Sirisha.M* et al. /International Journal Of Pharmacy&Technology
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ISSN: 0975-766X CODEN: IJPTFI
Available Online through Research Article www.ijptonline.com
FORMULATION, EVALUATION, MICROBIAL STUDY AND ACUTE TOXICITY STUDY OF LINSEED OIL OINTMENT USING DIFFERENT BASES
Sirisha.M*, Dr. Shyam Sunder.R2, Ashajyothi.V. 1ShadanWomens College of Pharmacy, Khairatabad, Hyderabad-4, Andhra Pradesh.
2Dean Faculty of Pharmacy, Osmania University. Email:[email protected]
Received on 02-07-2012 Accepted on 18-07-2012
Abstract:
Linseed, linumusitatissimum is used in ayuveda for many vata, pitta related disorders12,13. The lipid content of the seed
contains more than 55% of omega 3 fatty acid1,2,3 responsible for hypolipidaemic14,15, anticancer, anti inflammatory,
anti burn, anti rheumatic5,6 and also proven useful atherosclerosis11 and cardiac disorders16. Lignan content contains
cyanogenicglucosides and phyto oestrogens12,13. As it has anti-inflammatory9and anti burn activities12,13, preparation of
transdermal applications like ointments or creams make it much easier for application as well as for absorption of the
linolenic acid. Aim: This study was conducted with an aim of designing, formulation and evaluation of the Linn seed
oil ointment. Methods: Formulation of the ointment was based on incarporating different concentration of Linn seed oil
in the various ointment bases. Microbial study was performed by Cup plate method and Spread plate method.
Toxicological study was conducted based on the OECD guide lines for the acute toxicology. Results: It has been found
that the formulations having the formula as FORMULA 1E and FORMULA 2B were stable consisting of following
compositions: 1E) For 100gm of ointment: Linseed Oil (54%), Emulsifying wax (16%), White soft paraffin (10%),
Dibasic inorganic salt (20%) and tocopherol 2drops. 2B) For 100gm of ointment: Linseed Oil (50%), Paraffin wax
(17%), White soft paraffin (13%), Dibasic inorganic salt (20%) and tocopherol 2drops). They complied pH 7.45 and
7.9 respectively, and they complied all the required standard of physical and chemical parameter testing, they even
passed the microbial analysis and did not cause any skin toxicity. Conclusion: the above mentioned formulations
passed all the tests and complied all the standards. The accelarated stability studies have shown that the formulation
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with the above formula 2B was found to be most stable transdermal application.
Key words: Linn Seed oil, Formulation, Evaluation, Acute toxicity and Ointment.
Introduction:
Flax (Linumusitatissimum L.) is a broadleaf with very small, narrow leaves that are less than an inch long1,4. It is an
annual plant which is of 1.2 meters in height with slender stems. The oil has many varied applications; medicinal as
well as industrial. The anti cancer activity7,8 has been attributed to the LIGNANS present in the seeds, however the
LIGNANS are absent in the oil of linseed which has polyunsaturated fatty acids responsible for antihyperlipidaemic17,
anti inflammatory activity. Flax is an ancient crop that was used as a food source as far back as 3000 B.C. Flax has
been cultivated for more than 7,000 years in the Middle East as a source of linen fiber and for its oil. In Mexico, it is
used to treat burns, abscesses, cough, urinary tract inflammations, boils, swellings and gingivitis.
Materials and Method:
Formula 1: Ointment Prepared Using an Emulsion Base
1A) For 100 gm ointment: Linseed oil (35 %), Emulsifying wax (35 %), White soft paraffin (10 %), Dibasic inorganic
salt (20 %) and tocopherol 2drops.
Preparation of Ointment:
Emulsifying wax: It is a mixture of sodium lauryl sulphate or similar salts of sulphated higher primary aliphatic
alcohols and cetostearyl alcohol. Cetostearyl alcohol and sodium lauryl sulphate are taken in the ratio of 9: 1 in an
emulsifying wax10. Required quantities of ingredients as mentioned above in the formula are weighed by using an
electric balance. Cetostearyl alcohol and sodium lauryl sulphate are taken in two china dishes respectively and melted
by heating on a heating mantle. White soft paraffin is also taken in another china dish and melted. The weighed
quantity of linseed oil is taken in a beaker and the measured amount of Dibasic inorganic salt is taken and mixed in the
oil thoroughly. The melted white soft paraffin, cetostearyl alcohol and sodium lauryl sulphate are mixed. In a mortar,
the mixture of linseed oil and Dibasic inorganic salt is taken and the melted waxes are incorporated in small amounts
and triturated thoroughly until a homogenous mixture is obtained.
The formula 1A) was found to of very hard within 2days, due to the addition of equal amounts of active ingredient i.e.
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linseed oil and emulsifying wax. The hardness was due excess amounts of cetostearyl alcohol in the emulsifying wax.
Hence, there is a requirement of further improvisation of this formula.
1B) For 100gm ointment: Linseed oil (50%), Emulsifying wax (20%), White soft paraffin (10%), Dibasic inorganic
salt (20%) and tocopherol 2drops.
Preparation
Required quantities were weighed. Cetostearyl alcohol and sodium lauryl sulphate are taken in two china dishes
respectively and melted by heating on a heating mantle. White soft paraffin is also taken in another china dish and
melted. The weighed quantity of linseed oil is taken in a beaker and the measured amount of Dibasic inorganic salt is
taken and mixed in the oil thoroughly. The melted white soft paraffin, cetostearyl alcohol and sodium lauryl sulphate
are mixed. In a mortar, the mixture of linseed oil and Dibasic inorganic salt is taken and the melted waxes are
incorporated in small amounts and triturated thoroughly until a homogenous mixture is obtained and tocopherol was
added to prevent oxidation of the preparation. The formula 1B) was found to be stable with a good consistency; this
was due to the reduced amounts of emulsifying wax as compared to the formula 1A. But after 2 months the ointment
became very hard, which was not squeezable from the ointment tube. Hence, this formula 1 B was further improvised
to get an ointment of good consistency and better hardness.
1C) For 100gm of ointment: Linseed Oil (60%), Emulsifying wax (12%), White soft paraffin (8%), Dibasic inorganic
salt (20%) and tocopherol 2drops.
Preparation
The formulation was prepared following the same method as mentioned earlier. The formula 1 C) was found to be
stable but had a fluid like nature which was due to the excess amounts of linseed oil used.
1D) For 100gm of ointment: Linseed Oil (56%), Emulsifying wax (8%), White soft paraffin (16%), Dibasic inorganic
salt (20%) and tocopherol 2drops.
Preparation
The formulation was prepared following the same method as mentioned earlierThe formula 1D) was found to be fluid
in nature and had a lustrous look when applied to the skin.
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1E) For 100gm of ointment: Linseed Oil (54%), Emulsifying wax (16%), White soft paraffin (10%), Dibasic inorganic
salt (20%) and tocopherol 2drops.
Preparation
The formulation was prepared following the same method as mentioned earlier. The formula 1E) was found to be stable
and of good consistency and easily squeezable from the tube.
Formula 2: Ointment Prepared Using an Hydrocarbon Base
2A) For 100gm of ointment: Linseed Oil (60%), Paraffin wax (12%), White soft paraffin (8%), Dibasic inorganic salt
(20%) and tocopherol 2drops.
Preparation
The formulation was prepared following the same method as mentioned earlier. The formula 2 A) was found to be
stable. The consistency of the preparation can be improved.
2B) For 100gm of ointment: Linseed Oil (50%), Paraffin wax (17%), White soft paraffin (13%), Dibasic inorganic salt
(20%) and tocopherol 2drops.
Preparation
The formulation was prepared following the same method as mentioned earlier. The formula 2B) was found to be
stable and of very good consistency.
Formula 3: Ointment Prepared Using an Water Soluble Base
3A) For 100gm of ointment: Linseed Oil (60%), Polyethylene glycol (12%), White soft paraffin (8%), Dibasic
inorganic salt (20%) and tocopherol 2drops.
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Preparation
The formulation was prepared following the same method as mentioned earlier. The formula 3A) was found to be
unstable due to slight separation of oil.
3B) For 100gm of ointment: Linseed Oil (50%), Polyethlene glycol (16%), White soft paraffin (8%), Cetostearyl
alcohol (8%), Dibasic inorganic salt (20%) and tocopherol 2drops.
Preparation
The formulation was prepared following the same method as mentioned earlier. The formula 3B) was found to be fluid
in nature with a slight separation of oil and a granular feel.
3C) For 100gm of ointment: Linseed Oil (50%), Polyethlene glycol (12%), White soft paraffin (8%), Cetostearyl
alcohol (10%), Dibasic inorganic salt (20%) and tocopherol 2drops.
Preparation
The formulation was prepared following the same method as mentioned earlier. The FORMULA 3 consisting of
Polyethylene glycol is discarded. The hydroxyl groups of polyethylene glycol react with dibasic inorganic salt and
forms a complex and hence results in small precipitates like granules. Though changing the concentrations of the
ingredients used, it was found to be unstable. Hence this formulation was discarded.
Evaluation of Semisolid Preparations
Criteria for the evaluation of dermatological formulations: The pharmacists must observe physical, chemical and
biological parameters.
Physical Parameters
Physical parameters like Odour, Colour and Visual appearance, Rheological properties, Phase changes, Particle size
distribution, Texture feel and Particulate contamination were evaluated.
Chemical Parameters
Chemical parameters like Stability of the active ingredients and adjuvants, Loss of vehicle and other volatile
components, Ph and Release rate of medicaments (diffusion studies- invitro method) were studied.
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Biological Parameters
1. Bioavailability (absorption invivo method)
2. Microbial contamination and sterility
3. Irritant effect
Evaluation of ointment formula:
Physical parameters:
1. Odour : Characteristic linseed oil smell due to the absence of perfume
Colour: Light creamish
Visual appearance: Without any granules and of proper consistency
2. Rheological properties: The viscosity of the preparation is such that it is easily removed from the container.
3. Phase change: No phase change was observed. There was a uniform phase.
4. Texture feel: uniform lustrous oily appearance.
5. Particulate contamination: No particulate contamination as active ingredients are maintained without any
contamination and GMP are followed.
Chemical parameters
6. Loss of vehicle and other volatile components: No volatile components are used in the preparation hence
there is no loss of vehicle. The preparation is properly packed.
7. pH: It was found to be neutral within the range of 7.8 7.9
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8. Microbial contamination and sterility :
A nutrient agar medium was prepared with the following composition:NaCl (10gm), Peptone (10gm), Beef
extract (10gm), Agar (3-4) gm, Distilled water (100ml) All the above ingredients are taken in a conical flask
along with distilled water and stirred continuously till they dissolve. The petridishes were cleaned and sterilized
along with the medium. The medium is poured into petridishes under a laminar air flow maintaining sterile
conditions.
The preparation is placed onto the nutrient agar medium in two ways:
1. Cup plate method 2. Spread plate method The preparations were incubated on agar plate at 37C for three days and it was found that there was no growth
of micro organisms showing that the preparation is sterile.
Laminar air flow
After 24 hours: There was no growth of microorganisms (After 48hours)
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Cup-plate method Spread plate method
After 72hours: There was no growth of microorganisms
Cup-plate method Spread plate method
9. Irritant effect: This study was conducted after the approval of the ethics commette of the Shadan Womens
College of Pharmacy 412/cpcsea/file/no25/26/2010-awd. The study was conducted as per the OECD Guielines
for acute dermal toxicity testing. 2 month old healthy rabbit was taken. It was given staple food for a week and
accustomized to the lab environment. For the irritant effect test, the rabbits abdominal hair is shaved using a
razor, a 1 inch patch of hair is removed in the abdominal region and marked. On the shaved area, the ointment is
applied for testing the irritant effect and checked for every 24hrs for presence of redness, rashes, inflammation
or any hypersensitivity reaction. Absence of these symptoms proves that the preparation is non-irritant.
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Writhing reflex (falling asleep): There is no loss of Writhing reflex. No restlessness, lacrimisation was
observed. No mydriatic or meiotic effect. Normal pupil size was found. Corneal reflex (blinking) is not
observed.
Application of ointment on skin and eye
After 24 hours After 48 hours
No irritant effect
Stabilities Studies
The stability of pharmaceutical preparation should be evaluated by exposing the product to normal shelf conditions for
a year or extended periods ,the rate of decomposition is slow at room temperature and such a method is time consuming
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and uneconomical therefore in practice methods are devised to accelerate the rate of degradation by keeping the
product at higher temperatures. The formulations prepared were found to be stable at room conditions for a period of 2
months. There was no bleeding or separation of ointments.
Accelerated Stability Studies
The objective of accelerated stability studies is to predict the shelf life of a product by accelerating the rate of
decomposition, preferably by increasing the temperature. According to Arrhenius theory the rate of reaction is said to
double for each 10 degree rise in temperature. The prepared formulations were tested for accelerated stability. They
were kept in a hot air oven initially at 30C and there was an increase in temperature of 10 C every 10 mins until the
temperature reached 70C. It was found that there was no change in the formulations and were stable even at high
temperatures.
Results:
Formula 1: Ointment Prepared Using an Emulsion Base
For Formula 1A: After the formulation, the preparation was transferred into the aluminium tubes and closed properly
to avoid drying. The formula 1A) was found to of very hard within 2days, due to the addition of equal amounts of
active ingredient i.e. linseed oil and emulsifying wax. The hardness was due excess amounts of cetostearyl alcohol in
the emulsifying wax. Hence, there is a requirement of further improvisation of this formula.
For Formula 1B: After the formulation, the preparation was transferred into the aluminium tubes and closed properly
to avoid drying. The formula 1B) was found to be stable with a good consistency, this was due to the reduced amounts
of emulsifying wax (20%) as compared to the formula 1A(emulsifying wax -35%). But after 2 months the ointment
became very hard, which was not squeezable from the ointment tube. Hence, this formula 1B was further improvised to
get an ointment of good consistency and better hardness.
For Formula 1C: After the formulation, the preparation was transferred into the amberedcoloured bottle and closed
properly to avoid drying. In this formula, excess amount of linseed oil was used and the emulsifying wax was reduced
by 8% so as to improve the consistency and reduce the hardness of the preparation as the cetostearyl alcohol content of
emulsifying wax contributes much towards the consistency. The formula 1 C) was found to be stable but had a fluid
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like nature which was due to the excess amounts of linseed oil usage. Hence in order to reduce the fluidly nature and
improve its consistency further improvisation of this formula is required.
For Formula 1 D: After the formulation, the preparation was transferred into the amberedcoloured bottles and closed
properly to avoid oxidation. In this formula, the amount of linseed oil was reduced by 4% as compared to previous
formula to reduce the fluidly nature. The amount of white soft paraffin was increased by 8% i.e double the amount used
in the formula 1C to increase the body of the preparation. The formula 1D) was found to be fluid in nature and had a
lustrous look when applied to the skin. Hence this formula was further improvised to get a perfect preparation of good
consistency.
For Formula 1E: After the formulation, the preparation was transferred into the aluminium tubes and closed properly
to avoid oxidation. The amount of emulsifying wax was increased to 16 % i.e double the amount used in formula 1 D
so as to increase the hardness and reduce the fluidly nature of the preparation. The amount of white soft paraffin was
reduced by 6%. The formula 1E) was found to be stable for a period of 6months and was of good consistency and
easily squeezable from the tube.
Formula 2: Ointment Prepared Using an Hydrocarbon Base
For Formula 2 A: After the formulation, the preparation was transferred into the amberedcoloured bottles and closed
properly to avoid oxidation. The formula 2 A) was found to be stable. The hradness of the preparation can be increased
to get a good consistency of the ointment.
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For Formula 2 B: After the formulation, the preparation was transferred into the amberedcoloured bottles and closed
properly to avoid oxidation. The formula 2B) was found to be stable for about 6months and of very good consistency.
This was due to decrease in the amounts of linseed oil and increase in the paraffin wax amount compared to formula
2A.
Formula 3: Ointment Prepared Using an Water Soluble Base
For Formula 3A: After the formulation, the preparation was transferred into the amberedcoloured bottles and closed
properly to avoid oxidation. The formula 3A) was found to be unstable due to slight separation of oil. This was due to
the reaction of PEG, a water soluble base with the linseed oil. Hence, a further improvement of the formula is done
inorder to prepare an ointment using a water soluble base.
For Formula 3B: After the formulation, the preparation was transferred into the amberedcoloured bottles and closed
properly to avoid oxidation. The formula 3B) was found to be fluid in nature with a slight separation of oil and a
granular feel.
For Formula 3C: After the formulation, the preparation was transferred into the amberedcoloured bottles and closed
properly to avoid oxidation. The FORMULA 3 consisting of Polyethylene glycol is discarded. The hydroxyl groups of
polyethylene glycol react with dibasic inorganic salt and forms a complex and hence results in small precipitates like
granules. Though changing the concentrations of the ingredients used, it was found to be unstable. Hence this
formulation was discarded.
Conclusion
Transdermal applications made using linseed oil as the active principle by changing the formulations based on the
physical appearance and stability. It has been found that the formulations having the formula as FORMULA 1E and
FORMULA 2B were stable consisting of following compositions: 1E) For 100gm of ointment: Linseed Oil (54%),
Emulsifying wax (16%), White soft paraffin (10%), Dibasic inorganic salt (20%) and tocopherol 2drops. The
formulation was of liquid consistency but was stable even after a period of 6month. The pHof the preparation was
found to be 7.45.
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2B) For 100gm of ointment: Linseed Oil (50%), Paraffin wax (17%), White soft paraffin (13%), Dibasic inorganic salt
(20%) and tocopherol 2drops). The above two formulae were found to be stable. In these two formulations the
formula 2B was found to be of very good consistency and stable. Hence, the formula 2B was evaluated and tested for
stability. The formulation of formula 2B in which paraffin wax, a hydrocarbon base is used was found to be stable even
after a period of 6 months. The preparation is light creamish in colour. Has characteristic linseed oil smell. Has a pH of
7.9. The preparation had no irritant effect on the rabbits. There was no microbial contamination of the preparation.
Accelerated stability studies: The formulation was kept in hot air oven at a temperature of 30C initially and the
temperature was increased at 10 C every 10 mins until the temperature reached 70C. There was no change in the
formulation consistency and also in the physical appearance. Hence, it has been concluded that the formulation with
the above formula 2B was found to be a stable transdermal application.
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Corresponding Author: Sirisha.M*, Email:[email protected]