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Materials and Methods

In vitro and ex vivo analyses. Cell viability. 3T3-L1 cells were seeded into a 24-well plate and treated with different concentrations of caviar oil from 156.25 ppm to 20,000 ppm for 24 h. After removing the medium, 200 μl of 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added and incubated at 37˚C, 5% CO₂ for 4 h. Then, the agent was removed, and 200 μl of DMSO were added. Purple cell pellets were dissolved in dimethyl sulfoxide (DMSO) at room temperature for 10 min. Cell viability was assessed by measuring the optical density at 570 nm.

Cell culture and adipocyte differentiation. The mouse preadipocyte 3T3-L1 cell lines were seeded in a 6-well plate at a density of 2.5×10⁵ cells per well and cultured for 72 h in a Dulbecco’s Modified Essential Medium (DMEM) culture medium containing 10% bovine calf serum (BCS) and 1% Penicillin/Streptomycin (P/S) at 37˚C in 5% CO₂ to reach 100% confluence. The culture medium was then removed, and adipocyte differentiation was initiated using DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% P/S, 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX), 1 µM dexamethasone, and 10 μg/ml insulin for two days at 37˚C in 5% CO₂. During this period, the 3T3-L1 cell line was treated with various concentrations of caviar oil, including 625 ppm, 1,250 ppm, 2,500 ppm, 5,000 ppm, and 10,000 ppm. After the cells were transferred to a new culture in differentiation DMEM medium containing 10% FBS, 1% P/S, and 10 μg/ml insulin with different concentrations of caviar oil and incubated at 37˚C in 5% CO₂ for an additional two days. The cells were then cultured at 37˚C in 5% CO₂ for six days using normal DMEM medium supplemented with 10% FBS, and 1% P/S containing different concentrations of caviar oil. After that, the 3T3-L1 cell lines were washed three times with 1x PBS solution and fixed with 10% formal dehydrate at room temperature for 30 min. Following, the cells were rinsed three times with 1x PBS, and stained with Oil Red O working solution (ORO, Sigma Aldrich, Saint Louis, MO, USA) at room temperature for 20 min. Finally, cells were washed three times with 1x PBS solution, and the adipocytes were photographed at 200× magnification using an optical microscope Olympus CKX53 (Olympus, Tokyo, Japan) and dissolved lipid droplets in DMSO and measured absorbance at 520 nm with a Microplate spectrophotometer (BioTek, Winooski, VT, USA).

Evaluation of caviar oil absorption on human skin tissue. Human skin tissues (Dermalab™ FULL SKIN 2 cm×2 cm) were treated with saline (control group) and caviar oil (test group) and then recovered after 24 h and frozen. The recovered tissues were cryosectioned to a thickness of 8-10 μm and stained with Oil Red O solution, followed by nuclear staining using hematoxylin. Subsequently, the cross-sections of human skin tissue were observed at 200× magnification using an optical microscope (Zeiss Axio Observer 7, Oberkochen, Germany). The captured visual images were analyzed using Image J software (The National Institutes of Health, Bethesda, MD, USA). The stained skin area ratio was analyzed.

Preparation of treatment of human skin tissues with caviar oil. Human skin tissues collected after surgery of Korean women between the ages of 50 and 70 were used in the study with the approval of the KSRC Korea Skin Clinical Research Center IRB (IRB approval number: HBABN01-220801-HR-E0145-01). The human-derived skin tissues were washed twice with 1x PBS solution and then placed in a sterilized Petri dish. Human-derived skin tissues were punched by a Biopsy Punch (KAI Medical, Tokyo, Japan), and the skin tissues of all groups except for the untreated group were irradiated with an intensity of 200 mJ of UVB (BLX-LMC, Vilber Loumart, Collégien, France). After UVB irradiation, Transwell inserts (Corning, Corning, NY, USA) were attached to each 6-well plate, and skin tissues were placed on the Transwell insert. At this time, the positive control group (Ascorbic acid 1.00%), caviar oil groups at different concentrations of 1,000 ppm, 5,000 ppm, and 10,000 ppm were added to the DMEM culture medium containing 10% FBS, 1% P/S for 48 h.

Real time-PCR. After 48 h of cultivation, the crushed skin tissue was treated with Trizol and then reacted. Total RNA was extracted from the skin tissue using Total RNA extraction reagent (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. cDNA synthesis was performed from approximately 1 μg of total RNA according to the protocol provided by the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Specific primer pairs analyzed the expression of MMP-1, MMP-2, and MMP-9 were as follows: MMP-1 forward 5’-ACA GCC CAG TAC TTA TTC CCT TTG -3’, MMP-1 reverse 5’-GGG CTT GAA GCT GCT TAC GA -3’, MMP-2 forward 5’- CCC CAA AAC GGA CAA AGA G -3’, MMP-2 reverse 5’- CAC GAG CAA AGG CAT CAT CC -3’, MMP-9 forward 5’- CAC TGT CCA CCC CTC AGA GC -3’, MMP-9 reverse 5’- GCC ACT TGT CGG CGA TAA GG -3’. For quantitative PCR, TB Green^® Premix Ex Taq™ II (Takara Bio) was used on the ViiA™ Real-Time System (Thermo Fisher Scientific) with PCR condition following denaturation at 95˚C for 30 s, then 45 cycles of amplification at 95˚C for 5 s and 60˚C for 34 s. Relative mRNA expression values were determined using the comparative C_Tmethod, the 2^-ΔΔCt method.

Preparation of tissues for histological analysis. The tissue samples were prepared by fixing and embedding in paraffin blocks. Tissue slides were then obtained by sectioning the blocks at a thickness of 3 μm. These section slides were used for further histological examinations.

Hematoxylin and eosin (H&E) staining. After the hydration process, tissue slides were stained with Hematoxylin and Eosin solutions, and the results were quantified by examining the stained slides under a microscope.

Collagen I and III staining. After hydration, the antigen in samples was retrieved with 10 mM Sodium Citrate Buffer, and then an antibody (collagen type I and III) was treated. Afterward, coloring was performed using ABC reagent and DAB solution, and counterstaining was performed with hematoxylin.

Victoria Blue staining. After the hydration process, staining was performed using Potassium Permanganate-Sulfuric Acid Working Solution, followed by a reaction with Sodium Bisulfite 1%. Afterward, color development was induced using 70% alcohol, and counterstaining was performed with Nuclear Fast Red stain.

Clinical study. There were 5 clinical studies conducted following Good Clinical Practice (GCP), Ministry of Food and Drug Safety (MFDS) related regulations, and Korea Skin Clinical Research Center’s standard operating instructions and was registered with IRB approval number at HBABN01-220502-HR-E0101-01, HBABN01-220502-HR-E0102-01, HBABN01-220502-HR-E0104-01, HBABN01-220502-HR-E0105-01, and HBABN01-220502-HR-E0106-01. Each clinical study recruited over 20 participants in accordance with the recommendations of MFDS Guideline. Potential participants who meet the inclusion criteria and not the exclusion criteria were selected and provided with an explanation of the study purpose, methods, expected efficacy, and potential side-effects. Participants who expressed their intent to participate were required to complete a consent form for research participation and joined the study. For the test after four weeks of product use, subjects applied the product three times a day for four weeks (once in the morning, evening, and afternoon). During the study period, the researchers observed the skin condition of the participants, and in case of serious adverse reactions other than predictable ones, prompt and appropriate measures were taken to minimize possible adverse effects. In the event of an adverse reaction, the research director determined the relevance to the test product, and if the problem was due to the test product, the sponsoring institution assumed full responsibility and ensured the safety of the research subjects by providing appropriate compensation.

Study procedure. All evaluations in this study were conducted at the same measurement site. Study participants washed the test site during the visit and then stabilized for 30 min in a room with constant temperature and humidity (22 ± 2˚C, 50±5%). For the test after using the product once, participants washed their face, and then the researcher rolled the product onto the entire face and neck of the subjects and tapped lightly to absorb.

Measurement of six types of deep wrinkles. The selected forehead, glabellar, nasolabial, marionette, lip, and neck were assessed by topographic via Antera 3D^® CS (Miravex Limited, Dublin, Ireland) before and after using the product once and after four weeks of using the product. The wrinkle parameter values were analyzed using an analysis program.

Evaluation facial pillow marks relief effect. The effect of relieving facial pillow marks was assessed before, after a single product use, and after four weeks of product use. Pillow marks were artificially induced for 10 min on the selected cheek area at each time point. The Antera 3D^® CS (Miravex Limited) was utilized, and volume parameter values were analyzed.

Measurement of transepidermal water loss. Transepidermal water loss in the selected cheek area was measured three times before, after using the product once, and after four weeks of product use, and the average values were calculated. Transepidermal water loss was measured using Tewameter^® TM HEX (C+K, Köln, Germany).

Measurement of skin moisture. The skin moisture level of the selected cheek area was measured three times using Corneometer^® CM 825 (C+K) before, after one use of the product, and after four weeks of product use, and then the average value was analyzed.

Measurement of skin clarity (transparency). The skin clarity (transparency, surface reflectance value) of the selected cheek area was measured three times using Lumiscan™ (True systems, Anyang-si, Gyeonggi-do, Republic of Korea) at the time points before product use, after one use of the product, and after four weeks of product use, and the average value was calculated and analyzed.

Measurement of skin density. The dermal density of the cheek of participants was measured using the Ultrasound Probe of DermaLab^® Series SkinLab Combo (Cortex Technology, Aalborg, Denmark) before and after using the product for 4 weeks. The device displayed low density as a dark color and high density as a bright color through the signal strength.

Measurement of skin elasticity. The skin elasticity of the selected cheek area was measured using the Cutometer^® MPA580 (C+K) before, after one use of the product, and after four weeks of product use. The same posture and distance were maintained throughout the measurement process to ensure consistent measurements.

Measurement of five types of facial gloss (diamond zone radiance). The forehead left and right eye areas, and left and right mouth areas were selected for measurement before and after product use. The skin’s gloss was measured three times using the Skin-Glossymeter GL 200 (C+K), and the average value was analyzed. This device operates on the reflection principle. The measured value is expressed in Glossymeter units (GU).

Measurement of three types of eye area lifting. The selected eyelids, bags under the eyes, crow’s feet, and the bulldog area were photographed using Antera 3D^® CS (Miravex Limited) before using the product, after using the product once, and after four weeks of using the product. Lifting (skin filling) parameter values were analyzed using an analysis program.

Measurement of three-dimensional (3D) facial lifting. A 3D image of the subject’s face was captured using VECTRA H2 (Canfield, Parsippany-Troy Hills, NJ, USA) under the controlled conditions (consistent posture and distance) before, after product use one time, and after four weeks of product use. The length of each study area was analyzed using the captured 3D image and analysis software.

Evaluation of makeup entrapment relief effect. The participants’ facial areas were classified into 2 groups: a control group (which did not use Cavi Balm) and a test group (which used Cavi Balm). A makeup product (foundation) was applied to the test area. Antera 3D^® CS (Miravex Limited) was used to photograph the test area before and after a single use of the product of both the control group and the test product group. The skin volume parameter was measured using an analysis program.

Evaluation of skin tightening relief. Skin tightening relief was carried out by participants based on the degree of skin tightening before and after a single use of the product, as well as after four weeks of product use. The evaluation criteria involved measuring the degree of skin tightening (Grade 1: no tightening at all, ~Grade 7: severe tightening), with the average value of the calculated scale being analyzed.

Measurement of skin moisture under hot and cold air environmental conditions. The skin moisture content was measured on the forearm area (3×3 cm²) and compared between the control group (no application of Cavi Balm) and the Cavi Balm applied group. The hot/cold air environmental conditions (warm air: 40±3˚C, cold air: 20±3˚C) were achieved by using an electric heater (combined fan) from Cixi Pengxiang Electric Appliance Co., LTD, Zhejiang, PR China. The posture and distance of the participants were fixed to ensure consistency across participants. Warm air and cold air were applied to the forearm for 5 minutes each, and the total exposure time was 10 min. The moisture level of the skin in the selected forearm was measured three times using the Corneometer^® CM 825 (C+K) before, after using the product once, and after applying hot/cold air conditions, and the average value was analyzed.