The potential of Paeonia lactiflora pall seeds oil as a pure natural cosmetics raw material: In Vitro findings

As a traditional Chinese herbal medicine, Paeonia lactiflora Pall is rich in various active ingredients such as polysaccharides and total flavonoids while having ornamental value. It has potential application value in the development of food and cosmetics.


| Fatty acid content determination
The contents of fatty acids, such as linoleic acid and linolenic acid were determined using Agilent 8860 gas chromatography.Take 70 mg Paeonia lactiflora Pall seeds oil, weigh it accurately, put it in a conical flask, add 1 mL methanol solution of 0.5 mol/L potassium hydroxide, and heat it in a 60°C water bath for 30 min.After cooling, add 1 mL methanol solution of 10% boron trifluoride, and heat it again in a 60°C water bath for 15 min.After cooling, 5 mL of noctane was added, and 20 mL of saturated sodium chloride solution was added after full shaking, and then filtered for Gas chromatography (GC) analysis. 13Under the same extraction conditions, each sample was repeated three times, the test results were averaged (n = 3), and the content of each fatty acid was calculated by the corrected area normalization method.

| Cell culture
B16F10 cells were cultured in DMEM at 37°C with 5% CO 2 .
When the cells grew to about 80%, 0.25% trypsin was used for digestion and counted with a hand-held automatic cell counter.
Finally, a certain concentration of cell suspension was prepared for subculture.

| The effect of Paeonia lactiflora
Pall seeds oil on cell proliferation activity was determined B16F10 cells in the logarithmic phase were used to prepare a cell suspension at a concentration of (0.8 ~ 1.0) × 10 5 cells/mL.The cell suspension was seeded in a 96-well cell culture plate at 100 μL per well and cultured for 24 h.Then the culture medium in the wells was discarded, and 4%, 2%, 1%, and 0.5% Paeonia lactiflora Pall seeds oil were added to each well, with 5 parallel cells in each concentration.Cells cultured without samples were used as negative controls and incubated in a CO 2 incubator for 24 h.The cell culture plate was removed, and 10 μL of CCK-8 solution was added to each well.After 3-4 h of incubation in the incubator, the color change of the solution was observed, and the absorbance was measured at 450 nm wavelength of the microplate reader.The cell proliferation rate was calculated using the following formula:

| Melanin content determination
The melanin content was determined by NaOH lysis. 14B16F10 melanocytes were seeded in 6-well plates at a density of 10 × 10 4 cells/ mL.Then the culture medium in the wells was discarded, and 4%, 2%, 1%, and 0.5% Paeonia lactiflora Pall seeds oil were added to each well.
After 2 days of sample treatment, the supernatant was discarded and washed with PBS solution.Then 0.5 mL trypsin was added to each well to digest the cells, and 1 mL culture medium was added to terminate the digestion, and the cells in each group were counted.The cell suspension was centrifuged to discard the supernatant to obtain the precipitate, and then 1 mol/L NaOH solution containing 10% DMSO was added.After shaking for 5 min, the absorbance of the samples in each well was measured at 490 nm using a microplate reader.The relative melanin content can be calculated by the formula:

Collagenase inhibition rate
According to the experimental method established by Widowati et al. ( 2016) it was modified on this basis. 16In a 96-well plate, 50 μL paeoniflorin seed oil, collagenase solution (3 mg/mL) and Tris-HCl buffer (0.05 mol/L, pH 7.5) were added in turn.After shaking and mixing, the mixture was incubated at 37°C for 15 min, and 50 μL FALGPA (0.48 mg/mL) solution was added.The reaction was carried out at 37°C for 20 min, and the absorbance was measured at 345 nm.500 μg/mL EGCG was used as a positive control, and three parallel sets were set for each sample.The collagenase inhibition rate was calculated using the following formula: where: A Sample is the absorbance of the solution after all the substances are mixed; A Blank is the absorbance of the solution after mixing without adding the remaining substances of the sample.

| Eye irritation test
The in vitro experimental method of HET-CAM was used to replace the traditional animal experimental method, 18 and was carried out according to the "Test of chick embryo chorioallantoic membrane for cosmetic eye irritant/corrosive" (SN/T2329-2009).
The test was performed by reaction time method.The negative (0.9% NaCl) and positive control (0.1 mol/L NaOH) groups were set up at the same time.

| Statistical methods
Each experiment was repeated three times, and the mean value and standard deviation were recorded.SPSS 13.0 software was used for data processing and statistical analysis.p < 0.05 was defined as significant difference between samples, and Origin 8.0 software was used for drawing.

| Effect of Paeonia lactiflora Pall seeds oil on melanin content
Human skin color is mainly determined by the content and distribution of melanin in the skin.Mouse melanoma cell B16F10 has similar genes with human melanoma cells.It is a commonly used cell model for the study of human skin pigmentation metabolic diseases and is often used as a test cell for screening the efficacy of whitening agents. 20In Figure 3

| Inhibition rate of collagenase
Human collagen can speed up the metabolism of skin cells, protect cells from damage, and enhance the vitality of cells, so that the cells maintain a soft surface. 22Collagenase is a kind of Zn 2+ and Ca 2+ dependent matrix metalloproteinase, which can specifically cut off the triple helix structure of collagen molecule, further degrade collagen into peptides and amino acids, thereby destroying the normal metabolism of connective tissue and making skin inactive. 23The inhibitory effect of Paeonia lactiflora Pall seeds oil on collagenase is shown in Figure 5. Paeonia lactiflora Pall seeds oil in the concentration range of 0.5% to 4% has a certain inhibitory effect on collagenase.When the content of Paeonia lactiflora Pall seeds oil was 4%, the inhibition rate of collagenase was 73.38%, which was significantly higher than 71.38% of the positive control group (500 μg/mL EGCG) (p < 0.01).The results were consistent with those of elastase, which further indicated that Paeonia lactiflora Pall seeds oil had the possibility of developing into antiwrinkle and tightening cosmetic products.

| Inhibitory effect of Paeonia lactiflora Pall seeds oil on hyaluronidase activity
As the outermost layer of skin, extracellular matrix (ECM) is composed of proteins and fibroblasts.The degradation of ECM is closely related to the activity of hyaluronidase. 24Facial hyaluronic acid plays a role in avoiding water loss and participating in the production of ECM.Hyaluronidase can degrade hyaluronic acid, resulting in dry and sensitive skin.In addition, some studies have shown that the inhibition of hyaluronidase activity can reflect the anti-inflammatory activity of substances to a certain extent.The higher the inhibition rate of hyaluronidase, the stronger the anti-inflammatory activity, so as to improve capillary fragility and permeability, and effectively play a synergistic role in whitening and anti-wrinkling.Therefore, it is essential to effectively inhibit hyaluronidase activity. 25 Pall seeds oil is expected to be an additive for soothing cosmetics.

Pall seeds oil
The reaction of HET-CAM was observed within 5 min, and the toxic-

1 | 2 | 2 . 1 |
INTRODUC TI ON Paeonia lactiflora Pall, widely distributed throughout our country and with a rich planting history, possesses not only high ornamental value but also serves as a traditional Chinese herbal medicine.The seeds of Paeonia lactiflora Pall are abundant in flavonoids, phenolic acids, monoterpene glycosides, oligoliposis, and other bioactive compounds, showcasing promising properties in terms of antioxidant, anti-inflammatory, whitening, and anti-aging effects. 1 Paeonia lactiflora Pall seeds oil, an important woody edible oil in China, has attracted much attention due to its high nutritional value, and was included in the new resource food catalog by the Ministry of Health in 2011.At the same time, paeoniflora and Paeonia lactiflora Pall, both belonging to the genus Paeoniflora, have become the focus of attention.Some studies have shown that the main components of paeoniflora seeds oil and Paeonia lactiflora Pall seeds oil are similar, but the content is different. 2The Paeonia lactiflora Pall seeds oil extracted from the seeds is rich in nutritional polyunsaturated fatty acids, accounting for 90 ~ 95% of the total fatty acids.such as α-linolenic acid, linoleic acid, and oleic acid; account for 90%-95% of the total fatty acids, which has potential development and utilization value. 3All parts of Paeonia lactiflora contain rich polysaccharides, total flavonoids, paeoniflorin, and other chemical components.The Paeonia lactiflora Pall flower extract of paeoniflorin has been listed in the list of used cosmetic raw materials (2021 edition).Zhou et al., 4 used ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to identify and screen the active components in the extract of Paeonia lactiflora Pall flower.It was found that the extract of Paeonia lactiflora Pall flower had strong anti-glycosylation ability and significant inhibitory effect on tyrosinase and collagenase.It is a promising source for the development of natural skin health products.Čutović et al., 5 found that Paeonia lactiflora Pall petals are rich in phenolic acids and flavonoids, which have antioxidant and antibacterial effects, and have beneficial effects on the skin.Magid et al., 6 extracted and isolated 26 compounds from the flowers of Paeonia lactiflora Pall by liquid chromatography, which had significant free radical scavenging rate (DPPH) activity.The most active compound was identified as methyl gallate.Yang et al., 7 found that Paeonia lactiflora Pall seeds extract showed high antioxidant activity and could effectively inhibit the production of matrix metalloproteinases in keratinocytes, thereby inhibiting cell senescence.Yan et al., 8 examined the fatty acid composition, total phenolic content, and total flavonoid content of seeds from 17 different cultivars of Paeonia lactiflora Pall.Their findings revealed that the seed extracts of Paeonia lactiflora Pall exhibited robust antioxidant activity, as observed in their efficient scavenging of free radicals (DPPH) and overall antioxidant capacity (ABTS).Furthermore, several studies have determined that the seeds oil derived from Paeonia lactiflora Pall harbors a substantial quantity of tocopherols, with a particularly noteworthy presence of γ-tocopherol.Notably, γ-tocopherol demonstrates remarkable antioxidative prowess, contributing to an astonishing 92.97% of the overall tocopherol content. 9Paeoniflorin and oligomeric astragalus compounds contained in Paeonia lactiflora Pall seeds oil have also been confirmed to be anti-inflammatory and antibacterial.For example, resveratrol can reduce the exudation and production of inflammatory factors and play an anti-inflammatory role. 10At the same time, α-linolenic acid in Paeonia lactiflora Pall seeds oil can synthesize docosahexaenoic acid (DHA) in the human body to play an anti-dermatitis role. 11In recent years, the use of skincare products containing natural ingredients has gained significant popularity among individuals.With utmost emphasis on safety, the extraction of active components from plants has emerged as a primary focus in cosmetics research. 12The initial focus of research predominantly centered around the therapeutic properties of Paeonia lactiflora Pall root, disregarding the aerial portion, which was frequently discarded as refuse, leading to a significant wastage of resources.Currently, the nutritive benefits derived from Paeonia lactiflora Pall seeds oil have gained considerable traction in the realms of food and medicine fields.However, its incorporation into cosmetic products remains relatively infrequent.Therefore, this study initiates with the quantification of the fatty acid composition in Paeonia lactiflora Pall seeds oil.In order to study whether it has antioxidant, anti-inflammatory, whitening and anti-wrinkle potential, an in vitro cell model and biochemical assays were employed.Furthermore, the gentleness of Paeonia lactiflora Pall seeds oil was substantiated via HET-CAM assessment.These findings offer valuable data support for future research on the utilization of Paeonia lactiflora Pall seeds oil in cosmetics.MATERIAL S AND ME THODS Main materials and reagents Paeonia lactiflora Pall seeds oil; Mouse melanoma cells (B16F10 cells), DMEM medium, L-DOPA reagent, TritonX-100, DMSO, sodium hydroxide, fetal bovine serum, Zhejiang Tianhang Biological Technology Co., LtD.; Phosphate-buffered saline (PBS), penicillium streptomycin mixture, trypsin-EDTA digestion solution, CCK-8 reagent, Beijing Solarbio Technology Co., LtD.; N-Succinyl-Ala-Ala-Alap-nitroanilide (AAAPAN) and epigallocatechin gallate (EGCG), sigma, USA; N-[3-(2-Furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA), elastase, hyaluronidase, collagenase, Shanghai Yuanye Biotechnology Co., LtD.;Other routine reagents were analytically pure.

2. 3 . 5 |
Determination of Paeonia lactiflora Pall seeds oil inhibiting elastase and collagenase activity Elastase inhibition rate The elastase inhibition assay was performed according to the experimental method of Nema et al. (2013) with slight modifications. 1585 μL50 mmol/L Tris-HCl buffer (pH 8.0) was mixed with 15 μL Paeonia lactiflora Pall seeds oil, then 25 μL elastase solution (60 mU/ mL) was added and incubated at 25°C for 15 min.Then 25 μL of 1.015 mmol/L AAAPAN solution was added, and 15 min later, the absorbance value was measured at 410 nm.Both the elastase solution and AAAPAN solution were prepared with 50 mmol/L Tris-HCl buffer (pH 8.0). 100 μg/mL EGCG was used as a positive control and measured three times in parallel.The inhibition rate of elastase was calculated using the following formula: where: A is the absorbance of the reaction solution containing sample and enzyme; B is the absorbance of the reaction solution without enzyme; C is the absorbance of reaction solution without sample; D is the absorbance of the reaction solution without sample and enzyme.

3 | RE SULTS 3 . 1 |F I G U R E 1 1879 CHEN et al. 3 . 2 |Figure 2 .
Figure 2. It can be seen from the figure that the Paeonia lactiflora Pall seeds oil in the concentration range of 0.5%-4% has no effect on the proliferation of B16F10 cells, and the cell survival rate is also more than 90%, without obvious inhibitory and toxic effects on cells.The experimental results indicate that Paeonia lactiflora Pall seeds oil does not have an impact on the proliferation of B16 cells.
, for different concentrations of Paeonia lactiflora Pall seeds oil, the melanin content of 2% and 4% Paeonia lactiflora Pall seed oil was 85.60% and 91.90%, respectively, which was significantly lower than that of the control group (109.60%), and the inhibitory effect of 2% Paeonia lactiflora Pall seed oil on melanin was slightly better than that of 4% Paeonia lactiflora Pall seeds oil.These results indicate that Paeonia lactiflora Pall seeds oil can reduce cellular melanin synthesis at a certain concentration.Within the range of non-toxic cellular concentrations, Paeonia lactiflora Pall seeds oil demonstrated a significant reduction in cellular melanin synthesis.This suggests that Paeonia lactiflora Pall seeds oil has the potential to be utilized as an ingredient in whitening cosmetics.

3. 4 |F I G U R E 2
Effect of Paeonia lactiflora Pall seeds oil on inhibiting elastase and collagenase activities 3.4.1 | Inhibition rate of elastase Elastin is an important protein to maintain skin elasticity.Once it is lost and damaged, it will cause aging.Elastase is a kind of enzyme that causes the degradation of most extracellular matrix proteins, including elastin.Moreover, it can further activate the activity of other metalloproteinases and accelerate the degradation of dermal cell matrix, resulting in skin inactivity and wrinkles. 21Therefore, inhibition of elastase can slow down the loss of elastin resulting in skin aging, sagging and fine lines.The inhibitory effect of Paeonia lactiflora Pall seeds oil on elastase is shown in Figure 4.When the concentration of Paeonia lactiflora Pall seeds oil ranges from 0.5% to 4%, it has a certain inhibitory effect on elastase in vitro.When the content of paeoniflorin seed oil was 4%, the inhibition rate of elastase reached 60.90%.Compared with the positive control group (100 μg/ mL EGCG), the inhibition rate of elastase in the experimental group of 4% Paeonia lactiflora Pall seeds oil was significantly increased, TA B L E 1 Oil value of Paeonia lactiflora Pall seeds oil (n = 3).Effect of Paeonia lactiflora Pall seeds oil on proliferation activity of B16F10 cells.All the experimental data are showed as the mean ± standard deviation (n = 3).F I G U R E 3 Effect of Paeonia lactiflora Pall seeds oil on melanin content synthesis.All the experimental data are shown as the mean ± standard deviation (n = 3).***p < 0.001, suggest significant differences compared Control with sample group.#p < 0.05, suggest the 4% sample group was significantly different from the 2% sample group.with a significant statistical difference (p < 0.001).These results indicate that Paeonia lactiflora Pall seeds oil has a certain ability to maintain elastin, and has the potential to be used as an additive for anti-wrinkling and tightening cosmetics.
100 mg/ mL Centellaasiatica extract (Control 1) and 100 μg/mL EGCG (Control 2) were used as positive controls to evaluate the inhibitory ability of Paeonia lactiflora Pall seeds oil with different contents on hyaluronidase.The experimental results are shown in Figure 6.With the increase of the concentration of Paeonia lactiflora Pall seeds oil, the inhibition rate of hyaluronidase also increased.When the content of Paeonia lactiflora Pall seeds oil was 4%, the inhibition rate reached the highest of 35.70%, which was lower than that of Control 2 but significantly higher than that of Control 1.Consistent with the above results of elastase and collagenase inhibition rate, 4% Paeonia lactiflora Pall seeds oil had the best inhibitory effect; therefore, Paeonia lactiflora

F I G U R E 5 F I G U R E 6 F I G U R E 7 5 |
ity was graded and scored.The HET-CAM test results of Paeonia lactiflora Pall seeds oil are shown in Figure 7.The negative control group (0.9% NaCl) had no vascular bleeding, coagulation and fusion, and vascular maintenance normalization, while the positive control F I G U R E 4 Inhibitory effect of Paeonia lactiflora Pall seeds oil on elastase activity.All the experimental data are shown as the mean ± standard deviation (n = 3).***p < 0.001, suggest significant differences compared Control with sample group.Inhibitory effect of Paeonia lactiflora Pall seeds oil on collagenase activity.All the experimental data are shown as the mean ± standard deviation (n = 3).**p < 0.01, suggest significant differences compared Control with sample group.group (0.1 mol/L NaOH) had severe bleeding reaction with HET-CAM, indicating that the control groups of the two groups were set successfully and had comparative significance.The eye irritation of the test samples was judged by observing and recording the degree of bleeding, coagulation and vascular melting from the beginning of the test to the occurrence of each kind of bleeding, coagulation and vascular melting.Figure 7C shows that the results of the experimental group of Paeonia lactiflora Pall seeds oil were consistent with those of the negative control, and there was no vascular bleeding, coagulation and melting.Therefore, Paeonia lactiflora Pall seeds oil can be classified as non-irritating to the eyes and can be added to the cosmetic formula for skin care.4 | DISCUSS ION Our nation possesses not only abundant botanical resources but also boasts a longstanding history of traditional Chinese medicine, spanning thousands of years.The utilization of botanical ingredients in cosmetics has endured throughout history, and the domain of botanical cosmetics continues to exhibit promising prospects for development. 26This paper mainly studied the effect of Paeonia lactiflora Pall seed oil on reducing melanin content, inhibiting the activity of collagenase, elastase and other enzymes, and discussed the feasibility of Paeonia lactiflora Pall seed oil for cosmetics.As individuals' living standards continue to advance, fair and radiant skin is highly regarded, making the development of whitening products an ongoing pursuit within the cosmetics industry.Natural-based whitening products, known for their minimal side effects and low irritation, have gained popularity among consumers. 27The melanin content in Paeonia lactiflora Pall seed oil at concentrations of 2% and 4% is lower compared to the control group.Conversely, a decrease in the concentration below 2% leads to an increase in melanin content.These findings suggest that the concentration of Paeonia lactiflora Pall seed oil influences the formation of melanin content.Most of the natural plant extracts contain active ingredients such as total flavonoids and polysaccharides, which can play a certain degree of antioxidant and anti-wrinkle effects. 28Gallic acid (EGCG) has been proved to have good antioxidant and anti-allergy properties, and is widely used in cosmetics. 29EGCG was used as a positive control to evaluate the inhibition of elastase and collagenase in Paeonia lactiflora Pall seeds oil with different contents.The inhibition rate of 4% Paeonia lactiflora Pall seeds oil on elastase was 60.90%, and the inhibition rate on collagenase was 71.38%, which was significantly better than that of the positive control group EGCG.Morikawa et al., 30 determined the inhibition rate of collagenase activity by using the components isolated from the rhizomes of Coptis chinensis.It was found that each component had a significant effect on promoting collagen synthesis at an effective concentration of 10-30 μM, and had no cytotoxicity.The inhibitory activity on collagenase was comparable to that of EGCG (75.6 μM).These results suggest that Paeonia lactiflora Pall seeds oil may have potential advantages in anti-wrinkle and firmness.In addition, Paeonia lactiflora Pall seeds oil also has obvious inhibitory effect on hyaluronidase.Consistent with the results of elastase and collagenase, the higher the concentration of Paeonia lactiflora Pall seeds oil, the greater the inhibition rate of hyaluronidase.The inhibition rate of 4% Paeonia lactiflora Pall seeds oil was 35.70%, which was 2.95% lower than that of 100 μg/mL EGCG, but better than that of Asiatic pennywort herb.Silva et al., 31 further explored its potential value in cosmetic applications by studying the active compounds in kiwifruit leaves.The findings revealed that the kiwifruit leaf extract Inhibitory effect of Paeonia lactiflora Pall seeds oil on hyaluronidase activity.All the experimental data are shown as the mean ± standard deviation (n = 3).**p < 0.01, suggest significant differences compared Control with sample group.Irritant test of Paeonia lactiflora Pall seeds oil.All the experimental data are shown as the mean ± standard deviation (n = 6).(A) Negative control group; (B) Positive control group; (C) Paeonia lactiflora Pall seeds oil experimental group.exhibited a significant inhibitory effect on hyaluronidase activity.As the concentration of the extract increased, the inhibition rate correspondingly intensified.This outcome aligns with the hyaluronidase inhibition observed in the Paeonia lactiflora Pall seed oil case presented in this study.Finally, the HET-CAM test additionally verifies the safety and non-irritating nature of Paeonia lactiflora Pall seeds oil.Paeonia lactiflora Pall seeds oil has been found to effectively reduce the melanin content in B16F10 cells and inhibit the activities of elastase, collagenase, and hyaluronidase.Moreover, the ingredients of this oil are considered safe, making it a suitable option for individuals seeking an all-natural cosmetic additive.CON CLUS ION This study aimed to explore the in vitro effectiveness of Paeonia lactiflora Pall seeds oil.Paeonia lactiflora Pall seeds are abundant in linoleic acid and linolenic acid, which serve as fundamental components in the production of cosmetic oil ingredients.In addition, the results of cell proliferation experiment showed that the cell proliferation rate of B16F10 cells treated with different concentrations of Paeonia lactiflora Pall seeds oil was more than 90%, indicating that Paeonia lactiflora Pall seeds oil as skin care active also has certain safety.The results of HET-CAM also showed that Paeonia lactiflora Pall seeds oil had no eye irritation and has the potential to be further developed into skin care products.2% and 4% Paeonia lactiflora Pall seeds oil can significantly inhibit the production of melanin in cells, which has potential whitening effect.It is worth to note that 4% Paeonia lactiflora Pall seeds oil had the most outstanding effect on the inhibition rate of elastase, collagenase and hyaluronidase by measuring the inhibition rate of various enzymes, which were all higher than that of EGCG positive control group.These results suggest that Paeonia lactiflora Pall seeds oil may have potential effects on skin whitening, anti-inflammation, and delaying skin aging, and it is possible to add appropriate concentration of Paeonia lactiflora Pall seeds oil to various skin care products to exert its beneficial effects.