Beneficial Effects on Cosmetic Activity by Optimizing the Calamondin Orange Extraction Process

Abstract

This study expands the usage of Citrus madurensis (Citrofortunella microcarpa), which is a good source of skin whitening and antioxidants activation in cosmetics. The goal of this study is to provide cosmeceutical activity data about the extraction yield and total polyphenol of calamondin orange by optimizing the focused high ultrasound (INEFU), ultrasound extraction (UE), and water extraction (WE) conditions. Under optimal extraction conditions, which consisted of 1800 W for 45 minutes (INEFU) and 500 W for 45 minutes (UE), 21.55% (w/w) and 13.27% (w/w) of the highest extractions yield and total polyphenol content was obtained. For the skin whitening activity, tyrosinase inhibitory activity was observed at 69.28% in the INEFU extracts, which was 69.24% higher than that of the UE extracts (58.82%). To reduce melanin production in Clone M-3 cells, 86.9% melanin production was observed following the addition of control, WE relative to the control without a sample, and 81.2% and 78.9% were found in the UE and INEFU conditions, respectively. The highest total phenol secretion was conclusively obtained under the optimal conditions and resulted in a significant improvement of the cosmetic activities of C. madurensis (C. microcarpa)-based cosmeceutical ingredient.

Currently, ultrasound-assisted extraction has been applied to effectively extract natural components from different materials. ^1,2 Compared with the conventional hot water extraction method, ultrasound-assisted extraction can enhance the yield, save operation time, and streamline the process. ³ With rapid industrialization, environmental pollution is destroying the ozone layer and UV light exposure is increasing. In addition, people are becoming increasingly interested in living quality and longevity. Accordingly, the functional cosmetics market is expanding. ⁴ Orange have been so commonly used in functional foods, cosmetics, or medicines, which were ascribed to the valuable functional components. ⁵ Calamondin orange were rich in vitamin C, polyphenols, and trace elements. ⁶ Phenols are a class of plant compounds with the potential to eliminate free radicals because of their stable structure after free-radical capture. ⁷ Calamondin orange contains vitamin C, including tangeretin and sinensetin, in large quantities in the peel part of this citrus orange. ⁸ Pure juice extracted from calamondin orange resembles juice from shiikuwasha in color and flavor. ⁹ Moreover, the adulteration of fruit juices has been a serious economic problem. This problem has been detrimental to consumers and the food industry for many years. ¹⁰ Therefore, it is expected that healthy and eco-friendly products can be developed by applying natural cosmetic resources that have diverse bio-functions for the production of make-up. This study was conducted to provide relatively new cosmeceutical information about C. madurensis (C. microcarpa), which has been widely used as natural active components, and to increase its applicability by optimizing the focused high ultrasound (INEFU) extraction conditions. Table 1 shows the extraction yields and phenol content of the extracts under water extraction (WE), INEFU, and ultrasound extraction (UE) conditions. With respect to INEFU, the yield increased when the ultrasound watts treatment increased, and it was the highest at 1800 W. Therefore, it appears that a 45 minute process time at 1800 W would be considered as the most efficient extraction condition. The extract from the INEFU procedure had the highest yield ratio at 21.55% at the UE proceduce extraction. An ultrasonic wave energy of 120 kHz (1800 W) or higher could also increase the diffusion and solubility of the solvent, which resulted in an increase in the polyphenol content of the extracts. A similar UE extracts result was also reported elsewhere. ¹¹ The amount of total polyphenols in the WE, INEFU, and UE extracts was studied. INEFU extract showed significantly higher phenolic content than the WE and UE extracts (Figure 1). Melanin is produced from the biosynthesis of tyrosinase in skin cells, and it improves skin resistance to ultraviolet rays, dryness, and extreme temperatures; however, too much melanin production leads to pigmentation, such as freckles and liver spots, and to skin damage. Therefore, the reduction of melanin biosynthesis by tyrosinase has been used as an indicator of the whitening activities of natural products. ¹² As shown in Table 2, C. madurensis extracts had inhibition effects of 61.38 ± 0.95% (WE), 58.82 ± 1.13% (UE), and 69.24 ± 0.88% (INEFU) on the tyrosinase activity. The inhibition activity increased with the increase in the concentration. This result also indicates that the INEFU extract has a greater whitening effect by effectively reducing tyrosinase activity. In addition to the tyrosinase inhibition activity observation, the reduction in Clone M-3 cell melanin production after sample treatment has also been employed to measure the whitening activities of natural products. ¹³ Therefore, to examine melanin synthesis in Clone M-3 cells, the extracts were treated for 3 days at concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL. This result indicates that the extract from the INEFU process has great potential whitening activity because of the presence of ascorbic acid, a positive control. To inhibit melanin production (Table 2), 86.9% melanin production was observed following the addition of WE relative to the control without a sample, and 81.2% and 78.9% were found in the UE and INEFU conditions, respectively. Thus, it can be concluded that the highest extraction yield from the INEFU process results in the highest cosmetic activities relative to those from other processes by eluting the highest amount of the polyphenols components from C. madurensis. It appears that the treatment at INEFU, the focus high ultrasound for this INEFU process, effectively destroyed the hard cell walls, which resulted in increased solvent penetrability and the elution of its useful components. The 1800 W treatment, which was also determined to be the optimal focus on high ultrasonic wave intensity, increased the diffusion and solubility of the solvent and accelerated the elution of useful components from C. madurensis. This study was the first report to indicate that various cosmetic activities of C. madurensis (C. microcarpa) relating to the extraction yield and polyphenols content can be enhanced by optimizing the extraction conditions, such as the ultrasonic wave intensity, temperature, and treatment time. A treatment employing INEFU at 1800 W for 45 minutes produced the highest yield and polyphenols content as well as the highest whitening activity and the lowest melanin production. This finding indicates that focused high ultrasounds (INEFU) effectively destroyed the tissues and cell walls of the thin-surfaced C. madurensis (C. microcarpa), which resulted in an improved elution of the active cell components. It appears that the higher ultrasounds of the INEFU process especially loosened the thin tissues that prevent the elution of active components and increased the area of contact with the solvent to accelerate active component diffusion and elution. ¹¹ In addition, the high input energy produced by the cavitation from the ultrasonic device destroyed the inner tissues of the cells, which shortened the travel distance of the extracts, facilitated solvent diffusion, and increased solubility. The resulting cleavage of bonds between the atoms in the high molecular polymer might have contributed to the elution of substances that were not easily eluted using the existing methods. Therefore, the INEFU process can maintain the synergistic effect that improves both the extraction yield and the elution of polyphenols, which resulted in an increase of its cosmetic activities, such as whitening, and also expands the use of this natural polyphenols for multifunctional cosmetics.

Figure 1

Total phenolic contents of Citrus madurensis (Citrofortunella microcarpa) is means of 3 replicates ± standard deviation.

Table 1

Comparison of the Extraction Yields of the Citrus madurensis (Citrofortunella microcarpa) in Relation to Different Extraction Processes.

Extraction methods	Solvents	Extraction conditions			The yield of active components (%)
		A	B	C
INEFU	Water	1800	45	40	17.91 ± 0.52
UE	Water	500	45	40	14.11 ± 0.21
WE	Water	0	45	40	8.21 ± 0.84

A: Ultrasound power (W); B: Extraction time (min); C: Extraction temperature (°C).

Table 2

Comparison of Cosmetic Activities of the Citrus madurensis (Citrofortunella microcarpa) in Relation to Different Extraction Processes.

Cosmetic activities	Extraction methods
	WE	UE	INEFU
Tyrosinase inhibitory activity (%)	61.38 ± 0.95	58.82 ± 1.13	69.24 ± 0.88
Melanin production (%)	86.92 ± 0.78	81.20 ± 0.45	78.92 ± 0.36

WE, water extraction for 45 minutes at 40°C with water. UE, ultrasonication extraction for 45 minutes at 500 W at 40°C with water. INEFU, focused high ultrasonication extraction for 45 minutes at 1800 W at 40°C with water

Experimental

Sample Preparation

Citrus madurensis (C. microcarpa) were received from G-MARKET, Seoul, South Korea. Each sample was analyzed in triplicate. For the WE, INEFU (focused high ultrasound extraction), and UE, the samples were dried and ground from fruit parts. Citrus madurensis (C. microcarpa) powder was mixed with water at a ratio of 1:10. A UE extractor (Ilshin Lab, Daejeon, Korea) was used for 45 minutes at 500 W/vol. For the INEFU, 4 species were placed in a high ultrasound extractor (working volume of 1 L, Classys, Seoul, South Korea) and processed at frequencies of 1800 W/vol input energy for 45 minutes. Subsequently, all of the extracts were filtered by vacuum filtration and evaporated using a rotary evaporator (N–N series, Evela, Tokyo, Japan). The concentrates were freeze-dried and stored at −20°C before use.

Cell Lines and Culture Media

Melanin production was also tested using a mouse melanocyte, which is known as Clone M-3 (KCLB, Seoul, Korea). Melanocytes were cultured with 10% heat-inactivated fetal bovine serum (FBS) and Eagle's minimal essential medium (GIBCO, Grand Island, NY, United States) or Roswell park memorial institute 1640 (GIBCO), respectively. All other nutrients, including 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, gentamycin sulfate, and trypsin-Ethylenediaminetetraacetic acid (EDTA), were purchased from Sigma (St. Louis, MO, United States) as analyzed guaranteed or reagent grade.

Measurements of Extraction Yields and Polyphenol Content From Several Extraction Processes

The phenol concentration was quantified following the method previously described. ¹⁴ In brief, the Folin Ciocalteau method was utilized to determine the content of polyphenols in C. madurensis (C. microcarpa). A test sample (50 µL) was mixed with 2 mL 2% sodium carbonate and allowed to stand at room temperature (RT) for 2 minutes. During this time, 100 μL 50% Folin Ciocalteau reagent was added; the reaction mixture was kept undisturbed at RT for 30 minutes and readings were taken at 720 nm. Gallic acid was used as a standard for the calibration curve. The quantity of phenol present in the extract was expressed as Gallic acid equivalents.

Measuring Tyrosinase Activity Inhibition

The inhibition of tyrosinase activity has been shown to indicate skin whitening effects by the dopachrome method ¹⁵ : 150 µL of mushroom tyrosinase was mixed with 225 µL of 2.5 mM l-tyrosine, 225 µL of 0.4 M HEPES buffer (pH 6.8), and 300 µL of ethanol solution or 1 mg/mL C. madurensis (C. microcarpa) extracts and incubated for 15 minutes at RT. Then, the absorbance was measured at 475 nm, and the tyrosinase inhibition rates were calculated as follows:

$${\displaystyle \mathrm{T}\mathrm{y}\mathrm{r}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\mathrm{\%})=[(\mathrm{C}-\mathrm{D})-(\mathrm{A}-\mathrm{B})/(\mathrm{C}-\mathrm{D})]\times 100}$$

where A is the absorbance of the sample solution after the reaction, B is the solution with the sample before the reaction, C is the solution without the sample before the reaction, and D is the solution without the sample after the reaction, and 100% inhibition means that the enzyme activities were completely inhibited after addition of C. madurensis (C. microcarpa) extract samples.

Measurement of Melanin Production in Clone M-3 Cells

The amounts of melanin produced in Clone M-3 cells were measured using the following procedure ¹³ : 1 × 105 viable cells/well of Clone M-3 cells (KCLB 10053.1, Korean Cell Line Bank, Seoul, Korea) were inoculated into 96-well plates and then cultured in a CO₂ incubator (5%, 37°C) until 80% of the cells were attached to the wells. After 24 hours, each well was treated with C. madurensis (C. microcarpa) extracts at 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL or with ascorbic acid as a positive control for 24 hours. Then, the wells were washed with Phosphate-buffered saline and treated with trypsin-EDTA to detach and collect the cells by centrifugation at 5000 rpm for 10 minutes. The supernatant was removed, and the pellets were dried at 60°C. The melanin in the cells was obtained by placing the samples in a 60°C thermostatic bath and adding 100 µL of 1 M NaOH with 10% Dimethyl Sulfoxide (DMSO). The melanin quantity produced by the cells was calculated by measuring the absorbance at 490 nm in a microplate reader. Then, the relative concentration of melanin production from the cells was expressed as the ratio of the amounts of melanin after addition of the extract samples to the amounts without addition as a control by the following equation:

$${\displaystyle \mathrm{R}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{m}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}(\mathrm{\%})=(\mathrm{A}/\mathrm{B})\times 100}$$

where A is the amounts of melanin (μg/mL) after addition of the extract sample and B is the amounts of melanin produced without addition as a control (μg/mL).

Statistical Analysis

All data were expressed as mean ± standard deviation of 3 separate experiments. Design-Expert software (8.6 Statease Inc., Minneapolis, United States) was used for the experimental design and statistical analysis. Statistical significance was set at P < 0.05.