Tyrosinase and Elastase Inhibitory Activity (UMO Silica Powder)

KIWITA
Jan 15
4 min read

Updated: Jan 18

Test Report

Tyrosinase and Elastase Inhibitory Activities of

UMO Micron Powder

Evaluation

Client : APA Corporation Co., Ltd.

Test Material : UMO Micron Powder

Test Items : Cosmetic 3-Area Screening Set

〈Skin Whitening · Anti-aging · Antioxidant〉

Tyrosinase inhibitory activity

Elastase inhibitory activity

Test Date : March 16, 2020

Data Retention Period : 5 years after completion of the test

Report Structure

Summary
Test Objective
Test Overview
Materials and Methods　
4-1. Test Material　
4-2. Test Procedures　
4-2-1. Tyrosinase Inhibitory Activity　
4-2-2. Elastase Inhibitory Activity
Test Results　
5-1. Tyrosinase Inhibitory Activity　
5-2. Elastase Inhibitory Activity
References

1. Summary

UMO Micron Powder:

Significantly increased the tyrosinase inhibition rate compared with the control.
Significantly increased the elastase inhibition rate compared with the control.

Based on these results, UMO Micron Powder is considered to be an effective test material with skin-whitening and anti-aging properties.

2. Test Objective

To evaluate the tyrosinase inhibitory activity and elastase inhibitory activity of the test material and to verify its characteristics in the skin-whitening and anti-aging fields.

3. Test Overview

In aged skin, signs of aging such as spots, wrinkles, and sagging are commonly observed.The accumulation of melanin in the epidermis (spots) is strongly associated with tyrosinase activity in melanocytes.In addition, the reduction of skin elasticity (firmness), which contributes to wrinkles and sagging, is believed to be related to elastase activity, an enzyme that degrades elastic fibers (elastin) produced by dermal fibroblasts.

Therefore, in this study, the tyrosinase inhibitory activity and elastase inhibitory activity of the test material were evaluated to assess its potential application in anti-aging cosmetic formulations.

4. Materials and Methods

4-1. Preparation of Test Material

The test material was freshly prepared as a 10% solution using ultrapure water (CAS No. 7732-18-5, Wako, Japan).Serial dilutions were prepared using ultrapure water at a dilution ratio of 10 to obtain three concentrations:0.001%, 1%, and 10%.

Ultrapure water was used as the control.

4-2. Test Procedures

4-2-1. Tyrosinase Inhibitory Activity¹⁻³

The effect of the test material on tyrosinase activity was evaluated using Dihydroxyphenylalanine (DOPA) as the substrate.

Into a 1.5 mL tube (Cat. No. 0030125150, Eppendorf, Germany), the following were added:

80 μL of test material or control
160 μL of tyrosinase solution (40 units/mL; CAS No. 9002-10-2, Sigma-Aldrich, USA)
400 μL of 100 mM phosphate buffer (pH 6.8)

The mixture was vortexed and incubated at 23°C for 3 minutes.For blanks, 100 mM phosphate buffer was used instead of the tyrosinase solution.Three wells were used per treatment group.

After 3 minutes, 200 μL of 2.5 mM L-DOPA solution (CAS No. 59-92-7, Wako, Japan) was added and mixed.The mixture was centrifuged at 12,000 × g for 3 minutes, and the supernatant was collected.Aliquots of 210 μL were transferred to a 96-well plate (Lot No. 9107, Corning, USA), and absorbance at 490 nm (OD490) was measured using a microplate reader (SPARK®10M, TECAN, Switzerland).

The plate was incubated at 23°C for 10 minutes, and OD490 was measured again.

Tyrosinase activity and inhibition rates were calculated using the following equations:

Tyrosinase activity (%)

Tyrosinase inhibition rate (%)

Where:

Ab₀ / Ab₁₀: Blank OD490 before / after incubation
Ac₀ / Ac₁₀: Control OD490 before / after incubation
As₀ / As₁₀: Sample OD490 before / after incubation

4-2-2. Elastase Inhibitory Activity⁴⁻⁵

The effect of the test material on elastase activity was evaluated using N-Succinyl-Ala-Ala-Ala-p-Nitroanilide as the substrate.

Into a 1.5 mL tube, the following were added:

200 μL of test material or control
200 μL of elastase enzyme solution (1.25 μg/mL; CAS No. 39445-21-1, Sigma-Aldrich, USA)
400 μL of substrate solution (CAS No. 52299-14-6, Sigma-Aldrich, USA)

The mixture was vortexed and incubated at 37°C for 15 minutes.For blanks, 0.05 M Tris-HCl buffer was used instead of the elastase enzyme.Three wells were used per treatment group.

After 15 minutes, samples were centrifuged at 12,000 × g for 3 minutes, and the supernatant was collected.Aliquots of 200 μL were transferred to a 96-well plate, and absorbance at 415 nm (OD415) was measured.

Elastase activity and inhibition rates were calculated using the following equations:

Elastase activity (%)

(S−SB)(C−CB)×100\frac{(S - SB)}{(C - CB)} \times 100(C−CB)(S−SB)×100

Elastase inhibition rate (%)

(C−CB)−(S−SB)(C−CB)×100\frac{(C - CB) - (S - SB)}{(C - CB)} \times 100(C−CB)(C−CB)−(S−SB)×100

Where:

C / CB: Control OD415 / Control blank OD415
S / SB: Sample OD415 / Sample blank OD415

5. Test Results

Mean values, standard deviations, and bar graphs of the results are provided in the attached documents.

5-1. Tyrosinase Inhibitory Activity

The mean and standard deviation of tyrosinase activity and inhibition rates (with the control set as 100%) are shown in Table 1, and the bar graph of tyrosinase activity is shown in Figure 1.

5-2. Elastase Inhibitory Activity

The mean and standard deviation of elastase activity and inhibition rates (with the control set as 100%) are shown in Table 2, and the bar graph of elastase activity is shown in Figure 2.

6. References

(References 1–8 translated and preserved as cited)

Table 1: Tyrosinase activity and inhibition rates of the test material

Figure 1: Tyrosinase activity of the test material

n = 3, unpaired t-test, P < 0.05, *P < 0.01, ***P < 0.001

Table 2: Elastase activity and inhibition rates of the test material

Figure 2: Elastase activity of the test material