Evaluation of the Antioxidant Activity, Collagen Synthesis and Stability of Bright and Firm hybrid emulsion I

1 Department of Community and Family Medicine, Faculty of Medicine, Thammasat University, Pathumthani , Thailand 2 Faculty of Medical Technology, Nakhonratchasima College, Nakhonratchasima, Thailand 3 Faculty of Medicine, Burapha University, Chon Buri, Thailand 4 Kochicure Healthcare Co., Ltd, Bangkok, Thailand 5 Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, Umeå, Sweden

The Bangkok Medical Journal Vol. 15 The aim of this study is to evaluate antioxidant activity, collagen synthesis, and stability of bright and firm hybrid emulsion I.

Determination of pH
The pH meter was calibrated using standard buffer solution. A 0.5 g of the bright and firm hybrid emulsion I was weighed and dissolved in 50 ml of distilled water and its pH was measured. 4 Heating and cooling test The heating and cooling test was used for the stability determination of bright and firm hybrid emulsion I. The heating and cooling method was investigated using the modified method from Biskup et al. 5 (2013). Bright and firm hybrid emulsion I was kept at a temperature of 4ºC (48 hours) and a temperature of 45ºC (48 hours) for 7 cycles. The physical appearances of the bright and firm hybrid emulsion I such as color, smoothness, and pH were observed before and after testing.

Microbial growth test
The bright and firm hybrid emulsion I formulation was inoculated on the plate of Muller Hilton agar media by streak plate method. The plates were incubated at 37 ºC for 24, 48, 72, 96, 120, 144, and 168 hours. After incubation time, plates were taken out and checked for microbial growth by comparing it with the control. 4 Antioxidant methods 2,2′-Diphenyl-1-picrylhydrazyl (DPPH) method DPPH (2,2-diphenyl-1-picryl-hydrazyl) method is a rapid way to evaluate antioxidants by spectrophotometry. This assay measures the ability of antioxidants which will donate hydrogen (H) on DPPH. It is based on changing the DPPH free radicals which are purple to pale yellow. In brief, 0.22 mg/ml DPPH stock solution was prepared by accurately weighing 22 mg of DPPH in 100 ml of absolute methanol and allowed to settle overnight at 4 °C. To obtain DPPH working solution, the DPPH stock solution was diluted at 1:5 by adding 20 ml stock solution to 80 ml of methanol. To 1.9 ml of DPPH working solution, 100 µl of the final concentration of the extract and standard solution were added separately. Each solution was incubated in the dark at 37 ºC for 30 minutes. After incubation absorbance of the solution, it was measured at 517 nm by using UV-Vis spectrophotometer. Ascorbic acid was used as the reference standard compound. All determinations were carried out in triplicate. 6 Antioxidant capacity is expressed in mg Ascorbic acid equivalent /100g extract.
The percentage inhibition was calculated by using the following equation: 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) method ABTS method was evaluated for antioxidant activity. The experiment was performed according to Censi et al.(2018). 7 In brief, ABTS•+ stock solution was prepared by 1:1 chemical reaction of 15 mM ABTS and 5mM potassium persulfate in dark condition, overnight. To obtain ABTS•+ working solution, the ABTS•+ stock solution was diluted 1:50 with 5 mM phosphate buffer saline, pH 7.4. In the assay, either final concentration of the extract (10 µl), Trolox standard solution (10 µl) or ethanol (10 µl) for controls were mixed with ABTS•+ working solution (290 µl) in 96-well microplate. The microplate was incubated in the dark at 37°C for 6 minutes. All determinations were carried out in triplicate. After incubation, the absorbance of each well was determined at 734 nm by using a microplate reader. Background absorbance was corrected by subtracting the absorbance value of blank. The antioxidant activities are expressed in percentage inhibition and mg Trolox equivalent/100 g extract. The ABTS percentage inhibition was calculated by using the equation already described for the DPPH method. Antioxidant capacity is expressed in mg Trolox equivalent /100g extract.

Ferric reducing antioxidant power (FRAP) method
The FRAP method measures the antioxidant potential in a sample based on the ability of a compound to reduce the complex of ferric ion (Fe 3 +) and TPTZ to the ferrous form (Fe 2 +) in acidic condition. The reaction was monitored by determining the change of absorbance at 593 nm. This method was investigated using the modified method from A control x 100 Evaluation of the Antioxidant Activity, Collagen Synthesis and Stability of Bright and Firm hybrid emulsion I (v/v/v). This working solution was protected from light and prewarmed to 37°C before used. In the assay, the final concentration of the extract and standard solution were added to 2.25 mL FRAP working solution and 0.225 mL of deionized water. FeSO 4 •7H 2 O (Iron (II) sulfate heptahydrate) was dissolved in deionized water for using as standard solution.
The reaction mixture was shaken and incubated at 37°C for 5 min. Absorbance was measured at 593 nm by using the UV-Vis spectrophotometer. FRAP working solution with deionized water instead of a sample and was used as a blank. All determinations were determined in triplicate. Results are expressed in mg Iron (II) sulfate heptahydrate equivalent /100g extract.

Animals
In this study the histological analysis was investigated using the method of Bunman et al.

Animal preparation
Male Wistar rats were randomly divided into 2 groups of 6 animals, and assigned to receive 0.5 gram of cream base (control), and bright and firm hybrid emulsion I applied to the skin. Histological analysis evaluated collagen synthesis on day 28 post applied.
The Masson's trichrome examination Collagen synthesis was proved by skin stained with masson's trichrome. This study was investigated using the method of Chuenwattana et al. (2018). 3 Skin tissue sections were deparaffinized in xylene, hydrated, and stained in Weigert's iron hematoxylin solution for 10 minutes. They were washed and stained in acid fuchsin solution for 3 minutes, rinsed in distilled water, treated with phosphomolybdicphosphotugstic acid solution for 15 minutes, immediately submerged into aniline blue solution for 10 minutes, rinsed in distilled water, treated with acetic solution for 10 minutes, dehydrated in 95% alcohol, cleared twice in xylene, mounted with a cover slip, and observed under a light microscope.

Statistical analysis
The results are expressed as means ± standard deviation (SD). Stability studies and histological analysis were analyzed using descriptive statistics.

Results
Heating and cooling test of bright and firm hybrid emulsion I evaluated color, smoothness, and pH. The color of bright and firm hybrid emulsion I appeared as pearl like color. On 1 st to 7 th cycle, the color and pH of bright and firm hybrid emulsion I was unchanged when compared with the control group (Table 1). On 1 st to 7 th cycle, the physical of bright and firm hybrid emulsion I appeared smooth, the physical appearance was unchanged or no separation phase (Figure 2).
The microbial growth test was observed post incubate 24, 48, 72, 96, 120, 144, and 168 hours, respectively. Bright and firm hybrid emulsion I was not found to have a bacterial colony or no bacterial growth on media plates when compared with the control ( Table 2).  The antioxidant activity of bright and firm hybrid emulsion I was evaluated by 3 methods as DPPH, ABTS, and FRAP methods respectively. The result of antioxidant activity of bright and firm hybrid emulsion I had a percentage of inhibition 14.93 ± 0.39 ascorbic acid equivalent 0.53 ± 0.01 mg/100 g extract by DPPH method, percentage of inhibition 59.60 ± 6.21 Trolox equivalent 14.91 ± 1.50 mg/100 g extract by ABTS method, and Iron (II) Sulfate Heptahydrate equivalent 8.32 ± 0.44 mg/100 g extract by FRAP method, respectively ( Table 3).
The results from Masson's trichrome of rat skins staining can clearly differentiate important morphological keys for collagen synthesis assessment. Muscle, Hemoglobin, keratin and fiber are stained red color. Adipose tissue and cytoplasm are stained light red or pink. Cell nuclei show dark brown to black and collagen fiber is stained aniline blue. On day 28 post applied with bright and firm hybrid emulsion I showed higher levels of collagen fiber when compared to the control group ( Figure 2).

Discussion
Determination of pH, heating and cooling test, and microbial growth test were used for evaluation of the stability of a cosmetic creams. 8 The general properties of the bright and firm hybrid emulsion I had a pH that ranged between 5.5 and 6. This pH range is necessary and appropriate for skin pH and because the preservatives are effective at those levels. Study of the effect of temperature on the stability of the bright and firm hybrid emulsion I showed that the temperatures did not affect the physical appearance of the bright and firm hybrid emulsion I through the period of 7 cycles. The temperature ranged between 4°C and 45ºC and is necessary and appropriate for storage.
The study on the effect of light on the stability of the bright and firm hybrid emulsion I demonstrated that the physical appearances (color and solidity) of the bright and firm hybrid emulsion I were not changed throughout the period of 7 cycles. However, antioxidant ingredients such astaxanthin, ascorbic    acid, niacinamide etc. can degrade in light conditions, because the antioxidant ingredients are oxidized when they are exposed to light. 9 Therefore, bright and firm hybrid emulsion I is recommended to be kept under dark condition.
The microbial growth test was evaluated for preservative property, which control microbial growth. [10][11][12] Bright and firm hybrid emulsion I formulation added Caprylyl Glycol (1,2-Octanediol), Sodium Benzoate, and Potassium Sorbate as a preservative. The result demonstrated that bright and firm hybrid emulsion I formulation did not have microbial growth. Therefore, this preservative of bright and firm hybrid emulsion I can control microbial growth.
UV radiation from sun light damages the skin by increasing levels of free radicals. Cigarette smoke, pollution, stress, illnesses, and drugs etc. can increase levels of free radicals in the physical body. 7 For their elimination antioxidants contained in antioxidant creams are helpful. 13 The study demonstrated that bright and firm hybrid emulsion I can reduced free radical scavenging activity because it contained high active ingredients such as pseudoalteromonas ferment extract, chlamydomonas extract, hamamelis virginiana (witch hazel) extract, and astaxanthin etc.
Collagen and hyaluronan are synthesized in the fibroblast cells, which is the main component of connective tissue such as skin, tendon, etc., giving it strength and flexibility. The fibroblasts' proliferation is related to collagen content in skin. 14 Skin aging is also associated with loss of skin moisture. The key molecule involved in skin moisture is a glycosaminoglycan (GAG), hyaluronic acid (HA), or hyaluronan with a unique capacity to bind and retain water molecules. 15 From the results, the collagen of skin in the treated group on days 28 was higher than the control group. Therefore, bright and firm hybrid emulsion I may play an important role in fibroblasts proliferation, which increases collagen and HA synthesis in skin.

Conclusion
This study was able to show that bright and firm hybrid emulsion I not only displays cosmetic efficacy as an antioxidant effect and to accelerate collagen synthesis, but also has pleasant sensorial characteristics for cosmetic use.

Conflicts of interest
The authors declare that they have no conflicts of interest