INTRODUCTION:

Cosmetics include a range of products that are used to enhance ones visible personality. They are generally mixtures of chemical compounds, some being derived from natural sources, many being synthetic. The history of cosmetics spans 6000 years of human history and almost every society on earth (1). Cosmetics production is one of the biggest businesses in the world with an estimated revenue of more than 60 billion US dollars by 2016 in the United States of America alone (2).

For a business that touches almost everyone’s life, it is important to maintain a standard of safety, which is regulated by committees in each country. The industry comprises of skincare, hair care, colour (make-up), fragrances and toiletries (3). Natural products have been used for folk medicine purposes throughout the world for thousands of years. Many of them have beneficial properties such as antimicrobial, anti-inflammatory and cytostatic effects (4). Cosmetics like vanishing cream are made of waxes. stearic acid, potassium hydroxide, glycerine, water, sodium hydroxide, cetyl alcohol, propylene glycol, perfume and preservative. Soap consists of stearic acid, coconut oil, caustic potash, caustic soda, water, sodium hydroxide (NaOH), potassium hydroxide (KOH), glycerol, etc. (5). After preparation of these products, it is important to pack them properly and perform a quality control check. For creams, it is important to check the viscosity, the rate and temperature of solidifying, vanishing properties, microbial count, pH, irritation to skin, etc. Perfume, Colouring and flavouring agents may be added with natural extracts of different leaves, roots, fruits and flower (6). Algae are known to have the potential to produce maximum oil yield (7) and it is thus important to formulate a cosmetic product from algal oil. Algal oil is rich in omega fatty acids which represent an important structural component of human cell membranes. The omega fatty acids also play a key role in protecting skin from UV radiation and have good anti-oxidant properties (8). Michelia champaca L. is one plant whose essential oil is used in cosmetics for a long time now for its fragrance (9). The present study aims to utilize the algae oil and oil of Michelia champaca L. to formulate a cosmetic product and study the compounds and their properties.

MATERIALS AND METHODS:

Extraction of Algae and Michelia champaca Oil and its Gas Chromatography Mass Spectroscopy (GCMS) analysis:

Lipid extraction was performed using Bligh & Dyer Method 1959 for algal oil – Desmodesmus ultrasquamatus (10). The extracted lipids were weighed gravimetrically. The lipids were profiled for their fatty acid content using Gas Chromatography Mass Spectroscopy (GCMS) Gas Chromatography Mass Spectroscopy: Esterification reagent preparation: 0.6 N Methanolic HCl. For every 3mg of lipids, add 5ml of esterification reagent in the round bottom flask with lipids. The round bottom flask was subjected to 60^oC temperature for 2 hours in water bath to fasten the process of esterification. The esterified component was transferred to a test tube, to which 2 ml hexane was added. The solution was mixed thoroughly and the upper hexane layer was collected in separate glass vials and hexane was evaporated at room temperature. 1ml Chloroform was added to the remaining fraction of esterified component and was injected in Gas Chromatography Mass Spectroscopy (GCMS) machine Shimazdu GC-MS-QP2010 Ultra. The parameters of GC for the detection were as follows: Total flow 24.5 ml/min, Column flow 3.59 ml/min, Linear velocity 70.4, Purge flow 3.0, Total run time: 50.0mins, Carrier gas: Helium. The column used was 30 meter FAMEWAX Column (USP G16) from Restek Corporation. Oil from Michelia champaca L. was prepared using ethanol as a solvent. 1 gram of flower was weighed and 10 ml of 100% ethanol was used to crush the flower in a mortar and pestle with cool conditions maintained to avoid evaporation and loss of volatile compounds. The extract was transferred to a centrifuge tube of 15 ml volume and cell debris was separated by centrifugation at 5000 rpm for 10 mins. The supernatant was collected and transferred to a round bottom flask. Ethanol was separated from the oil component using a rotary evaporator. The obtained fraction was pure essential oil component. This oil component was diluted again to a concentration of 8mg/ml using ethanol for use during antibacterial assay of vanishing cream and soap. This component was subjected to esterification process as described above for analysis in GCMS.

Estimation of Saponification Value of Algae oil and Preparation of Vanishing Cream and Soap from algae and Michelia champaca oil:

For this purpose, the algae oil was compared to processed coconut oil (Parachute) and rice bran oil (Fortune) as control. 3 sets of beakers were prepared by weighing 1g each of Coconut oil, Algal oil, Rice bran oil in a 3 different beakers and dissolve each sets of oil in about 5ml of ethanol. Add 25ml of 0.5N alcoholic KOH each sets and mix well. Set up as the blank with all other reagents present except the fat. Place all the flasks in a boiling water bath for 1 hour. Cool the flasks to room temperature. Add 2 drops of phenolphthalein indicator to both the flasks and titrate with 0.5N HCl. The disappearance of pink indicates the end point. Note down the endpoint of blank and tests. The difference between the blank and test reading gives the number of millilitres of 0.5N KOH required to saponify 1g of fat. The saponification value was calculated using the formula: (Blank – Test) x 0.5 x 56 / Weight of oil (11). The formulation of vanishing cream was made after a series of trial and errors. Weigh the quantities of stearic acid, algae oil and coconut oil in separate beaker. Take Borax, KOH, Glycerol, Distilled water in another beaker and keep in boiling water bath at 80°C until the constituents dissolve. After the water solution has reached a temperature between 80° and 90°C, remove it from the heat and slowly pour the melted stearic acid mixture into the water solution a little at a time, stirring constantly. If you pour too fast or if you do not stir, your emulsion will be lumpy. Continue stirring until you have a smooth, uniform paste. The formulation of soap from various mixtures was finalized based on the saponification values and trial and errors as follows: Prepare 3 sets of breaker and Weigh 1g of Coconut oil, Algal oil, Rice bran oil in a 3 different beaker and keep in boiling water bath till the temperature reaches 43°C. Use the ingredients and amounts shown in Table.2. Label each mixture. 35% of water is added in total amount of mixture. Each of the oils requires a different amount of base to react completely based on the saponification values estimated. Keep mixture in boiling water bath till the temperature reaches 43°C. Once both the base solution and your oils are 43°C, you can begin slowly pouring the aqueous base solution into the oils while stirring. A magnetic stir bar may facilitate this process. Continue stirring until the mixture begins to thicken and you can see trails of your mixture on the surface upon lifting your spoon/stirrer above the surface. This stage is called ‘trace’ and some oils take longer than others to reach this stage. Once you have reached ‘trace’, you can add any fragrances or additives, mix thoroughly, then pour into your mold. Prepare your mold, anything that will hold its shape will do. You will want to either grease your mold with petroleum jelly or line with freezer paper, parchment paper, or plastic wrap. DO NOT use aluminum foil because it will react with your base and release hydrogen gas. A test tube cap was used here as a mold. The mixture gets completely saponified in 24-48 hours, at which point it can be removed from the mold. Though safe to use at this point, curing for 4-6 weeks will react any trace amounts of base and evaporate excess water, resulting in a smoother and longer lasting bar.

Coliform count and Antibacterial Assay:

Coliform count of the soap prepared was done using Nutrient Agar plates. Antibacterial assay was carried out by Agar Cup plate method 2 months after the soap was prepared. The soap was stored in a test tube cap covered with aluminum foil. Escherichia coli were incubated at 37 ± 0.1^oC for 24 h by inoculation into nutrient agar. The hard agar was prepared by dissolving 2.5 gm nutrient agar in 100 mL sterile distilled water. Soft agar was prepared by dissolving 1.0 gm agar in 100 mL sterile distilled water. All the media and equipment were sterilized by autoclaving at 120^oC 15 lbs pressure for 15 minutes. Hard agar was poured into sterile petri plates and allowed to solidify. Soft agar was allowed to stand until it achieved a temperature of approximately 40^oC. The inoculums of the respective bacterial strain was mixed with the soft agar and it was poured over the hard agar in the petri plate. It was allowed to solidify. Sterile borer (0.8 cm diameter) was used for making wells in the agar. Each petri plate was divided into 4 quadrants and a well was made in each quadrant. The soap solution was prepared by taking approximately 100 mg of soap and dissolving it in 1 ml of distilled water. 100 µl solution was used to test antibacterial effect on E. coli. Negative control was sterile saline and positive control was 0.1 µg/ml of penicillin disc. The plates were incubated for 24 h at 37±1^oC. Diameter of the Zone of inhibition (ZI) was recorded in millimeters using a scale and the experiment was performed in triplicates.

RESULTS:

Extraction of Algae and Michelia champaca Oil and its Gas Chromatography Mass Spectroscopy (GCMS) analysis:

Algae consisted of 21% of its total biomass content as lipid fraction. GCMS revealed this lipid to contain 0.98% Oxalic acid 0.55% Hexadecane, 1.56% Phytol, 1.50% 17-Octadecynoic acid, 0.90% Eicosane, 1.97% 2(3H)-Furan one, 5-hexyldihydro-, 23.60% Hexadecanoic acid, 10.24% 2(3H)-Furan one, 5-heptyldihydro-, 3.39% 9,12-Hexadecadienoic acid, 0.80% 1-Nonadecene, 2.06% 7,10,13-Hexadecatrienoic acid, 5.59% 4,7,10,13 Hexadecatetraenoic acid, 1.27% Heptadecanoic acid 16-methyl, 10.25% 9-Octadecenoic acid, 10.83% 9,12-Octadecadienoic acid, 2.15% Gamma-Linolenic acid, 18.36% 9,12,15-Octadecatrienoic acid, 2.51% Stearic acid, 0.51% Tetracosane. The GCMS analysis of Michelia champaca extract revealed presence of stigmasterol, Campesterol, β-sitosterol and Vitamin E. These compounds thus form a major part of the oil component of flower.

Estimation of Saponification Value of Algae oil and Preparation of Vanishing Cream and Soap from algae and Michelia champaca oil:

The saponification value estimated for each oil is the amount of KOH mentioned in Table.2. A series of trial and errors based on the saponification values of each oil vanishing cream and soaps were prepared. Table.1 depicts the proportions of each component required to prepare vanishing cream from Stearic acid, coconut oil with borax, algal oil and coconut oil without borax. With respect to coconut oil, the need of borax was due to the change in quantity of water and KOH used. In the set where very little water and no KOH was used, borax was used which helped in formation of cream. Algal oil required 0.2 gram of borax to form a cream, but did not require any glycerol. Also the amount of KOH required to form a cream was only 0.05 gm. Stearic acid being a standard single fatty acid, did not require borax.

Table.1. Formulation for preparing vanishing cream from stearic acid, coconut oil mixture and algae oil.

Ingredients	Mixture 1	Mixture 2	Mixture 3	Mixture 4
Oil	2 gm Stearic acid	2 gm Coconut oil	1 gm Algal oil	2 gm Coconut oil
KOH	0.1 gm	0.1 gm	0.05 gm	-
Glycerol	0.6 gm	0.6 gm	-	-
Borax	-	-	-	0.1 gm
Distilled water	6 ml	6 ml	3 ml	0.5 ml
Paraffin wax	-	0.2gm	-	0.2gm
pH	5.5	6.5	7.5	7.2

Table.2 depicts the proportions of each constituent that was required to form a soap from coconut oil, algal oil and rice bran oil. To make this soap, 8 mg/ml of Michelia champaca oil was also added. The concept of adding this oil to the soaps was to give the soap an antibacterial property as described by researchers (12).

Table.2. Formulation of Soap from Coconut, algal and rice bran oil.

Ingredients	Mixture1	Mixture 2	Mixture 3
Oil	10 gm Coconut oil	10 gm Algal oil	10 gm Rice bran
KOH	1.96 g	2.1 g	1.4 g
Distilled water	4 ml	4 ml	4 ml
Michelia champaca oil (8 mg/ml)	3 ml	3 ml	3 ml
pH	9	11	9

All the soap mixtures were checked for lather formation and washing off with water. The researchers conducted these checks by utilizing minimum quantity of products and no side effects were observed.

Coliform count and Antibacterial Assay:

The soap solution was prepared by adding 100 mg of soap to 1ml of distilled water. No coliform growth or any other bacterial growth was observed on plating the solution on nutrient agar plate. This solution was tested for antibacterial properties. A fresh soap solution was effective as the ZI recorded was 2.9 cm for E. coli. Similar soap solution was prepared after storing the soap at room temperature for 2 months and this time the ZI obtained was 1.2 cm. This shows a decrease in the antibacterial activity of the active compounds.

DISCUSSION:

10 gm or algae oil was obtained from just 50 grams of algal biomass and it was sufficient to produce a small bar of soap that is about 50 cm². The investment and time required to grow algae is far less than that required to grow coconut or rice. This makes algal oil a good alternative towards cosmetic production. Algae are rich in omega fatty acids and also chlorophyll. Both these molecules are naturally occurring and good anti-oxidant and UV protection properties. Borax that is used to prepare cosmetics as it helps in formation of creamy consistency, however excess exposure to borax can cause dry skin and rashes. Inhalation of borax causes cough, sore throat, nose bleeding. It can cause abdominal pain, headache, vomiting as well. Pregnant women are cautioned to stay away from exposure to borax (13). Algae oil solidifies at room temperature and easily forms a creamy texture and for this same reason paraffin wax was also not necessary. The soap formed using crude algal lipid was greenish black in colour compared to the routinely used white or coloured soaps. This is due to the high carbon content of algal oils. With addition of oil of Michelia champaca to the soap, a natural anti-bacterial soap can be produced with an attractive aroma. Michelia champaca is a plant that grows in abundance in India and it is essential to make full use of nature.

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12) URL:http://pesticideinfo.org/Detail_Chemical.jsp?Rec_Id=PC34355

Received on 08.11.2015 Accepted on 15.02.2016

Research J. Topical and Cosmetic Sci. 7(1): Jan.-June 2016 page 15-18

DOI: 10.5958/2321-5844.2016.00003.0