Abstract
Background
Angelica gigas Nakai is used as an herbal pharmaceutical material in Korea.
Aims
To investigate the anti‐wrinkle effects of A. gigas Nakai root extracts (ARE) using mineral‐rich water in in vitro and clinical trials.
Materials and methods
The cell viability of ARE was evaluated using a water‐soluble tetrazolium salt assay. After evaluating ARE's cytotoxicity, we used an enzyme‐linked immunosorbent assay kit to assess the effects of ARE on type I collagen in human dermal fibroblasts (HDFs). During a double‐blinded in vivo clinical study, participants were randomly assigned to use the sample and placebo formulations for the left and right sides of their face over an 8‐week period. We evaluated the anti‐wrinkle properties of the formulations using PRIMOS Lite and a global photodamage score.
Results
A. gigas Nakai root extracts cytotoxicity was evaluated in HDFs. We demonstrate that ARE increased type I collagen production by 40% at 50 μg/ml as compared with the control. The use of an ARE lotion significantly reduced the presence of crow's feet wrinkles in comparison with the use of the placebo after 8 weeks. Additionally, use of the ARE lotion led to decreased photodamage scores, indicating anti‐wrinkle effects.
Conclusion
The use of ARE with mineral‐rich water has anti‐wrinkle effects in in vitro and clinical trials.
Keywords: Angelica gigas Nakai root extracts, anti‐wrinkle effects, in vitro, in vivo, mineral‐rich water
1. INTRODUCTION
Skin senescence is a natural phenomenon of the aging process in humans. The aging of skin has various symptoms, such as dry skin; changes in skin thickness, skin tone, and skin elasticity; and wrinkles. 1 Aging is influenced by both intrinsic and extrinsic processes. The intrinsic skin aging process is genetic and causes morphological changes over time. The extrinsic skin aging process is caused by environmental factors, such as sun exposure or air pollution, and is characterized by wrinkles and pigmented lesions. 2 Skin contains dermal fibroblasts, which are differentiated mesenchymal cells that are primarily responsible for the organization of the dermis through the synthesis of dermal components, such as collagens. 3 In human skin dermis, a collagen‐rich extracellular matrix (ECM) is maintained by dermal fibroblasts. In addition, controlled degradation of the ECM is regulated through the modulation of the expression of matrix metalloproteinases (MMPs) in dermal fibroblasts. 4 MMPs play an important role in breaking down most substrate proteins that make up the ECM. 5
Angelica gigas Nakai, of the genus Umbelliferae, is used as compound pharmaceutical effects in Korea and northeastern China. 6 It is known to improve the appearance of aging skin by promoting blood circulation, providing ultraviolet protection, regenerating tissue, and counteracting the pigmentation changes caused by tyrosinase degradation. 7 The active component in A. gigas Nakai root extracts (ARE) is decursin or decursinol angelate. Purification or extraction of the active compounds may increase the efficacy of ARE. 8
The importance of water as a material for functional is increasing, not only as a role of quenching thirst. Mineral‐rich water is abundant in minerals and nutrients derived from volcanic rocks. 9 It is a resource that can only be obtained in Jeju island of South Korea and made by natural filtering of seawater in the volcanic rocks. It contains a large content of minerals such as sodium, manganese, zinc, calcium, potassium to exist in the form of a mixture of seawater and groundwater. 10 Mineral‐rich water in Jeju is also known to have moisturizing effects on the skin and increase the expression of membrane proteins involved in transport protein. 11
In a previous study, we reported improved biological effects on skin metabolism caused by the use of ground sea water with a high mineral composition. 12 In Jeju, we further demonstrated the biological efficiency of combining ARE with mineral‐rich water. 13 In this study, we aimed to investigate whether the use of ARE with mineral‐rich water could provide anti‐wrinkle effects in both in vitro and in vivo trials.
2. MATERIALS AND METHODS
2.1. Materials
A. gigas Nakai was purchased from Tojongherb Co., Ltd. A fibroblast basal medium (FBM) was purchased from Lonza. Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific. Penicillin‐streptomycin was purchased from Welgene. Water‐soluble tetrazolium salt (WST‐1) was obtained from DoGenBio.
2.2. Sample preparation
Ground A. gigas Nakai was cultured with mineral‐rich water at 5°C for 7 days in stationary condition. After removing the water, A. gigas Nakai was extracted with 70% ethanol at 60°C for 5 h. After adding 10% sodium bicarbonate solution in the same ratio, the mixture was concentrated using pressure reduction and then stirred at a high temperature. The extract was filtered using Whatman No. 2 filter paper, and the solvent was removed by decompression. This extract was frozen and stored at −20°C.
2.3. Cell culture
Human dermal fibroblasts (HDFs) were obtained from American Type Culture Collection. The HDFs were maintained in FBM containing 10% FBS and 1% penicillin‐streptomycin at 37°C in an incubator containing 5% CO2.
2.4. Cell viability
The effects of ARE on cytotoxicity were measured using a WST‐1 assay. WST‐1 produces a yellow‐colored water‐soluble formazan product upon the reduction by a cellular mechanism that occurs at the cell surface in nicotinamide adenine dinucleotide phosphate (NADPH) dependent manner in viable cells. 14 Cells were seeded in 96‐well plates (1.5 × 104 cells/well) for 24 h and were starved overnight in a serum‐free medium. After removing the media, the cells were treated with various concentrations of ARE and incubated for 24 h. After treatment, the cells were added to a WST‐1 reagent for 2 h. Then, the cells were measured for optical density at 450 nm for formazan analysis.
2.5. Measurement of type I procollagen
Type I procollagen was measured using the Procollagen Type I C‐peptide enzyme immunoassay kit (Takara Bio) according to the manufacturer's instructions. The absorbance of the treated samples was measured at 450 nm using a plate reader.
2.6. In vivo clinical trial
This study was performed at the Korea Institute of Dermatological Sciences in Korea in accordance with the Good Clinical Practice guidelines and approved by the Institutional Ethics Committee of the Institute (approval number: 1‐70005239‐AB‐N‐01‐201909‐HR‐300‐01). Volunteers were recruited for this clinical study of an ARE formulation to demonstrate its anti‐wrinkle effects in an in vivo test. Participants of this double‐blind clinical study were randomly assigned the sample and placebo formulations for use on the left and right sides of their face over an 8‐week period. Also, we were checked the reaction of skin in participants; erythema, edema, scaling, itching, stinging, burning, tightness, and prickling. The formulation compared various ingredients, including polysorbate 60, glyceryl stearate, cetyl ethylhexanoate, and squalene (Table 1).
TABLE 1.
Formulation of ARE‐containing lotion and placebo lotion
| Phase | INCI name | %W/W placebo | %W/W ARE lotion |
|---|---|---|---|
| A | Water | To 100 | To 100 |
| Disodium EDTA | 0.02 | 0.02 | |
| Glycerin | 4 | 4 | |
| Butylene Glycol | 6 | 6 | |
| Chlorphenesin | 0.2 | 0.2 | |
| B | Carbomer | 0.3 | 0.3 |
| C | Polysorbate 60 | 1 | 1 |
| Sorbitan Stearate | 0.3 | 0.3 | |
| Glyceryl Stearate | 0.5 | 0.5 | |
| Cetearyl Alcohol | 0.5 | 0.5 | |
| Caprylic/Capric Triglyceride | 3 | 3 | |
| Cetyl Ethylhexanoate | 2 | 2 | |
| Squalane | 1 | 1 | |
| D | Tromethamine | 0.24 | 0.24 |
| E | Phenoxyethanol | 0.2 | 0.2 |
| Caprylyl Glycol | 0.1 | 0.1 | |
| Ethylhexylglycerin | 0.1 | 0.1 | |
| F | Angelica gigas root Extract | — | 1 |
Abbreviations: ARE, Angelica gigas Nakai root extracts; INCI, international nomenclature of cosmetic ingredients.
2.7. Anti‐wrinkle assay by PRIMOS LITE
We evaluated the anti‐wrinkle effects of ARE using PRIMOS Lite (field of view 18 × 13; GFMesstechnik GmbH) and a VISIA‐CA (VISIA Complexion Analysis, Canfield Scientific, Inc.). To measure the effects of ARE using the PRIMOS Lite, we took pictures of the crow's feet wrinkles on skin at 0, 4, and 8 weeks. We analyzed skin wrinkle parameters (total roughness [R1], maximum roughness [R2], average roughness [R3], smooth roughness [R4], average of all heights and depths to the reference plane [R5]) with the PRIMOS Lite software (PRIMOS Lite version 5.8E) using 3D matching.
2.8. Anti‐wrinkle effect assay by global photodamage score
We also identified the anti‐wrinkle effects of ARE using a global photodamage score that was assigned based on a visual assessment. 15 At weeks 0, 4, and 8, dermatologists studied the photographs and scored them based on the severity of the wrinkles (0, none; 1, none/mild; 2, mild; 3, mild/moderate; 4, moderate; 5, moderate/severe; 6, severe; and 7, very severe).
2.9. Statistical analysis
The results are expressed as means ± standard deviation. The statistical analysis was measured using the SPSS software version 17.0 (SPSS Inc.) to determine the significance of the measured values using the test product. A repeated‐measures analysis of variance was conducted to analyze these results to compare placebo and ARE lotion group data to analyze the average of R1, R2, R3, R4, and R5 value, showing the degree of skin wrinkle. Statistical differences were considered significant at a p value of <0.05.
3. RESULTS
3.1. Cytotoxicity of ARE
To evaluate the cytotoxicity of ARE in fibroblasts, WST‐1 assays were performed using various concentrations using ELISA at 650 nm. ARE impacted cell viability in a dose‐dependent manner in fibroblasts. Cytotoxicity was maintained at over 90% when the cells were treated with sample doses of 50 μg/ml (Figure 1).
FIGURE 1.
Cell viability assay on the effect of Angelica gigas Nakai root extracts in human dermal fibroblasts
3.2. Effects of ARE on type I collagen
To determine the anti‐wrinkle effects of ARE in vitro, we demonstrated the synthesis of type I collagen in fibroblasts. The culture was treated with various concentrations of ARE for 24 h and compared with a control and transforming growth factor‐beta (TGF‐β) (10 ng/ml). The ARE slightly increased type I collagen production by 40% at 50 μg/ml as compared with the control (Figure 2).
FIGURE 2.
Effects of type I collagen on Angelica gigas Nakai root extracts in human dermal fibroblasts
3.3. Clinical trials by roughness analysis
The anti‐wrinkle effects of ARE were further evaluated in a clinical study of healthy women. This clinical test on human skin was conducted with volunteers who satisfied all the inclusion criteria and did not meet the exclusion criteria. Roughness parameters were calculated to determine the anti‐wrinkle effects of the ARE lotion compared with the placebo. The use of lotion with 0.025% ARE for 8 weeks decreased the appearance of crow's feet wrinkles significantly in comparison with the use of the placebo (p < 0.05; Figure 3). The changes in roughness parameters were analyzed based on the average R1, R2, R3, R4, and R5 values.
FIGURE 3.
Changes in roughness parameters in crow's feet wrinkles during the study (*p < 0.05) compared with the placebo
Prior to the treatment with ARE lotion and placebo, values of total skin roughness (R1) were 151.38 arbitrary unit (AU) and 153.46 AU, respectively. The value of R1 (ΔR1) for the ARE lotion group were decreased by 10.99 AU and 19.64 AU for 4 and 8 weeks, respectively. Maximal roughness (R2) was 144.90 AU and 145.83 AU in the ARE lotion and placebo groups. ARE lotion treatment decreased R2 values by 9.40 AU, 19.52 for 4 and 8 weeks, respectively. The values of average roughness (R3) were decreased 2.62 AU and 5.01 AU for 4 and 8 weeks compared to baseline in ARE lotion and placebo groups. Also, ARE lotion ameliorated the values of smooth roughness (R4), which means that ARE lotion improved loss of skin moisture, decreased 5.70 AU and 10.07 AU for 4 and 8 weeks. Average of all heights and depths to the reference plane were changed by the ARE lotion group, significantly. Meanwhile, No significant difference in R1 to R5 value was found at from 4 weeks and 8 weeks in placebo. We confirmed that all the parameter values improved at 8 weeks after ARE use compared with placebo use (Figure 4). These results suggest that the ARE lotion demonstrates anti‐wrinkle effects in human trials.
FIGURE 4.
Visual improvement in wrinkles after use of Angelica gigas Nakai root extracts (ARE) lotion for 8 weeks (participant No. 6)
3.4. Evaluation of photodamage score
Using a double‐blind method, dermatologists evaluated visual changes in the cutaneous condition of crow's feet wrinkles at 0, 4, and 8 weeks. Based on the photodamage scores, ARE had significant anti‐wrinkling effects. Based on the change of wrinkle score, skin treated with ARE lotion reduced the appearance of these wrinkle after 8 weeks of repeated application in comparison with initial value (Figure 5). These results suggest that the use of the ARE lotion decreased the photodamage score compared with the placebo over 8 weeks, indicating ARE's anti‐wrinkle effects.
FIGURE 5.
Effects of the placebo and Angelica gigas Nakai root extracts (ARE) lotion on the global photodamage score.
4. DISCUSSION
The A. gigas Nakai cultivated in Korea has more active compounds than those versions cultivated in China or Japan. 1 To improve the extraction effectiveness, ARE was cultured in mineral‐rich lava seawater from Jeju Island in Korea. Mineral‐rich water is reported by changes metabolites for mineral contents compared to distilled water. 16 Germinated barley extracts with mineral‐rich water in Jeju revealed to be associated with the increase of procyanidin and prodelphinidin, indicating the anti‐wrinkle activity. 9 It should be noted that the content of mineral water in Jeju affects the antioxidant activity and anti‐aging effects, means that its utilization value is higher than that of other water resources. 16 Previously, we confirmed the anti‐aging effects of A. gigas Nakai cultured in mineral‐rich water. 13 Our basic and clinical research have demonstrated the anti‐wrinkle properties of ARE.
The induction of MMPs and the reduction of collagen cause aging in skin. Aging of the skin is fundamentally related to reductions in the levels of type I collagen, the principal component of the dermal layer of the skin. 17 In this study, ARE induced the expression of type I collagen in a dose‐dependent manner (Figure 2). Furthermore, previous studies reported that the decursin in ARE suppressed the matrix metallopeptidase‐3 expression in HDFs. 13 The collagen fiber in the ECM comprises about 90% of the dermis. 18 Collagen fibers are degraded by diverse factors including generation of reactive oxygen species, photo‐aging. 19 Collagen may be metabolized by collagenases or matrix metalloproteinases (MMPs), which are endopeptidases a family of enzymes that breaks down collagen. 20 The synthesis of collagen is activated by the appearance of wrinkles to improve through collagen production or inhibition of MMPs. 21 We suggest that the increase in type I collagen is critical to the anti‐wrinkle effects of ARE in fibroblast.
Based on the assessment of the previous studies on ARE in fibroblast, we confirmed the anti‐wrinkle properties to check the roughness parameter and photodamage scores. The roughness parameters examined were R1 (depth of roughness), R2 (maximum roughness), R3 (mean depth of roughness), R4 (smoothness depth), and R5 (arithmetic average roughness). 22 Roughening during skin aging might be explained by the age of skin tissue components, which is associated with linear decreases in mechanical strength and skin moisture content. 23 The present study demonstrated that ARE has statistically significant anti‐wrinkle effects after 8 weeks as compared with the placebo (Figures 3 and 4). We confirmed decreases in photodamage scores of the ARE lotion compared with the placebo (Figure 5). No reactions to allergic or irritant contact dermatitis were observed after the test subjects were assessed (data not shown). In many previous studies, the extracts of natural plants have been reported to have potent anti‐wrinkle effects on human skin. 21 Furthermore, the photodamage scores show significant anti‐wrinkle efficacy in human participants after 8 weeks of ARE treatment. 24 The results demonstrated that the use of ARE with mineral‐rich water provides anti‐wrinkle effects in in vitro and clinical trials.
5. CONCLUSION
Our basic and clinical research have demonstrated the anti‐wrinkle properties of ARE. ARE induced the expression of type I collagen in a dose‐dependent manner without cytotoxicity. Based on the assessment of the previous studies on ARE in fibroblast, we confirmed the anti‐wrinkle properties to check the roughness parameter and photodamage scores. The present study demonstrated that ARE has statistically significant anti‐wrinkle effects after 8 weeks as compared with the placebo. We confirmed decreases in photodamage scores of the ARE lotion compared with the placebo. The results demonstrated that the use of ARE with mineral‐rich water provides anti‐wrinkle effects in in vitro and clinical trials.
CONFLICT OF INTEREST
The authors declare no competing interests.
AUTHOR CONTRIBUTIONS
JW Kang and HE Cho designed all experimental investigations and performed experiments and wrote manuscript. HM Choi and IC Lee designed the overall experiments together. All authors read and confirmed the final version of the manuscript.
ETHICAL APPROVAL
The human volunteers provided the written informed consent. The study was approved by the by the Institutional Ethics Committee of the Institute (No. 1‐70005239‐AB‐N‐01‐201909‐HR‐300‐01).
Kang J‐W, Cho H‐E, Choi H‐M, Lee I‐C. Anti‐wrinkle properties of Angelica gigas Nakai root extracts using mineral‐rich water. J Cosmet Dermatol. 2023;22:328–334. doi: 10.1111/jocd.15017
Jung‐Wook Kang and Hang‐Eui Cho contributed equally to this study as first authors.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.