Effects on Anti-Aging Related Gene Expression

Effect of GLM Concentrated Solution on Anti-Ageing Related Gene Expression

(Using the Real-Time PCR Method)

Requesting Company : APA Corporation

Test Substance : GLM Concentrated Solution

Test Item : Analysis of effects on anti-aging related gene expression

Test Date : August 20, 2019

Storage Period : 5 years after test completion

1. Summary

When GLM concentrated solution was added to cultured human dermal cells:

• The gene expression of Hyaluronan Synthase 1 (HAS1) was significantly increased.

• The gene expression of Hyaluronan Synthase 2 (HAS2) was significantly increased.

• The gene expression of Hyaluronidase 1 (HYAL1) was significantly decreased.

From these results, GLM concentrated solution is considered to promote hyaluronic acid synthesis in the dermis and suppress hyaluronic acid degradation, suggesting it has effective anti-aging properties.

2. Test Purpose

Hyaluronic acid produced by dermal fibroblasts is thought to be essential for maintaining skin moisture, firmness, and elasticity. Impairment of this function may cause wrinkles and sagging. Therefore, this test evaluated the effect of the test substance on the expression of genes related to anti-aging, including hyaluronic acid synthesis and degradation, using real-time PCR.

3. Test Overview

To maintain skin that is firm, elastic, moist, and resistant to wrinkles, hyaluronic acid produced by dermal fibroblasts is considered essential. Hyaluronic acid plays a key role in skin flexibility and moisture.

Wrinkles and sagging observed in aged skin are thought to result from increased degradation and alteration of hyaluronic acid due to aging, i.e., physiological aging.

In this test, the effects of the test substance on gene expression related to hyaluronic acid synthesis (HAS1, HAS2) and degradation (HYAL1) were evaluated using real-time PCR to assess anti-aging effects.

4. Materials and Methods

4-1 Cells

Human neonatal dermal fibroblast cell line NB1RGB (RIKEN BRC, Japan) was cultured in a CO₂ incubator (5% CO₂, 37°C) and used in this study.

4-2 Medium

Eagle's Minimal Essential Medium (EMEM, Wako, Japan) containing 10.0% (v/v) fetal bovine serum (FBS, Hyclone, UK) and 1.0% (v/v) antifungal/antibiotic solution (Invitrogen, USA) was used.

4-3 Test Substance

The test substance was prepared in purified water and serially diluted 3-fold to yield three concentrations (100%, 30%, 10%) for testing. Purified water was used as the negative control.

4-4 Genes

Gene expression analysis was performed using TaqMan Assay (Applied Biosystems, USA).

Anti-aging related genes:

1. Human Hyaluronan Synthase 1 (HAS1, Assay ID Hs00758053_ml) – codes for an enzyme produced by dermal fibroblasts responsible for synthesizing high-molecular-weight hyaluronic acid involved in skin moisture retention.

2. Human Hyaluronan Synthase 2 (HAS2, Assay ID Hs00193435_ml) – codes for an enzyme involved in hyaluronic acid synthesis by dermal fibroblasts.

3. Human Hyaluronidase 1 (HYAL1, Assay ID Hs00201046_ml) – codes for an enzyme involved in hyaluronic acid degradation.

Internal reference gene:

1. Human Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH, Assay ID Hs02786624_gl)

4-5 Test Setup

For gene expression analysis, three 35 mm dishes per treatment group were used, and the average values were calculated. All operations, unless otherwise noted, were performed at room temperature.

4-6 Test Method

4-6-1 Cell culture and test substance addition:

1. Seeded 5 × 10³ NB1RGB cells in 2 mL medium per 35 mm dish and cultured for 24 hours in a CO₂ incubator.

2. After 24 hours, removed the medium and added the test substance-containing medium. Cultured for another 24 hours.

4-6-2 RNA extraction, purification, and quantification:RNA was extracted using PureLink™ RNA Mini Kit (Invitrogen, USA) following standard protocols. RNA concentration was adjusted to 10 μg/mL using TE buffer.

4-6-3 Real-Time PCR gene expression analysis:Reverse transcription of RNA to cDNA was performed using SuperScript™ IV VILO™ Master Mix with ezDNase (Invitrogen, USA). Real-time PCR was performed using QuantStudio®3 (Applied Biosystems, USA), and gene expression was calculated using the ΔΔCt method, with statistical significance evaluated by paired t-test (P<0.05, P<0.01, P<0.001).

5. Test Results

Cₜ values, averages, standard deviations, and gene expression results are shown in graphs in the attached figures.

5-1 Effect on HAS1 gene expressionThe relative expression of HAS1 with test substance compared to control (set as 1) is shown in Table 1 and Figure 1.

5-2 Effect on HAS2 gene expressionThe relative expression of HAS2 with test substance compared to control (set as 1) is shown in Table 2 and Figure 2.

5-3 Effect on HYAL1 gene expressionThe relative expression of HYAL1 with test substance compared to control (set as 1) is shown in Table 3 and Figure 3.

6. References

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4. CL. Daly, et al., J. Invest. Dermatol., 73(1), 84-87, 1979

5. DL. Lee, et al., J. Dermatol. Sci., 83(3), 174-181, 2016

6. KT. Dicker, et al., Acta Biomater., 10(4), 1588-1570, 2014

7. S. Shuster, et al., Br. J. Dermatol., 93(6), 639-643, 1975

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12. R. Sten and IL. Maibach, Chin. Dermatol., 26(2), 103-122, 2008

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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抗老化関連遺伝子発現に及ぼす影響 - 株式会社APAコーポレーション

Real Time PCR法を用いた GLM濃縮溶液の抗老化関連遺伝子発現に及ぼす影響 試験依頼