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 OF MML OIL 1
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 2
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 3
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 4
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
(ml)
For blank
(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 5
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 6
Observation:
Sr. No For 1g oil
a (ml)
For 0.5g oil
a (ml)
For 0.25g oil
a (ml)
Blank
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 7
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 8
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 9
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 10
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:
Counts : 10
Average : 4.2323
Std. Dev. : 0.0013
Max. : 4.234
Min. : 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.
Sr. No pH
1 4.234
2 4.230
3 4.231
4 4.232
5 4.234
6 4.234
7 4.232
8 4.233
9 4.232
10 4.231
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 11
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
Counts : 10
Average : 1.491-0.012 (correction factor)
Std. Dev. : 0.001
Max. : 1.492 -0.012 (correction factor)
Min. : 1.489-0.012 (correction factor)
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
Sr. N0 Refractive index
1 1.492
2 1.491
3 1.492
4 1.492
5 1.492
6 1.489
7 1.491
8 1.492
9 1.490
10 1.489
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 12
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
Counts : 10
Average : 0.3721
Std. Dev. : 0.0037
Max. : 0.379
Min. : 0.369
Temp. : 29
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
Sr. No Opt. Rot
1 0.371
2 0.371
3 0.370
4 0.370
5 0.379
6 0.379
7 0.372
8 0.370
9 0.369
10 0.370
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 13
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
(seconds)
Time for water
(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 14
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 15
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 16
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)
The adjustment of pH to 7.3 ± 0.2 was done and it was sterilized.
Ingredients Quantity (g)
Pancreatic digest of gelatin 20
Lactose 10
Dehydrated ox bile 05
Bromocresol purple 10
Water q.s 1000 ml
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 17
(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 18
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 19
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 20
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 21
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
site
Control
Site
Test
site
Control
Site
Test
site
Control
Site
Test
site
Control
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
site
Control
Site
Test
site
Control
Site
Test
site
Control
Site
Test
site
Control
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 22
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
Weight
Relative
Weight
Absolute
Weight
Relative
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 23
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
Weight
Relative
Weight
Absolute
Weight
Relative
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 24
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 25
Croton Oil Induced Ear Edema
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
Control Standard TM (0.25mg/ml) TM (0.5mg/ml) TM (1mg/ml)
ControlStandardTM (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 26
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.
Statistically significant from control group *p value < 0.05 and ** p value < 0.01
Oxazolone Induced Ear Edema
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
Control Standard
ControlStandard
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%
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 27
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.
Statistically significant from control group ** p value < 0.01
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%
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 28
Oxazolone Induced Paw Edema
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Control Oxazolone Standard TM(0.25mg/ml)
TM (0.5mg/ml) TM (1mg/ml)
ControlOxazoloneStandardTM (0.25mg/ml)TM (0.5mg/ml)TM (1mg/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 29
Arachidonic Acid Induced Ear Edema
0
50
100
150
200
250
Control Standard TM (0.25mg /ml)
TM (0.5 mg/ml)
TM (1mg/ml)
ControlStandardTM (0.25 mg /ml)TM (0.5 mg /ml)TM (1mg /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 30
(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 31
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 32
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 33
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 34
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 35
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 36
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 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
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 42
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 43
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 44
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 45
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 46
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 47
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 48
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 49
PROCUREMENT / USAGE OF MAJOR EQUIPMENT
Equipment Make/Model Cost Date of
installation*/billing
Utilisation**
rate %
1 Polarimeter Timestar 14,006/- 25/3/2010 100
2 Abbe
Refractometer
Timestar 7,864/- 25/3/2010 100
3 Hot Air Oven Khera 29,363/- 26/3/2010 100
4 TLC Kit Loba 6,632/- 12/2/2009 100
5 Chromatography
Cabinet
Mohit 10,125/- 12/2/2009 100
6 Digital pH meter Mohit 22,781/- 7/2/2009 100
7 Hot Wire
Analgesiometer
Radiant type 8,201/- 26/3/2010 100
8 Melting Point
Apparatus
Kshistij 19,013/- 25/3/2010 100
9 Digital Screw
gauge
Rajesh 24,301/- 26/3/2010 100
10. Magnetic stirrer
with hot plate
Khera 4,252/- 29/3/2010 100
*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 50
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 51
STANDARDIZATION AND QUALITY CONTROL STUDIES OF MML OIL 52