Effect of Protease‐Treated Royal Jelly Extract on Facial Wrinkles: A Placebo‐Controlled, Double‐Blind, Parallel‐Group Study

TL;DR

A cream containing protease-treated royal jelly improved facial wrinkles and skin hydration in a clinical trial with Japanese women.

Contribution

This is the first clinical study demonstrating wrinkle-improving effects of protease-treated royal jelly.

Findings

pRJ cream reduced wrinkle depth and improved skin hydration compared to placebo.

pRJ cream increased dermal thickness and altered bacterial microbiome on cheeks.

pRJ cream upregulated COL17A1, a stem cell marker, in ex vivo studies.

Abstract

No clinical trials have reported on the wrinkle‐improving effects of cosmetics which activate stem cells in vitro.

To evaluate the anti‐wrinkle effects of protease‐treated royal jelly (pRJ)‐containing cream application to the skin.

Seventy healthy Japanese women concerned about skin aging were treated with pRJ‐containing cream or placebo for 12 weeks using the split‐face method in a placebo‐controlled, double‐blind, parallel‐group study.

The pRJ‐containing cream group showed an improvement in the maximum depth and average depth of the biggest wrinkle in the crow's feet wrinkle, along with increased water content of the stratum corneum and dermal thickness, compared to the placebo group. Significant decreases in the relative amount of bacterial microbiome of the cheeks were observed only in the pRJ‐containing cream group. In the ex vivo study, expression of COL17A1, epidermal stem cell function marker, was significantly up‐regulated with the use of the pRJ‐containing cream compared with the placebo cream.

This is the first study to demonstrate the wrinkle‐improving effects of pRJ. Our results suggest that pRJ not only controls stratum corneum water content, dermal thickness, and bacterial microbiome, but also affects stem cell competition and mesenchymal stem cell activation. pRJ can reduce or eliminate certain skin concerns of women by improving age‐related wrinkles.

Figures

Flow diagram of the double‐blind, split‐face, placebo‐controlled clinical trial. One hundred and twenty participants were screened for eligibility, 70 of whom underwent randomization. After the trial started, 6 participants dropped out. Data of 64 participants who completed the study were analyzed for efficacy and safety. Creams containing protease‐treated royal jelly (pRJ) and placebo are referred to as pRJ‐Cr and PLBO‐Cr, respectively.

Representative images showing changes in crow's feet wrinkles at baseline (Week 0, A) and after 12 weeks of creams containing protease‐treated royal jelly (pRJ‐Cr) application (Week 12, B). The figure demonstrates a visible reduction in wrinkles due to the application.

Representative images showing changes in skin texture at baseline (Week 0, A) and after 12 weeks of creams containing protease‐treated royal jelly (pRJ‐Cr) application (Week 12, B). The figure demonstrates a visible improvement in skin texture due to the application.

Effect of pRJ‐Cr on the expression of COL17A1 and TIMP1 in a human ex vivo skin model. (A–D) pRJ‐Cr or PLBO‐Cr was topically applied to the skin model twice daily for 24 h. COL17A1 (A and B) and TIMP1 (C and D) were detected by immunofluorescence with specific antibodies (red). Nuclei were visualized using DAPI (blue). Images were obtained and analyzed using BZ‐X800 microscope (Keyence, Tokyo, Japan). pRJ‐Cr application increased the expression of COL17A1 and TIMP1 compared to the PLBO‐Cr application. Three non‐consecutive cross‐sections were prepared for each tissue, and five images per section were randomly photographed for analysis. Representative images were shown. (E, F) Mean intensity was calculated as described in method section. Values are mean ± SD, p < 0.01 by Student's t‐test (vs. PLBO‐Cr). Creams containing protease‐treated royal jelly (pRJ) and placebo are referred to as pRJ‐Cr and PLBO‐Cr, respectively. DAPI, 4′,6‐diamidino‐2‐phenylindole; SD, standard deviation.

Tables

TABLE 1. Contents of the test product.

pRJ‐Cr

Royal Jelly Extract, Water, Butylene Glycol, Pentylene Glycol, Phenoxyethanol, Caprylic/Capric Triglyceride, Potassium Hydroxide, Acrylates/C10‐30 Alkyl Acrylate Crosspolymer, Carbomer

PLBO‐Cr

Water, Butylene Glycol, Pentylene Glycol, Phenoxyethanol, Caprylic/Capric Triglyceride, Potassium Hydroxide, Acrylates/C10‐30 Alkyl Acrylate Crosspolymer, Carbomer

TABLE 2. Participant background at the time of enrolment.

Number of participants	64
Sex	Female
Age (years) (Min ~ Max)	53.58	±	4.86
	44	~	60

TABLE 3. Change in parameters of the crow's feet wrinkle.

		Baseline	Week 6	Week 12	% Change
					Week 6/baseline	Week 12/baseline
Max. depth of the biggest wrinkle (μm)	pRJ‐Cr	147.69 ± 43.28	133.39 ± 45.47**	125.39 ± 42.26**	92.34% ± 25.48%	86.36% ± 20.41%##
	PLBO‐Cr	152.66 ± 49.43	145.47 ± 45.80	152.91 ± 43.54	97.83% ± 21.73%	104.61% ± 29.43%
Avg. wrinkle depth (μm)	pRJ‐Cr	31.70 ± 11.04	30.70 ± 12.03	32.16 ± 12.72	100.56% ± 32.32%	105.62% ± 42.70%
	PLBO‐Cr	35.98 ± 11.01	37.78 ± 11.80	39.09 ± 12.23	110.21% ± 36.58%	113.94% ± 36.65%
Avg. depth of the biggest wrinkle (μm)	pRJ‐Cr	35.83 ± 13.06	33.55 ± 15.17	33.72 ± 15.66	98.08% ± 39.91%	96.66% ± 36.88%#
	PLBO‐Cr	40.95 ± 13.14	42.06 ± 13.18	43.19 ± 13.78	108.24% ± 36.76%	112.73% ± 45.23%
Wrinkle volume (mm3)	pRJ‐Cr	1.89 ± 0.99	1.68 ± 0.95**	1.74 ± 0.97	92.42% ± 31.29%	96.07% ± 40.10%
	PLBO‐Cr	2.47 ± 0.96	2.34 ± 0.93	2.41 ± 0.92	98.87% ± 32.60%	103.63% ± 34.48%
% Wrinkle area	pRJ‐Cr	18.34 ± 0.95	16.72 ± 0.97**	16.62 ± 1.12**	91.27% ± 5.19%	91.27% ± 5.19%
	PLBO‐Cr	18.55 ± 0.52	16.64 ± 0.88**	16.83 ± 1.04**	89.68% ± 4.43%	90.72% ± 5.24%

TABLE 4. Changes in the water content of the stratum corneum and dermal thickness.

		Baseline	Week 6	Week 12	% Change
					Week 6/baseline	Week 12/baseline
The water content of the stratum corneum (A. U.)	pRJ‐Cr	50.07 ± 13.06	46.41 ± 10.76*	50.69 ± 11.51	96.56% ± 24.92%	106.37% ± 32.14%##
	PLBO‐Cr	49.90 ± 11.69	45.90 ± 13.30*	42.23 ± 11.75**	94.90% ± 30.62%	87.35% ± 28.03%
Dermal thickness (μm)	pRJ‐Cr	1331.20 ± 194.09	1311.73 ± 198.08	1366.86 ± 194.05*	98.79% ± 8.07%	103.10% ± 8.77%##
	PLBO‐Cr	1340.26 ± 188.88	1320.71 ± 187.11	1304.60 ± 164.55*	98.86% ± 8.52%	97.86% ± 8.31%

TABLE 5. Changes in the skin texture.

		Baseline	Week 6	Week 12
% Texture volume	pRJ‐Cr	25.58 ± 11.37	30.76 ± 9.56**	29.78 ± 8.72 *
	PLBO‐Cr	25.55 ± 11.83	28.82 ± 9.46	27.25 ± 10.12
Avg. texture depth (μm)	pRJ‐Cr	56.95 ± 2.93	57.65 ± 3.15	57.30 ± 2.92
	PLBO‐Cr	56.70 ± 2.99	57.51 ± 2.60*	57.66 ± 2.57*
Number of textures	pRJ‐Cr	0.78 ± 0.42	0.93 ± 0.38*	0.90 ± 0.33
	PLBO‐Cr	0.79 ± 0.46	0.85 ± 0.39	0.79 ± 0.35
% Volume	pRJ‐Cr	120.50 ± 42.82	133.87 ± 45.38*	135.30 ± 48.27**
	PLBO‐Cr	120.50 ± 50.60	131.94 ± 46.27	133.24 ± 53.28*
% Texture area	pRJ‐Cr	6.32 ± 2.82	7.57 ± 2.39**	7.34 ± 2.13*
	PLBO‐Cr	6.33 ± 2.95	7.09 ± 2.37	6.68 ± 2.49
Max. depth texture (μm)	pRJ‐Cr	89.98 ± 0.03	89.98 ± 0.08	89.99 ± 0.02*
	PLBO‐Cr	89.97 ± 0.07	89.98 ± 0.03	89.98 ± 0.04
Max. width texture (μm)	pRJ‐Cr	354.61 ± 47.96	352.69 ± 49.75	347.40 ± 54.96
	PLBO‐Cr	356.66 ± 55.94	357.26 ± 51.57	349.07 ± 55.53

TABLE 6. Changes in the stratum corneum condition obtained by tape stripping.

		Baseline	Week 6	Week 12
Projected area of the corneocyte (μm2)	pRJ‐Cr	754.91 ± 79.31	710.39 ± 89.71**	755.43 ± 98.28
	PLBO‐Cr	767.25 ± 99.16	728.90 ± 86.02**	740.61 ± 99.12
% Multi‐layer peeling	pRJ‐Cr	31.51 ± 8.65	13.46 ± 5.76**	17.57 ± 8.05**
	PLBO‐Cr	31.29 ± 7.77	12.39 ± 4.80**	18.25 ± 8.24**
% Nucleated cell	pRJ‐Cr	33.97 ± 14.70	28.23 ± 10.80**	25.70 ± 10.97**
	PLBO‐Cr	34.06 ± 16.89	27.40 ± 12.29**	25.21 ± 11.17**

TABLE 7. Diversity index in three major bacterial species of the skin bacterial microbiome.

		Baseline	Week 6	Week 12
Shannon Entropy	pRJ‐Cr	4.64 ± 1.30	4.95 ± 1.36*	4.91 ± 1.32
	PLBO‐Cr	4.70 ± 1.36	5.44 ± 1.28**,#	4.86 ± 1.35
Simpson's Index	pRJ‐Cr	0.82 ± 0.12	0.83 ± 0.12	0.83 ± 0.12
	PLBO‐Cr	0.82 ± 0.13	0.87 ± 0.11**	0.83 ± 0.12

TABLE 8. Changes of relative amount in the skin bacterial microbiome.

The relative amount (%)		Baseline	Week 6	Week 12
Cutibacterium acnes	pRJ‐Cr	32.39 ± 17.03	30.00 ± 15.90	28.27 ± 16.55*
	PLBO‐Cr	30.42 ± 18.25	26.00 ± 14.60*	29.19 ± 16.85
Staphylococcus epidermidis	pRJ‐Cr	4.26 ± 5.00	2.90 ± 3.60*	2.33 ± 3.59**
	PLBO‐Cr	3.93 ± 3.75	3.00 ± 2.90	2.82 ± 4.84
Staphylococcus aureus	pRJ‐Cr	2.32 ± 2.15	1.90 ± 2.20	1.56 ± 1.49**
	PLBO‐Cr	1.87 ± 1.37	2.10 ± 2.40	1.84 ± 2.44

TABLE 9. Stratified analysis of diversity index in the skin bacterial microbiome.

		Baseline	Week 6	Week 12
Shannon Entropy	pRJ‐Cr	4.55 ± 1.26	4.84 ± 1.40	4.96 ± 1.36*
	PLBO‐Cr	4.77 ± 1.34	5.51 ± 1.22**,#	4.85 ± 1.36
Simpson's Index	pRJ‐Cr	0.81 ± 0.13	0.82 ± 0.12	0.83 ± 0.12
	PLBO‐Cr	0.82 ± 0.13	0.88 ± 0.09**,#	0.83 ± 0.12

TABLE 10. Summary of adverse events.

	pRJ‐Cr		PLBO‐Cr
	Not relevant	Probably relevant	Not relevant	Probably relevant
Skin roughness	17	1	24	1
Redness	1	1	1
Irritation	1	3	1	4
Itchiness	4	1	2

Full text

Introduction

1

There are two major mechanisms underlying wrinkle formation that differ between the epidermis and the dermis. For example, wrinkles are induced by the loss of skin flexibility due to epidermal dryness or the loss of skin elasticity due to the degeneration of dermal extracellular matrix [1, 2].

Royal jelly, a creamy milky white substance that worker bees digest and subsequently decompose using pollen and nectar in the body, is secreted from their hypopharyngeal and large jaw glands. It contains unique components including 10‐hydroxy‐2‐decenoic acid and 10‐hydroxy decanoic acid, proteins, amino acids, vitamins, and minerals. Previous studies have shown that 10‐hydroxy‐2‐decenoic acid promotes production of filaggrin and collagen in vitro [3, 4]. Our clinical study suggested that applying an essence containing royal jelly ethanol extract significantly improves the water content of the stratum corneum on the cheeks compared to placebo [5]. However, its effect on wrinkles has not been verified.

On the other hand, a study involving protease‐treated royal jelly (pRJ) also reported a significant increase in the water content of the stratum corneum compared to placebo [6]. The pRJ was royal jelly treated with protease and filtered to remove insoluble matter, leaving only the water‐soluble components containing fatty acids, which decomposes proteins and is hypoallergenic. We also previously confirmed that pRJ maintains stem cell properties of epidermal keratinocytes in a three‐dimensional epidermal model [7]. Similarly, pRJ has been found to promote the secretion of collagen‐producing exosomes in adipose‐derived mesenchymal stem cells [8].

Furthermore, healthy skin has higher diversity of bacterial microbiome than skin with psoriasis or atopic dermatitis [9]. Royal jelly contains antibacterial peptides and has been reported to have antibacterial effects on Staphylococcus aureus ( S. aureus ), Pseudomonas aeruginosa , and Cutibacterium acnes (C. acnes) [10]. However, no studies have reported on the effects of royal jelly application on the skin bacterial microbiome, which might be involved in wrinkle formation.

Therefore, this placebo‐controlled, double‐blind, parallel‐group study firstly aimed to evaluate the impact of pRJ on wrinkles. Skin texture, epidermal turnover, and skin microbiome, which are known to be related to wrinkle formation, were also evaluated. Additionally, focusing on the activation of stem cells in the epidermis and dermis, we measured the water content of the stratum corneum and dermal thickness, which are the expected mechanisms for improving wrinkles and can indirectly reflect stem cell activation. Furthermore, we investigated type XVII collagen (COL17A1) expression ex vivo to confirm whether the results obtained in the clinical trial were associated with the effect of pRJ on epidermal stem cells.

Materials and Methods

2

Clinical Trial

2.1

Objective

2.1.1

We conducted the present study at the Research Center for Immunological Analysis Inc., between December 2022 and July 2023, recruiting healthy Japanese women aged 40–60 years.

Protocol

2.1.2

The Yamada Bee Company Ethics Committee reviewed and approved the study protocol on December 13, 2022. We conducted the study in accordance with the Declaration of Helsinki (revised at the 64th WMA General Assembly, Fortaleza, Brazil, October 2013) and the Ethical Guidelines for Medical and Biological Research Involving Human Subjects (Ministry of Health, Labour and Welfare, 2022). The study protocol was registered with the University Hospital Medical Information Network (UMIN 000049871). Written informed consent was obtained from the participants after fully explaining the purpose and methods of this study.

Participants

2.1.3

The Research Center for Immunological Analysis Inc. (Okayama, Japan) recruited the participants. The target numbers of participants for the screening, enrollment, and completion of the trial were 120, 70, and 60, respectively. We determined these patient numbers based on similar previous studies involving participants with age‐related wrinkles, where significant difference was observed between the test product and placebo [11]. Exclusion criteria were set based on a previous study [5].

The inclusion criteria were as follows: (1) individuals who suffered from skin aging issues, such as wrinkles, loss of firmness, and sagging skin; (2) healthy Japanese women aged 40–60 years at the time of providing consent; (3) individuals who agreed to comply with regulations during the study; and (4) individuals who fully understood the content of the study and provided written consent.

The exclusion criteria were as follows: (1) individuals who daily used cosmetics similar to the test product; (2) individuals who may exhibit skin allergic symptoms to the test product; (3) individuals who were currently receiving medications prescribed by the dermatologists or who planned to visit dermatologists during the study period (including those who were being treated for atopic dermatitis); (4) individuals with trauma or inflammation at the measurement site; (5) individuals undergoing hormone replacement therapy (e.g., estrogen preparation or progesterone preparations including drinks, patches, and ointments); (6) individuals with experience in esthetic medicine (i.e., hyaluronic acid injection) that affected the measurement site; (7) individuals who were unable to refrain from behavior leading to sunburn, such as visiting tanning salons and outdoor activities, during the study period; (8) individuals who could not avoid beauty care (e.g., epilation or beauty treatment) during the study period after providing consent; (9) individuals who could not maintain a regular lifestyle during the study period; (10) individuals who did not work on a day shift (those who work at night); (11) individuals whose alcohol intake was more than the equivalent of 500 mL beer/day; (12) individuals who had smoked for 3 months before providing consent, including electronic cigarettes; (13) individuals who participated in another clinical trial in the last 3 months; (14) individuals who worked for healthy food companies, cosmetic companies, and pharmaceutical companies; (15) individuals who used cosmetics, quasi‐drugs, drugs, or healthy food products that may affect the study; and (16) individuals who were judged to be unsuitable for enrollment based on other reasons suggested by the principal investigator.

Blinding and Allocation

2.1.4

The test products, a cream containing pRJ (pRJ‐Cr) or placebo cream (PLBO‐Cr), were identical in appearance. We blinded them using symbols “A” or “B,” and delivered to the Research Center for Immunological Analysis Inc. An individual not directly involved in this study conducted the blinding process. Participants were randomly assigned IDs (01–35 and 36–70); participants with IDs 01–35 applied test products A or B to the right or left side of the face, respectively. Meanwhile, IDs 36 to 70 applied test product A to the left side of the face and test product B to the right side. A key opening was conducted after the statistical analysis.

Test Product and How to Use

2.1.5

pRJ‐Cr containing pRJ (Lot. No. 210909), butylene glycol (Kokyu Alcohol Kogyo Co. Ltd.), pentylene glycol (Symrise AG), and phenoxyethanol (Yokkaichi Chemical Co. Ltd.) comprising > 0.018 w/v% 10‐hydroxy‐2‐decenoic acid were prepared by Yamada Bee Company Inc. (Table 1).

The participants applied approximately 1 g (five pushes) of pRJ‐Cr or PLBO‐Cr as the test product to one side of their face as described above, twice a day (morning and evening) for 12 weeks. The other test product was applied to the remaining half of the face under the same conditions. We evaluated the effects of pRJ‐Cr on the skin by comparing with those of PLBO‐Cr. If the participants usually used creams or emollients, they were replaced with the test products. The order of application of the skincare cosmetics was as follows: (1) face wash, (2) regular lotion, (3) regular essence or serum, and (4) test products. The number of days the test products were used and the amount used were recorded based on a diary and the remaining amount.

Endpoints

2.1.6

The primary endpoint was the objective evaluation of the crow's feet wrinkles by measuring the maximum depth of the biggest wrinkle. Secondary endpoints were the objective evaluation of the crow's feet wrinkles by measuring the average wrinkle depth, average depth of the biggest wrinkle, wrinkle volume, % wrinkle area, % skin texture, water content of the stratum corneum, dermal thickness, stratum corneum condition obtained by tape stripping, skin bacterial microbiome, and adverse events. We also performed photographic evaluation of the crow's feet wrinkles to confirm the participant's background at the time of enrolment.

Participants were instructed to remove their facial makeup and to wash their faces using soap without moisturizing ingredients. Subsequently, they acclimatized for 15 min in a room with constant temperature (21°C ± 1°C) and humidity (50% ± 5%) before measurement.

Objective Evaluation of the Crow's Feet Wrinkles

2.1.6.1

Based on the previous study of wrinkles [11], with the participant's eyes lightly closed, the skin analysis program SKINCAST (manufactured by RSI) was applied to the creases at the corners of the eyes, generating a replica of the skin. Wrinkles were aligned using bundled software to evaluate the same part of the replica on each observation day. We three‐dimensionally analyzed the replica using PRIMOS CR (Canfield Scientific Inc., Parsippany, NJ) to measure the following five items [11]:

• Maximum depth of the biggest wrinkle: The maximum depth of the biggest wrinkle within the analyzed area.
• Average wrinkle depth: The average depth of all wrinkles within the analyzed area.
• Average depth of the biggest wrinkle: The average depth of the biggest wrinkle within the analyzed area.
• Wrinkle volume: The sum of the volumes of all wrinkles within the analyzed area.
• % Wrinkle area: The area percentage of wrinkles in the analyzed area.

Skin Texture

2.1.6.2

We evaluated the skin texture of a replica of the cheek using SKINCAST and the reflective replica analysis system ASA‐03RXD (Asch Japan Co. Ltd.) for the following seven items, as reported previously [12]:

• % Texture volume: The volume percentage of skin texture within the analyzed area. ΣW'D′/XY/100 (W′: Width of the groove determined as texture [μm], D′: Depth of the groove determined as texture [μm], X: 4 Width of the analyzed area [mm], Y: Number of lines)
• Average texture depth: The average depth of skin grooves within the analyzed area.
• Number of textures: The number of skin textures within the analyzed area.N′/XY (N′: Average number of textures)
• % Volume: The volume percentage of all skin grooves within the analyzed area.ΣWD/XY/100 (W: Width of all grooves [μm], D: Depth of all grooves [μm])
• % Texture area: The skin texture area percentage within the analyzed area.ΣW'/XY/100
• Maximum depth texture: The maximum depth of skin grooves in the texture within the analyzed area.
• Maximum width texture: The maximum width of skin texture within the analyzed area.

Water Content of the Stratum Corneum

2.1.6.3

We used the Corneometer CM825 (Courage+Khazaka Electronic GmbH) to measure the moisture content of the stratum corneum, according to the previous study [11]. The measurement areas were the left and right cheeks (the intersection of the outer corner of the eye and nose). We calculated the average values by measuring five times and adopting three measured values, excluding the maximum and minimum values.

Dermal Thickness

2.1.6.4

We used the DermaLab (Cortex Technology) to evaluate the average dermal thickness (μm), by measuring three times using ultrasound at the same location on the cheek [13].

Stratum Corneum Condition Obtained by Tape Stripping

2.1.6.5

We collected the corneum layer of the cheek using corneum checker (Asch Japan Co. Ltd.), and imaged the BG‐stained corneocytes by an optical microscope (ECLIPSE E600W; Nikon Corporation), based on the previous similar research [14]. Thereafter, the projected area of the corneocyte, multi‐layer peeling rate, and % nucleated cell were measured using WinRoof V5.6 (Mitani Corporation).

Skin Bacterial Microbiome

2.1.6.6

We used World Fusion's S‐KIN kit, which consists of a swab soaked in physiological saline, to collect resident bacteria on the cheeks. The 16S rRNA V2‐3‐4‐6‐7‐8‐9 region sequences in the obtained DNA sample were amplified by Ion 16S Metagenomics Kit [15] and read by ION S5 sequencer. Low‐quality sequences and short reads (≤ 120 bp) were eliminated from analysis. The bacterial species were identified through 16S rRNA metagenomic analysis. We calculated diversity index (Shannon entropy and Simpson's index, representing alpha diversity) and relative amount as described previously [16].

Ex Vivo Study

2.2

Human ex vivo Skin Model

2.2.1

We purchased abdominal skin tissues of a healthy 39‐year‐old female donor with an 11‐mm diameter (NativeSkin©) from Genoskin (Toulouse, France) together with culture medium. Skin explants were cultured with 1 mL medium in a humidified incubator at 37°C with 5% CO_2_ [17]. We topically applied pRJ‐Cr or PLBO‐Cr (20 μL) onto the skin surface of area pre‐defined with a silicon ring twice daily for 24 h. Subsequently, tissues were harvested for immunofluorescence staining.

Histological Analysis

2.2.2

Histological analysis was performed according to the previous studies [18, 19]. Briefly, skin tissues were fixed with a cold 4% paraformaldehyde solution (Nacalai Tesque, Kyoto, Japan) overnight, and ethanol was replaced with G‐NOX (GenoStaff, Tokyo, Japan). We analyzed the expression of COL17A1 and tissue inhibitor of metalloproteinases 1 (TIMP1) using antibodies against COL17A1 (GT1273; rabbit mAb, geneTex, CA, USA) and TIMP1 (#8946; rabbit mAb, Cell Signaling, MA, USA), respectively. Briefly, sections (7‐μm thickness) were treated with 0.3% Triton X‐100 and 1% normal goat serum at room temperature (20°C ± 2°C) for 1 h. Subsequently, the anti‐COL17A1 primary antibody (1:100) or anti‐TIMP1 primary antibody (1:1000) was incubated with the sections overnight at 4°C. Sections were washed with Tween 20‐phosphate‐buffered saline (T‐PBS) and were incubated with goat anti‐rabbit IgG 555 (1:2000; Thermo Fisher Scientific, Inc., USA) for 1 h at room temperature. After washing with T‐PBS, the sections were stained with DAPI‐Fluoromount‐G (Southern Biotech, Birmingham, AL, USA). We obtained the images with an all‐in‐one fluorescence microscope (BZ‐X800; KEYENCE, Osaka, Japan) equipped with Plan Apochromat 20× objective (NA0.75, MRD00205; Nikon, Tokyo, Japan). Red fluorescence was detected using a TRITC filter (ex:545/25 nm, em:605/70 nm, dichroic:565 nm, OP‐87764; KEYENCE, Osaka, Japan). We prepared three non‐consecutive cross‐sections for each tissue, and five images per section were randomly photographed for analysis. Mean intensity was calculated as described previously [20].

Statistical Analysis

2.2.3

Values are presented as the mean ± standard deviation, unless indicated. Intragroup comparisons were performed using paired t‐test, and the significance level was corrected according to the number of tests using the Bonferroni method. Student's t‐test was used to compare mean values. We used JMP5.1 (SAS Institute Inc.) for all statistical analyses and GraphPad Prism version 7 (GraphPad Software) for the ex vivo study. All tests were two‐tailed, and statistical significance was considered at a level of 5%.

Results

3

Clinical Trial

3.1

Target Background

3.1.1

A total of 120 female participants underwent the screening test, with 50 found to be ineligible. Among the ineligible participants (n = 50), we excluded 46 participants for not meeting inclusion criteria, 1 by withdrawal, and 3 for other reasons. Remaining 70 participants were enrolled, of which 6 participants dropped out (5 by withdrawal and 1 by illness), and 64 participants completed the study. There were no participants with protocol deviations. Figure 1 shows the flow of the analysis, and Table 2 presents the background of the 64 participants at the time of enrollment.

Flow diagram of the double‐blind, split‐face, placebo‐controlled clinical trial. One hundred and twenty participants were screened for eligibility, 70 of whom underwent randomization. After the trial started, 6 participants dropped out. Data of 64 participants who completed the study were analyzed for efficacy and safety. Creams containing protease‐treated royal jelly (pRJ) and placebo are referred to as pRJ‐Cr and PLBO‐Cr, respectively.

Analysis Results

3.1.2

Primary Endpoint: Wrinkle

3.1.2.1

The maximum depth of the biggest wrinkle in the pRJ‐Cr group was 133.39 ± 45.47 μm after 6‐week application and 125.39 ± 42.26 μm after 12‐week application, which was significantly reduced compared to the depth at baseline (147.69 ± 43.28 μm, Table 3). Such improvements in the wrinkle depth were not found in the PLBO‐Cr group. Additionally, the % changes in both maximum depth of the biggest wrinkle (86.36% ± 20.41% vs. 104.61% ± 29.43%) and average depth of the biggest wrinkle (96.66% ± 36.88% vs. 112.73% ± 45.23%) at week 12/baseline showed significant improvements in the pRJ‐Cr group compared to those in the PLBO‐Cr group. Wrinkle volume in the pRJ‐Cr group was 1.68 ± 0.95 μm after 6‐week application, which was also significantly reduced compared to baseline (1.89 ± 0.99 μm). The % wrinkle areas in the pRJ‐Cr group were 16.72 ± 0.97 μm after 6‐week application and 16.62 ± 1.12 μm after 12‐week application, which was significantly improved compared to the baseline (18.34 ± 0.95 μm). However, this improvement was also seen in the PLBO‐Cr group. Figure 2 illustrates the representative images showing visible reduction in crow's feet wrinkles after applying pRJ‐Cr.

Representative images showing changes in crow's feet wrinkles at baseline (Week 0, A) and after 12 weeks of creams containing protease‐treated royal jelly (pRJ‐Cr) application (Week 12, B). The figure demonstrates a visible reduction in wrinkles due to the application.

Secondary Endpoints

3.1.3

Water Content of the Stratum Corneum and Dermal Thickness

3.1.3.1

When the values before and after 12‐week application were compared, the water content of the stratum corneum was maintained in the pRJ‐Cr group (50.07 ± 13.06 vs. 50.69 ± 11.51 A. U.), whereas we observed a significant decrease in the PLBO‐Cr group (49.90 ± 11.69 vs. 42.23 ± 11.75 A. U., Table 4). Moreover, we found a significant difference in the % change in the pRJ‐Cr group compared with the PLBO‐Cr group after 12‐week application (106.37% ± 32.14% vs. 87.35% ± 28.03%). Similarly, the dermal thickness significantly increased in the pRJ‐Cr group (1331.20 ± 194.09 vs. 1366.86 ± 194.05 μm) after 12‐week application, but decreased in the PLBO‐Cr group (1340.26 ± 188.88 vs. 1304.60 ± 164.55 μm, Table 4). Additionally, there was a significant difference in the % change of the pRJ‐Cr group compared to that of the PLBO‐Cr group after 12‐week application (103.10% ± 8.77% vs. 97.86% ± 8.31%).

Skin Texture

3.1.3.2

When comparing values before and after the 12‐week application, we found significant improvements in three factors (% texture volume, % texture area, and maximum depth texture) only in the pRJ‐Cr group after the 12‐week application (25.58 ± 11.37 vs. 29.78 ± 8.72, 6.32 ± 2.82 vs. 7.34 ± 2.13, 89.98 ± 0.03 vs. 89.99 ± 0.02, respectively, Table 5). The % volume significantly increased both in the pRJ‐Cr group (120.50 ± 42.82 vs. 135.30 ± 48.27) and PLBO‐Cr group (120.50 ± 50.60 vs. 133.24 ± 53.28) after the 12 weeks application. Average texture depth in the PLBO‐Cr group were 56.70 ± 2.99 μm before application and 57.66 ± 2.57 μm after the 12‐week application, which was significantly increased compared to the values at baseline. Figure 3 illustrates the representative images showing improvements in skin texture after applying pRJ‐Cr.

Representative images showing changes in skin texture at baseline (Week 0, A) and after 12 weeks of creams containing protease‐treated royal jelly (pRJ‐Cr) application (Week 12, B). The figure demonstrates a visible improvement in skin texture due to the application.

Stratum Corneum Conditions Obtained by Tape Stripping

3.1.3.3

We found significant improvements in two factors (% multi‐layer peeling and % nucleated cells) in both groups after 12‐week application (pRJ‐Cr group: 31.51 ± 8.65 vs. 17.57 ± 8.05 and 33.97 ± 14.70 vs. 25.70 ± 10.97, PLBO‐Cr group: 31.29 ± 7.77 vs. 18.25 ± 8.24 and 34.06 ± 16.89 vs. 25.21 ± 11.17, respectively, Table 6).

Skin Bacterial Microbiome

3.1.3.4

Both Shannon entropy and Simpson's index tends to have higher indices when the number of species is high and each species is equally present; however, Shannon entropy and Simpson's index are weighted toward rare species and major species, respectively. These parameters significantly increased in the PLBO‐Cr group only after 6‐week application compared to that at baseline (4.70 ± 1.36 vs. 5.44 ± 1.28 and 0.82 ± 0.13 vs. 0.87 ± 0.11, Table 7). Only Shannon entropy was significantly increased in the pRJ‐Cr group (4.64 ± 1.30 vs. 4.95 ± 1.36). Comparing the pRJ‐Cr and PLBO‐Cr groups, Shannon entropy in the PLBO‐Cr group was significantly up‐regulated only at week 6 (4.95 ± 1.36 vs. 5.44 ± 1.28). As to relative amount, significant decreases were observed in C. acnes, Staphylococcus epidermidis ( S. epidermidis ), and S. aureus only in the pRJ‐Cr group after 12‐week application (32.39 ± 17.03 vs. 28.27 ± 16.55, 4.26 ± 5.00 vs. 2.33 ± 3.59, and 2.32 ± 2.15 vs. 1.56% ± 1.49%, respectively, Table 8).

The above results were analyzed using data of participants whose application rate was ≧ 85% based on the number of days on which the test products were applied. When we performed the same analysis on participants who applied appropriate amount (≧ 85%) of the test products based on the remaining amount, in an intragroup comparison before and after application, significant increase was observed in the Shannon entropy only in the pRJ‐Cr group after 12‐week application (Table 9).

Adverse Events

3.1.4

The principal investigator assessed the adverse events using a four‐point scale (“relevant,” “probably relevant,” “probably not relevant,” and “not relevant”) (Table 10). A total of 41 adverse events were reported during the study; all were mild and transient. In six adverse events of pRJ‐Cr group, mainly skin roughness, redness, and irritation, causal relationship with the test products could not be ruled out (“probably relevant”). However, there was no difference in their incidence rate between the pRJ‐Cr and PLBO‐Cr groups. No participant discontinued the study due to adverse effects.

Five of the six “probably relevant” events in pRJ‐Cr group, except for one skin roughness spontaneously disappeared in one day. Remaining event lasted more than two days, but resolved spontaneously without medical intervention, and no long‐term adverse effects were observed. Thus, the 12‐week use of pRJ‐Cr appears well‐tolerated, with a safety profile comparable to that of the placebo.

Stability

3.1.5

The 10‐hydroxy‐2‐decenoic acid content of the remaining pRJ‐Cr after completing the test was at least 90% of its theoretical value, suggesting high stability.

Ex Vivo Study

3.1.6

Because stem cell competition through COL17A1 is known to diminish with aging [19], leading to the disruption of cell turnover and formation of wrinkles [21], we investigated the expression of COL17A1 in human skin ex vivo to confirm whether the results obtained in the clinical study were attributable to the effect of pRJ on epidermal stem cells. The immunostaining results showed that the pRJ‐Cr application increased the expression of COL17A1 compared to the PLBO‐Cr application (Figure 4A,B,E). COL17A1 was mainly expressed in the epidermal basement membrane, which is consistent with previous reports [22]. TIMP1, which inhibits proteolytic enzyme activity, has been shown to be involved in the inhibition of COL17A1 degradation [19]. pRJ‐Cr application also increased TIMP1 expression in the dermis compared to PLBO‐Cr application (Figure 4C,D,F).

*Effect of pRJ‐Cr on the expression of COL17A1 and TIMP1 in a human ex vivo skin model. (A–D) pRJ‐Cr or PLBO‐Cr was topically applied to the skin model twice daily for 24 h. COL17A1 (A and B) and TIMP1 (C and D) were detected by immunofluorescence with specific antibodies (red). Nuclei were visualized using DAPI (blue). Images were obtained and analyzed using BZ‐X800 microscope (Keyence, Tokyo, Japan). pRJ‐Cr application increased the expression of COL17A1 and TIMP1 compared to the PLBO‐Cr application. Three non‐consecutive cross‐sections were prepared for each tissue, and five images per section were randomly photographed for analysis. Representative images were shown. (E, F) Mean intensity was calculated as described in method section. Values are mean ± SD, *p < 0.01 by Student's t‐test (vs. PLBO‐Cr). Creams containing protease‐treated royal jelly (pRJ) and placebo are referred to as pRJ‐Cr and PLBO‐Cr, respectively. DAPI, 4′,6‐diamidino‐2‐phenylindole; SD, standard deviation.

Discussion

4

In this study, we examined the anti‐wrinkle effects and other skin benefits of applying pRJ‐containing cream to the face. The pRJ‐Cr group showed an improvement in the maximum depth and average depth of the biggest wrinkle in the crow's feet wrinkle, along with increased water content of the stratum corneum and dermal thickness, compared to the PLBO‐Cr group. Furthermore, the % texture volume, % texture area and maximum depth texture increased after application only in the pRJ‐Cr group. To the best of our knowledge, this is the first study to validate the anti‐wrinkle effects of pRJ in aged individuals with deep wrinkles. We considered parameters with significant differences at week 12 as meaningful. For parameters that exhibited significant differences at both week 6 and week 12 (e.g., maximum depth of the biggest wrinkle), the effect of pRJ was expected to appear at the early stage. On the other hand, although significant differences confirmed only at week 6 (e.g., wrinkle volume or number of textures) may be incidental, other possibility is that prolonged uptake of a single active material may lead to a plateau effect: in spite of an early significant effect, the difference sometimes becomes insignificant after a certain period of time [23, 24], probably due to maintain skin homeostasis. As described in the method section, we performed all measurements under controlled temperature and humidity conditions, and the study period (the end of January to the end of April for all participants) was fixed to minimize seasonal variation. Furthermore, all participants reside in the southern part of Okayama Prefecture, and their work patterns have not changed significantly. Thus, environmental variables are unlikely to affect the results.

The mechanism by which pRJ improve wrinkles seems to be in both the epidermis (increased water content of the stratum corneum) and dermis (increased dermal thickness). Previous reports suggested production of collagen in dermal fibroblasts is induced by royal jelly via mesenchymal stem cell‐derived exosomes and 10‐hydroxy‐2‐decenoic acid [4, 8]. Furthermore, our ex vivo study revealed that the application of pRJ‐Cr increased the expression levels of the epidermal stem cell marker COL17A1 and its regulator TIMP1, indicating that TIMP1 induction by pRJ controls COL17A1 expression, at least partly. Because COL17A1 plays essential roles in stem cell competition [18, 19], pRJ may maintain the number of stem cells, epidermal turnover, and skin homeostasis. Furthermore, pRJ also induces filaggrin via 10‐hydroxy‐2‐decenoic acid [3, 4]. These effects of pRJ may result in increased water content of the stratum corneum.

Furthermore, we firstly demonstrated that the application of pRJ‐Cr significantly reduced the amount of the three types of bacteria in the skin microbiome compared to those before application. Additionally, Shannon entropy significantly increased compared to that before application in participants who applied 85% or more of the test products. This increase may have been due to a decrease in the number of major bacterial species including C. acnes, S. epidermidis and S. aureus . Previous in vitro studies showed that application of royal jelly has antibacterial activity against C. acnes and other organisms [10]. Healthy skin has a higher diversity of bacterial microbiome than diseased skin [9]; hence, pRJ may lead to a healthy skin condition.

There are several limitations to this study: first, as our participants were Japanese women aged 44–60, the findings may not be generalizable to other populations. Although statistically significant, the number of samples determined was limited and insufficient to allow statistically based conclusions. In addition, because this study focused on the wrinkle‐improving effect of pRJ, the participants had relatively higher water content in the stratum corneum, and no significant improvement could be observed in intragroup comparisons. Hence, future studies should involve multi‐ethnic cohorts with a wider age range to validate the broader applicability of our results, and should include larger number of participants with lower baseline skin hydration to confirm the moisturizing effect of pRJ.

Second, while there was significant improvement in the maximum depth of the biggest wrinkle and the average depth of the biggest wrinkle in the pRJ‐Cr group, no significant difference was observed in the average wrinkle depth. Average wrinkle depth may not be able to reflect minor changes, and increasing number of participants may be necessary to observe statistical significance. These parameters are common endpoints used in previous studies. Additionally, for example, evaluation targeting changes in the maximum depth of the biggest wrinkle is a practical approach, which is an indicator of concern for participants. However, it has been unknown whether they are the most sensitive indicators of wrinkle change. Other parameters including maximum roughness depth and average roughness may also be included in the future studies. Moreover, it is not yet clear if microbiome changes we demonstrated are causal or correlative to wrinkle improvement from this study. Further interventional research (e.g., correlation analyses of longitudinal microbiome monitoring and skin condition during pRJ application) is needed.

Lastly, we conducted ex vivo studies using abdominal skin investigate the mechanisms behind the clinical trial results, based on previous studies [25]. However, facial skin samples were not available, and the effects of pRJ may differ between facial skin and abdominal skin. To demonstrate novel molecular mechanism of wrinkle formation, ex vivo studies of expression of COL17A1 and TIMP1 using facial skin or in vivo experiments using facial skin biopsy specimens would strengthen the conclusion.

In summary, the improvement of maximum depth and average depth of the biggest wrinkle in the crow's feet wrinkle was observed after 12 weeks of application of pRJ‐containing cream in the placebo‐controlled, double‐blind, parallel‐group study. pRJ can reduce or eliminate certain skin concerns of women by improving age‐related wrinkles and skin textures. Our results also suggested that pRJ not only controls stratum corneum water content, dermal thickness, and bacterial microbiome, but also affects stem cell competition and mesenchymal stem cell activation. The limitation of this study included selection of participants, sample size, parameter sensitivity, or mechanistic clarity. Further larger studies, including ex vivo studies using facial skin, will be needed to elucidate the detailed mechanism underlying the effects of pRJ.

Author Contributions

Shiho Ikegami: conceptualization, investigation, project administration, writing – original draft. Takashi Ito: investigation. Hideto Okamoto: investigation. Chizuru Fujikura: supervision, writing – review and editing. Hayate Itatani: conceptualization, supervision, writing – review and editing. Masayuki Yagi: conceptualization, writing – review and editing. Akio Ohkuma: investigation. Nobuaki Okumura: writing – review and editing. Ayanori Yamaki: conceptualization, supervision, writing – review and editing. Norihiro Shigematsu: supervision. Kayo Kunimoto: writing – review and editing. Masatoshi Jinnin: supervision, writing – review and editing.

Ethics Statement

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. This study was reviewed and approved by the Yamada Bee Company Ethics Committee on December 13, 2022 (2022011). The study protocol was registered with the University Hospital Medical Information Network (UMIN 000049871).

Consent

Fully explaining the purpose and methods of this study to the participants, written informed consent was obtained.

Conflicts of Interest

Ikegami, Ito, Okamoto, Fujikura, Itatani, Yagi, Okumura, Yamaki, and Shigematsu are employees of Yamada Bee Company, Inc.