Improving Cosmetic Activity by Optimizing Centella asiatica Extraction Process

Abstract

This study showed the use of Centella asiatica as a good source of compounds for skin therapy and antioxidant activation cosmetics. The goal of this study was to provide cosmeceutical activity data about the extraction yield and total phenol content of C. asiatica by optimizing the focused high ultrasound (INEFU) and ultrasound extraction (UE) conditions. Under optimal extraction conditions, that is, 1800 W for 45 minutes and 500 W for 45 minutes, 28.73% and 30.21% (w/w) of the highest extraction yields and polyphenol contents were obtained, respectively. For the skin whitening activity, tyrosinase inhibitory activity was observed at 61.52% in INEFU extracts, which was circa 20.3% higher than that of UE extracts (51.11%). To reduce melanin production in Clone M-3 cells, 86.9% melanin production was observed following the addition of control, water extraction relative to the control without a sample, and 79.6% and 74.5% were found in UE and INEFU conditions, respectively. In conclusion, the highest total phenol secretion was obtained under optimal conditions and resulted in a significant improvement in the cosmetic activities of C. asiatica extracts.

Currently, ultrasound-assisted extraction has been applied to effectively extract natural components from different kinds of materials. ^1,2 Compared with the conventional hot water extraction method, ultrasound-assisted extraction can enhance the extraction yield, save operation time, and streamline the operation 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 quality living and growing old in good shape. Accordingly, the functional cosmetics markets are expanding. ⁴ Response surface method has been applied widely to accelerate and optimize the operation process by saving time, energy, and raw materials. ^5,6 Centella asiatica, a member of the family Apiaceae, has been used by many cultures in Asia for over 2000 years as a medicinal plant. ⁷ Already a very important medicinal plant in the Orient medicine, ⁸ it is also now becoming very popular in the West and the East. ^9,10 The major bioactive chemical compounds in C. asiatica are triterpenes, alkaloids, volatile oils, flavonoids, and phenol glycosides, as reviewed by Songvut. ¹⁰ Centella asiatica has active compounds such as pentacyclic triterpenes and it is used in treating small wounds, burns, psoriasis, and scleroderma. ¹¹ Centella asiatica is also called guta kola. The function of guta kola involves increasing fibroblast production and synthesis of collagen and fibronectin. ^12,13 Brinkhaus et al reported that the application of C. asiatica showed a beneficial effect in reducing the progression of cellulite as well as resulting in significant improvement in 85% of the participants with no adverse reactions. ¹³ Moreover, researchers reported that it can improve the tensile strength of newly formed skin and reduce the inflammatory phase of hypertrophic scars and keloids.

Therefore, it is expected that healthy and eco-friendly products can be developed by applying natural derivative cosmetic resource that has diverse bio-functions for the production of make-up. This study was conducted to provide relatively new cosmeceutical information about C. asiatica, which has been used widely as a source of 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 contents of the extracts under water extraction (WE), focused high ultrasound (INEFU), and ultrasound extraction (UE) conditions. With respect to INEFU, the yield increased when the ultrasound watts treatment increased, and it was highest at 500 W. Therefore, it appears that a 45 minutes process time at 1800 W would be considered the most efficient extraction condition. The extract from INEFU procedure had the highest yield at 28.73% of the UE procedure. An ultrasonic wave energy of 120 kHz or higher could also increase the diffusion and solubility of the solvent, which resulted in an increase in the polyphenol contents of the extracts. A similar result was also reported in a previous study. ¹⁴ The amount of total polyphenols in the WE, INEFU, and UE extracts was studied. INEFU extract showed significantly higher phenolic content than WE and UE extracts (Figure 1).

Table 1

Comparison of the Extraction Yields of Centella asiatica in Relation to Different Extraction Processes.

Extraction methods	Solvents	Extraction conditions			The yield of active components (%)
		A	B	C
INEFU	Water	1800	45	40	11.89 ± 0.58
UE	Water	500	45	40	9.24 ± 0.88
Control	Water	0	45	40	5.23 ± 0.72

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

Figure 1

Total phenolic content of Centella asiatica is means of 3 replicates ± standard deviation.

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 skin damage. Therefore, the reduction in melanin biosynthesis by tyrosinase has been used as an indicator of the whitening activities of natural products. ¹⁵ As shown in Table 2, all the C. asiatica extracts generally had inhibition effects of 53.22% ± 0.78% (WE), 51.11% ± 1.02% (UE), and INEFU extract specifically inhibited circa 61.52% ± 0.72% of the tyrosinase activity in which INEFU extraction was 20.3% higher than 51.11% ± 1.02% in the UE extract. Its inhibition activity increased with an increase in concentration. This result also indicates that INEFU extract has a greater whitening effect by effectively reducing tyrosinase activity. In addition to the tyrosinase activity inhibition, the reduction in Clone M-3 cell melanin production after sample treatment has been employed to measure the whitening activities of natural products. ¹⁶ 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. However, INEFU extract had 74.51% inhibition ratio of melanin production compared to the production of melanin without any treatment as a control relative to 1.0 mg/mL concentrations. This result indicates that the extract from INEFU has great whitening activity potential because of ascorbic acid, which is a positive control. To inhibit melanin production (Table 2), 82.45% melanin was observed following the addition of WE relative to the control without a sample, and 79.61% and 74.51% were found in UE and INEFU conditions, respectively. Thus, it can be concluded that the highest extraction yield from INEFU process results in the highest cosmetic activities relative to those from other processes by eluting the highest amount of the polyphenols from C. asiatica. It appears that the treatment using 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 high ultrasonic wave intensity, increased the diffusion and solubility of the solvent and accelerated the elution of useful components from C. asiatica.

Table 2

Comparison of Cosmetic Activities of Centella asiatica in Relation to Different Extraction Processes.

	Extraction methods
	WE	INEFU	UE
Tyrosinase inhibitory activity (%)	53.22 ± 0.78	51.11 ± 1.02	61.52 ± 0.72
Melanin production (%)	82.45 ± 0.43	79.61 ± 0.11	74.51 ± 0.41

WE, water extraction for 45 minutes at 40°C with water.

UE, ultrasound extraction for 45 minutes at 500 W at 40°C with water.

INEFU, focused high ultrasound extraction for 45 minutes at 1800 W at 40°C with water.

This was the first study to indicate that various cosmetic activities of C. asiatica 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 highest whitening activity and lowest melanin production. This finding indicates that focused high ultrasounds effectively destroy the tissues and the cell walls of the hard-surfaced plant C. asiatica, which result in an improved elution of the active cell components. Moreover, focused high ultrasound loosens the hard tissues that prevent the elution of active components, and increases contact area with solvent to accelerate active component diffusion and elution. ¹⁷ In addition, the high input energy produced by the cavitation from the ultrasonic device destroys the inner tissues of the cells, which shortens the travel distance of the extracts, facilitates solvent diffusion, and increases solubility. The resulting breakage of bonds between the atoms in the high molecular polymer may contribute to the elution of substances that are not easily eluted using the conventional methods. Therefore, INEFU process can maintain the synergistic effect that improves both the extraction yield and the elution of polyphenols, which results in an increase in its cosmetic activities, such as whitening, and expands the use of this natural polyphenols as multifunctional cosmetics.

Materials and Methods

Sample Preparation

Centella asiatica was obtained from local farmers (Habcheon, South Korea), and dried and crushed for WE, concentrated focused high ultrasound extraction (INEFU), and ultrasonic extraction (UE). Centella asiatica powder was mixed with water at a ratio of 1:10 in glass lock. A UE extractor (Ilshin Lab, Daejeon, Korea) was used for 45 minutes at 500 W/vol. For the INEFU, C. asiatica samples 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 vacuum evaporator (Eyela, 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 (Korean Cell Line Bank, Seoul, Korea). Melanocytes were cultured with 10% heat-inactivated fetal bovine serum and Dulbecco's modified eagle medium (DMEM) (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.

Measurement of Extraction Yields and Polyphenol Contents 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 the C. asiatica extract. A test sample (50 µL) was mixed with 2 mL of 2% sodium carbonate and allowed to stand at room temperature (RT) for 2 minutes. During this time, 100 μL of 50% Folin Ciocalteau reagent was added; the reaction mixture was kept undisturbed at RT for 30 minutes and the 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.

Measurement of Tyrosinase Activity Inhibition

The inhibition of tyrosinase activity has been shown to indicate skin whitening effects by the dopachrome method ¹² : 150 µL of mushroom tyrosinase (150 U) 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. asiatica extract and incubated for 15 minutes at RT. Then, the absorbance was measured at 475 nm, and the tyrosinase inhibition rates were calculated as follows:

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

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 the addition of C. asiatica 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 procedures ¹⁵ : 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. asiatica 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 (PBS) 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 by 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:

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

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