Effect on Promotion of Hyaluronic Acid Production
- KIWITA
- Jan 15
- 5 min read
Effect of Umo Concentrated Solution on Promotion of
Hyaluronic Acid Production in Dermal Fibroblasts
Exam number: CAS19020503
Test Report
Client : APA Corporation Co., Ltd.
Test Substance : Umo Concentrated Solution
Test Item : Promotion of hyaluronic acid production in dermal fibroblasts
Test Date : February 5, 2019
Storage Period : 5 years after test completiont
Report Structure
1 Summary
2 Test purpose
3 Exam overview
4 Materials and test methods
4-1 cells
4-2 Medium
4-3 Test substance
4-4 Test configuration
4-5 Test operation
4-5-1 Cell culture
4-5-2 Promotion of hyaluronic acid production
5 Test results
5-1 Promoting hyaluronic acid production
6 References
1 Summary
The addition of umo concentrated solution significantly increased the hyaluronic acid production rate compared to the control. From the above, it was suggested that the concentrated solution of horse mackerel may be effective for anti-aging care.
2 Test purpose
It is believed that the production of hyaluronic acid by dermal fibroblasts is essential for maintaining skin that is firm, elastic, fresh, and wrinkle-resistant. Therefore, in this study, we will verify the effect of the test substance on hyaluronic acid production by fibroblasts.
3 Exam overview
The wrinkles, sagging, and fine lines observed in the skin of aging people are thought to be caused by decreased skin elasticity, decreased flexibility, and dryness 1-3). In particular, hyaluronic acid produced by dermal fibroblasts is thought to play an important role in skin flexibility and moisture. Therefore, in this study, the ability of the test substance to promote hyaluronic acid production in dermal fibroblasts was evaluated.
4 Materials and test methods
4-1 Cells
The dermal fibroblast cell line NB1RGB cells (RIKEN BRC, Japan) derived from human newborns were incubated in a CO2 incubator.
(CO2 concentration, 5%, 37°C) to conduct this test.
4-2 Medium
10.0% (v/v) Fetal Bovine Serum FBS, Cat No. SH30071.03, Hyclone, UK) and 1.0% (v/v) Antifungal
Eagle’s Minimal containing agent (Antibiotic-Antimycotic 100X, Cat No. 15240-062, Invitrogen, USA)
Essential Medium (EMEM, Cat No.051-07615, Wako, Japan) was used.
4-3 Test substance
Contains 30μM L (+) -ascorbic acid (CAS No. 50-81-7, Kanto Chemical, Japan) and 0.1% FBS
By EMEM
The test substance was diluted to give a total of 3 concentrations (0.03%, 0.3%, 3%) before use (final concentrations were 0.01%, 0.1%, 1%).
4-4 Test configuration
For calculation of hyaluronic acid production, 96-well plate (cell culture: Cat No. 3595,
Corning, USA Hyaluronic Acid Production: Cat No. 3855, Thermo scientific, USA) 3 wells were used. Also,
The test was conducted with one test substance group and one control group per plate. including test substance preparation;
Test operations were performed at room temperature unless otherwise stated.
4-5 Test operation
4-5-1 Cell culture
1) Seed NBIRGB cells at 2.0 × 104 cells/200 μL per well in a 96-well plate, and
Cultured in an incubator for 24 hours. In addition, to prevent drying during culture, 200 μL of Phosphate buffer saline (PBS(-), Cat No. 198601, Nissui, Japan) was added to wells not used for testing.
2) After 24 hours, add 100 μL of test substance and control to a 96-well plate and place in a CO2 incubator.
The cells were cultured for 48 hours.
(Final concentration of test substance: 0.01%, 0.1%, 1%).
EMEM containing 30 μM L-ascorbic acid and 0.1% FBS was used as a control.
3) After 48 hours, the culture supernatant was dispensed into a new 96-well plate and stored frozen (-80°C). Shown in 4-5-2
According to the method, the amount of hyaluronic acid was measured using Enzyme-Linked Immuno Sorbent Assay (ELISA)4), and the hyaluronic acid production promoting effect of the test substance was evaluated.
4-5-2 Hyaluronic acid production promotion effect evaluation 5-9)
Testing on the production amount of hyaluronic acid, which is said to hold water between collagen and elastin.
The efficacy of the substances was evaluated by sandwich ELISA.
1) 100μL of 5μg/mL Hyaluronan binding protein (HABP, Cat No. BC40, Hokudo, Japan) solution
was added to a high adsorption type 96-well plate and left standing at 4°C for 24 hours.
2) Remove the HABP solution and add 200 μL of 0.05% Tween20 in 1.5 M NaCl solution (Wash buffer, USA NaCl:
Washed three times with Cas No. 7647-14-5, Wako, Japan).
3) 100 μL of 5% BSA solution was added and left at 4°C overnight.
4) After removing the BSA solution and washing twice with 200 μL of Wash buffer, the plate was dried.
5) Add 1 volume of culture supernatant diluted 100 times with PBS (-) containing 0.5 M NaCl, 0.02% Tween20, and 1% BSA.
100 μL per well was added to a 96-well plate and left at room temperature for 2 hours.
6) After 2 hours, wash 4 times with 200 μL of Wash buffer, and add 100 μL of biotin-labeled HABP (Cat No.
BC41, Hokudo, Japan) solution was added and left at room temperature for 30 minutes.
7) After 30 minutes, wash 4 times with 200 μL Wash buffer, once with 200 μL ultrapure water, and add 100 μL strain.
Putavidin-labeled HRP solution was added and left at room temperature for 30 minutes.
8) After 30 minutes, wash 4 times with 200 μL Wash buffer and once with 200 μL ultrapure water, and add ABTS to 1 well.
100 μL was added per tube and left at room temperature for 10 minutes.
9) Shake the 96-well plate at 270 rpm for 30 seconds to even out the dye in the wells, then
Absorbance at 405 nm (OD405) was measured using a plate reader.
10) The hyaluronic acid production rate of the test substance was calculated using the OD405 of the control group as 100%.
5 Test results
The average value, standard deviation, and bar graph of the measurement results are shown in a separate sheet.
5-1 Evaluation of hyaluronic acid production promotion effect
The measured value of OD405 of each well and the hyaluronic acid production rate of the test substance when the hyaluronic acid production rate of the control was taken as 100% are shown in Table 1 and FIG.
6 References
1) Y. Hara., et al., Int. J. Cosmet. Sci., 39(1), 66-71,2017.
2) JW. Choi., et al., Skin Res. Technol., 19(1), 349-355, 2013.
3) T. Fujimura., et al., J. Dermatol. Sci., 47(3), 233-239, 2007.
4) S. I. Rennard., et al., Analyt Bioc., 104, 205-214, 1980.
5) K. Chichibu., et al., Clinica Chimica Acta., 181, 317-324, 1989.
6) H. Maeda., et al., Biosci. Biotechnol. Biochem., 63(5), 892, 1999.
7) JRM. Martins., et al., Anal. Biochem., 319, 65, 2003.
8) S. Haserodt., et al., Glycobiology, 21(2), 175-183, 2011.
9) H. Yuan., et al., Glycobiology, 23(11), 1270-1280, 2013.
Table 1 Effect of the test substance on hyaluronic acid production
Vertical axis shows hyaluronic acid production rate (%);
Horizontal axis shows test substance concentration (%)
Figure 1 Effect of test substance on hyaluronic acid production
n = 3, mean±s.d., unpaired t-test, P< 0.05, *P < 0.01, ***P< 0.001
s.d.: standard deviation
Note: This paper is translated from the following URL. The content is provided for reference on the scientific research of the raw material only. Whether APA raw materials are used or not, we hope this research will help increase understanding and awareness of body minerals.
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