FORMULATION OF CHEMICAL AND CRUELTY FREE VEGAN LIPSTICK AND ANALYZING ITS COMPONENTS

 

Rajgopal Balaguru ¹, Shirsha Bhowmick ², Dr. Priya Iyer ³

 

^1,2,3 Women’s Christian College, PG and Research Department of Biotechnology, Chennai-600006, India

 

1. INTRODUCTION

  Cosmetics have become a part of everyone's grooming routine, where lipstick promotes the mood of makeup.

 

1.1.    HISTORY OF LIPSTICK

Around 5000 years ago, the ancient Sumerian and Indus Valley men and women were possibly the first to invent and wear lipstick. Sumerians used crushed gemstones to decorate their faces, mainly on the lips and around the eyes. Egyptians like Queen Cleopatra crushed Red Carmine (bugs)to create a Red Shade.

 

 

1.2. INGREDIENTS USED IN MAXIMUM COMMERCIAL LIPSTICK

Unfortunately, lipstick contains some harmful ingredients like Carmine (Crushed cochineal beetle carcasses), Suet (hard white fat from the kidney and loins of cattle, sheep, etc.), Tallow (boiled fat from beef or sheep carcasses), Lead (optimized) amount, heavy metals such as nickel, copper, chromium, arsenic, and cobalt and some of these toxic substances get absorbed by the lips to stomach during the application of this lipstick. Therefore, people turn towards a solution for well-being and health, and vegan cosmetics are the answer! People tend to entrust plant-based products more than chemical ones because of the severe side effects related to the chemicals.

    

1.3. SOURCE OF PIGMENTATION

The first lipstick sample was prepared from the Beetroot (Beta vulgaris) in the Chenopodiaceae family, recently included in the Amaranthaceae family. The Red beetroot (Beta vulgaris) is the best source of betacyanin. The second lipstick sample is based on coffee decoction.

A coffee decoction is an extraction procedure, especially for water-soluble and thermostable constituents.

The third lipstick sample was prepared with yellow pigments of turmeric.

Turmeric has been used in Asia for centuries as a folk medicine, culinary spice, and dye. It has also been a part of Ayurveda, Siddha medicine, Unani, and Traditional Chinese Medicine

                 

2. MATERIALS AND METHODS

2.1. SAMPLE COLLECTION

Beet root extract, Coffee Decoction Turmeric extract, Olive Oil (Moisturizer)

Cocoa Butter, Shea Butter Candelila wax, Lavender essence for fragrance.

 

2.2. FORMULATION OF LIPSTICK

2.2.1.  SAMPLE A. BEETROOT   EXTRACT

Beetroot was washed thoroughly, cut into tiny pieces, and churned in the mixer grinder.

Now, with the help of a sieve, extract the fresh filtered juice. Then, diminish it by heating it in a pan to get a comparatively thicker solution.

Now, 5 ml of pigmented solution is collected. 2.5 ml of lime juice is added as a mordant. 5 ml of olive oil is added as a moisturizer. 2.5gm of Shea butter and 2.5gm of cocoa butter is added for richness.1.5 gm of Candelila wax (vegan) is used for binding the pigments and moisturizing agent.

After that, adding all ingredients to a beaker, it was melted in a microwave oven at 70°C for 1 minute, and immediately stirred with Lavender essence extensively with a glass rod, so that all the ingredients mixed homogeneously and let cool.

       

2.2.2.  SAMPLE B. COFFEE DECOCTION

Readymade liquid coffee decoction (Cothas Coffee) is used for the formulation of the second lipstick Then, diminish it by heating it in a pan to get a thicker solution.

Now, 5 ml of pigmented solution is collected. 5 ml of olive oil is added as a moisturizer.2.5gm of Shea butter and 2.5gm of cocoa butter are added for richness.1.5 gm of Candelila wax (vegan) is used for binding the pigments and moisturizing agent.

After that, adding all ingredients to a beaker, it was melted in a microwave oven at 70°C for 1 minute and immediately stirred extensively with a glass rod, so that all the ingredients were mixed homogeneously. Let it cool in a mold settle for a day at room temperature.

 

2.2.3.  SAMPLE C. TURMERIC EXTRACT

Turmeric is washed thoroughly, cut into tiny pieces, and churned in the mixer grinder. Now, with the help of a sieve, extract the fresh filtered juice.

Then, diminish it by heating it in a pan to get a comparatively thicker solution.

Now, 5 ml of pigmented solution is collected.5 ml of olive oil is added as a moisturizer. 2.5gm of Shea butter and 2.5 gm of cocoa butter are added for richness.

1.5 gm of Candelila wax (vegan) is used for binding the pigments and moisturizing agent.

After that, adding all ingredients to a beaker was melted in a microwave oven at 70°C for 1 minute, and immediately stirred with Lavender essence extensively with a glass rod, so that all the ingredients mixed homogeneously. Let it cool in a mold and settle for a day at room temperature.

 

3. QUALITATIVE AND QUANTITATIVE ANALYSIS

3.1. PRELIMINARY PHYTOCHEMICAL SCREENING

·        Test for Alkaloids

2ml of the sample was hydrolyzed with 5ml of 1% v/v Hydrochloric acid. This is then used for alkaloid detection. Hager’s Test: Few drops of the saturated aqueous solution of picric acid were added to the sample mixture. The development of a yellow precipitate confirms the presence of Alkaloids.

·        Test for Carbohydrates

The 2ml of the sample was dissolved in 5ml of distilled water.

Benedict’s Test: Few drops of Benedict’s reagent were added and kept in a boiling water bath for 10 to 15mins until colour develops. The development of the red colour indicates the presence of Carbohydrates.

·        Test for Proteins

Xanthoproteic Test: Few drops of    concentrated Nitric acid were added to 2ml of the sample. The development of yellow colour confirms the presence of Proteins.

·        Test for Amino Acid

Ninhydrin Test: 2ml of the sample was treated with 0.25% w/v Ninhydrin reagent and boiled for a few minutes. The development of the blue colour confirms the presence of Amino acids.

·        Test for Glycosides

2ml of the sample was hydrolysed with dilute Hydrochloric acid. This is then used for glycoside detection. Keller-Kilani Test: 2ml of Glacial acetic acid was added to the hydrolysed sample and 2ml of concentrated sulphuric acid was added on the sides of the test tube. The development of a brown colour ring at the interphase confirms the presence of Glycosides.

·        Test for Phytosterols

Salkowski’s Test: 10ml of chloroform was added to the sample and filtered. A few drops of concentrated sulphuric acid were added to the filtrate and were shaken well and left to stand. The development of golden yellow colour confirms the presence of Phytosterols.

·        Test for Saponins

Foam Test: Few drops of sodium bicarbonate were added to 0.5ml of the sample diluted in 2ml distilled water.

·        Test for Phenols

Ferric chloride test: Few drops of 1% Ferric chloride were added to the sample. The development of bluish/black colour confirms the presence of Phenols.

·        Test for Tannins

Ferric chloride test: 1ml of 5% ferric chloride solution is mixed with 0.5ml of the extract. The development of brownish colour indicates the presence of Tannins.

·         Test for Flavonoids

Alkaline Reagent Test: The sample was treated with a few drops of Sodium hydroxide. Development of intense yellow colour and becomes colorless on adding dilute hydrochloric acid confirms the presence of Flavonoids.

·        Test for Diterpenes

2ml of the sample was diluted in 5ml distilled water and 3-4 drops of copper acetate solution were added. The development of an emerald green colour confirms the presence of diterpenes.

 

1)    Estimation of protein by Lowry’s Method

    Lowry’s method was performed to verify the presence of protein in the plant extracts which are used to formulate the lipstick and the amount present in the lipstick. 

2)    Estimation of Carbohydrates by Orthotoludine Method

Orthotoludine method was performed to verify the presence of carbohydrates in the plant extracts which are used to formulate the lipstick and the amount present in the lipstick.

3)    Estimation Of Amino Acid By Ninhydrin Method

Ninhydrin method was performed to verify the presence of amino acids which are used to formulate the lipstick and the amount present in the lipstick.

4)    Estimation of Flavonoids

 Three test tubes were taken and labeled as U1 and U2 and Blank. The unknown (flaxseed aqueous extract) 1 and 2 ml were added to U1 and U2.A test tube containing 2 ml of distilled water alone served as a blank. To all the test tubes 1 ml of Aluminium chloride and 1 ml, Potassium acetate were added. 2.8 ml of distilled water was added to all the test tubes and incubated at room temperature for 45 minutes. The optical density was read at 420nm.The standard graph was calibrated and the number of carbohydrates in the given unknown solution was calculated.

5)    Estimation of Steroid

The unknown 0.5 and 1 ml were added to U1 and U2.3.5The unknown (flaxseed aqueous extract) 0.5 and 1 ml were added to U1 and U2.3.5 and 3 ml of Ferric chloride Acetic The test tubes were incubated at room temperature for 15 minutes. The optical density was read at 570nm.The standard graph was calibrated and the number of carbohydrates in the given unknown solution was calculated.

6)    Estimation of Glycosides

 The unknown (flaxseed aqueous extract) 0.5 and 1 ml was added to U1 and U2.7.5 and 7 ml of Ferric chloride Acetic acid was added to all the test tubes. 1 ml of picric acid and 1ml of 100% Sodium hydroxide were added to all the test tubes. The tubes were incubated at room temperature for 15 minutes. The optical density was read at 520nm.The standard graph was calibrated and the number of carbohydrates in the given unknown solution was calculated.

7)    Estimation of Alkaloids

5ml of aqueous extract of flaxseed was taken in a centrifuge tube and 2 ml of Dragendorff reagent was added. This mixture was centrifuged at 10000rpm for 30 minutes. The supernatant was removed, and the pellet was treated with ethanol. This mixture was centrifuged at 10000rpm for 10 minutes. The filtrate was discarded, and the pellet was treated with 2ml of Disodium sulfide solution and a few drops of concentrated nitric acid. This solution was diluted to 10 ml in a standard flask with distilled water.1 ml was taken from this standard solution and 5 ml of Thiourea solution was added. The optical density was read at 435nm.The standard graph was calibrated and the number of carbohydrates in the given unknown solution was calculated.

8)    Estimation of Saponins 

The powdered flaxseed was dissolved in 80% ethanol and 2 ml of this filtrate was taken in a test tube. 2 ml of Vanillin was added and mixed well. A few drops of concentrated Sulphuric acid were added. The test tube was kept in a boiling water bath at 60°C for 10 minutes. The optical density was read at 544 nm. The standard graph was calibrated and the amount of carbohydrates in the given unknown solution was calculated.

9)    Estimation of Terpenoids     

The powdered flaxseed was dissolved in ethanol and 2. 5 ml of this filtrate was taken in a test tube. 2.5 ml of Ammonium molybdate and 2.5 ml of concentrated Sulphuric acid were added. The test tube was incubated at room temperature for 30 minutes. 5 ml of Ethanol was added, and the optical density was read at 700nm. The standard graph was calibrated and the amount of carbohydrates in the given unknown solution was calculated. 

 

4. ANTIOXIDANT ASSAY

1.5 mo of 0.1mM DPPH solution and add 1.5 ml of extract. The mixture was shaken vigorously. Incubate at room temperature for 30 minutes in the dark. The reduction of DDPH free radical as measured by reading the absorbance at 517nm by a spectrophotometer. (-) Control: Without any extract, DPPH and Ethanol (+) Control: Ascorbic Acid The inhibition of the DPPH free radical in ℅ was calculated by:

The absorbance of Control -Absorbance of INHIBITION/ Absorbance of Control *10

 

 

 

 

5. ANTIMICROBIAL ACTIVITY                      

5.1. MATERIALS

The Petri plates were prepared with 20 ml of sterile Muller Hinton media. The strains (E. Coli, Klebsiella Pneumoniae, and Staphylococcus Aureus (Candida Albicans) that had been incubated for 24 hours were used for the assay. A sterile cotton swab was dipped into the bacterial suspension and then evenly streaked over the entire surface of a sterile Muller Hinton Agar plate to obtain uniform inoculum. Samples were added in different concentrations. The plates were incubated overnight at 37 C. The antimicrobial activity was determined by measuring the diameter of the zone of inhibition(mm).

        

6. ANTICANCER ACTIVITY

6.1. MATERIALS AND METHODS

6.1.1.  PRELIMINARY SCREENING

The preliminary screening for in vitro cytotoxicity was carried out in the Vero cell line with the synthesized nanoparticles, plant extracts, and lyophilized nanoparticles. Based on the initial assessment of non-toxicity, the assay was further checked with varying concentrations of the nanoparticles. The absorbance was read at 590nm with a reference filter of 620nm. The percentage of cell viability was calculated.

The formula for cell inhibition = (O.D. of treated cells/O.D. of control) 100.

Formula to check cell viability   = (100 – cell inhibition)  

 

6.1.2.  IN VITRO CYTOTOXICITY

The in vitro cytotoxicity of the 15 nanoparticles was tested in an epithelial cell lineage such as the Vero cell line through MTT assay. Further, the nanoconjugates NCC - (formulated based on the anticancer activity of NPS) nano-drug conjugates (NDC) and Chitosan Nano-drug were screened for in vitro cytotoxicity as per a similar protocol.

 The absorbance was read at 590 nm with a reference filter of 620 nm.

 The percentage of cell viability and IC so

were calculated.

 The formula for cell inhibition = (O.D. of treated cells/O.D. of control) x 100

 Formula to check cell viability (100-cell inhibition) (Loosdrecht et al., 1994)

 

% of inhibition= (Absorbance of control-Absorbance of treated cells)/absorbance of control *100

 

7.  RESULT AND DISCUSSIONS

7.1. QUALITATIVE ANALYSIS

1)    Test for Alkaloids

Alkaloids is present in the Lipstick Sample A from Beetroot extract as the yellow precipitate is observed.               

   

Alkaloid is present in the Lipstick Sample B from Coffee Decoction as the yellow precipitate is observed.

 

Alkaloid is present in the Lipstick Sample C from Turmeric extract.

as the yellow precipitate is observed.

  

 

2)    Test for Carbohydrates

  Carbohydrates are present in sample A from the Beetroot extract.

 

 

 Carbohydrates are present in the Lipstick Sample B from Coffee Decoction

  

 

  Carbohydrates are present in the    Lipstick. Sample C from Turmeric extract.

 

 

3)    Test for Proteins

    Proteins are present in the Lipstick Sample A from Beetroot extract as the yellow colour is observed.

 

 Proteins is present in the Lipstick Sample B from Coffee Decoction as the yellow colour is observed.

 

 Proteins are present in the Lipstick Sample B from Turmeric Extract as the yellow colour is observed.

 

4)    Test for Amino Acid

Amino acid is present in the Lipstick Sample A from Beetroot extract as the blue colour is observed.

 

 

Amino acid is present in the Lipstick Sample B from Coffee Decoction extract as the blue colour is observed.

 

 

Amino acid is present in the Lipstick Sample C from Turmeric extract as the blue colour is observed.

 

5)    Test for Glycosides

Glycosides are present in the Lipstick Sample A from Beetroot extract as there is the development of a brown colour ring at the interphase.

 

 

Glycosides is present in the Lipstick Sample B from Coffee Decoction as there is the development of a brown colour ring at the interphase.

  

 

Glycosides are present in the Lipstick Sample C from Turmeric Extract as there is the development of a brown colour ring at the interphase.

 

 

6)    Test for Phytosterols

Phytosterols are present in the Lipstick Sample A from Beetroot Extracts the golden yellow colour is observed.

Phytosterols are present in the Lipstick Sample B from Coffee Decoction as the golden yellow colour is observed.

 

 

Phytosterols are present in the Lipstick Sample C from Turmeric Extract as the golden yellow colour is observed.

  

 

7)    Test for Saponin

Saponin is absent in Lipstick   Sample A from Beetroot Extract

Saponin are absent in Lipstick Sample B from Coffee Decoction

Saponin are absent in Lipstick Sample C from Turmeric Extract

 

8)    Test for Tannin

Tannins is present in the Lipstick Sample A from Beetroot Extract as the development of brownish colour is observed.

 

 

 Tannins is present in the Lipstick Sample B from Coffee Decoction the development of brownish colour is observed.

 

 

Tannin is present in the Lipstick Sample C from Turmeric Extract  as the development of brownish colour is observed

 

 

9)    Test for Flavonoids

 Flavonoids are present in the Lipstick Sample A from Beetroot Extract

 

Flavonoids are present in the Lipstick Sample B from Coffee Decoction

 

 

Flavonoids are present in the Lipstick Sample C from Turmeric Extract

 

 

10) Test for Diterpenes

Diterpenes are present in the Lipstick Sample A from Beetroot Extract as emerald, green colour is observed.

 

 

 Diterpenes are present in the Lipstick Sample B from Coffee Decoction as emerald, green colour is observed.

 

 

Diterpenes are present in the Lipstick Sample C from Turmeric Extract as emerald, green colour is observed.

 

 

Figure 1

                                                                      

Figure 1 Research Gate by Samiullah, Farida Behlil, Farrukh Bashir (March 2015)

 

Table 1

Table 1 Qualitative Analysis
TEST	Beetroot	Coffee Decoction	Turmeric
ALKALOID	+	+	+
PHENOLS	-	-	-
TERPENOID	+	+	+
SAPONIN	-	-	-
FLAVONOID	+	+	+
TANNINS	+	+	+
GLYCOSIDES	+	+	+
PHYTOSTEROLS	+	+	+
CARBOHYDRATE	+	+	+
PROTEIN	+	+	+
AMINO ACID	+	+	+
DITERPENES	+	+	+
Research gate by Chika C. Nwankwo, Daodu Bamidele T (June 2021)

 

Table 2

Table 2 Quantitative Analysis
TEST	Beetroot	Coffee Decoction	Turmeric
ALKALOID	3.8	3.6	4
TERPENOID	3.5	3.9	3.8
FLAVONOID	2.4	2.2	2.4
TANNIN	2.8	2.8	2.7
GLYCOSIDE	4.8	4.2	4
CARBOHYDRATE	5.4	5.6	5
AMINO ACID	1.2	1.8	2.3

 

8. ANTICANCER TEST

CONTROL

BEETROOT EXTRACT=0.110

COFFEE DECOCTION=0.099

TURMERIC EXTRACT=0.112

 

SAMPLE

BEETROOT LIPSTICK=0.105

COFFEE DECOCTION LIPSTICK=0.111

TURMERIC LIPSTICK=0.104

 

BEETROOT LIPSTICK	95.45%
COFFEE DECOCTION LIPSTICK	112.12%
TURMERIC LIPSTICK	92.85%

 

10) Anti-Cancer Activity of Beetroot Extract=95.45%

11) Anti-Cancer Activity of Coffee Decoction=112.12%

12) Anti-Cancer Activity of Turmeric Extract=92.85%

 

     

 

     

 

  

 

9. ANTIOXIDANT ASSAY

ASCORBIC ACID	CONTROL	SAMPLE	INHIBITION%
0	O.414	0.315	23.91304348
20	O.414	0.376	9.178743961
40	O.414	0.308	25.60386473

 

Figure 2

                                                                     

Figure 2 Research Gate by Chika C. Nwankwo, Daodu Bamidele T (June 2021)

 

10. ANTIMICROBIAL ACTIVITY

 (DISC METHOD)

·        E. Coli - NO antimicrobial activity is seen

·        Klebsiella - NO antimicrobial activity is seen

·        Staphylococcus- NO antimicrobial activity is seen

·        Candida - NO antimicrobial activity is seen

 

11. STORAGE TEST

· STORAGE TEST FOR LIPSTICK SAMPLE A. (BEETROOT EXTRACT)

· The colour diminishes after 20 days. Risks of contamination prevail if not kept under refrigeration.

· STORAGE TEST FOR LIPSTICK SAMPLE B (COFFEE DECOCTION)

Being a Nude Shade, the colour stays intact for a minimum of 35-40 days at room temperature.

· STORAGE TEST FOR LIPSTICK SAMPLE C (TURMERIC EXTRACT)

· The colour stays intact for 35-40 days at room temperature. (Formulation and Evaluation of Herbal Lipsticks from Research Journal of Pharmacy and Technology by Nuha Rasheed,)

 

Syed Abdul Rahman, and Samreen Hafsa was also referred (Volume - 13, Issue - 4, Year - 2020)

 

11.1. SENSORY TEST

1)    SENSORY TEST FOR LIPSTICK SAMPLE A. (BEETROOT EXTRACT)

ODOUR-Smell of lavender Essence. No displeasing odour.

TOUCH- Smooth, semi-viscous.

PIGMENT- PINK SHADE

 

2)    SENSORY TEST FOR LIPSTICK SAMPLE B (COFFEE DECOCTION)

ODOUR-Smell of Cocoa Butter. No displeasing odour.

TOUCH- Smooth, semi-viscous.

PIGMENT- NUDE SHADE

 

3)    SENSORY TEST FOR LIPSTICK SAMPLE C (TURMERIC EXTRACT)

ODOUR-Smell of Lavender Essence. No displeasing odour.

TOUCH - Smooth, medium firm.

PIGMENT- LEMON YELLOW SHADE

 

12. CONCLUSION

The best thing about vegan or natural products is they are cruelty-free which intends products are free from animal testing and kill several thousand living creatures every year! Uric acid, insect secretion, diary extracts, and other undesirable chemicals like mica, and lead eventually get absorbed in the skin after application, which may lead to severe skin diseases, cancer, etc. In today’s harsh atmospheric conditions, many prefer organic, healthy ingredients. Plant extracts retain most of the nutritional value which benefits the skin. But on the grey side, chemicals, may not be conducive to the skin. By choosing a safer product like this we can actually contribute to the environment. Another significant aspect of the product is that it is pocket-friendly with better benefits. According to Future Market Insights report on Vegan Cosmetics Market, the vegan cosmetics market will exceed US $20 Bn by 2025. As the Veganism movement gains momentum in 2022, established firms will move toward the vegan cosmetic market. Some celebrities like, Harry Styles launched his gender-inclusive vegan beauty brand “Pleasing” in 2021. Currently, the company specializes in vegan nail polishes along with skincare products.

 

CONFLICT OF INTERESTS

None. 

 

ACKNOWLEDGMENTS

I would like to express my gratitude to my guide, Dr. Priya Iyer, for providing support and guidance. I gathered a lot of information about different techniques, product formulations, several types of assays and methods, and also the importance of including wellness in cosmetics, from my guide which will be very helpful for me in the future. I would also like to express my gratitude towards our other faculties, Dr. Anchana Devi for assisting us in performing Anticancer Activity, and Dr. Anitha J for extreme moral support and guidance. I also thank our laboratory assistant, Rekha Murali, Ma'am for helping me with daily laboratory schedules.

In the end, I would like to thank my parents. Without them, I would not have been able to complete this project.

 

REFERENCES

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Lwin, T., Myint, C. Y., Win, H. H., Oo, W. W., & Chit, K. (2020 December 31). Formulation and Evaluation of Lipstick with Betacyanin Pigment of Hylocereus Polyrhizus (Red Dragon Fruit). 10(4). https://doi.org/10.4236/jcdsa.2020.104022

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