Application of polygonatum sibiricum fermentation liquor in preparation of medicine for treating skin injury and skin inflammation

Abstract

The invention discloses application of polygonatum sibiricum fermentation liquor in preparation of a medicine for treating skin injury and skin inflammation, and belongs to the technical field of biology. According to the application of the polygonatum sibiricum fermentation liquor in preparation of the medicine for treating the skin injury and the skin inflammation, a zebra fish skin injury model caused by UV irradiation is adopted, and the effect of the polygonatum sibiricum fermentation liquor in skin care is evaluated; the fermented rhizoma polygonati can effectively improve fin damage, reduce oxidative stress by reducing ROS (reactive oxygen species) production, and improve the activity of SOD (superoxide dismutase) and CAT (catalase), reduce MDA (malondialdehyde) level and enhance antioxidant activity. Besides, the compound also can improve skin cell apoptosis induced by ultraviolet rays and regulate key inflammatory pathways, including PPAR-gamma, NF-kB and AP-1, so that the anti-inflammatory characteristics of the compound are highlighted. In addition, an SLS-induced zebra fish inflammation model is adopted, and the influence of the rhizoma polygonati fermentation liquor on inflammation is further studied. The research result emphasizes the potential of the natural component in the skin care formula.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to the application of fermented polygonatum sibiricum redoute liquid in the preparation of drugs for treating skin injuries and skin inflammation. Background Art

[0002] The skin plays a crucial role in many physiological processes and social interactions. At the same time, a series of factors, especially ultraviolet radiation, strongly promote skin aging, cause skin oxidative stress, and lead to skin morphological disorders, including uneven skin color, pigment disorders, skin roughness, and wrinkles. Cosmetics are important for regulating skin morphology. However, due to the effects of certain harmful chemicals and toxins on the skin, the resources of cosmetics have been a concern. Current efforts to mitigate the side effects of these ingredients in cosmetics include adding natural compounds extracted from plants to cosmetics, which provides a healthier choice for consumers. Increasing evidence confirms that plant cosmetics have the function of combating harmful factors such as skin aging, dryness, and environmental damage, thus improving the skin appearance. Nevertheless, little is known about the efficacy of many plant materials, which requires more evidence to clarify the role of these herbal products.

[0003] Due to the complex compound structure of plant materials, the effectiveness of natural products is also limited. Fermentation has been proven to be an effective strategy for improving plant-based foods. Compared with normal treatments, fermentation can significantly enhance the biological activity of traditional drugs. Zhao et al., (2021) believe that fermentation can improve antioxidant activity and bioactive compounds by decomposing plant cell walls. Majchrzak et al. (2022) showed that fermentation converts compounds with complex structures into simple structures, improving efficiency and bioavailability by changing epidermal compatibility and penetrating into the skin.

[0004] Polygonatum sibiricum redoute is an important multi-functional traditional Chinese medicine with great potential in the cosmetics industry, providing rich beneficial substances and a series of biological activities. Previous studies have shown that polygonatum sibiricum redoute and its close relative polygonatum cirrhifolium wall have skin care effects. A recent study by Wang et al. (2025) significantly improved the efficacy of polygonatum sibiricum redoute in skin care through fermentation technology.

[0005] Evaluating the efficacy of polygonatum sibiricum redoute and exploring its potential applications in the field of healthcare are crucial for expanding the use of traditional Chinese medicine and even the development of the traditional Chinese medicine or natural personal care industry. At the same time, the application of fermentation provides ideas for formulating appropriate ways to improve the efficacy of traditional Chinese medicine.

[0006] Therefore, providing the application of fermented polygonatum sibiricum redoute liquid in the preparation of drugs for treating skin injuries and skin inflammation is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides the application of polygonatum fermented liquid in the preparation of drugs for treating skin injuries and skin inflammation.

[0008] The present invention uses a zebrafish skin oxidative stress model induced by UV irradiation to study the skin care effect of polygonatum fermented liquid. Zebrafish are selected because they show the response of epidermal cells to caudal fin injury to illustrate the effect of drugs on tissue injury. In addition, zebrafish have the property of transparency, providing clear evidence for determining the effect of drugs on the skin. The objectives are: (1) to analyze the response of the fin morphology of zebrafish treated with polygonatum fermented liquid to UV irradiation; (2) to determine the antioxidant activity of polygonatum fermented liquid by analyzing reactive oxygen species (ROS) and antioxidant markers [superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT)] in zebrafish treated with polygonatum fermented liquid after UV irradiation; (3) to analyze the apoptosis of zebrafish skin cells treated with polygonatum fermented liquid under UV irradiation; (4) to study the effect of polygonatum fermented liquid on the expression levels of genes related to the inflammatory pathway in zebrafish, so as to clarify the mechanism of the effect of polygonatum fermented liquid on skin oxidative stress caused by UV irradiation; (5) to use a zebrafish skin inflammation model induced by sls to study the effect of polygonatum fermented liquid on skin inflammation and further test its therapeutic effect on the skin.

[0009] To achieve the above objectives, the present invention adopts the following technical solutions:

[0010] The application of polygonatum fermented liquid in the preparation of drugs for treating skin injuries.

[0011] Furthermore, the application of polygonatum fermented liquid in the preparation of drugs for treating skin inflammation.

[0012] Furthermore, the application of polygonatum fermented liquid in the preparation of drugs for reducing skin cell apoptosis.

[0013] Furthermore, the application of polygonatum fermented liquid in the preparation of antioxidant drugs.

[0014] Furthermore, the preparation method of the polygonatum fermented liquid is as follows:

[0015] (1) Crush and sieve polygonatum dried to constant weight to obtain polygonatum powder; add 200 g of polygonatum powder, 4 g of food-grade cellulase, and 4 g of food-grade pectinase to 2 L of distilled water preheated to 50 °C, and stir and enzymatically hydrolyze in a 50 °C water bath for 90 min; add 40 g of food-grade glucose to the enzymatically hydrolyzed solution, and after dissolution, pour the enzymatically hydrolyzed solution into a 5 L fermentation tank and sterilize it under high pressure;

[0016] (2) Inoculate 40 ml of the seed liquid of Lactobacillus plantarum NX-1 with an OD600 of 2.0 - 2.2 into the fermenter containing 2 L of the polygonatum enzymolysis solution in step (1), set the fermentation temperature at 37 °C, the rotation speed at 100 r / min, and the fermentation time at 2 days. After fermentation, obtain the primary polygonatum fermentation broth.

[0017] (3) Centrifuge the primary polygonatum fermentation broth obtained in step (2), take the supernatant for sterilization, and obtain the polygonatum fermentation broth.

[0018] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses the application of polygonatum fermentation broth in the preparation of drugs for treating skin injuries and skin inflammation. A zebrafish skin injury model induced by UV irradiation is used to evaluate the role of polygonatum fermentation broth in skin care; the fermented polygonatum can effectively improve fin damage, reduce oxidative stress by reducing ROS production, enhance antioxidant activity by increasing the activities of SOD and CAT and reducing the MDA level. In addition, it can also improve UV-induced skin cell apoptosis and regulate key inflammatory pathways, including PPAR-γ, NF-κB, and AP-1, highlighting its anti-inflammatory properties. In addition, an SLS-induced zebrafish inflammation model is used to further study the effect of polygonatum fermentation broth on inflammation. The research results emphasize its potential as a natural ingredient in skin care formulations. Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0020] Figure 1 It shows the effect of the polygonatum fermentation broth of the present invention on the relative caudal fin size of the UV skin injury zebrafish model; wherein, A: the relative fin size of zebrafish embryos recorded under a microscope, analyzed using Image J; B: quantification of the relative fin size of zebrafish embryos. Asterisks indicate statistically significant differences compared with the model group: *P < 0.05, P** < 0.01, P*** < 0.001.

[0021] Figure 2Effects of the fermented liquid of Polygonatum sibiricum Redoute on ROS production and antioxidant indexes (SOD activity, MDA level, CAT activity) in a zebrafish UV skin injury model; among them, A: Detection of neutrophils in zebrafish embryos; B: Quantitative analysis of fluorescence intensity in zebrafish embryos; Effects of the fermented liquid of Polygonatum sibiricum Redoute on SOD activity (C), CAT activity (D) and MDA level (E) in the UV skin injury zebrafish model; Asterisks indicate statistically significant differences compared with the model group: *P<0.05, P**<0.01, P***<0.001 (one-way ANOVA).

[0022] Figure 3 Effects of the fermented liquid of Polygonatum sibiricum Redoute on apoptosis of zebrafish embryo skin cells after UV irradiation; among them, A: Detection of apoptosis of zebrafish embryo skin cells by acridine orange fluorescence staining; B: Quantitative analysis of fluorescence intensity in zebrafish embryos; Asterisks indicate statistically significant differences compared with the model group: *P<0.05, **P<0.01, **P*<0.001 (one-way ANOVA).

[0023] Figure 4 Effects of the fermented liquid of Polygonatum sibiricum Redoute on inflammation-related genes PPAR-γ (A), NF-κB (B), iκbαa (C), AP-1 (D) in zebrafish embryos after UV irradiation; Asterisks indicate statistically significant differences compared with the model group: *P<0.05, **P<0.01, **P*<0.001 (one-way ANOVA).

[0024] Figure 5 Effects of the fermented liquid of Polygonatum sibiricum Redoute on the number of neutrophils in zebrafish embryos after UV irradiation; among them, A: Detection of neutrophils in zebrafish embryos; B: Quantitative analysis of neutrophils in zebrafish embryos. Asterisks indicate statistically significant differences compared with the model group: *P<0.05, **P<0.01, **P*<0.001 (one-way ANOVA). Detailed implementation manners

[0025] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0026] Raw Polygonatum sibiricum Redoute was purchased from Jiuhua Chinese Medicinal Materials Technology Co., Ltd., Qingyang County, Anhui Province; food-grade glucose was purchased from Qinhuangdao Lihua Starch Co., Ltd.; food-grade cellulase and food-grade pectinase were purchased from Henan Ming'ang Food Ingredients Co., Ltd.

[0027] The preservation number of Lactobacillus plantarum NX-1 is CGMCC No. 20109. See Patent 202010699291.3.

[0028] Observation, statistics and analysis

[0029] All data sets were statistically analyzed, and the means were expressed as mean ± SEM. The comparison of means between the control group and the model group was performed using a t-test. The comparison of means between the model group and the treatment group was performed using analysis of variance and Tukey's post-hoc comparison. * P < 0.05, ** P < 0.01, *** P < 0.001.

[0030] Example 1

[0031] (1) Strain activation and preparation of seed liquid:

[0032] Formulation of MRS medium: Peptone 10 g / L, beef powder 8 g / L, yeast powder 4 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate dibasic 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, made up to 1 L with pure water, and the pH value was adjusted to 5.7 ± 0.2.

[0033] According to the MRS medium formulation, prepare 2 bottles of 100 ml MRS liquid medium, sterilize at 118 °C for 15 min, and set aside after cooling; take 2 ml of the glycerol preservation solution of Lactobacillus plantarum NX-1 and inoculate it into the sterilized 100 ml MRS liquid medium, and place it in a 37 °C incubator for 8 h to obtain the first-stage mother liquor. Then take 2 ml of the first-stage mother liquor and inoculate it into a new 100 ml MRS liquid medium, and place it in a 37 °C incubator for 16 h to obtain the seed liquid (2.0 ≤ OD600 ≤ 2.2). The prepared seed liquid was put into a 4 °C refrigerator for later use.

[0034] (2) Preparation of polygonatum powder, enzymatic hydrolysis and sugar addition

[0035] The polygonatum is produced in Chizhou. Place 1 kg of fresh polygonatum in an oven at 50 °C until constant weight (about 72 h), then crush and sieve it (30 mesh) to obtain polygonatum powder. Weigh 200 g of polygonatum powder, 4 g of food-grade cellulase (enzyme activity 20,000 - 100,000 U / g), and 4 g of food-grade pectinase (enzyme activity 20,000 - 100,000 U / g), add them to 2 L of distilled water preheated to 50 °C, and stir and enzymatically hydrolyze in a 50 °C water bath for 90 min. Subsequently, add 40 g of food-grade glucose to the enzymatic hydrolysate, dissolve it, pour the enzymatic hydrolysate into a 5 L fermentation tank, and sterilize it at 121 °C under high pressure for 15 minutes.

[0036] (3) Inoculation and fermentation

[0037] Inoculate 40 ml of the seed liquid of Lactobacillus plantarum NX-1 into the above 2-L fermentation tank containing polygonatum enzymolysis solution by the flame inoculation method. Set the fermentation temperature at 37 °C, the rotation speed at 100 r / min, and the fermentation time at 2 days. After the fermentation is completed, the primary polygonatum fermentation liquid is obtained.

[0038] (4) Centrifugation

[0039] Pour the primary polygonatum fermentation liquid obtained in step (3) into a centrifuge bottle, centrifuge at 4000 r / min for 20 min, and pour the supernatant into a 5-L blue-capped bottle to obtain the polygonatum fermentation liquid.

[0040] (5) Sterilization

[0041] Put the polygonatum fermentation liquid obtained in step (4) into a vertical pressure steam sterilizer, sterilize at 118 °C for 15 min, and then cool for standby.

[0042] (6) Freeze-drying

[0043] Pour the sterilized polygonatum fermentation liquid obtained in step (5) into a freeze-drying tray, transfer it to a -80 °C refrigerator for pre-freezing. After pre-freezing, transfer it to a freeze-dryer for freeze-drying. Mix the freeze-dried solid with deionized water to form solutions with concentrations of 0.05, 0.10, and 0.20 mg / ml for subsequent experiments.

[0044] (7) Zebrafish embryo culture

[0045] In the zebrafish breeding system, maintain wild-type AB adult zebrafish at 28.5 °C, pH 7.5, a 14 / 10-hour (day / night) photoperiod, and a conductivity of 500 - 550 μS / cm. After that, obtain zebrafish embryos from these adult zebrafish by natural mating, and then place them in E3 water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) and culture them in an incubator at 28 °C.

[0046] Test Example 1 Fin morphology recording and microscopic observation

[0047] To study the effect of Polygonatum sibiricum fermented liquid on the fin morphology of zebrafish after ultraviolet-induced skin damage, zebrafish embryos at 8 hours post-fertilization were collected and incubated in 100 μM phenylthiourea (PTU) for another 40 hours until 48 hours. Then, healthy zebrafish embryos were collected and divided into a model group, three intervention groups (Polygonatum sibiricum fermented liquid at 0.05, 0.10, and 0.20 mg / ml), and an untreated control group. In the next 24 hours, zebrafish in the model group and the control group were incubated in 2 ml of E3 water; zebrafish in the intervention groups were incubated in 2 ml of the fermented liquid at three concentrations. After the 24-hour treatment, zebrafish in the model group and the intervention groups were placed under an ultraviolet lamp for 1.5 h at an irradiation intensity of 20 mW / cm 2 After the model was constructed, the liquid was changed again and the fish were placed in an incubator for 3 h. The treated zebrafish were collected, washed three times with E3 water, and the caudal fins of the zebrafish were observed under a microscope, and the size of the caudal fins was analyzed using Image J. The results are shown in Figure 1 .

[0048] UV irradiation had a significant effect on the fin morphology of zebrafish. The relative size of the fins in the model group was smaller than that in the untreated control group (P < 0.01)( Figure 1 A). After UV irradiation, the presence of Polygonatum sibiricum fermented liquid improved the fins of zebrafish. Compared with the model group, the relative area of the caudal fins was larger (P < 0.01)( Figure 1 A). Statistical analysis showed that the preparation concentration of the treatment group was positively correlated with the fin size( Figure 1 B). The relative caudal fin rate of the 0.20 mg / ml Polygonatum sibiricum fermented liquid was the highest, at 87.90 ± 2.91%, while the relative caudal fin rate of the 0.10 mg / ml Polygonatum sibiricum fermented liquid was 83.92 ± 2.19%, and the relative caudal fin rate of the 0.05 mg / ml Polygonatum sibiricum fermented liquid was 78.27 ± 3.15%( Figure 1 B).

[0049] Experimental Example 2 ROS Generation and Antioxidant Activity

[0050] (1) ROS Level

[0051] Zebrafish embryos at 8 hours post-fertilization were collected and incubated in 100 μM phenylthiourea (PTU) for 40 hours until 48 hours. Then, healthy zebrafish embryos were collected and divided into a model group, three intervention groups (Polygonatum sibiricum fermented liquid at 0.05, 0.10, and 0.20 mg / ml), and an untreated control group. In the next 24 hours, zebrafish in the model group and the control group were incubated in 2 ml of E3 water; zebrafish in the intervention groups were incubated in 2 ml of the fermented liquid at three concentrations. After the 24-hour treatment, zebrafish in the model group and the intervention groups were placed under an ultraviolet lamp for 1.5 h at an irradiation intensity of 20 mW / cm 2After the model was constructed, the medium was changed again and the samples were placed in an incubator for 3 h. The treated zebrafish were collected, the liquid in the 96-well plate was blotted dry, and 200 μL of 5 μM fluorescent dye DCFH-DA was added to each well. The zebrafish were incubated at 28 °C for 30 min to stain the neutrophils in the zebrafish body. Then, the DCFH-DA on the zebrafish embryos was removed with E3 water, and the zebrafish were anesthetized with 0.02% tricaine. The ROS level of the zebrafish embryos was observed under a microscope, and the fluorescence intensity was detected using Image plus 6.0.

[0052] (2) SOD activity, CAT activity, MDA level

[0053] The antioxidant activity of the fermented liquid of Polygonatum sibiricum was studied. Zebrafish embryos at 2 days post-fertilization (2 dpf) were collected and divided into a model group, three intervention groups (fermented liquid of Polygonatum sibiricum at 0.05, 0.10, and 0.20 mg / ml), and an untreated control group. In the next 24 h, the zebrafish in the model group and the control group were incubated in 2 ml of E3 water; the zebrafish in the intervention groups were incubated in 2 ml of the fermented liquid of Polygonatum sibiricum at three concentrations. After the 24-h treatment, the zebrafish in the model group and the intervention groups were irradiated under an ultraviolet lamp for 1.5 h, and the irradiation intensity was 20 mW / cm 2 After the model was constructed, the medium was changed again and the samples were placed in an incubator for 18 h. After the treatment, the zebrafish were washed twice with E3 water. The zebrafish embryos were transferred to a new clean test tube, and then the water in the tube was removed. Then, 400 μL of pre-cooled phosphate buffer solution (PBS) and zirconia beads were used to grind the zebrafish embryos in a tissue homogenizer. The collected homogenate was centrifuged at 4 °C for 10 - 15 min (10000 g) to obtain the supernatant. Then, the SOD activity of the zebrafish was measured using the WST-1 method with a catalase assay kit (G4306, Bioservice, Wuhan, China). The CAT level was detected using a catalase assay kit (G4307, Bioservice, Wuhan, China). The MDA level was detected using the TBA method with a catalase assay kit (G4302, Bioservice, Wuhan, China). The results are shown in Figure 2 .

[0054] The fermented liquid of Polygonatum sibiricum had a significant effect on reducing the ROS production induced by ultraviolet irradiation, and the fluorescence intensity of the model group was higher ( Figure 2 A). Statistical analysis showed that the fluorescence intensity of the model group (732.11%) was significantly higher than that of the untreated control group (100%) (P < 0.01), indicating that UV irradiation induced an increase in ROS production in zebrafish embryos ( Figure 2B). The presence of the polygonatum fermented liquid significantly reduced the production of ROS after UV irradiation. When the concentration of the polygonatum fermented liquid increased from 0.05 mg / ml to 0.20 mg / ml, the effect of the polygonatum fermented liquid was the best, at 151.01%, followed by the 0.10 mg / ml polygonatum fermented liquid with an effect of 179.86%, and the 0.05 mg / ml polygonatum fermented liquid with an effect of 385.49%( Figure 2 B). UV irradiation significantly reduced the activities of SOD and CAT (P<0.01, P<0.001); the zebrafish treated with 0.20 mg / ml polygonatum fermented liquid had the highest SOD activity, at 75.07 U / mgprot, while that of the 0.10 mg / ml polygonatum fermented liquid was 67.53 U / mgprot, and that of the 0.05 mg / ml polygonatum fermented liquid was 55.96 U / mgprot( Figure 2 C). Similarly, the zebrafish treated with 0.20 mg / ml polygonatum fermented liquid had the highest CAT activity, at 7.67 U / mgprot, while that of the 0.10 mg / ml polygonatum fermented liquid was 6.60 U / mgprot, and that of the 0.05 mg / ml polygonatum fermented liquid was 5.17 U / mgprot( Figure 2 D). UV irradiation significantly increased the MDA level (P<0.01; Figure 2 E). As the concentration of the polygonatum fermented liquid increased from 0.05 mg / ml to 0.20 mg / ml, the MDA level decreased (P<0.01)( Figure 2 E). The zebrafish in the model group had the highest MDA level, at 0.504 U / mgprot. At the same time, the treatment with 0.05, 0.10, and 0.20 mg / ml polygonatum fermented liquid significantly reduced the MDA level of zebrafish in the model group (0.396, 0.364, and 0.328 U / mg prot respectively) (P<0.05, P<0.01, P<0.01).

[0055] Experimental Example 3 Apoptosis of Zebrafish Embryo Skin Cells

[0056] Collect zebrafish embryos 8 hours after fertilization and incubate them in 100 μM phenylthiourea (PTU) for 40 hours until 48 hours to inhibit pigmentation. Collect the zebrafish and divide them into a model group, 3 intervention groups (polygonatum fermented liquid at 0.05, 0.10, and 0.20 mg / ml), and 1 untreated control group. In the next 24 hours, the zebrafish in the model group and the control group were incubated in 2 ml of E3 water; the zebrafish in the intervention groups were incubated in 2 ml of the polygonatum fermented liquid at 3 concentrations. After the 24-hour treatment, the zebrafish in the model group and the intervention groups were placed under an ultraviolet lamp for irradiation for 1.5 h, and the irradiation intensity was 20 mW / cm 2After the model construction was completed, the medium was replaced and the samples were placed in an incubator for 3 h. Then, the zebrafish were transferred onto a 96-well plate. The liquid in the 96-well plate was blotted dry, and 200 μL of 1 μg / mL acridine orange (AO) was added to each well. The samples were incubated at 28 °C for 30 min for acridine orange fluorescence staining. Subsequently, the AO on the zebrafish embryos was removed with E3 water, and the embryos were anesthetized with 0.02% tricaine. Then, microscopic observation was performed to observe the apoptosis of the skin cells of the zebrafish embryos, and the fluorescence intensity was detected using Image plus 6.0. The results are shown in Figure 3 .

[0057] Compared with the untreated group and the treatment group, the fluorescence intensity of the model group was significantly higher ( Figure 3 A). Statistical analysis showed that the fluorescence intensity of the model group (214.35%) was significantly higher than that of the untreated control group (100%) (P < 0.01), indicating an increase in the total number of apoptotic cells detected in zebrafish embryos induced by UV irradiation ( Figure 3 B). The presence of the fermented polygonatum sibiricum red juice significantly reduced the total number of apoptotic cells detected in zebrafish embryos after UV irradiation (P < 0.01) ( Figure 3 B). However, as the concentration of the fermented polygonatum sibiricum red juice increased from 0.05 to 0.20 mg / mL, the total number of apoptotic cells in zebrafish embryos decreased. Among them, the effect of the 0.20 mg / mL fermented polygonatum sibiricum red juice was the best, reaching 77.25%, followed by the 0.10 mg / mL fermented polygonatum sibiricum red juice reaching 87.16%, and the 0.05 mg / mL fermented polygonatum sibiricum red juice reaching 136.50% ( Figure 3 B). The results indicate that the fermented polygonatum sibiricum red juice can reduce the apoptosis of zebrafish embryo skin cells induced by ultraviolet rays, suggesting that polygonatum sibiricum may play a role in skin morphology regulation.

[0058] Example 4 Quantification of gene expression assays

[0059] To examine whether the fermented polygonatum sibiricum red juice has an effect on the expression of genes related to the inflammatory pathway in zebrafish, zebrafish embryos at 2 days post-fertilization were collected. The zebrafish were collected and divided into a model group, three intervention groups (0.05, 0.10, and 0.20 mg / mL of the fermented polygonatum sibiricum red juice), and one untreated control group. In the next 24 h, the zebrafish in the model group and the control group were incubated in 2 mL of E3 water; the zebrafish in the intervention groups were incubated in 2 mL of the fermented polygonatum sibiricum red juice at three concentrations. After the 24-h treatment, the zebrafish in the model group and the intervention groups were irradiated under an ultraviolet lamp for 1.5 h at an irradiation intensity of 20 mW / cm 2After the model was constructed, the medium was replaced and the samples were placed in an incubator for 18 h. Subsequently, the collected zebrafish were transferred to a new clean test tube. Total RNA was extracted using an RNA-easy extraction kit (Tiangen, China) according to the manufacturer's instructions. Then, contaminating genomic DNA was removed, and cDNA was synthesized using FastKing gDNA Dispelling RT SuperMix (Tiangen, China) according to the manufacturer's instructions. The mixture was composed of 4 μL of total RNA, 0.5 μg of FastKing gDNA Dispelling RT SuperMix, and H2O added to a final volume of 20 μL. Subsequently, the mixed solution was incubated at 42 °C for 42 min and then at 95 °C for 3 min. Quantitative reverse transcriptase real-time polymerase chain reaction (qPCR) was performed using gene-specific primers (Table 1). The qPCR mixture was as follows: 3 μL of cDNA sample, 10 μL of SYBR Green qPCR SuperMix, 6.2 μL of dH2O, and 0.4 μL of each primer. qPCR was performed for 40 cycles with the following temperature profile: 30 s at 95 °C, 10 s at 95 °C, and 3 s at 60 °C. The final melt curve was ramped from 60 °C to 95 °C, and each sample was run in triplicate. The expression of specific genes was analyzed by 2 -ΔΔCt method using a Real-Time PCR System (Tianlong, China), with β-actin as the internal reference. The results are shown in Figure 4 .

[0060] Table 1 Forward and reverse primers used for qPCR detection

[0061]

[0062]

[0063] UV irradiation and the treatment with Polygonatum sibiricum fermented liquid significantly regulated the expression of skin inflammation-related genes. Compared with the untreated control, the expression of PPAR-γ in zebrafish was significantly downregulated after UV irradiation, while the expression of PPAR-γ was significantly upregulated in the presence of Polygonatum sibiricum fermented liquid, and its effectiveness increased as the concentration of Polygonatum sibiricum fermented liquid increased from 0.05 mg / ml to 0.20 mg / ml ( Figure 4 A). Meanwhile, the expressions of NF-κB, iκbαa, and AP-1 in zebrafish were significantly downregulated after UV irradiation compared with the untreated control group. In the presence of Polygonatum sibiricum fermented liquid, the expressions of the three genes in zebrafish were all significantly upregulated ( Figure 4 B-D). Notably, compared with 0.05 and 0.20 mg / ml of Polygonatum sibiricum fermented liquid, 0.1 mg / ml of Polygonatum sibiricum fermented liquid showed better performance in downregulating NF-κB and AP-1 after UV irradiation ( Figure 4 B-D).

[0064] Experimental Example 5 Skin Inflammation

[0065] Under a stereomicroscope, healthy Tg(mpx:eGFP) zebrafish embryos at 8 hours post-fertilization were selected, added with 100 μM PTU solution, and incubated in a 28.5 °C biochemical incubator for 40 hours until 48 hours. The Tg(mpx:eGFP) zebrafish embryos were collected, 20 tails per well, with a total of 5 groups and 100 tails. The normal group was added with 4 mL of E3 culture water; the model group was added with 4 mL of 60 μg / mL SLS solution; the 0.05 mg / mL polygonatum fermented liquid group was added with 4 mL of 0.05 mg / mL polygonatum fermented liquid containing 60 μg / mL SLS; the 0.10 mg / mL polygonatum fermented liquid group was added with 4 mL of 0.10 mg / mL polygonatum fermented liquid containing 60 μg / mL SLS; the 0.20 mg / mL polygonatum fermented liquid group was added with 4 mL of 0.20 mg / mL polygonatum fermented liquid containing 60 μg / mL SLS; and they were incubated in the dark in a 28.5 °C biochemical incubator for 18 h. After the incubation, the zebrafish in each group were washed with E3 culture water, and neutrophils of zebrafish were observed and photographed under a fluorescence microscope. The results are shown in Figure 5 .

[0066] Compared with the untreated control group and the treatment group, a higher degree of fluorescence intensity was observed in the model group ( Figure 5 A). Statistical analysis showed that the intervention of polygonatum fermented liquid significantly reduced the number of neutrophils ( Figure 5 [[ID=...]]B). The concentration of polygonatum fermented liquid was positively correlated with the effect of polygonatum fermented liquid on zebrafish: the effect was the best when the concentration of polygonatum fermented liquid was 0.20 mg / ml (30.25), when the concentration was 0.10 mg / ml, the fluorescence intensity was 31.375, and when the concentration was 0.05 mg / ml, the fluorescence intensity was 34.625 ( Figure 5 B).

[0067] In this study, a zebrafish skin injury model induced by UV irradiation was used to evaluate the role of polygonatum fermented liquid in skin care. The fermented polygonatum could effectively improve fin damage, reduce oxidative stress by reducing ROS production, enhance antioxidant activity by increasing the activities of SOD and CAT and decreasing the MDA level. In addition, it could also improve UV-induced skin cell apoptosis and regulate key inflammatory pathways, including PPAR-γ, NF-κB and AP-1, highlighting its anti-inflammatory properties. In addition, a zebrafish inflammation model induced by SLS was used to further study the effect of polygonatum fermented liquid on inflammation. The research results emphasized its potential as a natural ingredient in skin care formulations.

[0068] These findings are consistent with previous studies on the benefits of fermentation in enhancing the bioactivity of plant-derived compounds. The fermented Polygonatum sibiricum Delar. ex Redoute extract offers a promising, natural alternative for cosmetic and therapeutic applications, improving skin elasticity and reducing harmful environmental impacts such as UV radiation. Future research can elucidate the mechanisms by using mammalian models and clinical settings to further explore its translational potential in dermatology and skin care.

[0069] The foregoing description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of polygonatum sibiricum fermented liquid in preparing drugs for treating skin injuries.

2. Application of polygonatum sibiricum fermented liquid in preparing drugs for treating skin inflammation.

3. Application of polygonatum sibiricum fermented liquid in preparing drugs for reducing skin cell apoptosis.

4. Application of polygonatum sibiricum fermented liquid in preparing antioxidant drugs.

5. The application according to any one of claims 1-4, characterized in that, The preparation method of the polygonatum sibiricum fermented liquid is as follows: (1) Crush and sieve polygonatum sibiricum dried to constant weight to obtain polygonatum sibiricum powder; add 200 g of polygonatum sibiricum powder, 4 g of food-grade cellulase, and 4 g of food-grade pectinase to 2 L of distilled water preheated to 50 °C, and stir and enzymatically hydrolyze in a 50 °C water bath for 90 min; add 40 g of food-grade glucose to the enzymatic hydrolysate, dissolve it, pour the enzymatic hydrolysate into a 5 L fermentation tank, and sterilize it under high pressure; (2) Inoculate 40 ml of the seed liquid of Lactobacillus plantarum NX-1 with an OD600 of 2.0 - 2.2 into the fermentation tank containing 2 L of polygonatum sibiricum enzymatic hydrolysate in step (1), set the fermentation temperature at 37 °C, the rotation speed at 100 r / min, and the fermentation time at 2 days. After fermentation, obtain the primary polygonatum sibiricum fermented liquid; (3) Centrifuge the primary polygonatum sibiricum fermented liquid obtained in step (2), take the supernatant and sterilize it to obtain the polygonatum sibiricum fermented liquid.

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