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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


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).


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


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


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).


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


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


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


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


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


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


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


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:


0.801 x 10-6 = K x 10

K = 0.801 x 10-7

Now, Kinematic Viscosity (v) of oil is:


= 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.


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)


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


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:


Determination for E. coli

(A) Culture media

(i)


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.


Pancreatic digest of gelatin 20

Lactose 10

Dehydrated ox bile 05

Bromocresol purple 10

Water q.s 1000 ml


(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


Peptone 05

Lactose 02

Disodium hydrogen phosphate 10

Sodium hydrogen selenite 04

Water q.s. 1000 ml



(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.


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



(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


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.


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


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


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


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.


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


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.


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%


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%


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


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)


(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


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.


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


HPTLC Fingerprinting:

Solvent system

Toluene, chloroform and ethyl acetate, (13:6:1, v/v).

HPTLC Condition as per Appendix F



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.


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







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


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.


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%.


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.


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.


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


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.


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.


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.



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