INTRODUCTION:

Environmental changes have the key role to reflect onto our skin and hair, its texture, its look, is appearance and the most its condition. The factors like temperature, weather condition presence of particulate pollutant and many-allied pollutant have direct or the indirect impact onto the health and beauty of hair and even onto the skin. In today’s competitive scenario we are least bothered to take personal care of our beauty. It is truly said that our outlook is our lookout. To cope up with chemical, mechanical and the environmental stress some type of nourishment is necessary. Hair is very delicate and can be damaged by various stresses which may be due to chemicals stress like hair waver, hair color, environmental stress like UV light, dry atmosphere, heating with dryer, physical stress like over brushing, blow-drying soaking wet hair.

Revitalizers^1-3are the agents which bring environmentally and chemically stressed hair to its original natural condition, enhance the follicular absorption, stimulate the hair growth where the hair loss is due to environmental stress, mechanical stress or chemically stress, nourishes the follicle so that it exhibits the properties this is generally required by the individual and gives the hair requisites lubricity and manageability.^3-5 Thus, taking this into consideration attempt has been made to make use of various revitalizing agent like Allantoin, milk casein, sodium PCA and arginine⁶ as these agent are more effective on to the hair as compare to other active ingredient specially used previously. But before adding these active directly into the cosmetic formulations like cream, lotion and gel it must be core with liposome technology, because it transport 100% revitalizer to the hair follicle and increased the stability into the product over a long period of time. Also since no much work has been done on incorporation of hair revitalizing agent-loaded liposome into cosmetic product the effects are made to evaluate its efficacy. Liposome^7-10is microscopic small vesicles (hollow spheres) consisting of one or several lipids bilayers that surround a watery nucleus. In the cosmetic field cream, lotion and gel have already absorption of active ingredient to the skin, but liposome technology penetrate the active ingredient to deeper layer of the skin hence enhanced follicular absorption thereby increased efficiency of the product formulation.

MATERIALS AND METHOD:

Milk casein was obtained from Clarion Casein Ltd., Aurangabad, allantoin and sodium PCA was obtained from Khusbu Beauty Care, Silvassa All other materials used were of commercial grade. First empty liposome vesicles were prepared using cholesterol and lecithin by film hydration technique in various combination. The ratio of cholesterol and lecithin was finally kept to 1:1. In this method, Soya-lecithin and cholesterol components in chloroform-methanol solvent mixture (2:1 v/v) was prepared first and then introduced into 250 ml round bottom flask. This flask was attached in rotary evaporator and rotated at 60 rpm for 15 min. The organic solvents were evaporated at about 30 ⁰C or above the transition temperature of the lipids used. The film so formed was hydrated using 25N calcium chloride and again it was rotated for another 30 min. or until all lipids has been removed from the wall of the flask and has given homogeneous milky white suspension free of visible particles. The suspension was allow to stand for a further 2 hours at room temperature or at a temperature above the transition temperature of the lipid in order to complete swelling process to give MLVs of entirely neutral lipid tend to be very tightly packed, multilayer assemblies with very little aqueous space between them the presence of negatively charged lipid in the membrane tends to push the bi-layers apart from each other and increased the volume of entrapment significantly. The placebo liposomes so prepared were optimized for its size. Then liposomes were loaded with the revitalizing agents under study using the same method as described previously. Four batches of active loaded liposome were prepared using 1:1 ratio of lecithin : cholesterol and varying concentration of active i.e. 0.25 g, 0.5 g, 0.75 g, 1.0 g were prepared. Same procedure was used for producing all the other actives.

Table 1 Prepared liposomes containing different conc. of revitalizing agent

Formula	F-I	F-II	F-III	F-IV
Lecithin	0.5 g	0.5 g	0.5 g	0.5 g
Cholesterol	0.5 g	0.5 g	0.5 g	0.5 g
Revitalizing agent	0.25 g	0.50 g	0.75 g	1.0 g

In this study various batches were taken for different concentration of revitalizing agent, but formula LL-II were observed 100% loaded active as compared to other three batches. In first batch of revitalizing agent in liposome concentration was not loaded to fulfill, as it was possible to load more active in liposomes. As with increasing concentration of active compared to LL-II and formula LL-III and LL-IV were extra loaded by due to extra loaded the boundary of liposomes was broken with all that consideration batch LL-II was selected for incorporation of all the active loaded liposomes in cream as the product is designed for after bath application or is leave-on type so it is the prerequisite that the product should be free from greasy look onto the hair, therefore O/W type of emulsion cream was prepared having following composition. All the ingredients were weighed accurately. Oil phase ingredient and aqueous phase ingredients were heated up to 75⁰C then add oil phase to water phase mixed well. Color was added to the formulation and mixed uniformly. The Perfume was added when the temperature of the system falls to 45⁰C and homogenized.

It shows the composition of various O/W crème’ prepared. formulation No. C-III was found to be stable so further it was taken for incorporation of liposome in this formulation only. The composition is shown in the table 2

Table 2 Composition of placebo cream formulation

Sr. No.	Ingredients	Quantity taken for l00g -III
1	Palmitic acid	10.00%	-	5.00%
2	Myristic acid	-	10.00%	5.00%
3	Cetyl alcohol	3.00%	3.00%	3.00%
4	Polyethylene Glycol 400	2.00%	2.00%	2.00%
5	Glycerin	2.00%	2.00%	2.00%
6	Methyl Paraben	0.1%	0.1%	0.1%
7	Propyl Paraben	0.05%	0.05%	0.05%
8	Orange peel oil (perfume’)	0.01%	0.01%	0.01%
9	Regular orange (color)	0.01%	0.01%	0.01%
10	Water	82.83%	82.83%	82.83%

By considering the stability factor for placebo crème’, the formulation C-III was selected for incorporation of active loaded liposome's in different concentration of 1.25%, 2.5%, 3.75% and 5% of each of the loaded liposomes. The composition is shown in the table. All the ingredients were weighed accurately. Oil phase ingredient and aqueous phase ingredients were heated up to 75⁰C then add oil phase to water phase mixed well. Color was added to the formulation and mixed uniformly. The Perfume was added when the temperature ofthe system falls to 45⁰C and homogenized. At about 40 ⁰C liposome loaded with active were added i.e., at low rate of shear. The prepared formulation was then evaluated further

In-vitro Evaluation –

The formulations so prepared were evaluated for organoleptic and asthetic properties, pH, total fatty matter¹¹ pH of the cream containing was determined and it was found to be 6.01 to 6.5

In vivo evaluation:

It was done for the prepared formulation12 In vivo evaluation comprises of testing on seven different individuals comprising the age group of 20-30 years having normal texture of hair but with environmentally, mechanically or chemically stressed hairs for lusture, manageability, ease of combing, measurement of hair and tensile strength The visual evaluation of luster was done considering the perception of individual client. The managibility of hair was measured on the basis of questionnaire filled up by the individual client. The dry combing analysis was done in the saloon by individual testing of the product on application on dry hair and the combing was thus analyzed and the findings were reported via questionnaire.

Table 3 : Formulation of Cream loaded with liposome

Sr. No.	Ingredients	Quantity taken for l00g
1	Palmitic acid	5.00%	5.00%	5.00%	5.00%
2	Myristic acid	5.00%	5.00%	5.00%	5.00%
3	Cetyl alcohol	3.00%	3.00%	3.00%	3.00%
4	Polyethylene Glycol 400	2.00%	2.00%	2.00%	2.00%
5	Glycerin	2.00%	2.00%	2.00%	2.00%
6	Methyl Paraben	0.1%	0.1%	0.1%	0.1%
7	Propyl Paraben	0.05%	0.05%	0.05%	0.05%
8	Orange peel oil (perfume’)	0.01%	0.01%	0.01%	0.01%
9	Regular orange (color)	0.01%	0.01%	0.01%	0.01%
10	Allantoin loaded liposomes	1.25 %	2.50%	3.75%	5.00%
11	Sodium PCA loaded liposomes	1.25 %	2.50%	3.75%	5.00%
12	Arginine loaded liposomes	1.25 %	2.50%	3.75%	5.00%
13	Milk casein loaded liposomes	1.25 %	2.50%	3.75%	5.00%
14	Water	77.83%	72.83%	67.83%	62.83%

Table 4 Asthetic properties of prepared liposome loaded creams

Sr. No	Parameter	Quantity of Active loaded liposome
1)	Appearance	Creamy	Creamy	Creamy	Creamy
2)	Color	Orange	Orange	Orange	Orange
3)	Consistency	Semi-Solid	Semi-Solid	Semi-Solid	Semi-Solid
4)	Spreadability	Good	Good	Good	Good
5)	Oily Feel	No	No	No	No

Table 5: pH of liposome loaded cream formulations

Sr. No	Parameter	Quantity of Active loaded liposome
1)	pH of crème’ loaded with liposome	6.01	6.33	6.48	6.50

Table 6:Total Fatty Matter of liposome loaded cream formulations

Sr. No	Parameter	Quantity of Active loaded liposome
1)	TFM of crème’ containing loaded liposome	12.99%	12.82%	12.67%	12.13%

Total fatty material was determined as per IS and it was found to be 12.13 to 12.99, similarly water content was also determined and was found to be 81.02 to 81.83

Tensile strength was determined by fabricating a simple assemply using laboratory utensil like glass pan, stand and weight box. the steps involved are given below:-

(1) One end of hair was tied to the rigid stand and the other end of the hair was tied to glass pan (of diameter 8 inches).

(2) Initially placed 50 gm weight then go on adding 10 gm, 20 gm followed by increment of 1 gm at a time.

(3) Noted the point at which the hair breaks. This point denotes the tensile strength of hair.

RESULTS and DISCUSSION:

The dry film hydration technique was used to prepare liposome of natural biodegradable phospholipid such as soya lecithin. The speed of stirrer and other conditions were maintained. The speed of stirring was found to be 60 rpm for optimum yield. The general procedure for the preparation of placebo liposome has been essentially the same as in all the batches with variation of ingredient in the formulation conditions. Five different formulations of placebo liposomes F1, F2, F3, F4, F5 were prepared with varying concentration of cholesterol i.e. 0.1 g, 0.2 g, 0.3 g, 0.4 g and 0.5 g and fixed concentration of lecithin (0.5 g) in 6 ml of chloroform : methanol mixture (2:1). The vesicles so obtained when seen under microscope shows the particle size ranging from 4.9-6.8 microns. This was due to the flexibility of the film formed by higher amount of lecithin On the other hand when the quantity of cholesterol (0.5 g) was kept constant with varying concentration of lecithin (0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g) in 6 ml of solvent mixture. The vesicular particle size ranges between 4.9 to 6.4. It was found that as the conc. of lecithin go on increasing the film rigidity also increases with reduction in particle size. This may the typical property of film rigidity of cholesterol. Thus, in both the cases because of film rigidity and the particles size obtained formulation f5 was selected for the incorporation of revitalizing agent into the vesicle and other formulation F1, F2, F3, and F4 were rejected as vesicles of desired particle size was not obtained.

By using 1:1 ratio of lecithin: cholesterol, active loaded liposomes were prepared with different concentration i.e., 0.25 g, 0.5 g, 0.75 g and 1.0 g of active in 0.5 g :0.5 g (lecithin: cholesterol) mixture. It was found that formulation f1 was not loaded properly so it was quite possible to add more amount of active to it. The loosening of vesicles in F3 followed by rupture in F4 of the vesicle indicates over loading. So F2 was finalized for the incorporation into the cream and other formulations F1, F3, F4 were rejected.

The simple emulsification technique was used to prepare crème’. The general procedure for preparation of placebo crème’ has been essentially the same as in all batches with variation of ingredients.

Three different batches of placebo crème’ F1, F2, F3 were prepared. In batch F1 the cream was stiffer because of palmitic acid, while formulation f2 was thinner because of myristic acid so F1 and F2 were rejected and formulation F3 was of appreciable aesthetic feel so it was selected for further incorporation of loaded liposomes. This formulation was considered as cream base in which the active loaded liposome was added in concentration of 1.25%, 2.50%, 3.75% and 5% respectively of each of liposome so as to make the liposome concentration in 5%, 10%, 15%, 20% respectively. Further the formulation of crème’ loaded with liposome were evaluated for pH, TFM, Water content and viscosity.

pH of all formulations measured and found to be slightly increased from the initial value and then become constant but was within the range 6.0 to 6.5.

The particle size of liposomes has more important in the cream, if the particle size is narrow, then the liposome disperse all over the cream, if the particle size is more and is not dispersed over the crème’, so the care should be taken that the liposome's are not broken by the blade of the stirrer.

In the analysis to Total Fatty Materials, according to IS specification the total fatty material of cream should be 12 to 18%, in present study have found to be between 12% to 13%.

In the analysis of water content, according to IS specification the water content should be 80 %. In present study have determined between 81% to 82%.

Viscosity of the cream should be 26000 to 36000cp and it is found to be 26952to 27204 cp at room temperature.

On the basis of stability for 48 hours at room temperature it was found that formulation f1 and f2 were thinner, while f4 was thicker with more stiff mass. So all the three batches of cream i.e. F1, F2 and F4 containing loaded liposome were rejected and only F3 was selected for In-vivo testing. In-vivo testing of cream shows that the product is non-irritant and has also improved the manageability, lustre, tensile strength, combing and hair length.

When evaluated for lusture, it was found that cream containing loaded liposomes shows better lusture

When hair length measurement was done the average length of hair by treatment was 695.75. When cream was applied it increased from 695.75 to 696.71.

When the tensile strength of hair shaft was measured in subjective study, for unapplied hair, it was found to be 0.7825 N/m². Which was increased to 0.7971 N/m² on application of cream.

On the basis of results obtained from subjective studies and on the basis of questionnaire it was concluded that,

The conditioning effect of hair has been increased which was marked by the development of uniform outer root sheath was developed onto the application on hair shaft after 15 days when seen under microscope.

The manageability of hair was found better after 15 days as compared to 1^st days and also before the application of the test product.

The wet and dry combing of hair was found better after 15 days as compared to 1^st days and also before the application of the test product.

The luster of the hair was found better as compare to the unapplied hair before the test started.

The tensile strength was increased, thus preventing the hair damage during combing.

Most important effect i.e. it revitalizing which bring back life to the dull and fatigue looking hair was seen in terms of increment in the hair length.

REFERENCES:

1) Maison G deNavvare The chemistry and Manufacture of Cosmetics, II Edition, Volume IV, pp 1098.

2) Idson B Update On Hair Conditioner Ingredients Cosmet. Toletries. , 1983

3) Randy Schueller, Perry Roamanowski Conditioning agents for hair and skin volume 21 Published by Marcel Dekker Inc. pp 281-300, 1999.

4) Takeo Mitsui, New Cosmetic Science Elsevier, 1992, pg 406-436

5) Wettering, P.J.J.M et al.: Acta Derm. Venereol. (Stockholm), 63, 315(1983)

6) Through internet website www.mamma.com

7) Through internet website www.liposomesite.com

8) Targeted and Controlled Drug Delivery : S. P. Vyas and R. K. Khar, pp. 181-193.

9) Encyclopedia, of Pharmaceutical Technology : Vol. 9, Edition-James Swarbrick and James C. Baylon

10) Novel Cosmetics delivery system by Shlomo Mugdussi, Elka Touitou Liposomes in cosmetics. Pg 233 -274 published by Marcel Dekker

11) Indian Standard : Guideline for hygienic manufacturer of cosmetics specification for skin cream, lotion and gel.

12) Louise B. Aust in cosmetic Claims substaintiation Vol. 18 published by Marcel Dekker, Inc. 1998 pp 25-26.

Received on 07.01.2010 Accepted on 20.02.2010

Res. J. Topical and Cosmetic Sci. 2(1): Jan. –June 2011 page 14-17