Materials and reagents
Ethanol was supplied by Thai Food and Chemical Co., Ltd., Bangkok, Thailand. All chemicals utilized in phytochemical screening were of analytical grade purchased from Sigma-Aldrich Corporation (Missouri, USA). Potassium bromide, FTIR grade, attained from Thermo Fisher Scientific (Massachusetts, USA). Folin-Ciocalteu was purchased from Loba Chemie (Mumbai, India). Gallic acid, sodium carbonate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), acetate buffer, ferric chloride hexahydrate, and bovine serum albumin were procured from Sigma-Aldrich Corporation (Missouri, USA). L-ascorbic acid was obtained from Chem-Supply Pty Ltd (Gillman, South Australia). Ultrapure water generated by GenPure equipment (TKA Wasseraufbereitungssysteme GmbH, Niederelbert, Germany) and ICP multi-element standard solution XIII obtained from Agilent Technologies (Santa Clara, USA) were utilized to determine heavy metal contents. Diclofenac diethylamine and microbiological media were acquired from Merck KGaA (Darmstadt, Germany). Iscove's Modified Dulbecco's Media (IMDM), fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Corporation (Missouri, USA). Dewaxed bleached shellac and polyvinylpyrrolidone (PVP) K30, USP grade were procured from Union Shellac Part., Ltd. (Bangkok, Thailand) and Shanghai Yuking Chemtech Co., Ltd. established by China Functional Polymer Industry Committee (Shanghai, China), respectively.
Extraction of plant materials
The dried aerial parts of A. paniculata and stems of T. crispa (Fig. 1) were collected from gardens in Nakhon Pathom, Thailand, in April 2020. Both plant specimens were substantiated using the key to species and description in the Botanical Garden Organization (BGO) plant database, Ministry of Natural Resource and Environment, Thailand [21]. Voucher specimens were deposited in the Faculty of Pharmacy, Silpakorn University, Thailand. Samples were separately ground into fine particles and sieved to obtain the particle size < 0.149 mm. Each sample was then macerated two times in 95% v/v ethanol at dried sample-to-solvent ratio of 1:5 g/ml, maceration time of 3 d, and maceration temperature of 25 ± 1°C with periodic agitation. The mixtures were independently filtered using Whatman no. 1 filter paper to collect the filtrates for subsequent evaporation of solvents using a rotary evaporator (R-100, Buchi, Japan) under reduced pressure at 45 °C to obtain A. paniculata and T. crispa ethanolic extracts. The extracts were dried to constant weight in a hot air oven (Heraeus, Hanau, Germany) at 50 ± 5°C and kept at -20°C until used.
Phytochemical investigation
Preliminary phytochemical screening tests in the 10 mg/ml ethanolic solutions of A. paniculata and T. crispa extracts were accomplished using the standard methods [22–25]. All chemical tests were executed in triplicate.
Tests for tannins [22, 23, 25]
Ferric chloride test: Several drops of 10% w/v ferric chloride solution were added to the sample solution. A brownish green color indicates the existence of tannins.
Lead acetate test: A few drops of 10% w/v lead acetate were added to the sample solution. The white precipitate was formed designating the presence of tannins.
Tests for glycosides [22]
2 ml of Glacial acetic acid and 1 ml of ferric chloride were transferred into 1 ml of sample solution and then 1 ml of concentrated sulfuric acid was added. The appearance of blue-green color represents the presence of glycosides.
Tests of reducing sugars [22, 24, 25]
Ten drops of each solution A and B were added to a test tube containing 2 ml of sample solution. After heating for 15 min at 60 ± 0.5°C, orange red precipitate or green suspension was formed stipulating the existence of reducing sugars.
Tests of alkaloids [22–25]
Dragendorff’s test: The sample solution was acidified with diluted hydrochloric acid. The mixture was heated on a water bath and then filtered through a Whatman no. 1 filter paper. Equal volumes of the resulting solution and Dragendorff’s reagent were reacted. The formation of an orange red precipitate indicates the existence of alkaloids.
Mayer’s test: Equal volumes of the resulting solution and Meyer’s reagent were mixed. The turbidity or a yellow precipitate indicates the presence of alkaloids.
Tests of saponins [22–25]
Frothing test: 5 ml of Distilled water was added to a test tube containing 2 ml of sample solution. The mixture was shaken for 5 min to observe the formation of 1-cm-thick layer of stable liquid foams.
Tests of terpenoids [22, 23, 25]
1.5 ml of Sample solution was mixed with 1 ml of chloroform and then 1 ml of concentrated sulfuric acid was slowly added to form a reddish-brown layer at the junction specifying the presence of terpenoids.
Tests for flavonoids [22–25]
Ferric chloride test: 2 ml of Sample solution was treated with 1 ml of 10% w/v ferric chloride solution. Formation of a wooly brownish precipitate indicates the presence of flavonoids.
Shinoda’s test: 1.5 ml of Sample solution was treated with 1 ml of methanol. The solution was warmed and magnesium ribbons was added. 5 Drops of concentrated hydrochloric acid were carefully added and orange or red color was observed for flavonoids.
Tests for steroids [22, 25]
1.5 ml of Chloroform was mixed with 1.5 ml of sample solution. 0.5 ml of Acetic anhydride and 1 ml of 10% w/v sodium hydroxide solution were added. After mixing and standing for 10 min, the appearance of a blue green ring indicates the presence of steroids.
Fourier-transform infrared spectroscopy (FTIR) analysis
With regard to produce potassium bromide (KBr) pellets of A. paniculata and T. crispa extracts, approximately 2 to 3 mg of each dried extract was amalgamated with 100 mg of dried KBr using a mortar and pestle and then the KBr/extract mixture was compressed into a thin transparent disc under a hydraulic press. The characteristic functional groups of both extracts were analyzed using a FTIR spectrometer (Thermo Electron Scientific Instruments Corporation, Madison, WI, USA) at the frequency region of 4000 − 400 cm− 1.
Total phenolic contents
Total phenolic contents of A. paniculata and T. crispa extracts were ascertained as mg of gallic acid equivalents per g of dried extract (mg GAE/g dried extract), in consonance with an improved Folin-Ciocalteu method [26]. A standard curve was created using gallic acid solutions which dissolved in methanol at concentrations between 20 and 100 µg/ml. For sample estimation, 50 µl of 1 mg/ml extract solution was completely mixed with 50 µl of 50% v/v Folin-Ciocalteu reagent at 25 ± 1°C for 5 min. The solution was combined with 100 µl of 7.5% w/v sodium carbonate solution and then incubated in the dark for 90 min at the same temperature. The absorbance at 765 nm wavelength was measured using a UV-visible spectrophotometer (Model U-2990, Hitachi, Japan).
Antioxidant activities
DPPH assay
Free radical scavenging activities of samples were assessed by a modified DPPH method [26]. L-Ascorbic acid in methanol was employed as a positive control and scavenging activities of samples were evaluated using their calibration curves and expressed as SC50 values (µg/ml), the concentration required to reduce the initial DPPH radical concentration by 50%. A volume of 300 µl of each sample solution at various concentrations from 1.25 to 10 mg/ml in methanol was pipetted into 2.7 ml of 0.5 mM DPPH methanolic solution. After mixing, the solutions were allowed to stand in the dark for 30 min at 25 ± 1°C. Absorbance values of solutions were read against a methanol blank at 515 nm using a UV-visible spectrophotometer (Model U-2990, Hitachi, Japan). The measurements were conducted in triplicate.
FRAP assay
Ferric reducing antioxidant power (FRAP) assay was carried out in keeping with our foregoing report [26]. In short, the FRAP reagent consisted of 10 mM TPTZ in 40 mM hydrochloric acid, 20 mM ferric chloride hexahydrate in ultrapure water, and 0.3 M acetate buffer (pH 3.6) in the volume ratio of 1:1:10. A volume of 90 µl of FRAP reagent was incubated with 30 µl of 1 mg/ml sample solution in 96-well plates in the dark at 37 ± 1 ℃ for 30 min. The absorbance values determined at 593 nm using a multimode microplate reader (VICTOR® Nivo™, Perkin Elmer, UK) were calculated by subtracting a reagent blank value. L-Ascorbic acid dissolved in methanol was used as a positive control and all determinations were attained in triplicate. FRAP values expressed as in micrograms of ascorbic acid equivalent per gram of a dried extract (µg AAE/g dried extract).
Anti-inflammatory activities
Inhibition of protein denaturation was determined according to our previous procedure [27] and the inhibitory activities were expressed as IC50 (mg/ml), the concentration of the extract producing 50% inhibition of the protein denaturation. Briefly, the reaction mixture was comprised of 0.2 ml of fresh egg albumin, 2.8 ml of phosphate buffered saline (pH 7.4), and 2 ml of sample solution with a concentration range varying between 0.2 and 4 mg/ml. All mixtures were incubated at 37°C ± 1°C for 15 min and then heated at 70 ± 1°C for 5 min. After cooling to room temperature, the absorbance values were determined at 660 nm using a UV-visible spectrophotometer (Model U-2990, Hitachi, Japan). Ultrapure water and diclofenac diethylamine were performed as negative and positive controls, respectively.
Quantification of heavy metals
After nitric acid assisted closed vessel microwave digestion, the concentrations of arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb) in samples were analyzed by inductively coupled plasma - mass spectrometry (ICP-MS) as described in our previous report [26]. The standard solutions at five different concentrations used for establishing the calibration curves for heavy metals were prepared by diluting an ICP multi-element standard solution XIII with 5% v/v nitric acid solution. All samples were digested by a microwave digester (Model ETHOS ONE, Milestone Corporation, Sorisole, Italy) and determined by an ICP-MS spectrometer (Model 7500ce, Agilent Technologies, Santa Clara, USA) in triplicate.
Microbial limit test
The microbiological examination including total aerobic mesophilic microorganisms (bacteria, yeast & molds), Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Clostridium spp. was done in accordance with the microbial enumeration test of the United States Pharmacopeia (USP) 41 [28].
Evaluation of the cytotoxicities of bitter extracts and nail lacquers containing bitter extract on human dermal fibroblasts
The cellular viability of human dermal fibroblasts was evaluated upon treatment with either bitter extract or bitter nail lacquer using MTT colorimetric assay [29]. ATCC® CRL-2076 cells (Manassas, VA, USA) were seeded at density of 1 × 104 cells/well in a 96 well plate and incubated with 100 µl supplemented IMDM until the confluency reached 80–90%. Samples were separately serially diluted with IMDM to obtain the appropriate concentration ranges for testing. After treatments, the cells were incubated for 24 h at 37°C, 5% CO2. 10 µl of MTT solution (5 mg/ml) was put into each well and incubated at 37°C for 2 h. The solution was then removed and 100 µl of DMSO was subsequently added to dissolve the formazan crystals, which are generated by mitochondria of viable cells. The absorbance was measured at 550 nm using a fusion universal microplate analyzer (Model A153601, Packard BioScience Company, Connecticut, USA) and the percentages of cell viability were calculated compared with untreated controls.
Formulation of nail lacquers
Preparation of extract-free nail lacquers
Film-forming solutions with various concentrations (10, 15, 20, 25, and 30% w/w) of shellac were prepared by dissolving different weights of dewaxed bleached shellac in the required amount of 95% v/v ethanol using a magnetic stirrer (Stuart Overhead Stirrer Model SS20, Staffordshire, UK) with rotational speed at 60 rpm. To allow for a comparison of drying time and weight gain (Table 1), three replicates (samples) were produced.
Table 1
Evaluation of drying time and weight gain of extract-free nail lacquers
Film-forming solution No. | Concentrations of shellac (% w/w) | Drying time (min) | Weight gain (g) |
1 | 10 | 15 | 0.0090 ± 0.0010 |
2 | 15 | 16 | 0.0153 ± 0.0006 |
3 | 20 | 17 | 0.0217 ± 0.0025 |
4 | 25 | 21 | 0.0347 ± 0.0067 |
5 | 30 | 26 | 0.0387 ± 0.0064 |
Preparation of nail lacquers containing bitter extract
The formulations containing either A. paniculata extract or T. crispa extract were performed as per formula delineated in Table 2. The mixture of shellac and bitter extract was dissolved in 95% v/v ethanol using a magnetic stirrer at a constant speed 60 rpm for at least 6 h until well combined. After mixing, the total volume of mixture was adjusted to the final desired amount by adding 95% v/v ethanol. The homogeneities, viscosities, and bitterness intensities of formulations containing bitter extract are shown in Table 2.
Table 2
Composition, homogeneity, viscosity, and bitterness of nail lacquers containing bitter extract
Formulation No. | Concentrations (% w/w) | Homogeneity* | Viscosity* | Bitterness* |
A. paniculata extract | T. crispa extract | Shellac |
1 | 10 | | 10 | +++ | + | + |
2 | 10 | | 15 | ++ | ++ | ++ |
3 | 10 | | 20 | ++++ | +++ | ++ |
4 | 10 | | 25 | + | +++ | + |
5 | 20 | | 10 | +++ | + | + |
6 | 20 | | 15 | ++ | ++ | +++ |
7 | 20 | | 20 | ++++ | +++ | ++++ |
8 | 20 | | 25 | + | ++++ | ++ |
9 | 30 | | 10 | Precipitation | + | + |
10 | 30 | | 15 | Precipitation | ++ | + |
11 | 30 | | 20 | Precipitation | +++ | + |
12 | 30 | | 25 | Precipitation | ++++ | + |
13 | | 10 | 10 | +++ | + | + |
14 | | 10 | 15 | +++ | ++ | ++ |
15 | | 10 | 20 | ++ | +++ | ++ |
16 | | 10 | 25 | + | +++ | + |
17 | | 20 | 10 | +++ | + | + |
18 | | 20 | 15 | +++ | ++ | +++ |
19 | | 20 | 20 | +++ | +++ | ++++ |
20 | | 20 | 25 | + | ++++ | + |
21 | | 30 | 10 | Precipitation | + | + |
22 | | 30 | 15 | Precipitation | ++ | + |
23 | | 30 | 20 | Precipitation | +++ | + |
24 | | 30 | 25 | Precipitation | ++++ | + |
*Grade offers four levels of quality: high (++++), moderate (+++), low (++), and very low (+) |
Development of nail lacquers containing bitter extract
The nail lacquers containing 20% w/w bitter extract and 20% w/w shellac in 95% v/v ethanol (formulations No. 7 and 19) were chosen for this study pursuant to their expected homogeneity, viscosity, and bitterness (Table 2). A study of a similar nail lacquer with added polypropylene glycol (PPG) in two concentrations (5% w/w and 10% w/w) showed that PG significantly reduced bitterness (data not shown). Nevertheless, PVP K-30 was used as a copolymer and cosolvent for enhancing the bitterness of nail lacquers and improving the solubility of bitter extracts. The formulation trials were carried out as per the formula described in Table 3. The mixture of shellac and PVP K-30 and the bitter extract were separately dissolved in 95% v/v ethanol in the required quantity using a magnetic stirrer with adjustable speed range of 50–60 rpm. Two resulting solutions were thoroughly mixed to ensure homogeneity and then made up to 100 g with 95% v/v ethanol. The developed nail lacquer was stirred until all parts of the solution were homogeneous and transferred to a tightly closed amber glass bottle with narrow mouth and plastic screw cap.
Table 3
Composition of developed nail lacquers containing bitter extract and copolymer
Formulation No. | Concentrations (% w/w) |
A. paniculata extract | T. crispa extract | Shellac | PVP K-30 |
7A | 20 | | 10 | 10 |
7B | 20 | | 15 | 5 |
19A | | 20 | 10 | 10 |
19B | | 20 | 15 | 5 |
Physicochemical and mechanical evaluation of developed nail lacquers [30]
Each prepared formulation was gently applied in the same direction on an acrylic fake nail with a brush. After hardening of a film at 25 ± 1°C without any materials adhering to the finger, drying time and weight gain were determined. Drying time or dry-to-touch time was measured using a stopwatch. Weight gain measurements were carried out by weighing the samples to the nearest 0.0001g with an analytical balance (Sartorius BP210S Electronic Balance, Sartorius Group, Göttingen, Germany) before and after applying formulations to each fake nail. The nail lacquer formulations were determined for their physical characteristics including homogeneity and color by visual inspection and pH values using a pH meter (SevenEasy, Mettler-Toledo, Switzerland). The viscosities of formulations were measured by using a Brookfield DV-III Ultra Programmable rheometer (Model RVDV-III Ultra, Brookfield engineering laboratories, Inc., Massachusetts, USA). All measurements were done in triplicate. A dial thickness gauge (Model G (0.01-10 mm), Peacock, Ozaki MFG. Co., Ltd., Tokyo, Japan) was used to measure dry film thickness. Tensile testing using a texture analyzer (TA.XT.plus, Stable Micro Systems Ltd., Surrey, UK) was performed to determine stress values of films formed by nail lacquers. The viscosity, pH, drying time, weight gain, film thickness, and stress of each developed formulation are provided in Table 4.
Table 4
Physicochemical and mechanical properties of developed nail lacquers containing bitter extract and copolymer
Formulation No. | pH | Viscosity (cP) | Drying time (min) | Weight gain (g) | Film thickness (mm) | Stress (N/mm2) |
7A | 5.09 ± 0.02 | 96.59 ± 6.88 | 8.00 ± 1.00 | 0.0234 ± 0.0022 | 0.9467 ± 0.2210 | 0.079 ± 0.013 |
7B | 4.96 ± 0.01 | 45.34 ± 4.72 | 6.33 ± 0.58 | 0.0183 ± 0.0022 | 0.6483 ± 0.1179 | 0.136 ± 0.047 |
19A | 4.66 ± 0.02 | 215.8 ± 6.34 | 10.67 ± 1.15 | 0.0488 ± 0.0012 | 0.7963 ± 0.0132 | 0.233 ± 0.050 |
19B | 4.52 ± 0.10 | 101.04 ± 3.64 | 7.67 ± 0.58 | 0.0124 ± 0.0007 | 0.6417 ± 0.0534 | 0.553 ± 0.255 |
The water resistance test was done by applying a tested nail lacquer onto a Teflon tray, leaving it to dry, peeling it off, cutting it to the same size, weighing each piece of film (known dry weight, Wo), placing the lacquer films in each testing basket, then immersing the baskets in distilled water at 25 ± 1°C using a disintegration tester (ZTx20 series, Erweka GmbH, Heusenstamm, Germany). The higher the percentage of the remaining weight in distilled water, the better the water resistance. In vitro bitterness release test was performed in simulated saliva (pH 6.8) at 37 ± 1°C according to the above method. The lower the percentage of the remaining weight in simulated saliva, the greater the bitterness release. The dried lacquer film was weighed and dedicated as dry weight after testing (Wt) in distilled water or simulated saliva and then the percentage of the remaining weight was calculated as illustrated in Eq. (1).
% remaining weight = (Wt / Wo) × 100 (1)
The percentages of the remaining weight obtained from water resistance and bitterness release tests are illustrated in Table 5.
Table 5
Percentages of the remaining weight of lacquer films after testing in distilled water (at 25 ± 1°C) and simulated saliva (at 37 ± 1°C)
Formulation No. | Water resistance test (in distilled water) | | Bitterness release test (in simulated saliva) |
Percentages of the remaining weight (%) | Time (min) | | Percentages of the remaining weight (%) | Time (min) |
7A | 0.00 ± 0.00 | 180 | | 0.00 ± 0.00 | 60 |
7B | 58.60 ± 12.37 | 180 | | 0.00 ± 0.00 | 40 |
19A | 10.86 ± 11.88 | 180 | | 0.00 ± 0.00 | 60 |
19B | 74.20 ± 2.31 | 180 | | 0.00 ± 0.00 | 40 |
Evaluation of film appearances and bitterness intensities in human volunteers
A total of 20 healthy participants (10 males and 10 females) ranging in age from 18 to 30 years volunteered to participate in the study. All of them were non-Muslims and had no prior history of allergic reactions to alcohol, food, medicines, natural extracts, and cosmetic ingredients. In addition, they were advised to steer clear of drinking (except water) and eating for a time no less than 1 h before starting the test. Each formulated nail lacquer was applied on participants’ thumb nails once a day. After the nail lacquer had dried, participants evaluated the film appearances and bitterness intensities of nail lacquer films by finger touching, visualizing, and sucking, in line with their own perceptions and then answered questionnaires (Fig. 2). Sensory assessments were performed in triplicate. The experimental protocol (REC 62.0912-038-4567) was approved by the Human Research Ethics Committee, Silpakorn University, Thailand.
Determination of stability
The developed nail lacquers were stored individually in tightly closed amber glass containers. The physical stability of samples was evaluated by heat-cool cycling for six cycles between temperature of 4 ± 1°C and 45 ± 1°C/75 ± 2% RH (relative humidity) with storage at each temperature for 24 h. The samples were then analyzed for their pH values, viscosities, phase separation, and colors (Table 6). DPPH free radical scavenging and protein denaturation inhibitory activities of developed formulations were determined, before and after the stability test (Table 7). In addition, heavy metal concentrations and microbial loads in developed formulations were also examined.
Table 6
pH Values, viscosities, phase separation, sediment volumes, and colors of developed nail lacquers after six cycles of heating/cooling treatment
Formulation No. | pH | Viscosity (cP) | Phase separation | Color |
7A | 5.07 ± 0.01 | 85.90 ± 9.41 | No separation | Dark green |
7B | 4.96 ± 0.01 | 31.07 ± 9.20 | No separation | Dark green |
19A | 4.57 ± 0.01 | 179.97 ± 12.44 | No separation | Dark brown |
19B | 4.52 ± 0.01 | 65.84 ± 9.37 | No separation | Dark brown |
Table 7
DPPH free radical scavenging and protein denaturation inhibitory activities of developed nail lacquers before and after six cycles of heating/cooling treatment
Formulation No. | DPPH radical scavenging SC50 | | Albumin denaturation IC50 |
Before | After | | Before | After |
7A | 1.46 ± 0.06 mg/ml | 2.11 ± 0.07 mg/ml | | 4.94 ± 0.18 mg/ml | 5.03 ± 0.20 mg/ml |
7B | 1.54 ± 0.08 mg/ml | 2.34 ± 0.10 mg/ml | | 5.09 ± 0.24 mg/ml | 5.18 ± 0.27 mg/ml |
19A | 0.82 ± 0.07 mg/ml | 1.92 ± 0.04 mg/ml | | 2.95 ± 0.12 mg/ml | 5.13 ± 0.24 mg/ml |
19B | 0.89 ± 0.09 mg/ml | 1.99 ± 0.08 mg/ml | | 3.06 ± 0.17 mg/ml | 5.34 ± 0.29 mg/ml |
Ascorbic acid | 4.47 ± 0.08 µg/ml | | ND |
Diclofenac diethylamine | ND | | 0.69 ± 0.01 mg/ml |
ND: not determined |
Statistical analysis
Experimental data is shown as mean ± standard deviation (SD) of three independent experiments. Statistical significance (p < 0.05) was estimated by one-way analysis of variance (ANOVA) using SPSS 16.0. The Duncan’s test was employed to measure specific differences between pairs of means.