1,3,4 Department of Basic Sciences, University of Health and Allied Sciences, PMB 31, Ho, Ghana
2,5 Department of Biomedical Sciences, University of Health and Allied Sciences, PMB 31, Ho, Ghana
6 Department of Pharmacognosy, School of Pharmacy, University of Ghana, Legon, Accra, Ghana
Plant extracts have been incorporated in the formulation of nail polish. Chromolaena odorata has been reported to exhibit antifungal and antimicrobial activities. In this study, the ethanolic extract of C. odorata has been incorporated into nail polish. Even though different extracts have been incorporated into nail polish formulations C. odorata has never been used in nail polish formulations. The study design involved the incorporation of C. odorata extract into nail polish formulations and comparing their performance with a known brand of nail polish sold on the market. The antimicrobial activity of the extract and the formulated nail polish were first determined by agar diffusion. Five (5) wells were created in each plate using a cork borer (No.3, 4 mm). 20 µL of 50% concentration of the ethanolic extract of C. odorata was dispensed into three wells representing triplicate repeat, 20 µL of tetracycline and Nystatin (positive control) were dispensed in the middle well (bacteria and fungus respectively) and 20 µL of dilute ethanol (2 mL of ethanol: 2 mL of distilled water) was dispensed in the last well (negative control). The set up was incubated at 37 °C for 24/ 48 h and the zones of inhibitions recorded. The plates were then checked for the presence or absence of growth in the Nutrient agar or SDA. The extract showed good antimicrobial activity when tested against Staphylococcus aureus (NCTC 12493) (8.60 ± 2.85 mm), (nail paronychia), Candida albicans (ATCC 90028) (Onycholysis and Onychomycosis) (18.33± 0.63 mm), and Streptococcus pyogenes (Clinical). (14.60± 0.39 mm). The formulated products exhibited good antimicrobial activity, were stable over the testing period, and showed good functionality when subjected to different stability tests.
Nail cosmetics have gained popularity due to advertisements, online presence and social media networks leading to increased consumer appetite for these products (Koch et al, 2019). Nail cosmetics are mainly used for adorning and enhancing the appearance of nails but sometimes this comes with some negative side effects, including dermatitis; nail discoloration, and other nail infections [1]. Nail polish, hardeners, moisturizers, and prostheses are the four broad categories of nail cosmetics. Nail polish are mostly applied as basecoats or topcoats of both toenails and fingernails [2]. These basic components include plasticizers and resins, solvents, film-forming agents and pigments [3]. Nitrocellulose is added to nail polish formulations to enhance their hardness, water resistance, viscosity and because they dry rapidly to create a hardened thin film [4]. Placticizers and resins are used to enhance adhesion, toughness, brightness, flow quality and flexibility of a nail polish. Examples of plasticizers include alkyl resins, acrylates, vinyl, or polyesters [5]. The chemical composition of most additives in cosmetic products affect the properties of the final cosmetic product developed [6].
C. odorata is a perennial shrub that grows abundantly in Asia and sub-Saharan Africa, it has been reported to be used in the treatment of many ailments and disease conditions such as diabetes, malaria, wounds, inflammation and fever. It has antidiabetic, anticancer, anti-inflammatory, antimicrobial, antiparasitic, antinociceptive, antipyretic and wound healing activities [7] C odorata have antibacterial, antispasmodic, antiprotozoal, antifungal, antihypertensive, anti-inflammatory, astringent, diuretic, and hepatotropic effects, which makes it suitable for use in cosmetic products [8]. The presence of saponins, phenols, and tannins in the aqueous and ethanolic leaf extracts of C. odorata have been reported. The extracts have been found to exhibit hemolytic, anti-inflammatory, antioxidative, immunostimulant, and antibacterial properties [9]. The ethanolic extract of C. odorata has been reported to improve cardiac conditions by lowering blood pressure, boosting circulation, and preventing the buildup of arteriosclerosis plaque and blood clots [10]. A bioprocess for the synthesis of ethanol using mixed invasive species (including C. odorata) has been reported. A composite biomass of the weeds upon acid hydrolysis, alkaline delignification and enzymatic hydrolysis gave pentose-rich and hexose-rich hydrolyzates, which were each fermented under sonication. The phenolic extract of C. odorata has been found to inhibit protein fibrillation and oxidation in vitro because of the presence of phenols, flavonoids and terpenoids [11].
In this study, we present the formulation of herbal nail polish incorporated with the ethanolic extract of C. odorata, evaluation of their properties and as well as their antimicrobial activity. This work reports the first incorporation of C. odorata extracts into nail polish formulations.
MATERIAL AND METHOD
Extraction of plant samples
Dried leaves of C. odorata were obtained from the Ho municipality, Volta Region, Ghana. The botanical identity of the sample was confirmed by experts from the School of Basic and Biomedical Sciences of the University of Health and Allied Sciences before being transferred to the laboratory. The extraction procedure followed previously published methods with slight modifications [12]. The dried sample was pulverized and weighed. About 700 g of the sample was macerated in 2 L of ethanol (95 % v/v) for a week. It was then filtered and the solvent removed by rotary evaporation, after which the extract obtained was allowed to sit for one week to evaporate all the residual solvent to yield a constant weight of extract. The yield of the extract was calculated and screened for its phytochemical constituents.
Phytochemical Screening
The phytochemical tests of the ethanolic extract of C. odorata was determined according to the standard methods [13-15].
Standard Reagents
The selected chemicals used in this study include: nitrocellulose (10%) (film-forming agent) was obtained from Abro chemicals, ethyl acetate (solvent), (Merck Chemicals), camphor (plasticizer), castor oil (resin), oil-based colorant (blue, yellow, and red), polyethylene glycol (thickener) were purchased from Unique Fragrances (Kasoa, Ghana). Ethanol, sodium hydroxide, hydrochloric acid, sodium chloride and Potassium Mercuric Iodide were obtained from Merck Sigma, Germany. The plant sample (dried C. odorata) was obtained from the Ho municipality, Volta Region, Ghana. Two antibiotic agents; Tetracycline and Nystatin were used as positive controls in the microbial analysis whilst ethanol and ethyl acetate were used as negative controls.
Test organisms
The test organisms used in this study included methicillin-resistant Staphylococcus aureus (NCTC 12493), (nail paronychia), Candida albicans (ATCC 90028) (Onycholysis and Onychomycosis), and Streptococcus pyogenes (Clinical) (nail psoriasis) are all known to cause nail infections. These microorganisms were obtained from the Microbiology unit of the School of Basic and Biomedical Sciences, University of Health, and Allied Science (UHAS) and were activated by sub-culturing them on the nutrient agar (Oxoid, United Kingdom) for 24 hours at 37 °C in an incubator after which they were made ready for the microbial analysis.
Preparation of plant and nail polish samples
A stock solution of the extract was prepared by weighing 1 g of the extract into two clean sterile 15 mL falcon tubes containing 5 mL of ethanol. From this stock solution, an aliquot of 2 mL was transferred into two clean and sterile 15 mL falcon tubes, 2 mL of ethanol was added and used for further antimicrobial analysis. An aliquot of 1 mL of the stock solution (100%) were used to prepare 100 mg/mL of each sample.
Antimicrobial determination of plant extract
The antimicrobial activity of the ethanolic extract of C. odorata was determined using both the Kirby-Bauer agar well diffusion and the broth micro-dilution methods. From the Kirby-Bauer agar well diffusion, the zones of inhibition were measured. The Kirby-Bauer agar well diffusion involved a test agar plate with a standardized concentration of test organisms and either antibiotic disc or wells with antibiotic agents placed on the lawn of microorganisms. After overnight incubation, the diameter of the zone of inhibited growth were measured. The method used was based on reported work with slight modifications [16-19]
Broth microdilution method of plant extract
The minimum inhibitory concentrations (MICs) of the extract were determined by the micro broth dilution method using the 96 well microtiter plates per the method according to the Clinical and Laboratory Standards Institute [20]. The method used was consistent with reported work with slight modification [21-24]. A 50% concentration of the stock solution of the extract was prepared as earlier described. Ten different concentrations were obtained by serial dilution, an aliquot of 100 µL of double-strength Mueller Hinton broth (for bacterial strains) and Brain-Herat Infusion broth (for fungal strain) (Oxoid Limited, United Kingdom) were distributed into each 96-well plate (Cito test Labware Manufacturing Co. Ltd, Jiangsu, China) and mixed with 100 µL of the plant extract to prepare well concentrations ranging from 0.1–50.0 mg/mL. Wells 11 and 12 were designated positive control (Broth + organism only) and negative control (Broth with no organism) respectively for each microbial strain on each column. This was followed by the addition of 100 µL of each of the 0.5 McFarland standardized test organisms at a concentration of 105 CFU after which the plates were subjected to incubation at 37 ? for 24−48 hours for bacterial and fungal strains respectively. The MIC values were then evaluated by adding 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye and kept in the incubator for 30 min after which the MIC was observed as the least concentration which does not change color from yellow to red. The experiment was performed in triplicate and their averages were recorded as the MICs.
Determination of Minimum Bactericidal Concentrations (MBC) and Minimum bactericidal Concentrations/ Minimum Inhibitory Concentration (MBC/MIC)
The MBC/MIC was determined to establish whether the ethanolic extract of C. odorata could destroy the microbial cells. Aliquots from each well from the susceptibility tests were transferred to plates with Nutrient agar and then incubated for 24–48 hours at 37 °C. The plates were then examined for growth on Nutrient agar [25].
Nail polish formulation
The preparation of the different nail polish formulations in this study was according to the method reported with slight modifications [26].
Formulation of antimicrobial nail polish with C. odorata leaves
Two different formulas were used in preparing the herbal nail polish with C. odorata leaves. Table 1 below shows the first formula for the preparation of herbal nail polish with C. odorata leaves and ones without the plant extract. The formula below was used for the first nail polish formulations with the C. odorata extract (CO1, CO2, CO3, CO4, and CO5) and the ones without C. odorata extract (WCWCO and CWCO), 10 g of camphor was grounded into powder and dissolved in 140 mL of ethyl acetate with stirring. 20 g of nitrocellulose, 30 g of castor oil and 10 g polyethylene glycol were added and stirred until all the components were dissolved.
The formulated stock nail polish in the beaker was then dispensed into 20 mL nail polish containers. Different amounts (0.1 g to 0.5 g) of the C. odorata extract were weighed and incorporated into five different formulations. A red colourant was then added to six of the formulations including the five formulations containing the plant extract. The nail polish formulation with colourant (red) but without C. odorata extract (CWCO) and a nail polish formulation with no colourant nor C. odorata extract (WCWCO) were used as controls for the study.
Table 1: Formula for antimicrobial nail polish with C. odorata leaves extract (CO1, CO2, CO3, CO4, and CO5) and without extract (CWCO and WCWCO)
|
FORMULA |
QUANTITY |
|
Nitrocellulose (10% nitrocellulose) |
20 g |
|
Ethyl acetate |
140 mL |
|
Camphor |
10 g |
|
Castor oil |
30 g |
|
Colorant (Red) |
q. s |
|
Polyethylene glycol |
10 g |
|
Acheampong leaves |
Varying amounts |
|
CO1 |
0.1 g |
|
CO2 |
0.2 g |
|
CO3 |
0.3 g |
|
CO4 |
0.4 g |
|
CO5 |
0.5 g |
|
Controls |
Varying amounts |
|
WCWCO |
No plant extract |
|
CWCO |
No plant extract |
The second formula used in formulating the herbal nail polish with C. odorata leaves and ones without the plant extract are indicated in Table 2. The formula below was used for the formulation of COb1, COb2, COb3, COb4, and COb5 with varying amounts of C. odorata extract incorporated. The formulations without the extract were (CWCOb and WCWCO2), 19 g of camphor was grounded into powder and dissolved in 70 mL of ethyl acetate with stirring, and 60 g of nitrocellulose was added and stirred to obtain a clear solution.
The formulation was dispersed into 10 mL containers and the different amounts of extract of C. odorata incorporated. The formulated nail polish was then dispensed into 10 mL containers, different amounts of the C. odorata extract was incorporated into the relevant sample. The blue colour was incorporated and mixed thoroughly to obtain COb1, COb2, COb3, COb4, and COb5. Two control formulations were prepared one contained only the colourant (blue) without C. odorata extract (CWCOb), the other nail polish had no colourant nor the C. odorata extract (WCWCO2).
Table 2 Formula for antimicrobial with C. odorata leaves extract (COb1, COb2, COb3, COb4, and COb5) and without extract (CWCOb and WCWCO2)
|
FORMULA |
QUANTITY |
|
Nitrocellulose (10% nitrocellulose) |
60 g |
|
Ethyl acetate |
70 mL |
|
Camphor |
19 g |
|
colorant (blue) |
q.s. |
|
C. odorata leaves |
Varying amount |
|
COb1 |
0.1 g |
|
COb2 |
0.2 g |
|
COb3 |
0.3 g |
|
COb4 |
0.4 g |
|
COb5 |
0.5 g |
|
Controls |
Varying amount |
|
CWCOb |
No plant extract |
|
WCWCO2 |
No plant extract |
Evaluation of Herbal Nail Polish
The nail polish formulations were evaluated for their functionality and stability using the Bureau of Indian standards. They were also tested against some microorganisms that cause nail infections (methicillin-resistant Staphylococcus aureus (NCTC 12493), (nail paronychia), Candida albicans (ATCC 90028) (Onycholysis and Onychomycosis), and Streptococcus pyogenes (Clinical) (nail psoriasis) These evaluations were conducted as a quality control measure on the nail polish to understand their antimicrobial properties.
Functionality tests
Stability tests
This is the measure of the durability of the nail polish. All the nail polish formulations were taken through accelerated stability tests where they were stored at a temperature of 25 º ± 2 °C and 37 oC ±2 °C, after 1 month, the formulations were inspected for their organoleptic characteristics (color, smell, texture, and consistency).
Bureau of Indian Standards
Smoothness to flow
About 1 mL of each nail polish formulation and marketed nail polish were pipetted on a glass slide of area 137.81 cm2 and raised vertically. The smoothness of flow was observed visually and determined by comparing it with a marketed nail polish used as a standard [27].
Gloss
The gloss of a nail polish refers to how shiny and smooth it looks when observed visually. The gloss of formulated nail polish samples and the marketed nail polish were determined by applying a film each at the same consistency with the nail polish brush on a glass slide of area 137.81 cm2 and observed visually using the marketed nail polish as a standard of comparison [27]
Drying time
The optimal drying time of an ideal nail polish is between 1 to 2 minutes without developing bloom. A film of the same consistency for each nail polish sample and the marketed nail polish was applied on a glass slide of area 137.81cm2 with the help of the nail polish brush. The time required to form a dry touch film at room temperature was recorded using a stopwatch. It was then compared with the marketed nail polish [28]. Figure 1 gives the pictorial representation of sample CO3 undergoing drying.
Figure 1 Drying of CO3 nail polish sample
Non-volatile content
The empty clean sterile petri dish plate for the nail polish samples were weighed first and denoted as W1. About 3 mL of each nail polish sample including the marketed nail polish were pipetted into the weighed clean sterile petri dish plates and their weight were noted as W2. The difference (W2-W1) was the actual weight of the nail polish samples. The petri dishes plates with the nail polish samples were placed in a hot air oven at 105 ± 2 °C for 1 hour. The plates were then removed, allowed to cool and weighed, W3. The difference in weight (W3-W1) for each nail polish sample was calculated and the non-volatile content was expressed in percentages (Rasheed, et al, 2012). Figure 2 (a, b) shows pictorial presentations of some stages in the non-volatile content test.
Figure 2 Non-volatile tests for (a) C. odorata (red), and (b) C. odorata (dark blue color
In-vitro adhesion test
An area of 3.6 cm by 2.4 cm was marked with a marker and a rule on a glass slide of area 11.45 cm by 12.45 cm. A few drops of each nail polish sample as well as the marketed nail polish weighing 0.0645 g were applied on the marked area on the glass slide and spread evenly with the nail polish brush. The films were allowed to dry at 25 ± 2 °C for 24 h. The entire film was covered with cellophane tape and pressed manually with the thumb. The tape were then removed quickly and the area of film that peeled off was calculated and given in percentage [29]. Figure 3 shows a pictorial presentation of some of the nail polish samples spread in the defined area and dried at room temperature for 24 h in the in vitro adhesion tests.
Figure 3 Some nail polish samples spread on an area of 8.64 cm2 on glass slides drying at room temperature for 24 h for in-vitro adhesion tests.
Water resistance
This is the measure of the resistance of the formulated nail polish film towards water permeability. The empty clean microscope slides were weighed and dried. A few drops of the nail polish was applied on the dried microscope slides to get 0.0605 g (W1) and allowed to dry at 25 ± 2 °C. The dried slides were immersed in a water bath at 37 °C, removed after 24 h, wiped with tissue paper, and reweighed (W2). The difference in weight were calculated. The higher the increase in weight the lower the water resistance of the nail polish sample (hence water permeability was high) and vice versa. Water resistance tests were not conducted for WCWCO and CWCO due to insufficient quantity left after the preceding tests. Figure 4 shows a pictorial presentation of some nail polish samples after drying at room temperature to be used for the water resistance tests.
Figure 4: Nail polish samples with C. odorata leaves being dried at room temperature for the water resistance tests.
RESULTS
Percentage yields of the plant extract
The percentage yields of the ethanolic extracts of C. odorata leaves (Figure 5) were determined from the formula:
Percentage yield (C. odorata) = Total mass of the ethanolic C. odorata extract (g)/Mass of plant material (dried) used for the extraction (g) x 100%
Percentage yield = (57.34g ÷ 669.84g) × 100%
Percentage yield = 8.5 %
The determination of the percentage yield gives an idea about the extractability of the plant studied under different conditions. It can be affected by the season during which the plant were picked or even the time of the day [30-33]. The reported yield of C. Odorata ranged from 8.42 to 11.2 % [34]. The ethanolic extract obtained from this work is 8.5% which is within the range reported in the literature.
Phytochemical composition of the plant extract
The phytochemical constituents of the ethanolic extract of C. odorata leaves revealed the presence of flavonoids and saponins. (Table 3)
Table 3: Phytochemical screening of C. odorata leaves extract
|
Plant Constituent |
Test/ Reagent |
Ethanolic AL extract |
|
Alkaloids |
Mayer’s test |
- |
|
Flavonoids |
Alkaline Reagent Test |
+ |
|
Saponins |
Foam test |
+ |
|
Tannins |
Gelatin test |
- |
|
Reducing Sugars |
Benedict’s test |
- |
Key, (+) = presence of the phytochemical; (-) = undetected
Determination of Minimum Bacteriostatic Concentration (MBC) and Minimum Bacteriostatic Concentration/Minimum Inhibitory Concentration (MBC/MIC)
The antimicrobial studies of the ethanolic C. odorata extract was evaluated through the broth micro-dilution method to obtain the MICs and MBCs. Table 4 gives the MIC, MBC and MBC/MIC concentrations of the plant extract.
Table 4: Antimicrobial activity (MIC analysis) of the extract of C. odorata against strains that cause nail infections
|
Organisms |
MIC |
MBC |
MBC/ MIC |
Comment |
|
C. albicans |
0.49 |
1.563 |
3.19 |
Fungicidal |
|
S. aureus |
12.50 |
25.00 |
2.00 |
Bactericidal |
|
S. pyrogenes |
6.25 |
25.00 |
4.00 |
Bactericidal |
Kirby-Bauer agar well diffusion method of plant extract C. odorata Leaves
The ethanolic extract of C. odorata leaves was tested against selected microorganisms. The extract of C. odorata showed the highest activity against Candida albicans followed by Streptococcus pyogenes and the least microbial activity against Staphylococcus aureus. The positive control used for the bacterial stains (tetracycline) showed good antimicrobial activity against Staphylococcus aureus (22 ± 2.08 mm) but Streptococcus pyogenes showed resistance to it as well as resistance to the negative control (ethanol). Candida albicans was resistant to the negative control and the positive control (Nystatin). Table 5 shows the zones of inhibitions of agar disc diffusion for the ethanolic extract of C. odorata leaves against the selected organisms. The zones of inhibition of C. odorata leaves extract against the selected organisms (Figure 6)
Table 5 Zones of inhibitions of agar well diffusion for the ethanolic extract of C. odorata leaves against test organisms
|
Zones of inhibitions/ mm Mean/ Standard dev. |
|||
|
Plant Extract |
Candida albicans |
Streptococcus pyrogenes |
Staphylococcus aureus |
|
|
18.33± 0.63 |
14.60± 0.39 |
8.60 ± 2.85 |
(a) (b) (c)
Figure 6 Zone of inhibition for the ethanolic extract of C. odorata against (a) C. albicans (b) S. pyrogenes (c) S. aureus (PC: Nystatin, PC: Tetracycline, NC: Ethanol)
Formulated antimicrobial nail polish with C. odorata
a) First formulation
The pictures of the formulated nail polish samples tested in this work. (Figure 7)
Figure 7 Formulated nail polish (red) with C. odorata and the controls without the extract
(b) Second formulation
The formulated nail polish (blue) with the C. odorata leaves and ones without the plant extract together with the marketed nail polish (Figure 8a and 8b)
(a) (b)
Figure 8 (a, b) Formulated nail polish (blue) with C. odorata, with color, colorless and standard nail polish.
Evaluation of Herbal Nail Polish
The formulated nail polish were subjected to preliminary evaluation tests which included functionality tests, the Bureau of Indian standards and antimicrobial studies against microorganisms that cause nail infections
Functionality Tests
Stability tests
The formulated nail polish showed good stability when stored at 37±2 °C. After 1 month, the formulated nail polish samples were inspected for their organoleptic characteristics (color, smell, texture, and consistency) and there was no significant change in these characteristics.
Bureau of Indian Standards
Smoothness to flow
The smoothness of flow for all the nail polish samples showed a satisfactory flow property when compared to the marketed nail polish, however, the flow of the formulated nail polish samples was faster than the marketed nail polish with formulations CO1b, CO2b, CO3b, CO4b, CO5b, WCWCO2 and CWCOb giving a rough film texture when felt with the fingers.
Gloss
The gloss of the nail polish samples and the marketed nail polish were determined by applying a film at the same consistency with the nail polish brush on a glass slide of area 137.81cm2 and observing them visually. Table 6 gives a summary of the results for the gloss property of the nail polish samples.
Table 6 Results of the gloss property of the nail polish samples.
|
Nail polish samples |
Gloss property |
Comment |
|
StandarD nail polish |
Pass |
Shiny and wet-looking |
|
CO1 |
Pass |
Shiny and wet-looking |
|
CO2 |
Pass |
Shiny and wet-looking |
|
CO3 |
Pass |
Shiny and wet-looking |
|
CO4 |
Fail |
Dark, rough with particles of plant extract |
|
CO5 |
Fail |
Dark, rough with particles of plant extract |
|
CO1b |
Satisfactory |
Losses gloss upon drying |
|
CO2b |
Satisfactory |
Losses gloss upon drying |
|
CO3b |
Satisfactory |
Losses gloss upon drying |
|
CO4b |
Satisfactory |
Losses gloss upon drying |
|
CO5b |
Satisfactory |
Losses gloss upon drying |
|
WCWCO |
Pass |
Shiny and wet-looking |
|
WCWCO2 |
Satisfactory |
Losses gloss upon drying |
|
CWCO |
Pass |
Shiny and wet-looking |
|
CWCOb |
Satisfactory |
Losses gloss upon drying |
Drying time
The drying times for the nail polish formulations were found to be between 131.01 ± 6.25 seconds and 2606.00 ± 1.12 seconds on average at room temperature. The marketed nail polish showed an average drying time of 82.36 ± 6.30 seconds Table 7 gives the average drying time of the nail polish samples.
Table 7: Drying time of the formulated nail polish samples
|
Nail polish samples |
Drying time(s) Mean/Standard dev. |
|
Standard nail polish |
82.36 ±6.30 |
|
CWCOb |
131.01 ±6.25 |
|
CO5b |
181.11 ±6.10 |
|
CO4b |
214.51 ±6.00 |
|
CO3b |
222.90 ±5.90 |
|
CO1b |
391.00 ±5.48 |
|
WCWCO2 |
522.19 ±5.09 |
|
CO2b |
634.00 ±4.74 |
|
CO5 |
1024.00 ±3.59 |
|
CO4 |
1160.00 ±3.19 |
|
CO3 |
1164.00 ±3.17 |
|
CO2 |
1284.00 ±2.82 |
|
CO1 |
1962.00 ±0.70 |
|
WCWCO |
2361.00 ±0.39 |
|
CWCO |
2606.00 ±1.12 |
Non-volatile content
This test was done to check the quantity of the non-volatile content in the nail polish formulations since the solvent used was volatile. The non-volatile content in percentage terms for the formulated nail polish formulations were found to be in the range of 29.52±1.40% and 52.24 ± 9.11% with the least non-volatile content observed in CO1b and the highest observed in CO5b. Table 8 illustrates the non-volatile content of the nail polish samples expressed in percentages Mean/Standard dev.
Table 8 Non-volatile content of the nail polish samples expressed in percentages Mean/Standard dev.
|
Nail polish Samples |
Percentage non-volatile content |
|
SNP |
18.90±6.31% |
|
CO1 |
39.06±3.01% |
|
CO2 |
36.54±1.85% |
|
CO3 |
37.76±2.40% |
|
CO4 |
39.55±3.23% |
|
CO5 |
43.91±5.25% |
|
CO1b |
29.52±1.40% |
|
CO2b |
39.71±3.31% |
|
CO3b |
36.96±2.04% |
|
CO4b |
33.98±0.66% |
|
CO5b |
2.24±9.11% |
|
WCWCO |
21.18±5.30% |
|
WCWCO2 |
27.40±2.38% |
|
CWCO |
20.73± 5.47% |
|
CWCOb |
28.60±1.83 % |
In vitro adhesion
In vitro adhesive strength of nail polish samples was determined by the film peel-off test with the nail polish spread evenly on a constant area of 8.64cm2. The percentage of film peel-off in the nail polish samples was found to be in the range of 2.90± 3.32% to 93.17± 4.48%. Table 9 gives the results of the in vitro adhesion (peel-off test) of the nail polish samples expressed in percentages. Percentage peel off = (area of peel off /area of spread) × 100%. Figure 9 gives the pictures of the in vitro adhesion results of some nail polish samples.
(a) (b) (c)
Figure 9 In-vitro adhesion tests reults of (a) Standard nail polish (SNP) (b) CO3 (c) CO4
Water resistance
Water resistance tests were conducted to evaluate the water resistance of prepared nail polish samples. All the nail polish samples showed a decrease in weight after the water resistance test indicating a satisfactory water resistance and lower water permeability except for CO3 and CO1 that showed poor water resistance resulting in increased weight. CO4b showed excellent water resistance due to a decreased weight after the water resistance tests while the other nail polish samples showed good water resistance due to moderate decrease in weight after the water resistance tests. The results of the water resistance tests are given in Table 10.
Table 10 Results of water resistance for the nail polish samples Mean/Standard dev.
|
Nail polish samples |
Difference in weight (g) |
Water resistance |
|
CO4b |
0.0347 ±0.016 |
Excellent |
|
CO1b |
0.0321 ±0.014 |
Good |
|
CO2 |
0.0297 ±0.011 |
Good |
|
WCWCO2 |
0.0291 ±0.011 |
Good |
|
CWCOb |
0.0285 ±0.010 |
Good |
|
CO4 |
0.0275 ±0.009 |
Good |
|
CO3b |
0.0265 ±0.008 |
Good |
|
CO5b |
0.0244 ±0.006 |
Good |
|
CO2b |
0.0235 ±0.005 |
Good |
|
CO5 |
0.0220 ±0.003 |
Good |
|
SNP |
0.0201 ±0.002 |
Good |
|
CO1 |
0.0232 ±0.088 |
Poor |
|
CO3 |
0.0704 ±0.09 |
Poor |
Antimicrobial property determination of the formulated nail polish
Kirby-Bauer agar well diffusion method
The nail polish samples (CWCO and WCWCO) formulated using the first formula and without the plant extract were tested against the test organisms as described previously and they showed variable antimicrobial activities. With WCWCO showing no activity against S. pyrogenes. The nail polish sample showed the highest antimicrobial activity against C. albicans (23 ± 1.09 mm) and the lowest activity against S. aureus (7.33 ± 2.00 mm) the results showed good antimicrobial activity against most of the selected strains. C. albicans and S. pyrogenes were resistant to both the positive and negative controls. S aureus however, was susceptible to the positive control (22 ± 1.09 mm), Figures 10 and Figure 11 give the results of the antimicrobial activity of WCWCO against the test organisms. Figures S1, S2 and S3 give the zones of inhibition of the ethanolic C. odorata extract, WCWCO and CWCO when tested against S. aureus, C. albicans, and S. pyogenes.
CWCO gave the highest anti-microbial activity against S. aureus (8.60 ± 0.40 mm) and followed by C. albicans (8.33 ± 0.47 mm) and the lowest activity was observed for S. pyrogenes (2.33 ± 1.92 mm) with all organisms resistant to the negative control as shown in Figure 11 below.
(a) (b)
Figure 10: Zone of inhibition of WCWCO nail polish sample when tested against C. albicans and S. aureus select set of organisms (NC: negative control (ethyl acetate) PC: Positive control (Tetracycline)
(a) (b) (c)
Figure 11: Zone of inhibition of CWCO nail polish sample when tested against (a) S. aureus (b) C. albicans and (c) S. pyrogenes.
Determination of MBC and MBC/MIC Concentration
The antimicrobial studies of nail polish samples (CO1b, CO2 CO3b, CO4b, CO5b, CWCOb and WCWCO2) were evaluated using the broth micro-dilution methods and the MICs and MBCs results were obtained. The nail polish samples formulated with C. odorata and a red colorant interfered with the red dye used in the MICs analysis hence the blue coloured nail polish was used.
Table 11 gives the MIC, and MBC results for all the nail polish formulations and controls against C. albicans, S. aureus and S. pyrogenes.
Table 11 MIC and MBC results for all the nail polish formulations and controls against C. albicans, S. aureus and S. pyrogenes.
|
Organisms |
|
MIC |
MBC |
MBC/MIC |
Comment MBC/MIC |
|
C. albicans |
CO1b |
25 |
25 |
1.00 |
Fungicidal |
|
|
CO2b |
25 |
50 |
2.00 |
Fungicidal |
|
|
CO3b |
50 |
50 |
1.00 |
Fungicidal |
|
|
CO4b |
50 |
50 |
1.00 |
Fungicidal |
|
|
CO5b |
50 |
>50 |
>1.00 |
Fungicidal |
|
|
CWCOb |
50.00 |
50.00 |
1.00 |
Fungicidal |
|
|
WCWCO2 |
50.00 |
>50.00 |
>1.00 |
Fungicidal |
|
S. aureus |
CO1b |
25 |
50 |
2.00 |
Bactericidal |
|
|
CO2b |
50 |
50 |
1.00 |
Bactericidal |
|
|
CO3b |
50 |
50 |
1.00 |
Bactericidal |
|
|
CO4b |
50 |
50 |
1.00 |
Bactericidal |
|
|
CO5b |
50 |
>50 |
>1.00 |
Fungicidal |
|
|
CWCOb |
25.00 |
50.00 |
2.00 |
Bactericidal |
|
|
WCWCO2 |
25.00 |
50.00 |
2.00 |
Bactericidal |
|
S. pyrogenes |
CO1b |
25 |
50 |
2.00 |
Bactericidal |
|
|
CO2b |
25 |
50 |
2.00 |
Bactericidal |
|
|
CO3b |
50 |
>50 |
>1.00 |
Bactericidal |
|
|
CO4b |
50 |
>50 |
>1.00 |
Bactericidal |
|
|
CO5b |
50 |
>50 |
>1.00 |
Bactericidal |
|
|
CWCOb |
50.00 |
50.00 |
1.00 |
Bactericidal |
|
|
WCWCO2 |
50.00 |
>50.00 |
>1.00 |
Bactericidal |
Key: Values are in µL/mL, MIC: Minimum Inhibitory Concentration; MBC: Minimum Bactericidal Concentration. Experiment was carried out in triplicate.
Reference range for MBC/MIC: The antimicrobial activity of the nail polish samples were classified as bactericidal/fungicidal if the ratio of MBC/MIC was less than or equal to 4, and as bacteriostatic/fungistatic if it was more than 4 [35].
DISCUSSION
Herbal cosmetics are natural and devoid of any potentially dangerous synthetic compounds that could endanger human health. These herbal cosmetics are used substantially in various permissible range of cosmetic ingredients to provide specific cosmetic health benefits and are termed cosmeceuticals [36]. This has created an increasing demand for herbal cosmetics since they comprise a remarkably diverse array of bioactive substances with a variety of unique pharmacological and technical applications, including natural antioxidants, natural preservatives and natural colorants [37]. These herbal cosmetics have also been tested and proven to have less allergic reactions on the skin, hair and nails compared with synthetic ones with profound allergic reactions and irritations to the body and being environmentally unfriendly [38]. We therefore decided to formulate herbal nail polish with antimicrobial properties against some nail infection causing organisms using the ethanolic extract of C. odorata. The plant extract was tested for its antimicrobial activity against the selected organisms that cause nail infections to justify it inclusion in the nail polish formulations. The antimicrobial activity of the C. odorata leaf extract using agar well diffusion showed a significant activity with the highest activity observed against C. albicans, followed by S. aureus and S. progenies. The results corresponds to a study that assessed the antimicrobial activity of C. odorata leaves against organisms that cause wound infections, this showed substantial antimicrobial activity against S. aureus [39] and C. albicans when the effect on the plant extract against some human pathogens were assessed [40]. The results of the antimicrobial susceptibility tests of the same ethanolic plant extract also showed higher activity against C. albicans and S. pyogenes with moderate activity observed for the benzene, ethyl acetate, chloroform, and water extracts against the same microorganisms [41]. Above all S. aureus was susceptible to the Tetracycline but S. pyrogenes was resistant this was also observed for C. albicans to Nystatin. All the organisms were resistant to the negative control except S. aureus. Comparing the MICs and MBC/MIC of C. odorata leaf extract [100 mg/mL] against the organisms in the study, the extract was fungicidal against C. albicans with the extract being the best at the lowest concentrations. All the nail polish formulations were subjected to stability, functionality, and bureau of Indian standard tests. The stability tests were used to determine the shelf life and storage condition of the nail polish samples and the results indicated that the nail polish formulations with the herbal extract or without, showed good stability when it was stored at a temperature of 37 ±2 °C. After 1 month, the formulated nail polish samples were inspected for their organoleptic characteristics (color, smell, texture, and consistency and there was no significant change in the organoleptic characteristics which also correlates with a study of formulated medicated nail polish samples with tolnaftate [42], and were followed up with the bureau of Indian Standards tests. Smoothness of flow for all the nail polish samples showed satisfactory flow compared to the marketed nail polish however the rate of flow of the nail polish samples were faster than the marketed nail polish due to low viscosity of the nail polish. Formulations with C. odorata leaves (CO1, CO2 and CO3) passed the gloss test with a shiny look when compared with the marketed nail polish but two formulations (CO4 and CO5) failed the test with dark particles of the plant extracts on the surface when applied on the glass slide, this may be due to the higher amounts of the extract used in the formulation which did not dissolve completely. Formulations from the second formula (CO1b, CO2b, CO3b, CO4 and CO5b) showed satisfactory gloss properties when compared to the marketed nail polish. The controls of the first formulation (CWCO and WCWCO) had similar gloss in comparison to the marketed nail polish whilst the samples of the second formula (CWCOb and WCWCO2) showed satisfactory gloss property. Drying time for all the nail polish formulations were found to be between 131.01 ± 6.25 seconds and 93.98 ± 20.47seconds on average under room temperature as compared to the marketed nail polish 82.36 ± 6.30 seconds, however there was a decrease in drying time as the amount of C. odorata extract increased showing that the plant extract increased the viscosity of the formulations making them dry faster. The nail polish formulations were subjected to non-volatile content test and it was observed that for the formulation with C. odorata (CO1, CO2, CO3, CO4 and CO5) the non-volatile content was between 36.54 ± 1.85% and 43.91 ± 5.25% with CO2 being the lowest and CO5 the highest. For the second formulation with C. odorata leaf extract, (CO1b, CO2b, CO3b, CO4b and CO5b) the non-volatile content was between 29.52 ± 1.40% and 52.24 ± 9.11%. The controls were also between 21.18 ± 5.30% and 28.60 ± 1.83% with the marketed nail polish sample containing between 18.90 ± 6.31% non-volatile content. It can therefore be observed that the non-volatile content decreases as the amount of the plant extract increases with a few inconsistencies (Table 11), which is contrary to a reported study where the polymer concentration increases as the non-volatile content increases [43].
In vitro adhesive strength of nail polish samples was determined by film peel off test. The percentage of film peel-off in the nail polish samples was found to be in the range of 2.90 ± 3.32% to 93.17± 4.48%. Comparing the nail polish formulations that showed the best adhesions, CO3, CO4, and CO5 showed excellent adhesion to the glass slide with no significant peel-off, also CO2 and CO1 showed very good adhesion with an area of peel-off being 0.25 cm2 and 0.5 cm2 which was slightly better than standard nail polish peel off with peel off area of 0.6 cm2. The least adhesion with the highest peel off % was seen in CO3b and the highest adhesion but the lowest peel off % was seen in CO2. It was also observed that the higher the area of peel off the lower the adhesion of the nail polish samples. Water resistance of prepared nail polish formulations were evaluated using the water resistance test. The amount of water absorbed by the nail polish samples after keeping in water bath at 37 °C for 24 hours was found to be generally low among the nail polish samples except for CO3 and CO1 which showed a higher increase in weight after the water resistance test with CO3 having higher increase in weight (0.1309 ± 0.18 g) than CO1 (0.0837 ± 0.09 g). Thus, CO3 had a poorer water resistance than CO1. Amongst the nail polish samples with a decreased weight after the water resistance test, the marketed nail polish sample (SNP) showed the highest decrease in weight (0.0404 ± 0.007 g) and therefore gave a good water resistance. CO4b showed the smallest decrease in weight (0.0258 ± 0.028 g) amongst the formulated nail polish samples hence had excellent water resistance. In all, CO3 showed the highest increase in weight and hence the lowest water resistance whilst CO4b showed the lowest increase in weight and hence the highest water resistance. The antimicrobial activity of the nail polish samples without the plant extract using the first formula (CWCO and WCWCO) showed substantial antimicrobial activity against C. albicans, S. aurues and S. progenes. CWCO nail polish sample [100 mg/mL] showed the highest zone of inhibition in mm from the agar diffusion assay against S. aureus. All organisms were resistant to ethyl acetate the negative control throughout the study. The WCWCO nail polish sample showed a higher activity against C. albicans followed by S. aureus but showed no activity against S. pyrogenes. The MIC analysis showed CO1b exhibit a similar antimicrobial activity against C. albicans, S. aureus and S. pyrogenes with an MIC of 25 µL/mL. CO2b gave an MIC of 25 µL/mL for C. albicans and S. pyrogenes and an MIC of 50 µL/mL for S. aureus. CO3b, CO4b, CO5b gave MICs of 50 µL/mL against C. albicans, S. aureus and S. pyrogenes. Comparing these results to the MIC of the C. odorata extract, it was observed that C. odorata leaves give MIC as low as 0.49 µL/mL, 12.50 µL/mL and 6.25 µL/mL to kill 50% of the organisms whilst the nail polish samples gave MICs of 25 and 50 µL/mL for activity against the same organisms. The nail polish samples without the plant extract (CWCOb and WCWCO2) gave MICs of >50 µL/mL whilst CWCOb gave an MIC of 25 µL/mL against S. aureus. It can be observed that generally the higher the amount of the plant extract the lower the antimicrobial activity which is contrary to a reported study [44], where a mixture of herbal plants (lemon grass, garlic etc) were used in very small amounts in nail polish formulations against C. albicans and discovered that as the concentration of the herbal extracts increases the antimicrobial activity also increased. This suggest that there is a threshold concentration for optimum activity beyond which the extract in the presence of other constituents of the formulation lead a lower activity. Using the extract alone the activity increases with increasing concentration but in the presence of nitrocellulose, ethyl acetate, camphor, castor oil, the colourant and polyethylene glycol the activity of the C. odorata leaf extract is suppressed.
CONCLUSION
C. odorata leaf extract is widely used as topical treatment for wounds, nail infections and in other cosmetics in wound- healing balms with promising antimicrobial activity. From the results obtained it can be concluded that the formulations showed satisfactory stability and functionality. They showed good antimicrobial activity against most of the organisms that cause nail infections but the extract showed a higher activity as well as the controls for both formulation except S. pyrogenes in the first formulation. The findings from this work has the potential to change how nail polish products are marketed. With these products they can be marketed for their wound healing properties as well. The incorporation of the extracts into the nail polish reduced the antimicrobial activity in some cases. It is suggested that for future work the compounds in C. Odorata should be isolated, characterized and tested for their suitability as additives in the formulated nail polish.
Authorship Contributions
Concept Design: F.O., Literature search: F.O., S.B., Data Collection: S.B., D.N., C.K., Analysis or Interpretation of results: F.O., S.B., D.N., Writing: Review and editing: F.O., S.B., D.N., J.W.A.J., E.Q., C.K.,
ACKNOWLEDGEMENTS
The authors would like to than the University of Health and Allied Sciences for the use of their facilities.
Ethical statements (or Informed Consent in case of human study):
The project did not require any ethical clearance because it did not involve human subjects
Conflict of Interest statement
The authors no conflict of interest in this project.
Consent to Publish declaration: Not applicable
Consent to Participate declaration: Not applicable.
Funding Declaration
The authors declare that this study received no financial support.
REFERENCES
Felix Odame, Shadrach Bortier, David Neglo, Justice Wiston Armstrong Jonathan, Eunice Quaynor, Cindy Kitcher, Development and Evaluation of Herbal Nail Polish Using the Ethanolic Extract of Chromolaena odorata, Int. J. of Pharm. Sci., 2026, Vol 4, Issue 5, 1233-1251. https://doi.org/10.5281/zenodo.20055688
10.5281/zenodo.20055688
