jurnal semisolid

Sirisha.M* et al. /International Journal Of Pharmacy&Technology

IJPT | July-2012 | Vol. 4 | Issue No.2 | 4577-4590 Page 4577

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



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.



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


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


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.



1E) For 100gm of ointment: Linseed Oil (54%), Emulsifying wax (16%), White soft paraffin (10%), Dibasic inorganic


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.



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.



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



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)



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.



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



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



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.



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.



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]

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