**1. Introduction**

Emulsions are the most common form of skin care products [1]. A variety of cosmetic emulsions are utilized for functional applications, such as sebum control, skin whitening, and UV protection [2]. Cosmetic emulsions are mainly classified as oil-in-water (O/W) [3], water-in-oil (W/O) [4], or water-in-silicone (W/S) emulsions [5,6]. Emulsions applied in

**Citation:** Chaiwong, N.;

Phimolsiripol, Y.; Leelapornpisid, P.; Ruksiriwanich, W.; Jantanasakulwong, K.; Rachtanapun, P.; Seesuriyachan, P.; Sommano, S.R.; Leksawasdi, N.; Simirgiotis, M.J.; et al. Synergistics of Carboxymethyl Chitosan and Mangosteen Extract as Enhancing Moisturizing, Antioxidant, Antibacterial, and Deodorizing Properties in Emulsion Cream. *Polymers* **2022**, *14*, 178. https://doi.org/10.3390/ polym14010178

Academic Editors: Lorenzo Antonio Picos Corrales, Angel Licea-Claverie and Grégorio Crini

Received: 14 November 2021 Accepted: 6 December 2021 Published: 3 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

cosmetic applications, both O/W and W/O types, need to satisfy several requirements, such as having the rheology for skin, feeling good on the skin, having good spreadability, and long-term physical stability under various conditions. The ingredients are safe and do not cause skin irritation or any harmful effects [1]; O/W emulsions are the most commonly used in the cosmetic industry. Many cosmetic industries produce a wide variety of beauty products to care for and to avoid excessive sweating and body odor [7]. Deodorants are one of the cosmetic preparations containing substances or ingredients able to eliminate or reduce body odor [8]. Body odor is caused by the growth of microorganisms, and odors are associated with perspiration and its breakdown by bacteria in the armpits, feet, or any other part of the body [9]. Many products claim to have skin benefits, such as anti-aging [10], skin tightening, and moisturizing activity [11]. In general, the preparation of deodorant is carried out using emulsion systems, where the active ingredients are mixed with waxes, oils, and silicones and produced in the desired form, such as deodorant cream, gel, roll-ons, and sticks [12].

Carboxymethyl chitosan (CMCH) is a polymer synthesized by introducing a carboxymethyl group into the main structure of chitosan, achieved by carboxymethylation of the hydroxyl and amine of chitosan, [13] and shows a potential application in cosmetics [14]. The water-soluble property of carboxymethyl chitosan provides conclusive insights into the utilization of its properties of biocompatibility, biodegradation, biological activity, and low toxicity [15]. CMCH in the cosmetics industry seems to be a promising avenue to boost its application as a multifunctional ingredient. Different aspects of CMCH have been applied in five major directions, as a moisture retention agent, antimicrobial agent, antioxidant agent, delivery system, and naturally derived emulsion stabilizer [14]. However, there are many herbs which have antimicrobial properties, this being a primary prerequisite for the development of deodorant formulations.

The use of mangosteen extract (ME) as a raw material to be used as an active ingredient for the preparation of cosmetic products has been evaluated by Ghasemzadeh et al. [16]. It contains the active ingredients xanthones, tannins, and proanthocyanins, which are predominant in mangosteen. In addition, ME has also demonstrated antibacterial properties, reduced acne inflammation, prevented acne, and also contains antioxidants which help to firm the skin and reduce melanin production in the skin, which whitens the skin [17]. ME is made from *Garcinia mangostana* with a standardized solvent extraction process; the product is easy to use and can be mixed with all types of cosmetics [16]. The mangosteen pericarp contains many compounds with outstanding antioxidant, anti-inflammatory and antibacterial properties, especially for the bacteria *Propionibacterium acnes* and *Staphylococcus epidermidis*. The solid waste obtained after extraction of these compounds from the pericarp is also ideal for the production of useful activated carbon [18], while wastewater generated from the extraction process can be efficiently treated with synergistic catalysts [19]. Pothitirat et al. [20] revealed that ME can inhibit the cause of acne and reduce acne rash. Deodorants are substances applied to the body in order to affect body odor caused by bacterial growth and the smell associated with bacterial breakdown of sweat in armpits, feet, and other areas of the body. Ham et al. [21] also reported properties that help in the elimination of body odor. ME is commonly used in various products, such as body cleansing products, soaps, shower creams, facial cleansers, acne treatment products, acne gel, serum, and facial cream [22]. Industries have also been promoted with specially developed synthetic cosmetics as the main ingredient. There are many herbs that have antimicrobial properties, which must be a key factor for improving the deodorant property. Again, herbal formulas require deodorizing properties with activity close to synthetic formulas. However, the synergistic effect of antibacterial activity and deodorizing and moisturizing performance has not been fully investigated. Therefore, this research aimed to determine the effect of CMCH with ME used as moisturizing, antioxidant, antibacterial, and deodorant agents by studying various parameters, then developing final products and evaluating the efficacy of the cream emulsion system.

#### **2. Materials and Methods**

#### *2.1. Materials*

High-molecular-weight native chitosan (H, 310–375 kDa) with a degree of deacetylation above 90% was purchased from Kritnarong Limited Partnership, Phitsanulok, Thailand. Ethanol, methanol, isopropanol, sodium hydroxide, and glacial acetic acid were purchased from RCI Labscan (Bangkok, Thailand). Monochloroacetic acid and trans-2-nonenal were obtained from Merck KGaA (Darmstadt, Germany). Aluminium chlorohydrate, ceteareth-25, glyceryl monostearate, propylene glycol, and stearyl alcohol were bought from Thai Poly Chemicals (Samutsakhon, Thailand). All other reagents were of analytical grade. Six species of bacteria: *Corynebacterium* spp. (TISTR 1259), *Staphylococcus epidermidis* (TISTR 518), *Staphylococcus aureus* (ATCC 25923), *Bacillus subtilis* (DMST 15896), *Pseudomonas aeruginosa* (TISTR 781), and *Escherichia coli* (ATCC 25922) were obtained from Thailand Institute of Scientific and Technological Research (Pathum Thani, Thailand).

#### *2.2. Synthesis of CMCH*

H-CMCH was synthesized following the method of Chaiwong et al. [23]. Chitosan flake was ground and sieved to obtain a particle size under 60 mesh (Endecotts, UK). The chitosan (25 g) was suspended in 50% (*w*/*v*) sodium hydroxide solution (400 mL), and 100 mL of isopropanol was added and mixed well at 50 ◦C for 1 h. Monochloroacetic acid (50 g) was dissolved in isopropanol (50 mL) and gradually dropped into the reaction for 30 min, and the system was left to stand in reaction at 50 ◦C for 4 h. The reaction was stopped by adding 70% (*v*/*v*) methanol. The pH of the sample was later adjusted to 7.0 by 1% (*v*/*v*) glacial acetic acid. From that point, the solid was separated and washed with 70% (*v*/*v*) ethanol 5 times, 250 mL each time, and finally washed with 250 mL of 95% (*v*/*v*) ethanol for desalting and dried in a hot air oven (ED56, Binder GmbH, Tuttlingen, Germany) at 80 ◦C for 12 h. The functional groups of high-MW native chitosan and H-CMCH were measured using a Fourier transform infrared spectrometer (Frontier, PerkinElmer, Waltham, MA, USA) in the range of 500–4000 cm−<sup>1</sup> as shown in Figure S1. Chitosan was converted to CMCH. The –COO groups enhanced the hydrophilic properties of the CMCH. A certain amount (10 g) of H-CMCH powder was dissolved in 20 mL of deionized water. The suspension was mechanically stirred at 50 ◦C for 10 min by following the method of Rachtanapun et al. [15] for use as a moisturizing agent.

#### *2.3. ME Preparation*

Mangosteen fruits were purchased from a local market in Fresh Fruits Market, Chiang Mai, Thailand. Mangosteens with reddish purple skin were selected at the fifth color level according to the Thai Agricultural Standard for Mangosteen (TAS 2-2013) color index [24]. The mangosteen fruits were rinsed with distilled water to remove impurities such as dust before the pericarp was separated from the fruits manually. The mangosteen pericarp was chopped into small pieces and dried at 60 ◦C. The dried mangosteen pericarp was ground into powder using a blending machine. The powdered mangosteen pericarp (5 g) was thoroughly extracted using a sonication bath (SB25-12 DTD, Ningbo SCIENTZ Biotechnology Co., Ltd., Zhejiang, China) with 50% ethanol (200 mL). The filtrates were concentrated by a rotary evaporator (R-250, Buchi, Flawil, Switzerland) at 50 ◦C to give a crude extract (200 mg). The ME was kept in air-tight amber bottles and stored at −4 ◦C until use [20].

#### *2.4. Deodorant Cream (O/W) Preparation*

Cream base was prepared using an emulsification technique according to the method of Kassakul et al. [25]. First, all ingredients were weighed accurately by a calibrated analytical balance as shown in Table 1. The oil phase was prepared by mixing aluminium chlorohydrate, stearyl alcohol, ceteareth-25, glyceryl monostearate, and mineral oil and then heating to 70 ◦C. The aqueous phase was prepared by dissolving glycerin and propylene glycol in distilled water in a beaker and heating to 75◦C. Both phases (oil and aqueous) were heated up to the same temperature (45 ◦C) in a water bath; the aqueous phase was added into the oil phase gradually with stirring. Then, ME was added, and different concentrations of H-CMCH, including 0.5, 1.0, 1.5, 2.0, and 2.5% (H1, H2, H3, H4, and H5) were added into the cream base. Finally, the weight of cream was 100 g, and 0.01 mL of perfume was added into the mixture with continuous stirring for 20 min until the cream cooled to 25 ◦C.

**Table 1.** Deodorant cream formulas with different content of H-CMCH (0.5, 1.0, 1.5, 2.0, and 2.5% (*w*/*v*)).


#### *2.5. Degree of Skin Moisturizing (DM)*

The degree of skin moisturizing of the deodorant cream was examined with 0.5, 1.0, 1.5, 2.0, and 2.5% (*w/v*) of H-CMCH on pig skin and compared with untreated skin, water, and propylene glycol. The pig skins were prepared from the back side of the pig ear obtained from local market sources (Chiang Mai, Thailand). The samples were washed and cleaned, with removal of the fat layer, prior to cutting into 3 × 3 cm pieces. Each sample (100 µL) was applied to the skin surface. The skin without any substance was used as a control. Skin moisturizing was measured before application to samples and after application at 0, 15, and 30 min intervals using a Corneometer® (Courage-Khazaka Electronic GmbH, Cologne, Germany). Before applying the sample and recording the parameter, the pig skins were kept at 25 ◦C for 30 min. This method was adapted from Kassakul et al. [25]. The degree of skin moisturizing (%) was tested in triplicate to detect random error and calculated using Equation (1).

$$\text{DM} \ (\%) = \frac{\text{Mi} - \text{Ma}}{\text{Mi}} \times 100 \tag{1}$$

where *M*<sup>i</sup> is the initial moisturizing content before the sample was applied to the skin and *M*<sup>a</sup> is moisturizing content after the sample was applied to the skin.

#### *2.6. Accelerated Stability Study*

The deodorant creams with different concentrations of H-CMCH (0.5, 1.0, 1.5, 2.0, and 2.5% (*w*/*v*)) were centrifuged (Universal 320R, Hettich, Tuttlingen, Germany) at 6000× *g* for 20 min. Accelerated stability tests were performed at both 4 ◦C and 45 ◦C for 24 h in an Incucell incubator (MMM Medcenter Einrichtungen GmbH, München, Germany) for 6 cycles. The physicochemical characteristics of the creams—including visual appearance, pH using a pH meter (FiveEasy F20, Mettler Toledo, Greifensee, Switzerland), viscosity

using a Brookfield viscometer (DV-II+, Brookfield, Middleboro, MA, USA), and color L\*, a\*, b\* using a colorimeter (CR-410, Konica-Minolta, Tokyo, Japan)—were monitored at every cycle. Total color difference (∆E) was calculated for each sample and each cycle following the strategy of Tkacz et al. [26].

#### *2.7. Deodorizing Activity*

The deodorizing activity of the developed deodorant cream was evaluated against the odor component trans-2-nonenal, following the method of Ham et al. [21] by using solidphase microextraction and gas chromatography. The dilute deodorant cream solutions of different concentrations (1–100 mg/mL) were prepared by dissolving the extract in 0.2 M potassium phosphate buffer solution (pH 7.4). An aliquot (1 mL) of the dilute deodorant cream solutions was mixed with aqueous solution (100 µL) containing the odor compound: 10 ppm of trans-2-nonenal or solution. The mixture of deodorant cream and odor substance was placed in a vial (20 mL), which was tightly sealed with a cap furnished with PTFE/silicone septa (Supelco, Bellefonte, PA, USA). The sample vial was then placed in a stirring water bath at 35 ◦C for 10 min to achieve phase equilibrium, and then the odor substance in the headspace of the vial was taken by a SPME fiber during additional stirring for 5 min at 37 ◦C. Carboxen/polydimethylsiloxane (Carboxen/PDMS; 75 µm film thickness) was used for detecting trans-2-nonenal. After the adsorption of the odor substance, the fiber was removed from the vials and immediately inserted into the injector of a gas chromatography system for quantitative analysis. The odor compounds were desorbed from the fiber by heating at 250 ◦C for 2 min in the gas chromatography system.

Gas chromatography was carried out using a gas chromatography flame ionization detector (GC-2010 Series, Shimadzu, Santa Clara, CA, USA) equipped with a flame ionization detection system. The oven temperature for trans-2-nonenal analysis was programmed at 50 ◦C for 2 min, from 50 ◦C to 200 ◦C at a heating rate of 8 ◦C/min, 200 ◦C for 2 min, and finally 250 ◦C hold for 1 min. Injector and detector temperatures for the analysis of trans-2-nonenal were also 250 ◦C. The samples were injected in a spitless mode using nitrogen as the carrier gas (1 mL/min) at a volume of 1.0 µL. Deodorizing activity (%) was calculated by Equation (2).

$$\text{Decoderizing activity} \left( \% \right) = \frac{H\text{n} - H\text{c}}{H\text{n}} \times 100 \tag{2}$$

where *H*<sup>n</sup> is the headspace amount of the odor substance (trans-2-nonenal) and *H*<sup>c</sup> is the headspace amount of the deodorant cream.

#### *2.8. Antioxidant Properties*

The optimal cream formula was selected based on skin moisturizing and deodorizing properties and a stability test for antioxidant activity. These properties were compared in the prototype cream formula (no ME and H-CMCH). Solutions of the developed deodorant cream (stock: 5 mg/mL in distilled water) at different concentrations of 1, 2, 3, 4, and 5 mg/mL were prepared and used for DPPH, ABTS, and FRAP assays.

#### 2.8.1. DPPH Radical Scavenging Activity

The ability of antioxidants to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was tested by using the modified method of Surin et al. [27] and Phimolsiripol et al. [28]. After that, 100 µL of the stock samples (as described above, concentrations: 1, 2, 3, 4, and 5 mg/mL) were blended with 100 µL of 0.2 mM DPPH reagent (Sigma-Aldrich, Singapore) and incubated at 25 ◦C for 30 min in the dark. Absorbance was measured at 517 nm in a 96-well microplate reader (SpectraMax® i3x, Molecular Devices, San Jose, CA, USA). The radical scavenging activity of the sample was calculated based on gallic acid (Sigma-Aldrich, Darmstadt, Germany). Results were expressed as milligram gallic equivalent per gram of sample (mgGAE/g sample). The percentage of DPPH radical

scavenging activity can be calculated as shown in Equation (3) before plotting of IC<sup>50</sup> against respective concentration.

$$\% \text{DPPH radical inhibition} = \frac{A\mathbf{c} - A\mathbf{s}}{A\mathbf{c}} \times 100 \tag{3}$$

where *A*<sup>c</sup> is absorbance of the DPPH solution and *A*<sup>s</sup> is absorbance of different concentrations of samples.

#### 2.8.2. ABTS Radical Scavenging Activity

2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity was tested according to the method described by Surin et al. [27,29] and Ruksiriwanich et al. [30]. ABTS (Sigma-Aldrich, Singapore) reagent solution was freshly prepared by mixing 7 mM of ABTS solution with 2.45 mM of potassium persulfate (Sigma-Aldrich, Singapore). ABTS powder and potassium persulfate powder were individually dissolved in water to the required concentration and then combined in a bottle. After 16 h of incubation in the dark at 25 ◦C, the resultant dark blue color of the ABTS reagent solution was diluted with ethanol until the absorbance reading reached 0.7 ± 0.2. The solutions of H-CMCH were prepared as described previously in 2.6. Each sample solution (0.5 mL at concentrations: 1, 2, 3, 4, and 5 mg/mL) was mixed with 1.0 mL of ABTS stock solution and incubated for 6 min in the dark. Absorbance was measured at 734 nm in the 96-well microplate reader. The ABTS radical scavenging activity was expressed as milligram gallic equivalent per gram of sample (mgGAE/g sample). The percentage of ABTS radical scavenging activity can be calculated as shown in Equation (4) with plotting of IC<sup>50</sup> against respective concentration.

$$\% \text{ ABTS radical inhibition} = \frac{A\mathfrak{c} - A\mathfrak{s}}{A\mathfrak{c}} \times 100\tag{4}$$

where *A*<sup>c</sup> is absorbance of the ABTS solution and *A*<sup>s</sup> is absorbance of different concentrations of samples.

#### 2.8.3. Ferric Reducing Antioxidant Power (FRAP)

The ferric reducing antioxidant power (FRAP) assay was carried out according to the technique of Surin et al. [31]. The FRAP reagent was prepared by mixing 25 mL of 0.3 M acetate buffer (pH 3.6), 2.5 mL of 4,6-tripyridyl-s-triazine (TPTZ) (Sigma-Aldrich, Darmstadt, Germany) solution in 40 mM HCl (RCI Labscan, Bangkok, Thailand), and 2.5 mL of 20 mM ferrous sulphate (Loba Chemie, Mumbai, India). Then, 50 µL of sample (5 mg/mL) was mixed with 950 µL of FRAP reagent and incubated in the dark for 30 min. Absorbance was measured at 593 nm in a 96-well microplate. The ferric reducing antioxidant power of the sample was determined based on ferrous sulphate (Merck KGaA, Darmstadt, Germany). Results were expressed as ferrous sulphate equivalent antioxidant capacity (FEAC) with µmol Fe2+/g sample.

#### *2.9. Antibacterial Properties*

The antibacterial properties on six species of bacteria—*Corynebacterium* spp., *S. epidermidis*, *S. aureus*, *B. subtilis*, *P. aeruginosa*, and *E. coli*—were tested using the agar well diffusion method by Bai-Ngew et al. [32]. For this step, a good representative formula was compared with the prototype cream formula (no ME and H-CMCH). The bacterial culture was swabbed on sterile nutrient agar plates. Subsequently, filter paper discs (6 mm in diameter) were dipped into the prototype deodorant cream, developed deodorant cream (stock 5 mg/mL in distilled water), and positive control (10 mg/mL Streptomycin). The plates were incubated at 37 ◦C for 18–24 h in an upright position. The experiment was carried out in triplicate, and the inhibition zone was recorded and expressed in millimeters.

## *2.10. Statistical Analysis*

All data were analyzed by one-way ANOVA. Mean comparison was performed by Duncan's multiple range tests with significance level *p* < 0.05. Statistical analyses were performed with the SPSS 17.0 (SPSS Inc.; IBM Corp.; Chicago, IL, USA).

#### **3. Results and Discussion**

#### *3.1. Effect of H-CMCH Synthesis*

The yield, moisture content, water solubility, viscosity, and pH of H-CMCH were 45.36%, 5.56%, 89.5%, 360 cP, and 7.33, respectively. Solubility is a significant property of CMCH that measures its resistance to water. The H-CMCH improved water solubility by about 89% when compared to chitosan. The solubility and conformation of CMCH are a result of the deacetylation, pH, and MW of native chitosan. The solubilization process of CMCH is related to functionalized polymers and different types of chemical and physical interactions, such as hydrogen bonds, hydrophobic interactions, and van der Waals forces. High water solubility suggests that CMCH is moisture absorbent and has a greater ability to bind with water than chitosan. However, the solubility of chitosan was relatively poor in water and organic solvents, which resulted in limitation in its uses. At pH < 6.0, chitosan is positively charged (–NH<sup>3</sup> + ), with increased solubility in water. As the pH increases, chitosan loses its charges, due to protonation, while the amino groups decrease as the solutes begin to precipitate. Chemical modification of amino groups and hydroxyl groups using a carboxymethylation reaction results in a large number of water-soluble chitosan derivatives [14]. This causes the increase of hydrated water molecules around the chains of CMCH than surround the chitosan chains, resulting in higher water solubility. The results are also consistent with the report of Siahaan et al. [33], who found that temperature and NaOH concentration affected CMCH synthesis, and Rachtanapun et al. [34] in carboxymethyl bacterial cellulose. The interactions between NaOH and monochloroacetic acid resulted in reduced CMCH forming and lower solubility. The mitigation in solubility might stem from the loss of free amino functional groups that enhance the hydrophobic nature of the compounds [35]. The greater solubility of L-CMCH and M-CMCH resulted in a decrease in viscosity, but H-CMCH showed higher viscosity. This could be explained as follows, CMCHs with longer chains or higher MW were contributing to the gel. H-CMCH is an effective water-soluble polymer with high viscosity which could be successfully utilized in pharmaceuticals and cosmetics as an emulsion stabilizer and thickening agent. Thanakkasaranee et al. [36] also reported that the yield of CMCH was also dependent on the concentration of NaOH, MW of chitosan, solvent, and reaction temperature. In addition, the solvent ratio and the processing also affect the yield and antioxidant activities [37].

#### *3.2. Degree of Skin Moisturizing (% DM)*

The degree of skin moisturizing indicates the water-holding capacity of the skin, which can be tested by the Corneometer® method. The Corneometer® measures the changes in electrical capacitance related to the moisture content of the skin before and after applying the solutions [25]. The degree of skin moisturizing of the deodorant cream with different concentrations of H-CMCH (0.5, 1.0, 1.5, 2.0, and 2.5% (*w*/*v*)) was examined on pig skin and compared with untreated skin, water, and propylene glycol at 15 and 30 min as presented in Figure 1.

**Figure 1.** DM (%) as affected by time (15 and 30 min) and different treatments (skin, DI, PG, H1, H2, H3, H4, and H5) on pig skin. Different lowercase letters (a,b,c...) indicate significant differences between solutions at 15 min and different uppercase letters (A,B,C...) indicate significant differences between solutions at 30 min. **Figure 1.** DM (%) as affected by time (15 and 30 min) and different treatments (skin, DI, PG, H1, H2, H3, H4, and H5) on pig skin. Different lowercase letters (a,b,c...) indicate significant differences between solutions at 15 min and different uppercase letters (A,B,C...) indicate significant differences between solutions at 30 min.

The degree of moisturizing at time 15 and 30 min showed that the degree of skin moisturizing of the solutions decreased with increasing time after applying solutions. At the same time, the degree of skin moisturizing of all treatments showed a significant difference after applying between 15 and 30 min (*p* < 0.05). Applying H-CMCH solution for 15 min gave a higher degree of skin moisturizing than 30 min, showing the degree of skin moisturizing of untreated skin, water, propylene glycol, H1, H2, H3, H4, and H5 solutions applied on pig skin for 30 min were significantly decreased compared to 15 min. This confirms that the H-CMCH solution provided a good moisture absorption. In fact, the skin moisturizing effects appeared to decrease with increasing time due to a lack of mechanisms to maintain skin moisturizing and the dryness of pig skin cells [38]. The higher MW CMCH also had superior moisture retention capacity. Kassakul et al. [25] found that 0.2% *Hibiscus rosa-sinensis* mucilage as a natural ingredient provided good results for skin moisturizing after applying for 30 min, improving by about 130%. The results showed that moisturizing products could increase the water content of the skin while maintaining softness and smoothness [39]. Chaiwong et al. [23] reported, after applying solutions containing different MW of water-soluble CMCH (L-CMCH, M-CMCH, H-CMCH), that the moisture content of the skin increased. The mechanism of the moisturizing effect is based on the formation of a water film on the skin surface after dissolution of CMCH, and a subsequent stage of water evaporation could further prevent water evaporation from the skin [40]. Positive electrical charges and relatively high MW facilitate prolonged skin adherence [14]. Our results also showed that H-CMCH decreased the loss of water while The degree of moisturizing at time 15 and 30 min showed that the degree of skin moisturizing of the solutions decreased with increasing time after applying solutions. At the same time, the degree of skin moisturizing of all treatments showed a significant difference after applying between 15 and 30 min (*p* < 0.05). Applying H-CMCH solution for 15 min gave a higher degree of skin moisturizing than 30 min, showing the degree of skin moisturizing of untreated skin, water, propylene glycol, H1, H2, H3, H4, and H5 solutions applied on pig skin for 30 min were significantly decreased compared to 15 min. This confirms that the H-CMCH solution provided a good moisture absorption. In fact, the skin moisturizing effects appeared to decrease with increasing time due to a lack of mechanisms to maintain skin moisturizing and the dryness of pig skin cells [38]. The higher MW CMCH also had superior moisture retention capacity. Kassakul et al. [25] found that 0.2% *Hibiscus rosa-sinensis* mucilage as a natural ingredient provided good results for skin moisturizing after applying for 30 min, improving by about 130%. The results showed that moisturizing products could increase the water content of the skin while maintaining softness and smoothness [39]. Chaiwong et al. [23] reported, after applying solutions containing different MW of water-soluble CMCH (L-CMCH, M-CMCH, H-CMCH), that the moisture content of the skin increased. The mechanism of the moisturizing effect is based on the formation of a water film on the skin surface after dissolution of CMCH, and a subsequent stage of water evaporation could further prevent water evaporation from the skin [40]. Positive electrical charges and relatively high MW facilitate prolonged skin adherence [14]. Our results also showed that H-CMCH decreased the loss of water while elevating skin humidity. The higher apparent viscosity of H-CMCH can improve stability and enhance skin hydration. In fact, H-CMCH was superior to untreated skin, water, and propylene glycol in terms of degree of skin moisturizing effect. The higher concentrations of H-CMCH also indicated potential for film forming and multilayer coating of the skin. Subsequently, it could be used in cosmetic preparations, with further studies suggested to test skin irritation in human subjects.

elevating skin humidity. The higher apparent viscosity of H-CMCH can improve stability and enhance skin hydration. In fact, H-CMCH was superior to untreated skin, water, and propylene glycol in terms of degree of skin moisturizing effect. The higher concentrations

Subsequently, it could be used in cosmetic preparations, with further studies suggested

to test skin irritation in human subjects.

#### *3.3. Effect of Accelerated Stability Study* incubator. The temperature was alternated between 4 °C and 40 °C every 24 h during any period of time. This method is commonly used during the initial stage of developmental period of time. This method is commonly used during the initial stage of developmental

3.3.1. Visual Appearance

3.3.1. Visual Appearance

*3.3. Effect of Accelerated Stability Study*

*3.3. Effect of Accelerated Stability Study*
