The evaluation of antioxidant and antityrosinase efficacy of Carissa carandas fruit extracts and the development of a preliminary skincare product

Nadechanok Jiangseubchatveera1, Charinrat Saechan2, Nattawut Leelakanok3, Arpa Petchsomrit4* 1Division of Pharmacognosy and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Burapha University, Chonburi, Thailand. 2Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Thailand. 3Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Burapha University, Chonburi, Thailand. 4Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Burapha University, Chonburi, Thailand.


INTRODUCTION
Even though historical cosmetic toner was unpopular because of the irritation caused by skin drying, toner has become favorable these days because of the advancement in the formulating technologies that enhance the oil removal capability of the toner, retain the skin moisture, and refine the care of the skin simultaneously. Botanical products are one of the preferred ingredients used in the toner. Apart from the fact that consumers perceive botanical ingredients as safe and nature friendly, natural ingredients are usually a mixture of chemicals that allow various biological activities. Antioxidants derived from natural sources have become extensively studied and used in the cosmetic industry, especially for skincare products.
Free radicals are unstable molecules produced in the biological processes by cells, for example, superoxide, peroxyl, alkoxylate, and hydroxyl. The radicals create the oxidative stress that involves in the development of several diseases (Phaniendra et al., 2015) and skin aging (Poljšak and Dahmane, 2012). Plantderived compounds such as phenolic compounds are oxidant scavengers that are ubiquitously found in nature (de Lima Cherubim et al., 2020). Carissa carandas Linn. is a plant that originated in Asia and is commonly found in Thailand, and its fruits contain a high amount of antioxidant substances such as anthocyanin (Le et al., 2019;Sueprasarn et al., 2017) and vitamin C. Phenolics in ripe fruit, for example, isoamyl alcohol, benzyl acetate, lupeol, oxalic acid, tartaric acid, citric acid, malic acid, malonic acid, and glycolic acids, also exhibited antioxidant activity (Khunchalee, 2019). Antioxidant screening of C. carandas fruit was measured from ethanol (Khunchalee, 2019), methanol (Ondee, 2019), and hexane (Normaizatul Afizah et al., 2016) extracts. There is limited data on the water extract of this fruit (Le et al., 2019;Sueprasarn et al., 2017). Therefore, in this study, C. carandas fruit aqueous extract was screened for antioxidant and antityrosinase capacity (Khunchalee, 2019) and fabricated as skincare toner. In addition, the toner preparation was evaluated for its appearance and stability.

Sample preparation and extraction
C. carandas ripe fruits were collected from Tambon Banlao, Muang District, Chaiyaphum Province. The fruits (625 g) were washed with water, air-dried under the shade at room temperature, then homogenized with 600 ml of distilled water, and filtered through Whatman No. 1 filter paper (GE Healthcare Life Sciences, Marlborough, MA) to remove plant residues. The water extract was put into a -80°C freezer. The frozen sample was lyophilized by freeze dryer (Labconco™ FreeZone™ -105°C 4.5 l Benchtop Freeze Dry Systems, Kansas City, MO) for 3 days at a processing temperature of -103°C and constant pressure in the drying chamber at 47.0 Pa. The lyophilized extract was stored at 4°C for future studies.

DPPH radical scavenging activity
The antioxidant potential of the fruit extract was determined using the DPPH method as described previously (Jiangseubchatveera et al., 2017). In brief, the extract was serially diluted with ethanol to a concentration of 1-10 mg/ml. A fresh ethanolic solution of DPPH at a concentration of 66 μg/ml was prepared. 180 μl of DPPH solution was added to 20 μl of the fruit extract in a 96-well microtiter plate and stored in the dark for 30 minutes at room temperature. Then, the absorbance (A) was measured at 520 nm by using a microplate reader (Multimode Microplate Reader, FLUOStar Omega 2020, Ortenberg, Germany). Trolox and ascorbic acid were used as positive controls. All measurements were performed in triplicate. The capability to scavenge the DPPH radical was calculated using the following equation:

DPPH scavenging activity (%) = [(A control -A sample )/A control ] × 100
A control is the absorbance of the blank (ethanol), while A sample is the absorbance of the sample or the standard. The inhibitory concentration (IC 50 ) values showed the concentration of the sample which was able to scavenge 50% of DPPH free radicals.

ABTS radical scavenging activity
The antioxidant activity by ABTS radical cation was carried out according to Jiangseubchatveera et al. (2017). The ABTS •+ was generated by the reaction between 7 mM ABTS in water and 2.4 mM potassium persulfate solution at a ratio of 2:1. The mixture was then stored in the dark for 16 hours at room temperature. For the assay, the freshly prepared ABTS ·+ solution was diluted with distilled water to obtain the absorbance of 0.70 ± 0.02 at 734 nm using a spectrophotometer (Hitachi U2900, Japan). After that, 20 µl of the fruit extract was mixed with 2 ml of diluted ABTS •+ solution and incubated at room temperature for 5 minutes before the measurement of the absorbance at 734 nm. All the measurements were performed in triplicate. Trolox and ascorbic acid were used as positive controls. The percentage inhibition of free radical (I%) was calculated using the following equation: A control is the absorbance of all reagents except the sample or standard and A sample is the absorbance of the sample or standard. The ABTS of the sample was expressed as mg Trolox equivalent antioxidant capacity (TEAC)/g extract and mg ascorbic acid equivalent antioxidant capacity (AEAC)/g extract, respectively.

Antityrosinase activity
Antityrosinase activity of C. carandas extract was determined using the dopachrome method (Saghaie et al., 2013). The samples were prepared in water at the final concentration of 10 and 50 mg/ml. Twenty microliters of each sample was mixed with mushroom tyrosinase (40 µl, final concentration 500 U/ml), followed by the addition of phosphate buffer (100 µl, 20 mM, pH 6.8). After the incubation at 37°C for 10 minutes, L-DOPA (40 µl, 0.85 mM) was added to the reaction mixture and incubated under the same condition for 20 minutes. The formation of the dopachrome was measured for the visible light absorbance at 492 nm using a microplate reader (Multimode Microplate Reader, FLUOStar Omega 2020, Germany). Kojic acid was used as a positive control. All measurements were carried out in triplicate. The percent inhibition of tyrosinase activity was calculated according to the following equation: where A control is the absorbance of the control solution without kojic acid (or the extract), A sample is the absorbance of kojic acid (or the extract) solution with tyrosinase, and A blank is the absorbance of kojic acid (or the extract) solution without tyrosinase.

Development of the fruit extract skincare product
The fruit extract was incorporated into the skincare toner. The formulation of the toner-based consisted of 70% sorbitol solution (4%), propylene glycol (2%), glycerin (1%), Tween ® 20 (1%), phenoxyethanol (1%), allantoin (0.25%), and purified water. To fabricate the preliminary formulation of the fruit extract toner, the toner base was mixed with the fruit extract (1 g). The physical appearances and stability properties of the toner were then evaluated.

Physical characteristics
The physical appearances, color, and phase separation of the fruit extract toner were observed after the preparation for 24 hours. The pH values were determined by a pH meter (Mettler-Toledo, Columbus, OH).

Stability study
The stability test was evaluated by two types of assays including a centrifugation assay (Charoenjittichai et al., 2016) and a six-cycle acceleration test. The formulations were centrifuged at 3,500 rpm for 30 minutes at room temperature and observed for the phase separation (Labofuge™ 400 Centrifuges, Thermo Fisher Scientific, Waltham, MA). The acceleration test, six cycles of a heating-cooling cycle, was carried out. Each cycle consisted of storage at 4°C for 48 hours and 45°C for 48 hours. At the end of this experiment, physical appearances, pH values, and antioxidant capacity of the toner were determined.

Effects of light and temperature for the storage condition
The effects of light and temperature on the final product were observed for a month. To determine the effect of light, the fruit extract toner was stored at ambient temperature (25°C) in dark and light conditions. Also, the toner was kept at 4°C to evaluate for the effect of temperature. The physical properties and the antioxidant activity were assessed.

Extraction yield
The ripe fruit of C. carandas was lyophilized to provide red extract with a 6.83% (w/w) yield. The yield in this study was higher than that reported in a previous study (Khunchalee, 2019) which was 5.76 and 2.54% yield using 70 and 100% ethanol as solvents, respectively.

Antioxidant activity
DPPH and ABTS assays were commonly used for the evaluation of the antioxidant activity of plant extracts. The extract showed the concentration-dependent inhibition of radical scavenging activity. The IC 50 value of the fruit extract from the DPPH assay still showed less activity than that of Trolox and ascorbic acid (Table 1). According to the ABTS assay, the extract also showed free radical scavenging ability (Table 1). These results revealed that the extract of C. carandas fruit had antioxidant activity. The antioxidant potential of C. carandas fruit extract is from its phenolics and anthocyanins (Chodok and Khumkhom, 2020;Dhar et al., 2017;Le et al., 2019;Sueprasarn et al., 2017). In recent studies, the C. carandas fruit extracts from ethanol, methanol, and hexane fractions also presented the antioxidant abilities by DPPH and ABTS assays (Khunchalee, 2019;Le et al., 2019;Madhuri and Neelagund Shivayogeeswar, 2019;Normaizatul Afizah et al., 2016;Sueprasarn et al., 2017). Moreover, Madhuri and Neelagund Shivayogeeswar (2019) reported that the methanolic extract of this fruit exhibited antioxidant power using ferric-reducing antioxidant power assay (FRAP). Additionally, our results are in agreement with those reported by Chodok and Khumkhom (2020) who found a strong correlation between total phenolic content and FRAP assay. Therefore, our findings agree with other studies that strongly supported the antioxidant activities of C. carandas fruit extracts.

Antityrosinase activity
The percentage inhibition of the fruit extract at a concentration of 1, 2, and 5 mg/ml was 6.55 ± 0.16, 25.80 ± 0.11, and 83.37% ± 0.52%, respectively, shown in Table 2. Among these, the tyrosinase inhibitory effect of the extract was in a dosedependent manner. The IC 50 value of the extract and kojic acid was exhibited in Table 2. In this case, the antityrosinase activity of the fruit extract could be attributed mainly to its phenolic compounds (Chodok and Khumkhom, 2020;Dhar et al., 2017). The phenolics of plants have been reported as a source of various pharmacologically active compounds. The hydroxyl groups of phenolics inhibited tyrosinase by copper chelating in the active site (Panzella and Napolitano, 2019).

Physicochemical characteristics
The toner from the fruit extract was pink and translucent. The pH value of the freshly prepared extract toner was 3.3, which was improper for the skin application. Therefore, triethanolamine was used to adjust the pH value to be approximately 5.5, mimicking the skin pH. The color of the toner changed to orange-pink but it was still transparent (Fig. 1).

Stability study
The characteristics of cosmetic and pharmaceutical products can be commonly influenced by environmental factors.  These factors, such as temperature, pH, light, air, and humidity, contribute to phase separation, color and viscosity changes, loss of activities, and active ingredients degradation (Liu et al., 2019;Restu et al., 2015). Moreover, centrifugation is helpful in the determination of the preliminary shelf life of a product (Khan et al., 2013). The physical appearances of the toner from the fruit extract under centrifugation and accelerated conditions were evaluated. After the centrifugation and acceleration test, there were no phase separation and aggregation. Their color was not significantly changed (orange-pink color), and the pH values remained approximately 5.5. The antioxidant activity of the toner determined using DPPH and ABTS assays was not significantly different in both before and after the heating-cooling tests (Table  3). These outcomes indicated that the toner from the fruit extract was stable; therefore, it could be used as an effective antioxidant skincare product.

Effects of light and temperature for storage
After a month of all storage conditions, the physical properties of the toner remained unchanged. The effects of light and temperature on the antioxidant activity of the extract toner were evaluated using DPPH and ABTS radical scavenging assays which were the general method used to determine the antioxidant potential (Ratz-Lyko et al., 2012). The antioxidant properties of the toners are presented in Table 3. For the storage at 4°C and under the light and dark conditions at 25°C, the antioxidant efficiency of the product obtained by both DPPH and ABTS assays was not significantly changed (one-way analysis of variance, p > 0.05). Besides, the increasing temperature slightly decreased the ability of antioxidants as reported by Réblová (2012). However, the cosmetic product was not normally stored in a hot environment. Consequently, the prepared toner does not require light protection and could be stored at ambient temperatures.

CONCLUSION
This study found that C. carandas water extract had antioxidant and antityrosinase activities. The toner formulation containing C. carandas fruit extract was orange-pink, translucent, and stable. Moreover, light and temperature had an insignificant effect on the antioxidant activity of the toner. These results indicated that C. carandas extract can be utilized as an antioxidant from a natural source for further cosmetic purposes, especially in skincare.

AUTHORS' CONTRIBUTION
The authors confirm contribution to the paper as follows: study conception and design: Nadechanok Jiangseubchatveera and Arpa Petchsomrit; data collection:  Nadechanok Jiangseubchatveera; analysis and interpretation of results: Nadechanok Jiangseubchatveera, Charinrat Saechan, and Arpa Petchsomrit; draft manuscript preparation: Nadechanok Jiangseubchatveera, Charinrat Saechan, Nattawut Leelakanok, and Arpa Petchsomrit. All authors reviewed the results and approved the final version of the manuscript.