surface area) of each rabbit. The left side (about 6 cm2) served as a test site, while the right side as a control site. Rabbits were caged individually and left undisturbed for 24 h. On day 1 of the test period, 1 g/kg and 5g/kg of the oil was evenly applied to the shaved area and the skin was covered with gauze patch, plastic sheet and a non-irritating adhesive plaster. The rabbits were then returned to their cages. After 24 h of exposure period, the coverings were removed and the test site was rinsed with distilled water. The animals were examined for the presence of erythema and edema according to Draize dermal irritation scoring system (0: no erythema or no edema; 1: barely perceptible erythema or edema; 2: well defined erythema or slight edema; 3: moderate to severe erythema or moderate edema; 4: severe erythema or edema) at grading intervals of 1, 24, 48 and 72 h [Draize, 1959]. In parallel, cage side observations were made daily for signs of clinical toxicity throughout the test period.
Primary Irritation Index (PII) was calculated by dividing the sum of erythema and edema scores of the grading intervals with the number of test sites (6) and multiplying by the number of grading intervals (4). The extract was then classified according to Draize method of classification using the PII scoring as mildly irritant (if PII < 2), moderately
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 20 Erythema scores of MML oil at dose 1 gm/kg for acute Skin Irritation Test on rabbits
Rabbit (R) Erythema Scores
1 h 24 h 48 h 72 h
Test Control Test Control Test Control Test Control
site Site site Site site Site site Site
R1 0 0 0 0 0 0 0 0
R2 0 0 0 0 0 0 0 0
R3 0 0 0 0 0 0 0 0
R4 0 0 0 0 0 0 0 0
R5 0 0 0 0 0 0 0 0
R6 0 0 0 0 0 0 0 0
Erythema scores of MML oil at dose 5 gm/kg for acute Skin Irritation Test on rabbits
Rabbit (R) Erythema Scores
1 h 24 h 48 h 72 h
Test Control Test Control Test Control Test Control
site Site site Site site Site site Site
R1 0 0 0 0 0 0 0 0
R2 0 0 0 0 0 0 0 0
R3 0 0 0 0 0 0 0 0
R4 0 0 0 0 0 0 0 0
R5 0 0 0 0 0 0 0 0
R6 0 0 0 0 0 0 0 0
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 21 Acute Dermal Toxicity Test (Limit Test)
This test was performed on rats based on OECD guideline number 404 [OECD, 1981a]. Hair was clipped from the back of each rat (weighing 200 300 g), approximately 10% of the total surface area, and then each rat was caged individually and left undisturbed for 24 h. Thereafter, the oil (5000 mg/kg) was evenly applied on the shaved area and immediately covered with gauze and protected using a non-irritating adhesive plaster. The rats were then returned to their cage. Following 24 h after application; the covering was removed carefully and cage side observation was made daily, but weight measurement was taken weekly for 15 days. Observation included evaluation of skin and fur, eyes, respiratory effects (salivation, diarrhea and urination), and central nervous system effects (tremors and convulsion, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength and bizarre behaviour). By the 15th day rats were humanely killed and organs were carefully taken out and weight measurements were taken.
Absolute and relative organ weights of Male rats (n = 5) that received acute treatment of MML oil at dose 5000 mg/kg
Organ Control MML oil
Absolute Relative Absolute Relative
Weight Weight Weight Weight
Brain 1.73+0.05 0.63+0.02 1.71+0.13 0.64+0.06
Heart 1.25+0.16 0.46+0.04 1.24+0.18 0.46+0.05
Liver 8.80+0.26 3.23+0.03 8.40+0.14 3.13+0.02
Spleen 0.71+0.14 0.26+0.05 0.68+0.11 0.25+0.03
Lung 1.41+0.17 0.52+0.03 1.39+0.16 0.52+0.02
Kidney 1.79+0.12 0.66+0.04 1.69+0.05 0.63+0.03
Testis 2.40+0.16 0.88+0.05 2.38+0.15 0.89+0.04
Body weight 272.3+16.1 268.00+19.14
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 22 Absolute and relative organ weights of Female rats (n = 5) that received acute treatment of MML oil at dose 5000 mg/kg
Organ Control MML oil
Absolute Relative Absolute Relative
Weight Weight Weight Weight
Brain 1.70+0.24 0.75+0.04 1.68+0.22 0.75+0.03
Heart 1.26+0.12 0.55+0.01 1.25+0.16 0.56+0.02
Liver 7.31+0.15 3.21+0.07 7.43+0.12 3.31+0.05
Spleen 0.89+0.06 0.39+0.03 0.85+0.07 0.38+0.04
Lung 1.51+0.14 0.66+0.03 1.48+0.16 0.66+0.05
Kidney 1.61+0.16 0.71+0.04 1.64+0.11 0.73+0.03
Ovaries 0.086+0.005 0.038+0.002 0.082+0.004 0.036+0.001
Body weight 227.4+20.15 224.7+16.12
BIOLOGICAL EVALUATION
The MML oil has been evaluated for anti-inflammatory activity by using the following models:
A. Croton oil induced ear edema
B. Oxazolone-induced ear edema in mice
C. Oxazolone-induced paw edema in mice
D. Arachidonic acid (AA)-induced mouse ear edema
Croton oil induced ear edema (Tonelli 1965)
This method is basically meant to assess the antiphlogistic and thymolytic activity of topically applied steroids or NSAIDS.
The effect is determined by expressing the increase in weight of the treated ear as percentage of the weight of the contralateral control ear.
The difference of both weights is divided by the 100 times weight of control ear.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 23 The simple difference between control and treated ears is also calculated and statistically evaluated.
Hydrocortisone (1 mg/ml) is an effective standard drug.
Procedure For Albino mice/NMRI mice the croton oil composition is: Croton oil 1 part + ethanol 10 parts + pyridine 20 parts + ethyl ether 69 parts For Wistar rats the composition is : Croton oil 4 parts + ethanol 10 parts + pyridine 20 parts + ethyl ether 66 parts. The testing material (TM) and standard drugs were dissolved in the above mentioned compositions. The mice used were Albino strain while the rats were Wistar. Six animals were used for controls and each test group. The test compounds were dissolved in a concentration of 1 mg/ml for mice. On both sites of the right ear 0.01 ml of composition in mice or 0.02 ml in rats applied locally. Controls receive only the irritant composition The left ear remains on untreated. The irritant was applied under ether anesthesia. Four hr after local application the animals were sacrificed under anesthesia. Both ears were removed and discs of 8 mm diameter are punched. The discs were weighed immediately and weight difference between the treated and untreated ear is recorded. The calculations were made as described above
Group Ear edema Edema Inhibition (%) Control 0.0725+0.0054 - Standard Drug 0.0245 +0.0031** 66.2% TM (0.25mg/ml) 0.0622+0.0024* 14.2% TM (0.5mg/ml) 0.0522+0.0035** 28.0% TM (1mg/ml) 0.0362+0.0026** 50.1% Statistically significant from control group *p value < 0.05 and ** p value < 0.01
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 24 Croton Oil Induced Ear Edema Control 0.09 Standard 0.08 TM (0.25mg/ml) 0.07 TM (0.5mg/ml) 0.06 TM (1mg/ml) 0.05 0.04 0.03 0.02 0.01 0 Control Standard TM (0.25mg/ml) TM (0.5mg/ml) TM (1mg/ml)
Oxazolone-induced ear edema in mice (Vogel 2002)
General
The oxazolone induced ear edema model in mice is a;
delayed contact hypersensitivity or Delayed Type Hypersensitivity (DTH) model
that permits the quantitative evaluation of the
Topical, and systemic anti-inflammatory activity of test compound following topical administration.
Procedure
Mice (22-25g; either sex) are employed.
Before each use a fresh solution of oxazolone (2%) was prepared in acetone.
The mice were sensitised by application of 0.01 ml of the acetone solution on the inside of both ears under ether anaesthesia.
The mice were challenged after 8 days later again under anaesthesia by applying 0.01 ml of 2% oxazolone solution to the inside of right ear (control group).
The test groups were treated with 0.01 ml of oxazolone solution in which different concentrations of TM have been dissolved.
Positive control group was treated with oxazolone solution + reference drug.
Groups of 6 animals were treated with the irritant alone or with the solution of the test compound.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 25 The left ear remains untreated.
The maximum inflammation occurs 24 h later.
At this time the animals are sacrificed under anaesthesia and disc of 8 mm diameter was punched from both sides.
The weight difference was taken as the measure of inflammatory edema in different groups.
Average values of the increase of weight in the discs were calculated for each treated group and compared statistically with the control group.
A 0.003% solution of hydrocortisone used as topical drug.
Groups Edema Edema inhibition % Control 0.0132+0.0005
Std. Drug 0.0074+0.0002** 43.9% 0.0124+0.0004 06.0%
0.0105+0.0003** 20.5%
0.0093+0.0003** 29.5%
Statistically significant from control group *p value < 0.05 and ** p value < 0.01
Oxazolone Induced Ear Edema
0.016 0.014 0.012 Control 0.01 Standard 0.008 0.006 0.004
0.002 0 Control Standard
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 26 Oxazolone-induced paw edema in mice (Griswold 1974)
General
This is one of the most commonly employed technique.
Phlogistic agent employed : Oxazolone
Usually, the volume of the injected paw is measured before and after the application of the irritant.
The paw volume of the treated animals is compared to the controls using;
a simple plethysmometer (volume displacement)
The volume of the inflamed paw depends upon the irritant used.
The edema could be short or long lasting.
Procedure
Male or female mice were starved overnight with water.
The test groups were treated with the TM in different doses.
Thirty min. later, 3% solution of oxazolone applied to the left paw of mice.
The paw volume is measured as mentioned above;
Immediately and 3 h after topical treatment.
The inflamed paw was immersed in mercury in a part of the plethysmometer and the volume of mercury displaced in the equipment is measured every time.
A 0.003% solution of hydrocortisone used as topical standard drug.
Groups Paw volume(ml) Edema inhibition %
Control 0.85+0.02 -
Oxazolone induced 1.38+0.03 - Standard Drug 1.15 +0.03** 43.4%
TM(0.25mg/ml) 1.24 +0.03** 26.4%
TM(0.5mg/ml) 1.20 +0.02** 34%
TM(1mg/ml) 1.18 +0.02** 38.3%
Statistically significant from control group ** p value < 0.01
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 27 Oxazolone Induced Paw Edema
1.6
1.4 Control 1.2 Oxazolone 1 Standard 0.8 TM (0.25mg/ml) TM (0.5mg/ml) 0.6 TM (1mg/ml) 0.4
0.2
0 Control Oxazolone Standard TM TM (0.5mg/ml) TM (1mg/ml) (0.25mg/ml)
Arachidonic acid (AA)-induced ear edema (Young et. al 1984, Crummey et. al 1987)
Procedure
Mice (22-25g; either sex) were employed.
The test groups were treated with the TM in different doses (1, 0.5, 0.25mg/ear)
Thirty min. later, arachidonic acid (2mg/ear) applied.
The reference groups were treated with Phenidone (1mg/ear).
The thickness of the ears was measured before and 1 hr after induction of edema using a micrometer.
The edema was measured as indicated above and expressed as an increase in the ear thickness due to AA application.
Group %I
Control 221+10 -
Standard 109+7** 51
TM (0.25mg/ear) 202+10 9
TM (0.5mg/ear) 159+9** 28
TM (1mg/ear) 143+18** 36
Statistically significant from control group ** p value < 0.01
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 28 Arachidonic Acid Induced Ear Edema
250
200 Control 150 Standard TM (0.25 mg /ml) 100 TM (0.5 mg /ml) TM (1mg /ml) 50
0 Control Standard TM (0.25 TM (0.5 mg TM (1mg mg /ml) /ml) /ml)
Histology
Ear samples (were fixed in 4% neutral buffered formalin. Each sample was cut longitudinally into equal halves, one of which was embedded in paraffin wax, sectioned at 3 4 Am and stained with haematoxylin eosin. A representative area was selected for qualitative light microscopic analysis of the inflammatory cellular response with a 40X objective. To minimise a source of bias, the investigator did not know the group that he was analyzing.
(a) (b)
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 29
(c) (d)
(e) (f) Effect of MML oil on histopathological changes in croton oil induced ear edema. Plate (a) shows normal architecture of the covering dermal and epidermal layers as well as subcutaneous tissue of the skin. Plate (b) shows ear tissue from croton oil alone treated exhibiting massive neutrophil infiltration with extravasations of red blood cells and edema in the dermal layer. Plate (c) exhibits standard drug. Plate (d), (e), (f) corresponding to MML oil 0.25mg/ml, 0.5mg/ml and 1mg/ml respectively; show less neutrophil infiltration, collagen fiber dispersion and edema
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 30 CHROMATOGRAPHY STUDIES
Thin Layer Chromatography:
Principle
Separation by TLC is effected by the application of the mixture as a spot onto sorbent that has been applied to a backing plate. The plate is then placed into a tank with sufficient suitable solvent to just wet the lower edge of the plate-sorbent but not enough to wet the part of the plate where the spots were applied, the solvent front then migrates up the plate through the sorbent by capillary action, a process known as development. of the separated substances. It is given in the form of the Rf value
Rf = Distance (starting zone-substance zone) Distance (starting zone-solvent front) Equipment
1. Balance
2. Thin layer applicator
3. Hot air oven
4. TLC solvent tank
Reagents
1. Mixture of Toluene, chloroform and ethyl acetate, (13:6:1, v/v)
2. Silica gel G
3. Iodine
Procedure
1. Arrange the TLC plates on the TLC applicator.
2. Weighed 40g of silica gel into a conical flask.
3. Add 80-85 ml distilled water and close the flask with a rubber stopper.
4. Shake vigorously for 5-7 min. The slurry was ready for spreading when particles swell and become more viscous.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 31 5. Pour the complete slurry into the spreader at once and move the spreader forward with uniform force.
6. Leave the coated plates undisturbed for 1h at room temperature.
7. Activate the coated plates by keeping them in an oven at 1000C for 1h.
8. Applied the spot of the oil with the help of the capillary tube 1cm above the bottom edge.
9. Saturate TLC chamber 2-3 hr with solvent system, Toluene, chloroform and ethyl acetate, (13:6:1, v/v).
10. Keep the spotted plate in the chamber for developing.
11. Remove the plate when the solvent front was run up to 10cm from the spot.
12. Dry the plate and placed in Iodine chamber until coloured spots developed.
Calculation
The Rf value is calculated using a scale by measuring the distance covered by the spots and solvent front.
TLC observations for MML oil (Iodine chamber)
Spot No. Colour of Spot Rf Value 1 Yellow 0.27
2 Yellow 0.41
3 Yellow 0.50
4 Yellow 0.60
5 Dark brown 0.70
6 Dark Brown 0.86
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 32 HPTLC Fingerprinting:
Solvent system
Toluene, chloroform and ethyl acetate, (13:6:1, v/v).
HPTLC Condition as per Appendix F
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 33
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 34 Gas Chromatography:
Principle
The separation of compounds in a gas chromatograph depends on the difference in partition coefficients between the liquid (stationary) and gaseous (mobile) phases of the constituents in a mixture. The constituents with a high affinity for the stationary phase will tend to remain in it for a long time. These will travel slowly through the column. Those with little or no affinity for the stationary phase will travel faster and get eluted quickly.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 35 Gas Chromatography Mass Spectroscopy (GCMS):
Conditions
The GCMS data were obtained on a Shimadzu QP-2000 instrument at 70 eV and 2500C. GC Column: ULBON HR-1 equivalent to OV-1, fused silica capillary 0.25 mm x 50 M with thickness 0.25 micron. Initial temperature was 1000C for 6 minutes and then heated at the rate of 100C per minute to 2500C. Carrier gas (helium) flow: 2 ml per minute. Peak No. R. Time Compound Identified 1 6.06 Heptan-2-ol 2 6.16 Heptan-3-ol 3 6.50 Carvone 4 6.73 Piperitone 5 6.83 3-methyl furan 6 7.03 3-cyclopentene-1,2-diol 7 10.26 Pent-2,4-dien-1-ol
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 36 STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 37
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 38
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 39
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 40
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 41 Bibliography:
Anonymous (1996): Indian Pharmacopoeia, Ministry of Health and Family Welfare, Controller of Publication, New Delhi, pp. A50- 52, A93- A99.
Auletta C.S. (1995): Acute, subchronic, and chronic toxicology. In: Derelanko, M.J., Hollinger, M.J. (Eds.), Handbook of Toxicology. CRC Press Inc., London, pp. 51 162.
Crummey A, Harper GP, Boyle EA, Mangan FR (1987): Inhibition of arachidonic acid-induced ear oedema as a model for assessing topical anti-inflammatory compounds. Agents Actions 20:69 72
Draize J.H. (1959): The appraisal of chemicals in foods, drugs and cosmetics. The Association of Food and Drug Officials of the United States, p. 46.
Griswold DE, DiLorenzo JA, Calabresi P (1974): Quantification and pharmacological dissection of oxazolone-induced contact sensitivity in the mouse. Cell Immunol 11:198 204
OECD (2004): Acute dermal toxicity fixed dose procedure. In: OECD Guidelines for Testing of Chemicals, No. 434. Organization for Economic Cooperation and
Development, Paris, France.
L Tonelli G, Thibault, Ringler I (1965): A bio-assay for the concomitant assessment of the antiphlogistic and thymolytic activities of topically applied steroids. Endocrinology 77:625 630
Vogel H.G. (2002): Oxazolone-induced ear edema in mice. Drug Discovery and
Evaluation: Pharmacological Assays. Springer-Verlag Berlin Heidelberg, Ed-2, 756-757
Young JJ, Spires DA, Bedord CJ, Wagner B, Ballaron SJ, DeYoung LM (1984): The mouse ear inflammatory response to topical arachidonic acid. J Invest Dermatol 82:367 371
WHO (1998): Quality control methods for medicinal plants. World Health Organization, Geneva
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 42
DETAILED ANALYSIS OF RESULTS
Physiochemical parameters:
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 results (Acid value = 8.08) indicate the amount of fatty acids present in the MML oil, which in turn indicates the degree to which the fat can rancidify. Since, fats in the oil can undergo rancidity, where the triglycerides are converted to fatty acids and glycerol, causing an increase in acid value.
The saponification value is an index of mean molecular weight (or chain length) of the fatty acids of the glycerides. Lower saponification value indicates higher molecular weight of fatty acids and vice-versa. The saponification value of MML determined is 194.95.
The Ester value of the oil determined is 186.87, whereas, the hydroxyl value revealed a reading of 36.13.
The iodine value indicates the degree of unsaturation of the oil. Since the presumed Iodine Value of MML oil was unknown, three quantities of the oil were taken for determination of Iodine value, and Iodine value was found to be 39.49 for 1g, 63.65 for 0.5g and 93.20 for 0.25g of oil, respectively.
Peroxide value as per the procedure gave results which were not satisfactory; therefore the procedure was amended to obtain the Peroxide value of oil to be 2.2.
The MML oil was further investigated for Density (wt/ml), Relative density, pH, Refractive index, Optical rotation and Kinematic viscosity, which gave the results as 0.9078 wt/ml, 0.9114, 4.2323, 1.491, 0.3721, 32.0410-6 m2/sec or 32.04 centistokes, respectively.
The above physiochemical parameters were executed keeping in view the procedures given by Indian Pharmacopeia, 1996.
Microbial contamination:
The WHO guidelines (1998) were adopted to determine any microbial contamination. No such contamination was revealed in the MML oil under study.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 43 Biological Studies:
The MML oil has been evaluated for anti-inflammatory activity by using the following models:
A. Croton oil induced ear edema
It was observed that the application of croton oil to ears caused massive increase in weight of the ear punch compared to unchallenged ears. Pre-treatment of ears with the three dose levels of MML oil, 0.25, 0.5 and 1mg/ml significantly reduced the increase of punch weight by 14.2%, 28.0% and 50.1% respectively. In addition standard drug produced significant reduction of edema by 66.2%. This activity could be justified by the remarkable cutaneous absorption of the MML oil.
B. Oxazolone-induced ear edema in mice:
When assayed with contact-delayed mouse hypersensitivity, induced by oxazolone, MML oil inhibited significantly both the hypersensitivity induction and the inflammation phase at dose of 0.5mg/ml and 1mg/ml up to 20.5% and 29.5, respectively. However standard drug reduced the edema by 43.9%
C. Oxazolone-induced paw edema in mice:
Topical application of oxazolone to mice paw resulted in severe inflammation and significant increase in the mean volume of the challenged paw compared to that of untreated paw. Pre-treatment of mice with MML oil at dose of 0.25, 0.5 and 1mg/ml significantly inhibited the oxazolone induced increase in edema volume of the paw by 26.4%, 34% and 38.3%, respectively. Similarly hydrocortisone treated group showed significant anti-edema effect (43.4%).
D. Arachidonic acid (AA)-induced mouse ear edema:
In the arachidonic acid induced ear edema, MML oil at dose of 0.5 and 1mg/ear significantly reduced the ear thickness by 28% and 36%, while standard drug reduced ear thickness by 51%.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 44 All results are reported as mean + S.E.M. The statistical analysis was performed using one-way analysis of variance (ANOVA). The significant difference between control and experimental groups were assessed by Dunnetts student test. A probability level of less than 5% (p value < 0.05) was considered significant. All statistical analysis were performed using InStat3 software for window.
Toxicological studies:
A. Skin Irritation Test:
The oil was found to have moderate irritation effect on rabbits, as the PPI was calculated to be less than 2.o. There was no irritation or any sign of erythema. Grading for the test was continued after 72 hours, day 7, 10, and 14. No observable erythema was seen in the observation did not reveal any observable sign of systemic toxicity in all rabbits.
B. Acute Dermal Toxicity:
The result of the Acute dermal toxicity studies indicate that MML oil at dose of 5000 mg/kg did not produce any sign of toxicity or death in rats. No significance difference in body weight gain of the surviving animals was noted between the control or treated group over the period of observation. No statistical difference was observed between the relative organ weight in the control and treated group. This study suggests a LD50 above 5000 mg/kg, hence MML oil can be considered practically non-toxic.
Chromatography studies:
A. Thin Layer Chromatography (TLC):
The MML oil revealed six spots, by using the solvent system toluene: chloroform: ethyl acetate (13:7:1), developed in iodine chamber, with Rf values 0.27, 0.41, 0.50, 0.60, 0.70, and 0.86, respectively.
B. High Performance Thin Layer Chromatography (HPTLC):
The MML oil revealed six spots, by using the solvent system toluene: chloroform: ethyl acetate (13:7:1), using UV at 254 nm, with Rf values 0.13, 0.16, 0.33, 0.41, 0.47, and 0.57. Whereas, when scanned at 366 nm, MML oil revealed 9 spots, with Rf values 0.13, 0.15, 0.26, 0.38, 0.41, 0.47, 0.55, 0.59, and 0.88, respectively.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 45 C. Gas Chromatography (GC):
When the MML oil when subjected to Gas chromatography, 25 peaks were observed, as per the spectra annexed hereto.
D. Gas Chromatography Mass Spectroscopy (GCMS):
The MML oil revealed seven peaks, by using GCMS, with retention time 6.06, corresponds to Heptan-2-ol, 6.16 relates to Heptan-3-ol, 6.50 corresponds to carvone, 6.73 represents to piperitone, 6.83 corresponds to 3-methyl furan, 7.03 corresponds to 3- cyclopentane-1,2-diol, and 10.26 which relates to pent-2,4-dien-1-ol, as per spectra annexed hereto.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 46
CONCLUSIONS SUMMARIZING ACHIEVEMENTS AND
SCOPE FOR FUTURE WORK
The present study of MML oil embodies the physicochemical, biological, microbiological, toxicological and chromatographic studies, keeping in view the aims and objectives of the project assigned. These studies will serve as standard reference and the standard operating procedures to be adopted in future for the MML oil.
The physicochemical parameters performed will give an idea about the quality of the MML oil. i.e. The acid value indicates the amount of fatty acids present in the MML oil, The saponification value is an index of mean molecular weight (or chain length) of the fatty acids of the glycerides, which was determined to be 194.95. The Ester value of the oil determined is 186.87, whereas, the hydroxyl value revealed a reading of 36.13. The iodine value indicates the degree of unsaturation of the oil and Iodine value was found to be 39.49 for 1g, 63.65 for 0.5g and 93.20 for 0.25g of oil, respectively. The Peroxide value as per the procedure gave results which were not satisfactory; therefore the procedure was amended to obtain the Peroxide value of oil to be 2.2. The MML oil was further investigated for Density (wt/ml), Relative density, pH, Refractive index, Optical rotation and Viscosity, which gave the results as 0.9078 wt/ml, 0.9114, 4.2323, 1.491, 0.3721, 32.0410-6 m2/sec or 32.04 centistokes, respectively.
The MML oil was evaluated for microbial count, no such contamination was revealed in oil under study, hence within permissible limits.
The Toxicology studies show the safety of the oil for future use for the benefit of humans and animals in pharmaceutical preparations.
The MML oil has been screened for anti-inflammatory activity by using the following four models viz. Croton oil induced ear edema, Oxazolone-induced ear edema in mice, Oxazolone-induced paw edema in mice, Arachidonic acid (AA)-induced mouse ear edema. All results are reported as mean + S.E.M. The statistical analysis was performed using one-way analysis of variance (ANOVA). The significant difference between control and experimental groups were assessed by Dunnetts student test. A probability level of less than 5% (p value < 0.05) was considered significant. All
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 47 statistical analysis were performed using InStat3 software for window. These results help us to conclude that the MML oil possess anti-inflammatory activity.
The chromatography studies performed like TLC, HPTLC fingerprint profile, GC, GCMS will serve as a reference for quick quality control approval of the MML oil.
Thus, the generated data can be used for determining the correct identity and purity of the MML oil, in future, and detect any adulteration as well. The stated results will help in manufacturing a suitable herbal formulation with the MML oil, as the future scope of work.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 48
PROCUREMENT / USAGE OF MAJOR EQUIPMENT
Equipment Make/Model Cost Date of Utilisation** installation*/billing rate % 1 Polarimeter Timestar 14,006/- 25/3/2010 100
2 Abbe Timestar 7,864/- 25/3/2010 100 Refractometer
3 Hot Air Oven Khera 29,363/- 26/3/2010 100
4 TLC Kit Loba 6,632/- 12/2/2009 100
5 Chromatography Mohit 10,125/- 12/2/2009 100 Cabinet
6 Digital pH meter Mohit 22,781/- 7/2/2009 100 7 Hot Wire Radiant type 8,201/- 26/3/2010 100 Analgesiometer
8 Melting Point Kshistij 19,013/- 25/3/2010 100 Apparatus
9 Digital Screw Rajesh 24,301/- 26/3/2010 100 gauge
10. Magnetic stirrer Khera 4,252/- 29/3/2010 100 with hot plate
*The equipments were installed much before the date of billing, so that the experimental work does not hamper, and payment was processed only after receiving due permission of extension from CCRAS, and thus enabling the release of funds.
** The equipments purchased have been used for the project work, as well they are being used by the PG/PhD research students, which are available to them during working hours on all working days.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 49
MANUSCRIPT FOR PUBLICATION
Standardization and quality control studies of MML oil was awarded to Prof. Surendra Kr Sharma, Principal Investigator, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science & Technology, Hisar-125 001.
The objective of the project was:
- To carryout standardization of MML oil
- To perform Toxicological studies
- To evaluate biological activity to ascertain anti-inflammatory activity
The various physicochemical properties like acid value, saponification value, ester value, iodine value, hydroxyl value, peroxide value, refractive index, optical rotation, weight per ml, relative density, pH and viscosity were determined as per official method, to establish standardization procedures for the MML oil. The microbiological contamination studies were performed as per WHO guidelines, and no contamination was revealed in oil under study. Also the Toxicological studies, which include the Sighting study, Skin irritation test and Acute dermal toxicity test, were performed as per OECD Guideline 434 (2004), which showed the safety of the oil. The biological screening was carried out for anti-inflammatory activity, by using four animal models viz croton oil induced ear edema, oxazolone induced delayed type hypersensitivity model, oxazolone paw edema model, and arachidonic acid induced ear edema, which ascertains a significant anti-inflammatory activity of the MML oil. The TLC, HPTLC, GC, and GCMS were performed on MML oil which will serve as a reference for quick quality control.
Thus, the generated data can be used for determining the correct identity and purity of the MML oil, in future, and detect any adulteration as well. The stated results will help in manufacturing a suitable herbal formulation with the MML oil, as the future scope of work.
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 50
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 51
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 52