Extraction, Biological Activity, Formulation, and Evaluation of Bitter Herbs as Anti-Nail-Biting Lacquers

Background: Nail biting can increase the risk of infection and abnormal-looking nails. The purpose of the present investigation was to formulate and evaluate anti-nail-biting lacquers consisting of bitter herbal extracts. Methods: The hydroalcoholic extracts obtained from Andrographis paniculata and Tinospora crispa were determined for phytochemical constituents, total phenolic contents, antioxidant activities, anti-inammatory activities, and cytotoxicities. Anti-nail-biting lacquers were prepared by using herbal extracts (bittering agent), shellac (lm forming polymer), ethanol (volatile solvent), and other indispensable additives with continuous stirring. Thus, attempts to enhance the lm property and bitterness is accomplished by using polyvinylpyrrolidone K30 as a copolymer and varying concentrations of herbal extracts. Good accepted formulations were established for drying time, pH, viscosity, smoothness of lm, lm strength, water resistant, and solubility in simulated saliva and then evaluated their bitterness in human volunteers. Results: Phytochemical constituents including tannins, glycosides, reducing sugars, alkaloids, terpenoids, and avonoids were found present in both extracts while saponins was only detected in A. paniculata extract. Although T. crispa extract exhibited a signicantly higher (p < 0.05) total phenolic content and antioxidant activity than A. paniculata extract, it showed lower protein denaturation inhibition property than A. paniculata extract. Because of the potentials of both extracts without cytotoxicity, anti-nail-biting lacquers containing either A. paniculata extract or T. crispa extract were developed and evaluated. Drying time of formulations were 6-11 min with visually seen glossiness of formulation. Formulations of the nail lacquer showed good pH, viscosity, smoothness of lm, lm strength, water resistant, and solubility in simulated saliva. The formulations displaying no signicant cytotoxicity effect on CRL-2076 cells were assessed on healthy human volunteers to compare bitterness and lm characteristics. Conclusion: The optimized formulation containing A. paniculata one-way

Results: Phytochemical constituents including tannins, glycosides, reducing sugars, alkaloids, terpenoids, and avonoids were found present in both extracts while saponins was only detected in A. paniculata extract. Although T. crispa extract exhibited a signi cantly higher (p < 0.05) total phenolic content and antioxidant activity than A. paniculata extract, it showed lower protein denaturation inhibition property than A. paniculata extract. Because of the potentials of both extracts without cytotoxicity, anti-nail-biting lacquers containing either A. paniculata extract or T. crispa extract were developed and evaluated. Drying time of formulations were 6-11 min with visually seen glossiness of formulation. Formulations of the nail lacquer showed good pH, viscosity, smoothness of lm, lm strength, water resistant, and solubility in simulated saliva. The formulations displaying no signi cant cytotoxicity effect on CRL-2076 cells were assessed on healthy human volunteers to compare bitterness and lm characteristics.
Conclusion: The optimized formulation containing A. paniculata extract could successfully achieve good lm forming property and bitterness release which is considered promising for stopping nail biting.

Background
Onychophagia (nail biting) means putting one or several ngers in the mouth and biting on nail with teeth. It is a chronic behavioral disorder in children and adults that commonly co-occur with thumb and nger sucking [1]. These behaviors probably associated with psychiatric disorders can cause paronychia, onycholysis, onychomycosis, gingivitis, temporomandibular dysfunction, oral carriage of Enterobacteriaceae, parasitic infection, and some negative social impacts [2][3][4][5].
There are several approaches to cease from nail biting and nger sucking, such as painting a bitter tasting lacquer containing denatonium benzoate and sucrose octa-acetate onto the nails [2] and utilizing a nonremovable reminder, e.g. wristband, nger guard, and glove [6]. However, bitter nail lacquers containing denatonium benzoate or sucrose octa-acetate for utilize as nail-biting and thumb-sucking deterrents cannot be generally recognized as safe and effective because there is lack of information acquired from adequate and well-controlled, double-blind studies [7]. As in the previous study, although the method of applying a distasteful lacquer exhibited higher drop-out rate than the method of wearing a non-removable reminder, it was more effective to quit the nail-biting habit by examining only non-dropouts [6]. This study indicated that the bitter nail lacquer was a potential alternative to prevent the nail-biting habit.
Currently, the pitfalls of anti-nail-biting lacquers were unclear toxicity of synthetic polymers and bitter substances, apply several times daily due to easy to wash off, and cross contamination of nail lacquers during cooking and eating [6]. Consequently, anti-nail-biting lacquers mainly composed of natural edible ingredients have been developed in order to reduce the toxicity, enhance bitterness, improve water resistance and dissolution in simulated saliva, and also increase other biological activities.
A chronic habit of nail biting or nger sucking causes dry and peeling skin and leads to in ammation of the skin surrounding the nail [8]. Plenty of unpalatable herbs grown throughout Southern and Southeastern Asia exhibited various biological effects, especially antioxidant and anti-in ammatory activities. Andrographis paniculata, commonly known as king of bitters was found to possess antioxidant activities associated with an increase in the activity of antioxidant enzymes including catalase, glutathione S-transferase, and superoxide dismutase [9]. It could also safeguard cutaneous cells from in ammation [10]. Tinospora crispa was reported to have antioxidant activity through radical scavenging and metal chelating mechanisms and anti-in ammatory activity via increasing intracellular expressions of cytokine, INF-g, IL-6, and IL-8 [11]. Antioxidant and anti-in ammatory activities of extracts from A. paniculata and T. crispa were associated with the contents of phenolics and other phytochemicals [9,11]. In this study, herbal extracts obtained from A. paniculata and T. crispa were selected as active ingredients in anti-nail-biting lacquers because of their bitterness and biological activities. One purpose of this study was to determine antioxidant and anti-in ammatory activities of selected herbal extracts.
A coating of nail lacquer can protect thin, friable, vulnerable, and irregular nails along with giving a sustainable exterior layer in order to make them look stronger and more beautiful [12]. The chemical properties of nail lacquers are established on polymerization, evaporation, and adhesion [13]. Monomers form strong bonds with other molecules through polymerization reactions resulting cross-linking of polymer lms. After applying a nail lacquer, a solid polymer lm is formed by solvent evaporation. A lm forming polymer can adhere to the nail plate.
Nail lacquers generally consists of lm-forming polymers, volatile solvents, plasticizing agents, and dyes [14,15]. Coating formulas often contain pliable resins so as to amplify adhesion and offer glossy appearance. Because of optimum drying time, solvents including ethyl acetate, butyl acetate, and isopropyl alcohol are normally employed for dissolving various resins and other components of nail lacquers [15,16]. Plasticizers such as camphor, triphenyl phosphate, trimethyl pentanyl diisobutyrate, and acetyl tributyl citrate can enhance exibility and durability to the lms. Dyes or colorants are coloring substances dissolved in nail lacquers and adsorbed onto the nail plates to which they are applied [14,15].
With regard to help people safely utilize nail lacquers, it is important to study the materials engaged in the production of nail lacquers. Synthetic resins (e.g. toluenesulfonamide-formaldehyde, polyvinyl butyral, and polyester resins) may lead to paronychia, onycholysis, and onychodystrophy [17]. Toxic plasticizers (e.g. phthalate and organophosphate) and harmful solvents (e.g. toluene and formaldehyde) have been prohibited or restricted from use in nail lacquers because of their adverse effects on skin, reproductive system, embryonic development, thyroid gland, and central nervous system [18]. People may have a considerable risk of being exposed to detrimental polymers, plasticizers, and solvents in nail lacquers especially if they are swallowed. Therefore, this study was carefully selected the safe ingredients, aiming for the safety and quality of nail lacquers.
Natural lac is a resin secreted by lac insects (e.g. Laccifer Lacca Kerr and Kerria Lacca,) [19]. Seed lac derived from stick lac is processed into shellac, a natural gum resin, by handcrafted, heat or solvent methods. The special properties of shellac include energetically favorable adhesion to the surface, water protection, and shimmering appearance. Furthermore, shellac has been accepted by US Food and Drug Administration (US FDA) and European Food Safety Authority (EFSA) to utilize in pharmaceuticals and food [19,20]. According to the consumer product safety, synthetic resins have been superseded by durable natural polymers, especially shellac, which could be developed to improve drying time, adhesiveness, and exibility of nail lacquers [15]. In case of solvent selection, ethanol could be employed as a solvent in nail lacquers because of its rather low toxicity compared with other organic solvents used for nail lacquers and e cacy to dissolve various active constituents in herbal extracts.
The objective of this study was to develop non-toxic anti-nail-biting lacquers containing bitter herbal extracts obtained from A. paniculata and T. crispa. Not only the selection of ingredients but also the evaluation of products is essential for product safety and quality. Therefore, this study was invented to determine the quality control parameters (e.g. drying time, pH, viscosity, smoothness of lm, lm strength, water resistant, and solubility in simulated saliva) of formulated nail lacquers and then evaluate the bitterness in human volunteers.

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 ne 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 sampleto-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 ltered using Whatman no. 1 lter paper to collect the ltrates 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][23][24][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][23][24][25] Dragendorff's test: The sample solution was acidi ed with diluted hydrochloric acid. The mixture was heated on a water bath and then ltered through a Whatman no. 1 lter 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][23][24][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.
Formation of a wooly brownish precipitate indicates the presence of avonoids.
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 avonoids.
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 Scienti c 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-

Anti-in ammatory activities
Inhibition of protein denaturation was determined according to our previous procedure [27] and the inhibitory activities were expressed as IC 50 (mg/ml), the concentration of the extract producing 50% inhibition of the protein denaturation. Brie y, 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.

Quanti cation 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 ve 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 broblasts
The cellular viability of human dermal broblasts 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 × 10 4 cells/well in a 96 well plate and incubated with 100 µl supplemented IMDM until the con uency 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% CO 2 . 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 Page 9/27 60 rpm. To allow for a comparison of drying time and weight gain (Table 1), three replicates (samples) were produced. 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 nal 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). 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 signi cantly 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. 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 lm at 25 ± 1°C without any materials adhering to the nger, drying time and weight gain were determined. Drying time or dry-to-touch time was measured using a stopwatch. Weight gain  Table 4. The water resistance test was done by applying a tested nail lacquer onto a Te on tray, leaving it to dry, peeling it off, cutting it to the same size, weighing each piece of lm (known dry weight, W o ), placing the lacquer lms 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 lm was weighed and dedicated as dry weight after testing (W t ) in distilled water or simulated saliva and then the percentage of the remaining weight was calculated as illustrated in Eq. (1).
% remaining weight = (W t / W o ) × 100 (1) The percentages of the remaining weight obtained from water resistance and bitterness release tests are illustrated in Table 5.

Evaluation of lm 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 lm appearances and bitterness intensities of nail lacquer lms by nger 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.

Plant extracts
Extraction yields and chemical constituents The extraction yields of A. paniculata and T. crispa ethanolic extracts were 7.75 ± 0.32% w/w and 3.50 ± 0.23% w/w, respectively. Appearances of both extracts were of dark greenish brown mass, as illustrated in Fig. 3. The extracts showed the presence of tannins, glycosides, reducing sugars, alkaloids, terpenoids, and avonoids except steroids. However, saponins were found in A. paniculata extract but absent in T. crispa extract.
The structures of compounds in A. paniculata and T. crispa extracts were identi ed by comparing their FTIR spectra with previously reported data. The FTIR spectra of both extracts (Fig. 4)  characteristic of aporphine alkaloids [32]. The results of this study were consistent with several earlier investigations showing that terpenoids and alkaloids are the most common bitter compounds found in A. paniculata and T. crispa, respectively [9][10][11].

Total phenolic contents and biological activities of bitter extracts
The total phenolic contents of A. paniculata and T. crispa extracts were evaluated based on the standard curve of gallic acid y = 0.0296x + 0.0796; R 2 = 0.9997. The total phenolic contents of T. crispa extract (64.43 ± 3.77 mg GAE/g dried extract) was found to be signi cantly higher (p < 0.05) than that of A. paniculata extract (21.05 ± 1.94 mg GAE/g dried extract). A. paniculata and T. crispa extracts obtained with 95% v/v ethanol contained greater amounts of phenolic compounds than previously reported extracts prepared with 70% v/v methanol (7.78 mg GAE/g dried extract) [33] and 80% v/v ethanol (29.83 ± 2.14 mg GAE/g dried extract) [34], respectively. Therefore, 95% v/v ethanol seemed to be an appropriate solvent for extraction of natural phenolics from A. paniculata and T. crispa. The lower value of albumin denaturation IC 50 indicates the higher ant-in ammatory activity. It was observed that T. crispa extract with IC 50 value of 0.90 ± 0.01 mg/ml displayed signi cantly stronger (p < 0.05) anti-in ammatory activity than A. paniculata extract with IC 50 value of 1.03 ± 0.16 mg/ml, while diclofenac diethylamine is 0.68 ± 0.00 mg/ml as a comparison.
Many earlier studies showed the positive correlation between total phenolic content, antioxidant activity, and anti-in ammatory property [33][34][35]. In consistent with previous studies, the present study revealed that T. crispa extract with higher total phenolic content exhibited stronger antioxidant and antiin ammatory activities than A. paniculata extract with lower total phenolic content.

Heavy metals and microbial loads of bitter extracts
Maximum limits of Hg (1 mg/kg), Pb (20 mg/kg), As (5 mg/kg), and Cd (5 mg/kg) in cosmetics have been set by Association of South East Asian Nations (ASEAN) guidelines [36]. The concentrations of Pb and Cd in A. paniculata extract were 0.010 ± 0.001 mg/kg and 0.002 ± 0.002 mg/kg, respectively, but Hg and As were not detected. The heavy metal concentrations found in T. crispa extract were: Hg = 0.002 ± 0.002 mg/kg, Pb = 0.010 ± 0.009 mg/kg, and Cd = 0.001 ± 0.003 mg/kg, but As was not found. The concentrations of heavy metals in both extracts were below the permissible values.
ASEAN microbiological limits in products for children under 3 years, eye area and mucous membranes were: total aerobic mesophilic microorganisms not more than 500 colony forming unit per gram (cfu/g) and speci ed pathogens including P. aeruginosa, S. aureus, C. albicans absent in 0.1g of test sample [36].
Owing to Thailand local concerns, the extracts were performed the additional test for Clostridium spp. A. paniculata and T. crispa extracts showed total aerobic mesophilic microorganisms below 10 cfu/g, but all speci ed pathogens including P. aeruginosa, S. aureus, C. albicans, and Clostridium spp. were not detected in any of the samples analyzed. The results were consistent with the microbiological requirements. In conclusion, both extracts were safe from harmful heavy metals and pathogens.

Cytotoxicities of bitter extracts
This experiment was performed to assess the cytotoxic activities of bitter extracts at various concentrations on CRL-2076 human dermal broblasts. The percentages of cell viability upon 24-h treatments with A. paniculata and T. crispa extracts are illustrated in Figs. 5A and 5B, respectively. The viability of cells treated with A. paniculata (0.05 mg/ml) and T. crispa (0.63 mg/ml) extracts was not signi cantly different from the control. Their % survival values were 92% and 83%, respectively. Thus, the results showed that A. paniculata and T. crispa extracts at the concentration ranges of 0.03-0.05 mg/ml and 0.16-0.63 mg/ml, respectively, exhibited low levels of cytotoxicity upon 24-h incubation.

Developed nail lacquers
Formulation development of nail lacquers The extract-free nail lacquers were prepared by dissolving shellac in 95% v/v ethanol to obtain lmforming solutions in the concentration range of 10-30% w/w. As delineated in Table 1, the shellac concentration affects the drying time and weight gain. The results revealed that the higher the shellac concentration, the longer the drying time, the greater the weight gain. Film-forming solution No. 5 composed of shellac concentration up to 30% w/w produced a very thick lm with the highest weight gain that was prone to peeling. Furthermore, its drying time was too long and it was too viscous to apply. Therefore, lm-forming solution No. 5 was cut off from the next experiment.
The nail lacquers containing A. paniculata extract (formulations No. 1-12) and T. crispa extract (formulation No. [13][14][15][16][17][18][19][20][21][22][23][24] were performed by a simple mixing technique and their homogeneities, viscosities, and bitterness intensities were evaluated at room temperature. The results are depicted in Table 2  Physicochemical and mechanical evaluation of developed nail lacquers . It was seen that as the PVP K-30 concentration increases from 5% w/w to 10% w/w and the shellac concentration decreases from 15% w/w to 10% w/w the weight gain increases (Tables 3 and 4). The results revealed that non-volatile content of nail lacquers varied depending on the type and concentration of polymer used. The thicknesses of the lms obtained from all developed formulations were varied in the range between 0.6417 ± 0.0534 and 0.9467 ± 0.2210 mm ( Table 4). The thickness of lm was found to be consistent for all formulations. It can be concluded that the variation of lm thicknesses was solely affected by the ratio of shellac : PVP K-30 and the amount of herbal extract employed in nail lacquers. The results also revealed that lm thickness decreased as the percentage of shellac increased, perhaps because of the lower solid contents of shellac [19] and T. crispa extract compared to PVP K-30 and A. paniculata extract, respectively. The lm obtained from the formulation No. 7A (20% w/w A. paniculata extract, 10% w/w shellac, and 10% w/w PVP K-30) had the greatest thickness value (0.9467 ± 0.2210 mm).
Tensile strength designated as a stress is estimated as units of applied force per area (N/mm 2 ). Measured stress values of lms derived from developed nail lacquers ranged from 0.079 ± 0.013 to 0.553 ± 0.255 N/mm 2 ( Table 4). The lm stress increased with increasing the proportion of shellac (Tables 3 and 4). If lm stress is too high, it can lead to lm cracking [39]. Accordingly, lower stress value might possibly lead to higher mechanical exibility of lms. respectively. Although, the formulation No. 7A was the least water-resistant nail lacquer (remaining weight 0.00 ± 0.00%), it was able to withstand distilled water for up to 180 min that fell within acceptable criteria.
Bitterness release test was conducted in simulated salivary uid pH 6.8 and the results are illustrated in  Heavy metals and microbial loads of developed nail lacquers The contents of Pb (0.001 ± 0.004 mg/kg) and Cd (0.001 ± 0.005 mg/kg) in developed formulations were lower than the permissible limits of ASEAN guidelines for cosmetics [36] while Hg and As were not detected. Total aerobic mesophilic microorganisms and speci ed pathogens of developed formulations were within ASEAN microbiological limits. Therefore, all developed nail lacquers were found to be safe and acceptable, especially in children.

Stability studies
The stability studies were obtained to nd out the alteration of pH values, viscosities, phase separation, colors, and biological activities. The results are shown in Tables 6 which indicates that there were no major changes in physical appearances and pH values except for a signi cant decrease in viscosities. After six cycles of heating/cooling stability testing, the developed nail lacquers clumped into precipitates but they were more uniformly distributed after hand-shaking. Therefore, all developed formulations might require gentle shaking before use.
DPPH free radical scavenging and protein denaturation inhibitory activities of developed formulations subjected to accelerated stability were investigated and compared with freshly prepared nail lacquers. The results obtained are detailed in Table 7. All developed formulations showed signi cant increase (p < 0.05) in DPPH radical scavenging SC 50 and albumin denaturation IC 50 without exhibiting microbial growth on the investigation. Even though their biological activities and viscosities were reduced, they were within the acceptable ranges. The results of stability studies showed that there were no serious stability problems of all developed formulations. The recommended storage condition for the developed formulations was 4°C in order to preserve their biological activities.

Conclusions
Anti-nail-biting lacquers containing bitter herbal extracts were successfully prepared using shellac and PVP K-30 as lm formers. Formulation No. 7A containing 20% w/w A. paniculata extract, 10% w/w shellac, and 10% w/w PVP K-30 was the most appropriate formulation in relation to preventing nail biting. A. paniculata and T. crispa extracts loaded in formulations at 20% w/w possessed antioxidant and antiin ammatory properties, harmonizing the traditional medicine practice. After six cycles of heating/cooling treatment, the analysis of all developed formulations has revealed the existence of biological activities.
Moreover, all developed formulations had gratifying appearances with consistency and without the tendency of separation.