Safety Validation of Plant-Derived Materials for Skin Application

Abstract

The cosmetic industry faces a critical need to balance commercial innovation with scientific validation, especially regarding the safety and efficacy of raw materials. Plant-derived materials (PDMs) offer a promising alternative to animal-derived ingredients in cosmetics, particularly due to their safety and compliance with vegan and ethical standards. Unlike compounds such as polydeoxyribonucleotide (PDRN), which is derived from the testis or seminal fluid of Salmonidae species and raises concerns regarding its origin, sustainability, and consumer acceptability, PDMs provide a cleaner, ethically preferable profile. In this study, we evaluated 50 PDM candidates using in vitro cell viability, wound healing, and immunocytochemistry assays, along with primary skin irritation tests in human participants. None of the samples showed harmful effects. Notably, sample Nos. 38 and 42 demonstrated significant wound-healing capacity and upregulated filaggrin expression without causing notable irritation in clinical testing. These findings support the biological activity and safety of specific PDMs as functional cosmetic ingredients. This study presents scientifically validated evidence for plant-based alternatives to animal-derived materials and offers a new milestone in the shift toward sustainable and ethical cosmetic development. By bridging the gap between consumer demand and scientific rigor, this study provides a robust platform for future innovations in vegan cosmetics.

1. Introduction

To date, ongoing and intense competition over patents and innovations continues to shape the cosmetic industry and field of biomedical science [1]. However, commercial interests in the cosmetics sector often outweigh academic pursuits, making issues of safety and tolerability critically important [2]. Although frequently overlooked, these aspects demand closer academic scrutiny. Despite the significance of these concerns, the current body of literature remains limited, with most discussions emerging from news reports, patents, and commercial articles rather than from peer-reviewed scientific publications. Therefore, these issues must be substantiated through academic validation. The aim of this study was not only to address the commercial and patent-related challenges associated with cosmetic ingredients but also to contribute scientifically relevant evidence that can support the broader application of such materials. Given the increasing development of cosmetic products derived from plant-based sources [3,4], this study focused on raw plant-derived materials (PDMs), such as polydeoxyribonucleotide (PDRN), botanical extracts, and related derivatives.

PDRN is a DNA fragment typically extracted from the testes or semen of salmon or trout, with a molecular size ranging from 200 to 1500 bp (approximately 50–1500 kDa) [5,6]. It activates adenosine A_2A receptors upon topical application, stimulating wound healing, anti-inflammation, and tissue regeneration pathways [7,8], which has led to its growing use in cosmetics and biomedicine. However, several issues remain unresolved. PDRN is often labeled as semen-derived [9], though it is extracted from testicular tissue, raising concerns about purity and accurate classification. Ethical and regulatory concerns also arise due to its animal origin, especially among consumers preferring vegan or cruelty-free products. In addition, some extraction processes use phenol-based reagents that are incompatible with vegan standards and claims about using ultra-short DNA fragments often lack solid scientific background. Plant-derived extracts, in contrast, generally raise fewer ethical concerns and have been studied for toxicity and biocompatibility [10,11], offering a relatively robust body of literature that supports their use in cosmetic formulations [12,13,14]. However, not all plant extracts have been thoroughly investigated; rigorous testing is still required to confirm their safety and efficacy as cosmetic ingredients.

Finally, PDMs, such as exosomes and peptides, have garnered increasing attention as cosmetic ingredients [15,16,17,18]. Although several studies have evaluated their safety, further scrutiny, particularly of exosomes, is warranted. Exosomes, by nature, exhibit limited stability and are typically difficult to preserve beyond two weeks at –80 °C [19,20]. Despite this, many cosmetic companies incorporate exosomes into formulations that are stored at room temperature, often alongside other ingredients. This practice raises serious concerns regarding the authenticity and functional integrity of exosome-containing cosmetic products. Even when advanced preservatives are included in the formulations, whether the structural and functional properties of exosomes are maintained under non-ideal storage conditions remains uncertain. In our preliminary studies, we observed that plant-derived exosomes retained their stability only when extracted from freshly harvested plants and stored at −80 °C for up to one week. In contrast, their stability significantly declined when stored at elevated temperatures, including at room temperature and 40 °C, suggesting that exosomes marketed for cosmetic use under such conditions may not retain their original characteristics.

Therefore, we argue that commercially available exosome-based products, particularly those stored at room temperature, should be re-evaluated to confirm their stability and efficacy. Despite these uncertainties, PDMs generally present fewer safety and ethical concerns than animal-derived alternatives. In this study, we aimed to examine the safety profiles of plant-derived cosmetic ingredients, including exosomes, PDRNs, and extracts, and provide scientific evidence supporting their potential as viable and safe alternatives in cosmetic formulations. We hope that this research will contribute to both academic discourse and industrial practices by reinforcing the credibility and applicability of plant-based materials in the cosmetics sector.

2. Materials and Methods

2.1. Chemicals and Reagents

All chemicals and reagents used in this study were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) unless otherwise specified. Plant materials were obtained either from farms under contract with our affiliation or through online commercial vendors.

2.2. Callus Induction, Culture, and Extraction

In this study, various plant species were utilized, each requiring slightly different culture media compositions and cultivation protocols. Nevertheless, the general procedure for plant induction and culture was largely consistent across species. Specific media formulations for each plant are shown in the Supplementary File (Table S1). When seeds were used, they were first rinsed with sterilized water and germinated until the development of plantlets or, in some cases, until flowering. Subsequently, leaves or flower petals were immersed in 70% ethanol for surface sterilization, followed by several washes with distilled water. The sterilized tissues were then treated with 0.2% sodium hypochlorite solution (concentration adjusted depending on plant species) and washed again with distilled water. Under sterile conditions, the petals or other plant tissues were cut into small segments (approximately 0.5–1 cm) and cultured in Murashige and Skoog (MS) medium (M0222; Duchefa, Haarlem, The Netherlands) supplemented with plant growth regulators, specifically auxins and cytokinins. Cultures were maintained in darkness at 25 ± 2 °C to induce callus formation. The initial calli were subcultured and allowed to proliferate in the same Petri dishes for 2–3 weeks. Once the calli reached an appropriate size for extraction, they were harvested, rinsed with sterile distilled water, and subjected to a high-temperature drying oven for a minimum of three days. The dried calli were then stored in a dark and dry environment until further use. Calli that were dried at temperatures exceeding 80 °C for more than three days were confirmed to have been completely dehydrated. These dried calli were then subdivided, vacuum-sealed, and subjected to extraction. The designated amount (2 g/mL; the optimized extraction concentration was determined based on findings from previous studies [13,21]) was extracted at 121 °C for 20 min.

2.3. Production of Polydeoxyribonucleotide

The PDRN extraction process was conducted under uniform conditions across all plant species used in this study. Initially, plant-derived calli were thoroughly rinsed to eliminate any residual culture medium and then rapidly frozen in liquid nitrogen to facilitate mechanical disruption. Subsequently, approximately 100 g of fresh callus tissue was resuspended in a lysis buffer containing 0.5 M sodium chloride (S6546, Sigma-Aldrich) and 5% detergent solution (ALPICARE GL612, COSFINE Inc., Gunpo-si, Republic of Korea). To promote efficient nucleic acid release, homogenization was carried out with the addition of 200 µL of RNase A (19101, QIAGEN, Hilden, Germany), enabling simultaneous physical and chemical lysis. The homogenized mixture was incubated at 65 °C for 1 h to ensure complete lysis. Following incubation, the lysate was centrifuged at 4500 rpm for 20 min using a Supra R12 centrifuge (Hanil, Seoul, Republic of Korea). The supernatant was then collected and filtered through Miracloth (475855-1R, Merck, Darmstadt, Germany) to remove cellular debris. For DNA precipitation, ethanol (99%, 64-17-5, Merck) was added at a 1:1 volume to the filtrate, and the solution was incubated overnight at −80 °C. The DNA pellet was subsequently collected by centrifugation, washed with 80% ethanol, air-dried, and resuspended in nuclease-free water. To produce PDRN, the isolated genomic DNA was subjected to sonication at 28 kHz for 90 to 120 min to induce fragmentation (SFX550, Branson Digital Sonifier, Geneva, Switzerland). The extent of DNA fragmentation was assessed via agarose gel electrophoresis. Additionally, the purity and concentration of the resulting PDRN were confirmed using spectrophotometric analysis with a Nanodrop (DS-11, Denovix, Wilmington, DE, USA) instrument.

2.4. Exosome Isolation

The culture medium in which the callus was cultured was sequentially filtered using basic mesh filters (110 mesh, Supply Filter TECH, Daejeon, Republic of Korea) to remove large debris. Subsequent filtration was carried out using suction-based 0.45 μm and 0.22 μm membrane filters (FJ25ASCCA004FL01 and FJ25ASCCA002DL01, GVS Life science, Bologna, Italy). Thereafter, exosomes were isolated using the Exofilter bottle top kit (60250, Microgentas, Seoul, Republic of Korea) according to the manufacturer’s protocol, employing suction filtration. The captured exosomes were then washed using the elution buffer provided in the kit, ultimately yielding a purified exosome-containing solution. The obtained exosome preparation was validated using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM).

2.5. Assessment of Cell Viability

The cytotoxicity of each PDM was assessed using the Cell Counting Kit-8 (CCK-8) assay. Keratinocytes (ATCC, Manassas, VA, USA) were seeded in 96-well plates at a density of 5 × 10⁴ cells per well and allowed to stabilize for 24 h. Following cell attachment, serum starvation was performed using serum-free medium to synchronize the cells. Subsequently, the cells were treated with PDMs (1, 5, and 10%, or 0.5, 1, 2, and 5 ppm), including PDRN, extracts, and exosomes, at concentrations determined from preliminary studies and previous publications [21,22] and, again, incubated for 24 h. Control groups were treated with the corresponding solvents used for each PDM. After treatment, 1× CCK-8 solution (CCK-3000, Donginbio, Seoul, Republic of Korea) diluted in serum-free medium was added to each well and incubated for another 24 h. Cell viability was then quantified by measuring absorbance at 450 nm using a spectrophotometer. The percentage of viable cells was calculated using the following formula:

Cell Viability (%) = (Absorbance of treated cells/Absorbance of control cells) × 100

2.6. Wound-Healing Assay

PDMs were employed in a wound-healing assay to evaluate their potential wound-healing effects on skin cells. Some of these assessments have also been conducted in our previous publications [22]. Initially, keratinocytes were seeded at a density of 1 × 10⁶ cells per well to reach approximately 90% confluency and were cultured for 24 h to stabilize the cells. Subsequently, a horizontal scratch was made at the center of each well using a scratcher (201925, SPL life Sciences, Pocheon-si, Republic of Korea). This time point was designated as 0 h (Day 0). Thereafter, PDMs were administered to the cells and cultured for an additional 24 h. A total of 100 ng/mL of epidermal growth factor was applied for the positive control. After 24 h, the extent of cell migration (healed area) was quantified by measuring the area covered by migrating cells using ImageJ software (version 1.46R; National Institutes of Health, Bethesda, MD, USA).

2.7. Immunocytochemistry

After the treatment with PDMs, the cells were thoroughly washed with PBS and subsequently fixed with 4% paraformaldehyde for 2 h at room temperature. After fixation, the cells were rewashed with PBS. Permeabilization was then performed using 1% Triton X-100 in distilled water for 1 h at 37 °C. The cells were again washed with PBS, followed by a blocking step using 2% bovine serum albumin in 1% PBS for 2 h. Thereafter, the cells were incubated overnight at 4 °C with a primary antibody against filaggrin (FLG, 1:200, PA5-115235, Thermo Fisher Scientific, Waltham, MA, USA). After incubation with the primary antibody, the cells were washed several times with PBS and then incubated with a secondary antibody (Anti-rabbit; 1:200, ab6717, Abcam, Waltham, MA, USA) for 2 h at room temperature in the dark. Subsequently, the cells were counterstained with 5 μg/mL Hoechst 33342 for 5 min. After final PBS washes, the cells were mounted with coverslips and observed under a fluorescence microscope. Fluorescence images were acquired and analyzed using ImageJ software (version 1.46R; National Institutes of Health, USA).

2.8. Primary Irritant Patch Test

2.8.1. Test Subjects

Inclusion Criteria for Study Participants

(1)

Healthy adult men and women between the ages of 20 and 59, with no acute or chronic physical illnesses, including skin diseases;

(2)

Individuals who have received sufficient explanation from the principal investigator or a designated researcher regarding the study and have voluntarily signed the informed consent form;

(3)

Individuals who are able to undergo follow-up observation throughout the study period.

Exclusion Criteria for Study Participants

(1)

Pregnant or lactating women, or women who are likely to become pregnant;

(2)

Individuals who have used topical steroids for the treatment of skin diseases for more than one month;

(3)

Individuals who have participated in a similar study within the past four weeks;

(4)

Individuals with sensitivity or allergic reactions to cosmetics, pharmaceuticals, or daily sun exposure;

(5)

Individuals with irritation or severe allergic reactions to adhesive tapes;

(6)

Individuals with sensitive or hypersensitive skin;

(7)

Individuals with moles, acne, tattoos, scars, erythema, telangiectasia, or burn marks on the test site that could interfere with the study.

2.8.2. General Method

At least 30 participants were recruited for the patch test, which was conducted under controlled environmental conditions, with the upper back of each participant selected as the test area. The study was conducted under controlled temperature and humidity conditions (temperature: 21.0–22.2 °C; humidity: 47.0–57.0%). For the patch application, the test area was first cleansed with purified water and then allowed to air dry for 5 min. An IQ chamber containing 20 μL of the test product was then applied and secured to the designated area under occlusion for 24 h. Upon removal of the IQ chamber, the test site was photographed, and the degree of skin irritation was evaluated (single-blinded) by a trained researcher at the following two time points: 30 min and 24 h post-removal. Macroscopic evaluation of the test area was conducted in accordance with the criteria established by the International Contact Dermatitis Research Group (ICDRG;

Table 1. International Contact Dermatitis Research Group (ICDRG) grading criteria.

Skin Irritation Index	Criterion
0.00–0.75	No irritation
0.76–1.50	Low irritation
1.51–2.50	Slight irritation
2.51–4.00	Moderate irritation
4.01–5.00	Severe irritation

). As shown in Table 1, the final values are obtained by first categorizing the criteria presented in

Table 2. Skin irritation criterion.

Grade	Symbol	Criteria
0	-	No reaction	-
1	+	Uncertain positive reaction	Slight erythema
2	++	Mild positive reaction	Erythema, infiltration, or papules
3	+++	Strong positive reaction	Erythema, infiltration, papules, and small vesicles
4	++++	Very strong positive reaction	Severe erythema, infiltration, papules, and irregular vesicles
IR	IR (irritant reaction)	Irritant reaction	Single erythema or papule without edema or infiltration

by grade and then applying the corresponding values for each grade to the formula provided below.

$$\mathrm{Skin}\mathrm{irritation}\mathrm{index}={\displaystyle \frac{{\left(\frac{\sum _{\mathrm{i}=1}^{\mathrm{n}}\mathrm{N}\mathrm{o}. \mathrm{o}\mathrm{f} \mathrm{R}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{r}\mathrm{s}}{\mathrm{n}}\right)}_{30 \mathrm{m}\mathrm{i}\mathrm{n}}+{\left(\frac{\sum _{\mathrm{i}=1}^{\mathrm{n}}\mathrm{N}\mathrm{o}. \mathrm{o}\mathrm{f} \mathrm{R}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{r}\mathrm{s}}{\mathrm{n}}\right)}_{24 \mathrm{h}}}{\mathrm{N}\mathrm{o}. \mathrm{o}\mathrm{f} \mathrm{e}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}}$$

2.9. Statistical Analysis

Statistical analyses were conducted using SigmaStat software (ver. 19, 25) (SPSS Inc., Chicago, IL, USA). All experiments were performed with a minimum of three biological and technical replicates. Data are expressed as the mean ± standard error of the mean. Prior to statistical testing, datasets were evaluated for normality and homogeneity of variance. For comparisons involving three or more groups, one-way analysis of variance (ANOVA) was used for normally distributed data, while the Kruskal–Wallis test was applied to non-normally distributed data. When ANOVA indicated significance, appropriate post hoc tests were performed. Depending on the equality of variances, either Duncan’s multiple range test (for equal variances) or Dunnett’s T3 test (for unequal variances) was employed. Statistical significance is indicated by asterisks above the bars in the figures.

3. Results

3.1. Samples

All samples listed in

Table 3. List of PDMs used in this study.

Sample No.	Sample Type	Name of Material	International Nomenclature of Cosmetic Ingredients (INCI)
1	Callus	Aloe vera phytoplacenta extract	Aloe Barbadensis Phytoplacenta Extract
2	Callus	Aloe vera callus extract	Aloe Vera Callus Extract
3	Callus	Angelica keiskei callus extract	Angelica Keiskei Callus Extract
4	Callus	Artemisia princeps callus extract	Artemisia Princeps Callus Extract
5	Callus	Aster spathulifolius callus extract	Aster Spathulifolius Callus Extract
6	Callus	Camellia japonica callus extract	Camellia Japonica Callus Extract
7	Callus	Camellia japonica phytoplacenta extract	Camellia Japonica Phytoplacenta Extract
8	Callus	Camellia sinensis callus culture extract	Camellia Sinensis Callus Culture Extract
9	Callus	Campanula punctata callus extract	Campanula Punctata Callus Extract
10	Callus	Centella asiatica callus extract	Centella Asiatica Callus Extract
11	Callus	Dryas octopetala callus cultureextract	Dryas Octopetala Callus Culture Extract
12	Callus	Glycine max callus culture extract	Glycine Max (Soybean) Callus Culture Extract
13	Callus	Gynostemma pentaphyllum callus extract	Gynostemma Pentaphyllum Callus Extract
14	Callus	Leontopodium alpinum callus culture extract	Leontopodium Alpinum Callus Culture Extract
15	Callus	Lilium candidum callus culture extract	Lilium Candidum Callus Culture Extract
16	Callus	Myrothamnus flabellifolia callus culture extract	Myrothamnus Flabellifolia Callus Culture Extract
17	Callus	Neofinetia falcata callus culture extract	Neofinetia Falcata Callus Culture Extract
18	Callus	Opuntia ficus-indica callus culture extract	Opuntia Ficus-Indica Callus Culture Extract
19	Callus	Oryza sativa callus culture extract	Oryza Sativa (Rice) Callus Culture Extract
20	Callus	Orostachys japonica callus extract	Orostachys Japonica Callus Extract
21	Callus	Panax ginseng callus culture extract	Panax Ginseng Callus Culture Extract
22	Callus	Rhizophora mangle callus culture extract	Rhizophora Mangle Callus Culture Extract
23	Callus	Rosa damascena callus culture extract	Rosa Damascena Callus Culture Extract
24	Callus	Salicornia herbacea callus culture extract	Salicornia Herbacea Callus Culture Extract
25	Callus	Solanum lycopersicum callus culture extract	Solanum Lycopersicum (Tomato) Callus Culture Extract
26	EV	Camellia sinensis callus extracellular vesicles	Camellia Sinensis Callus Extracellular Vesicles
27	EV	Centella asiatica callus extracellular vesicles	Centella Asiatica Callus Extracellular Vesicles
28	EV	Glycine max callus extracellular vesicles	Glycine Max Callus Extracellular Vesicles
29	EV	Panax ginseng adventitious root extracellular vesicles	Panax Ginseng Adventitious Root Extracellular Vesicles
30	Filtrate	Eryngium maritimum callus culture filtrate	Eryngium Maritimum Callus Culture Filtrate
31	Filtrate	Eryngium maritimum callus Culture Filtrate Liposome	Eryngium Maritimum Callus Culture Filtrate
32	PDRN (Callus)	Adenium obesum Sodium DNA	Sodium DNA
33	PDRN (Plant)	Brassica oleracea Sodium DNA	Sodium DNA
34	PDRN (Plant)	Camellia japonica Sodium DNA	Sodium DNA
35	PDRN (Plant)	Centella asiatica Sodium DNA	Sodium DNA
36	PDRN (Callus)	Glycine max Sodium DNA	Sodium DNA
37	PDRN (Callus)	Gynostemma pentaphyllum Sodium DNA	Sodium DNA
38	PDRN (Plant)	Houttuynia cordata Sodium DNA	Sodium DNA
39	PDRN (Callus)	Lavandula angustifolia Sodium DNA	Sodium DNA
40	PDRN (Callus)	Leontopodium alpinum Sodium DNA	Sodium DNA
41	PDRN (Plant)	Melaleuca alternifolia Sodium DNA	Sodium DNA
42	PDRN (Callus)	Morinda citrifolia Sodium DNA	Sodium DNA
43	PDRN (Plant)	Narcissus tazetta Sodium DNA	Sodium DNA
44	PDRN (Plant)	Oryza sativa Sodium DNA	Sodium DNA
45	PDRN (Plant)	Panax ginseng Sodium DNA	Sodium DNA
46	PDRN (Plant)	Pinus densiflora Sodium DNA	Sodium DNA
47	PDRN (Plant)	Rosa damascena Sodium DNA	Sodium DNA
48	PDRN (Callus)	Rosa damascena (callus) Sodium DNA	Sodium DNA
49	PDRN (Plant)	Solanum lycopersicum Sodium DNA	Sodium DNA
50	PDRN (Callus)	Vitis vinifera (callus) Sodium DNA	Sodium DNA

were produced by our laboratory. Some of these samples had already undergone testing and have been included in previously submitted or published manuscripts. Others represent earlier developments that had successfully passed preliminary evaluations (Figure 1a). Based on this, a subset of the remaining samples was randomly selected for further assessment, including fundamental analyses, such as cell viability, wound healing, and primary irritant patch testing. The samples that had already been published, mentioned, or previously studied are appropriately cited in the present manuscript. The samples used for each assay were as follows: cell viability—Nos. 7, 12, 27, 28, 33, 34, 35, 38, 41, 42, 43, 45, and 48 (Figure 2); wound healing—Nos. 28, 33, 34, 35, 38, 41, 42, 43, 45, and 48 (Figure 3); and immunocytochemistry—Nos. 33, 34, 35, 38, 41, 42, 43, 45, and 48 (Figure 4). The number of samples selected for each assay varied due to differences in the experimental objectives, sample availability, and material constraints. Some samples were excluded from specific assays either because they had already been evaluated in previous studies or were reserved for future research. Moreover, certain tests, such as immunocytochemistry, required stricter quality or concentration standards, which further limited the number of applicable samples. Sample Nos. 30 and 31 were used in a previous study [23]. To maintain their integrity and freshness, exosomes were freshly isolated and promptly utilized after extraction, either under refrigerated or frozen conditions. Samples Nos. 26, 27, 28, and 29 represent exosomes extracted from plant callus culture media, as indicated in Table 3. Following extraction, the presence and concentration of these exosomes were confirmed using NTA, with the average particle count calculated as 1.17 × 10⁹ ± 4.87 × 10⁷ particles/mL (Figure 1b). Additionally, the presence of exosomes was further verified through TEM (Figure 1c).

3.2. In Vitro Assessments of Plant-Derived Materials

Along with cell viability assessment, the wound-healing assay demonstrated that the tested samples were nontoxic (Figure 2a,b) and did not impair the wound-healing process. In addition, some of the samples significantly increased cell viability and promoted the wound-healing process (p < 0.05). Specifically, sample Nos. 41 and 43 showed significantly enhanced wound closure compared with that of the control (p < 0.05; Figure 3a). For instance, sample No. 38, which contained Melaleuca alternifolia and PDRN, markedly increased the healed area, indicating a strong pro-regenerative effect (p < 0.05; Figure 3b). These results suggest that the selected samples are not only biocompatible but also promote positive effects on skin cells.

Although cell viability and wound-healing assays provide morphological information, they are limited in their ability to assess molecular changes. To further investigate the effects at the protein level, we performed immunocytochemistry to evaluate the expression of FLG, a key protein involved in skin barrier function and integrity. All tested samples, except for sample No. 35, showed a significantly increased FLG fluorescence in-tensity compared with that of the control (p < 0.001; Figure 4a). As shown in Figure 4b, representative fluorescence images of samples Nos. 42, 43, and 48 highlight the differential expression levels of FLG. Collectively, these findings indicate that the selected samples not only maintained or enhanced cellular morphology but also exerted beneficial effects at the molecular level.

3.3. Clinical Tests

3.3.1. Study Participants

A minimum of 30 study participants, including both male and female individuals, were randomly selected. The average age was approximately 38.2 ± 11.6 years. The participants were categorized and selected based on skin-type criteria, as follows: dry, oily, normal, intermediate-oily, and intermediate-dry. PDN was applied to the skin of the participants using an IQ chamber, and their reactions and symptoms were subsequently monitored and evaluated.

3.3.2. Evaluation

None of the PDMs tested induced clinically significant irritation during the human trials. Therefore, we confirmed that all the samples tested were generally safe. Although the differences were not statistically significant, a few samples caused slight irritation in a limited number of individuals.

Specifically, treatment with sample Nos. 4, 8, 18, 20, and 22 resulted in a Skin Irritation Index of 0.36 or 0.72 in one or two subjects, respectively. In the case of sample No. 6, one subject exhibited an SII value of 0.9. Nevertheless, these values fall within the “No Irritation” range according to our evaluation criteria and, therefore, cannot be considered as indicative of irritation. Furthermore, follow-up monitoring confirmed that the mild reaction observed in the participant exposed to sample No. 6 resolved spontaneously. This mild response was observed in only one of the 32 participants, and statistical analysis confirmed that the overall effect was not significant. The clinical results corresponding to all samples described in Table 1 are detailed in

Table 4. Irritation index of each PDM.

Sample No.	Name of Materials	No. of Participants	Index	Irritation
1	Aloe vera phytoplacenta extract	32	0.00	None
2	Aloe vera callus extract	31	0.00	None
3	Angelica keiskei callus extract	31	0.00	None
4	Artemisia princeps callus extract	31	0.36	None
5	Aster spathulifolius callus extract	32	0.00	None
6	Camellia japonica callus extract	31	0.9	None
7	Camellia japonica phytoplacenta extract	31	0.00	None
8	Camellia sinensis callus culture extract	31	0.36	None
9	Campanula punctata callus extract	32	0.00	None
10	Centella asiatica callus extract	32	0.00	None
11	Dryas octopetala callus cultureextract	33	0.36	None
12	Glycine max callus culture extract	32	0.00	None
13	Gynostemma pentaphyllum callus extract	32	0.00	None
14	Leontopodium alpinum callus culture extract	31	0.00	None
15	Lilium candidum callus culture extract	31	0.00	None
16	Myrothamnus flabellifolia callus culture extract	31	0.00	None
17	Neofinetia falcata callus culture extract	32	0.00	None
18	Opuntia ficus-indica callus culture extract	31	0.36	None
19	Oryza sativa callus culture extract	31	0.00	None
20	Orostachys japonica callus extract	31	0.36	None
21	Panax ginseng callus culture extract	32	0.00	None
22	Rhizophora mangle callus culture extract	31	0.72	None
23	Rosa damascena callus culture extract	30	0.00	None
24	Salicornia herbacea callus culture extract	32	0.00	None
25	Solanum lycopersicum callus culture extract	32	0.00	None
26	Camellia sinensis callus extracellular vesicles	32	0.00	None
27	Centella asiatica callus extracellular vesicles	32	0.00	None
28	Glycine max callus extracellular vesicles	31	0.00	None
29	Panax ginseng adventitious root extracellular vesicles	32	0.00	None
30	Eryngium maritimum callus culture filtrate	32	0.00	None
31	Eryngium maritimum callus Culture Filtrate Liposome	32	0.00	None
32	Adenium obesum Sodium DNA	33	0.00	None
33	Brassica oleracea Sodium DNA	33	0.00	None
34	Camellia japonica Sodium DNA	33	0.00	None
35	Centella asiatica Sodium DNA	33	0.00	None
36	Glycine max Sodium DNA	33	0.00	None
37	Gynostemma pentaphyllum Sodium DNA	33	0.00	None
38	Houttuynia cordata Sodium DNA	33	0.00	None
39	Lavandula angustifolia Sodium DNA	33	0.00	None
40	Leontopodium alpinum Sodium DNA	33	0.00	None
41	Melaleuca alternifolia Sodium DNA	33	0.00	None
42	Morinda citrifolia Sodium DNA	33	0.00	None
43	Narcissus tazetta Sodium DNA	33	0.00	None
44	Oryza sativa Sodium DNA	33	0.00	None
45	Panax ginseng Sodium DNA	33	0.00	None
46	Pinus densiflora Sodium DNA	33	0.00	None
47	Rosa damascena Sodium DNA	33	0.00	None
48	Rosa damascena (callus) Sodium DNA	33	0.00	None
49	Solanum lycopersicum Sodium DNA	33	0.00	None
50	Vitis vinifera (callus) Sodium DNA	33	0.00	None

. In conclusion, all 50 PDMs tested were demonstrated to be safe, with no evidence of irritation. Thus, the safety of all the tested samples was confirmed.

4. Discussion

Although PDRN extracts and other derivatives obtained from plants have been sporadically reported in the literature, comprehensive data substantiating the biological effects of these compounds remain lacking. Within the cosmeceutical industry, information related to such materials is often disseminated through patents, news articles, and media coverage, rather than through peer-reviewed scientific publications. Consequently, critical discussions and controversies surrounding these substances frequently emerge in non-academic contexts, limiting the depth of scientific discourse. Scholarly references supporting the safety and manufacturing of plant-derived PDRN and related compounds through clinical approaches remain scarce, with most sources restricted to patent literature. Nonetheless, some scientific journals have reported on the safety and basic efficacy [24,25], such as cell viability or the mitigation of specific physiological conditions, of plant-derived extracts, PDRNs, and exosomes [22,26,27,28]. In addition, a limited number of studies have investigated flavonoids and secondary metabolites extracted from plant cells or plant-origin cell cultures [29,30]. Plant-derived exosomes, peptides, and peptide mimetics have also been examined in relevant biological assays [31]. Despite these efforts, the broader academic community has yet to systematically address these materials or their implications. Therefore, this study was designed to clarify and provide evidence-based insights regarding the biological potential of PDMs. Major and minor concerns related to these materials were addressed through the data-driven findings presented in this investigation.

Although the trade of cosmetic raw materials has been active for many years, a growing demand for documentation that certifies the safety of these ingredients has emerged since 2024. This trend has significantly intensified in 2025, reaching a peak, with increasing challenges in cross-border transactions, particularly in Japan, disrupting the smooth flow of raw material trade. We acknowledge the importance and necessity of these regulatory measures. Notably, countries such as the United States and China have begun implementing their own safety and regulatory frameworks, and similar actions are expected to be initiated in Europe shortly. Beginning with international agreements, such as the Nagoya Protocol, adopted in 2014, the demand for fair trade practices and safety assurance in the sourcing and utilization of cosmetic ingredients has steadily increased and continues to gain momentum (while not extensively documented in previous studies, our practical observations indicate that this statement is grounded in our direct experience and current practical circumstances, rather than supported by academic literature, as no scholarly references were found to support it). Therefore, as this shift begins to occur in the cosmetics industry, we strongly emphasize the importance of correcting the misuse or dissemination of inaccurate information, particularly regarding PDMs. We agree that proper clarification is essential in this context.

Accordingly, we believe that generating and sharing reliable safety data should serve as both a foundation and prerequisite for trading raw materials, conducting academic research, and validating efficacy through experimental studies. From this perspective, we advocate that, at least, to some extent, ensuring the safety of such materials must be considered as a necessary and integral part of the process. As previously mentioned, PDMs are extracted from various types of plants and utilized in multiple forms, such as PDRN, oils, or cosmetic formulations [32,33,34]. A growing number of reports have supported the safety of these applications. Although it would be valuable to review such safety data individually, our goal was to provide a foundational reference through our study. Specifically, we aimed for researchers working with PDMs to regard our findings as a milestone in the field, helping to establish a baseline understanding of safety that can inform future research and development.

In our previous study, we investigated the effects of a Lavandula angustifolia callus extract on cellular systems [21]. We evaluated its efficacy not only in vitro but also through clinical studies and molecular analyses. In particular, we elucidated how the Lavandula angustifolia callus extract activates the NRF2 signaling pathway and demonstrated its functional relevance. The results confirmed that the extract enhances anti-aging, antioxidant capacity, and skin barrier function via NRF2 signaling. Similarly, we developed exosome- and liposome-based delivery systems using Eryngium maritimum callus culture filtrates and applied them to skin models [23]. The results showed improved skin permeability, and the filtrate itself exhibited strong efficacy, even under in vitro conditions. In addition, we assessed the efficacy of Hibiscus-sabdariffa-derived PDRN and confirmed its positive effects both in vitro and at molecular levels [22]. Through stepwise investigations of each PDM, we progressively verified their efficacy and safety.

This study serves as a foundational reference to facilitate future research and promote effective communication within the cosmetics industry. By integrating our findings with those of previously published studies, we anticipate that these extracts are not only safe but also highly effective. Therefore, rather than questioning the safety of individual PDMs, many users have reported experiences suggesting that these materials go beyond mere safety—they are beneficial. Although previous studies have evaluated the toxicity of plant extracts (not from calli but directly from plant tissues), some of these studies showed weak toxicity. However, it is worth noting that those results were based on oral administration in mice [35] or were limited to moderate skin irritation [36], which differs significantly from our experimental context. Even in cases where toxicity has been reported, it is often attributed to extracts derived from plants that contain toxic compounds [37] or vesicles during transportation [38] and, thus, should be considered exceptions. In the case of PDRN, few studies have indicated its toxicity. On the contrary, PDRN is known to promote wound healing and exert anti-inflammatory effects primarily through activation of the adenosine A_2A receptor [39]. Despite these beneficial effects, comprehensive evidence of its safety remains insufficient. In summary, the safety of PDMs has been partially supported by previous studies, and the present study further confirms their safety through a systematic evaluation.

5. Conclusions

In the cosmetic raw material industry, a growing demand for safety assurances similar to those provided by CIR reports has emerged. Although not all PDMs examined in this study were individually tested, most were evaluated using various experimental approaches. Based on these assessments, while direct comparative data are not presented in this study, it may be inferred based on our previous related work [22,40] and practical experience that plant-derived substances could be safer than their animal-derived counterparts. However, further comparative studies are needed to substantiate this assumption. In particular, PDRN—extracted and fragmented using a vegan-compatible method—holds a greater promise for application in the cosmetic industry than their animal-derived alternatives. Furthermore, other substances, such as plant extracts, exosomes, and peptides, have demonstrated favorable safety profiles for topical application, either alone or in combination with other formulations. We acknowledge the necessity of continued clinical and in vitro testing to ensure that a wider range of samples meet the safety standards. However, the 50 PDMs utilized in this study were developed over an extended period. Although clinical trials were conducted for all of them, comprehensive in vitro revalidation of each PDM was not feasible. Therefore, additional validation is warranted. Nonetheless, the findings presented in this study provide preliminary evidence that most plant-derived derivatives exhibit acceptable safety profiles. We anticipate that these results will support more confident safety claims within the cosmetics industry.