A cyanobacterial extract with photothermal damage repair effects, its preparation method and application

Abstract

This application relates to a cyanobacteria extract with photothermal damage repair effects, its preparation method, and its application, belonging to the field of cosmetic raw material technology. The cyanobacteria extract provided in this application can be used to balance the inflammatory and repair signaling pathways of skin cells after photothermal damage, and can be used to prepare skin care products with soothing, anti-inflammatory, and barrier-repair-promoting effects. It can synergistically balance the pro-inflammatory damage signaling pathway TLR4 / NF-κB axis and the survival-promoting repair signaling pathway HSP90 / PI3K-Akt axis simultaneously activated in skin cells after photothermal damage, targeting multiple points. This can more effectively control excessive inflammation, reduce secondary damage, and enhance the cell's self-repair ability. It is suitable for various scenarios such as sunburn, post-laser surgery, and thermal environment damage, and has significant technical advantages compared to existing technologies, with broad application prospects and huge market value.

Description

Technical Field

[0001] This application relates to a cyanobacterial extract with photothermal damage repair effects, its preparation method and application, belonging to the fields of biotechnology and cosmetic raw materials technology. Background Technology

[0002] When skin is exposed to environments such as ultraviolet rays, lasers, and high temperatures, photothermal damage occurs, characterized by an imbalance in cellular stress, inflammatory cascade responses, and tissue repair processes. Current technologies for repairing photothermal skin damage, such as sunburn, post-laser treatment, and thermal burns, primarily employ the following strategies: 1) Single anti-inflammatory ingredients, such as dipotassium glycyrrhizate and bisabolol, mainly targeting the terminal stages of the inflammatory response; 2) Physical cooling ingredients, such as menthol and alcohol, which produce a cooling sensation by stimulating TRPV receptors, but may exacerbate skin sensitivity; 3) Barrier repair ingredients, such as ceramides and cholesterol, focusing on repairing the stratum corneum structure; 4) Common algae extracts, such as spirulina and chlorella extracts, primarily providing antioxidant support.

[0003] We now have a deeper understanding of the signaling pathways involved in skin damage. Existing research has clearly shown that photothermal damage can simultaneously activate the TLR4 / NF-κB inflammatory axis, which drives inflammation clearance but may lead to overreaction, as well as the PI3K-Akt repair axis, which can promote cell survival and proliferation, and the TRPV thermosensory channel, which mediates the burning pain sensation.

[0004] Current skincare products targeting photothermal damage primarily focus on single anti-inflammatory (e.g., dipotassium glycyrrhizate) or moisturizing / repairing (e.g., ceramides) active ingredients, lacking strategies for systematically regulating the complex cellular signaling network following injury (especially the balance between inflammation and repair signals). Therefore, there is an urgent need in this field for novel active ingredients capable of synergistically regulating key inflammation and repair pathways after photothermal damage to achieve more efficient and safer post-injury care.

[0005] While existing algal extracts (such as spirulina and chlorella extracts) have been reported to have antioxidant and anti-inflammatory properties, there is a lack of products that employ a synergistic regulatory strategy for the three key nodes of TLR4, NF-κB, and HSP90. Summary of the Invention

[0006] The existing technology has the following main drawbacks, which the present invention aims to address: 1) Existing products mostly adopt a simple combination of "anti-inflammatory + repair", which fails to achieve a dynamic balance between inflammation and repair at the signal network level and lacks source regulation of key receptors (TLR4) and thermal sensory channels (TRPV) in the early stage of injury. 2) Physical cooling ingredients such as menthol can produce an immediate cooling sensation, but may irritate sensitive skin and have no substantial repair effect; barrier repair ingredients take effect slowly and cannot meet the immediate soothing needs in the early stages of damage. 3) Algae from common environmental sources have limited activity, and their metabolite profiles generally lack in-depth exploration of the special value of cyanobacteria in skin repair in extreme saline lake environments. 4) Efficacy claims lack systemic biological basis. Most product efficacy claims are based on in vitro antioxidant or single anti-inflammatory experiments, lacking a mechanism explanation of complete signaling pathways.

[0007] To address the aforementioned issues, a cyanobacterial extract with photodamage-resistance properties and its applications are provided. This extract can be used to balance the inflammatory and repair signaling pathways of skin cells after photothermal damage, and can be used to prepare skin care products with soothing, anti-inflammatory, and barrier-repair-promoting effects. It can synergistically balance the pro-inflammatory damage signaling pathways (TLR4 / NF-κB axis) and the survival-promoting repair signaling pathways (HSP90 / PI3K-Akt axis) simultaneously activated in skin cells after photothermal damage, targeting multiple points. This can more effectively control excessive inflammation, reduce secondary damage, and enhance the cell's self-repair ability, showing significant technological advantages compared to existing products.

[0008] This application provides a method for preparing a cyanobacterial extract with photothermal damage repair effects, the preparation method comprising the following steps: 1) Cultivate cyanobacterial strains to obtain wet algal sludge; 2) After resuspending the obtained wet algal mud in water, cell wall disruption was performed using a repeated freeze-thaw method; 3) Place the wet algae mud after cell wall disruption in a temperature-controlled water area and add cellulase for extraction; 4) After adding phosphate buffer to the extraction system, repeat the treatment with short-term low-temperature cycling; 5) After extraction, microfiltration and ultrafiltration membranes are used to remove cell residues and enrich cyanobacterial active substances in the range of 10-30 kDa. The resulting solution is then separated by isoelectric point adjustment and finally concentrated by evaporation to obtain the cyanobacterial extract.

[0009] Optionally, step 5) includes: cooling to 2-6°C after extraction, removing large particulate impurities through a sieve, and then removing cell debris through a 0.45μm microfiltration membrane; removing macromolecular polysaccharides and protein aggregates through a 40-50kDa ultrafiltration membrane; and then fractionating and retaining the permeate through a 10-30 kDa ultrafiltration membrane to enrich cyanobacterial active substances in the range of 10-30 kDa. The pH of the system was adjusted to 4.5-5.5, and the mixture was allowed to stand at 4°C for 30-60 min to allow some impurities, proteins, or pigments to selectively precipitate. The precipitate was removed by centrifugation, and the resulting target component solution was concentrated using a vacuum rotary evaporator at 40-50°C to obtain the cyanobacterial extract.

[0010] In this application, through specific separation and purification processes, the resulting extract can achieve synergistic regulation of three key nodes in a damage event: TLR4, NF-κB, and HSP90. This has significant advantages over the single-target strategies in existing technologies.

[0011] After repeated experiments and explorations, it was found that the above preparation process is crucial for obtaining high-purity active ingredients that simultaneously possess the three key nodes. In particular, enriching cyanobacterial active substances in the range of 10~30 kDa can effectively contain active ingredients that synergistically regulate the three key nodes TLR4, NF-κB, and HSP90. Combined with other purification steps, the purity of the extract can be improved, resulting in better effects.

[0012] Optionally, step 4) includes: adding 0.01~0.05 mol / L phosphate buffer to the extraction system, rapidly reducing the temperature of the extraction system from 38~45℃ to 10~15℃ 10~15 min before the end of the extraction process, maintaining this temperature for 3~5 min, and then restoring the system temperature to 30~40℃. The above short-term low-temperature cycling treatment can be repeated 1~2 times.

[0013] Researchers found that the above treatment improves the stability of the active ingredients, which is crucial for the subsequent extraction of high-purity and highly active components. In particular, the treatment process conditions significantly enhance the stability of the active ingredients in the extract, allowing the active ingredients that synergistically regulate the three key nodes TLR4, NF-κB, and HSP90 in the extract to achieve optimal effects. Verification showed that without step 4), the stability of the active ingredients in the obtained extract is poor, and the efficacy is significantly reduced.

[0014] Optionally, step 3) includes: placing the broken-cell wet algal mud in a temperature-controlled water bath reactor, adding deionized water at a solid-liquid ratio of 1:(8~10) for extraction, with an extraction temperature of 38~45℃, a stirring speed of 200~300 rpm, and an extraction time of 2~4 h; wherein, at the initial stage of extraction, 0.1~0.3wt% cellulase is added for mild enzymatic hydrolysis for 30~60 min, and then the extraction system is subjected to low-intensity pulsed electric field treatment, with an electric field strength of 1~3 kV / cm, a pulse width of 20~40μs, and a treatment time of 1~3 min.

[0015] The extraction method described in this application can effectively extract key active ingredients, allowing for their full release. This extraction process is the optimal solution obtained by researchers after repeated experiments and comparisons. Changing the process parameters significantly reduces the extraction efficiency of the active ingredients, leading to a substantial decrease in the yield of the extracted product.

[0016] Optionally, step 2) includes: resuspending wet algae mud and deionized water at a ratio of 1:(3~10) and homogenizing at 8000~10000 rpm for 2~3 min in a high-speed homogenizer; The freezer was then frozen at -15 to -25°C for 6 to 8 hours, and then thawed in a water bath at 30 to 35°C. During the thawing stage, 0.2 to 0.4 mol / L NaCl solution was added, and then the freezer was diluted with deionized water to restore the isotonic environment. This freeze-thaw-osmosis cycle was repeated 3 to 5 times.

[0017] The above processing steps can achieve efficient cell wall disruption through repeated freeze-thaw cycles. The above process conditions are also the best solution after repeated experiments and explorations by the researchers. They can better cooperate with the subsequent active ingredient extraction steps, with a cell wall disruption rate of >95%, thus effectively ensuring that the subsequent active ingredient extraction steps can fully extract the active ingredients that synergistically regulate the three key nodes TLR4, NF-κB, and HSP90.

[0018] Optionally, before repeated freezing, low-power pulsed ultrasound treatment is performed at a frequency of 20-25 kHz and a power of 300-400W, with intervals of 3-10 seconds, for a total treatment time of 8-10 minutes.

[0019] The cell wall breakage rate can be further improved by adding low-power pulsed ultrasonic treatment.

[0020] This application provides a cyanobacterial extract with photothermal damage repair function, which is prepared according to the above-mentioned preparation method of the cyanobacterial extract with photothermal damage repair function.

[0021] This application provides the application of the above-mentioned cyanobacterial extract with photothermal damage repair effects in the preparation of cosmetic products with heat damage repair effects.

[0022] Optionally, the thermal damage repair is achieved by increasing the expression level of HSP90.

[0023] Optionally, the thermal damage repair is achieved by increasing the expression level of HSP90, thereby improving the stability of Akt protein.

[0024] The beneficial effects of this application include, but are not limited to: The extract of this application achieves skin photothermal damage repair through a multi-target synergistic mechanism: 1) It gently regulates TLR4 receptor activity. The special polysaccharides in the extract can competitively bind to the TLR4 receptor, inhibiting excessive damage signal input from upstream, avoiding the defect of simply inhibiting TLR4 in existing technologies that may affect normal immune function; 2) It precisely regulates the intensity and duration of NF-κB signal. By regulating the phosphorylation and degradation rate of IκBα protein, it allows NF-κB to be moderately activated in the early stage of injury to clear necrotic tissue, while promoting its timely regression in the later stage of inflammation, thus solving the problem of existing anti-inflammatory components. "One-size-fits-all" inhibition may affect tissue clearance; 3) Stabilize and support the function of HSP90 molecular chaperone. Small molecule peptides in the extract can enhance the binding stability of HSP90 with client proteins such as Akt, strengthen the PI3K-Akt repair pathway, and naturally balance the inflammatory response through the negative feedback regulation of NF-κB by Akt; 4) Regulate TRPV1 channel activity for immediate relief. Lipid components in the extract can regulate the calcium ion permeability of TRPV1 channels, producing a physical cooling sensation while avoiding neurogenic inflammation that may be caused by irritating components such as menthol.

[0025] The extract provided in this application is the first to achieve synergistic regulation of three key nodes in a single injury event: TLR4, NF-κB, and HSP90. This offers significant advantages over existing single-target strategies and provides both immediate and long-term benefits. Immediate effects include a 5°C reduction in skin surface temperature, increased skin moisture content, and enhanced thermal protection of HSP90-stabilized AKT protein. In the medium to long term, TRPV regulation significantly reduces burning sensation, and continued use can delay the rise of melanin in the skin. Furthermore, derived from cyanobacteria, it is non-irritating and non-allergenic, offering a safety advantage that is significantly superior to chemical cooling ingredients. It is suitable for various scenarios such as sunburn, post-laser treatment, and damage from thermal environments. Attached Figure Description

[0026] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 This is a graph showing the relative expression rate of TLR4 involved in Test Example 1 of this application; Figure 2 This is a graph showing the relative expression rate of NF-κB involved in Test Example 1 of this application; Figure 3 This is a graph showing the upregulation rate of HSP90 gene expression involved in Test Example 2 of this application; Figure 4 This is a graph showing the absolute fluorescence intensity results of HSP90 involved in Test Example 2 of this application; Figure 5This is a graph showing the relative expression rate of FGF13 in Test Example 3 of this application; Figure 6 This is a graph showing the results of the rate of change of transdermal water loss in the skin involved in Test Example 4 of this application (the vertical axis represents the rate of change of transdermal water loss in the skin). Figure 7 This is a graph showing the change rate of skin stratum corneum moisture content in Test Example 4 of this application (the vertical axis represents the change rate of skin stratum corneum moisture content). Figure 8 This is a graph showing the results of the immediate cooling effect involved in Test Example 4 of this application (the vertical axis represents temperature). Figure 9 This is a graph showing the results of the skin color regulation effect involved in Test Example 4 of this application (the vertical axis represents the skin color a* rate of change). Figure 10 The image shows the test results of skin sensitivity changes involved in Test Example 4 of this application (a is the blank control group, and b is the sample group of 1% cyanobacteria extract aqueous solution of this application). Detailed Implementation

[0027] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0028] The present application solution will be described below through specific embodiments.

[0029] Example 1: Preparation of Cyanobacteria Extract With cyanobacterial strains (Nostoc) Nostoc sp. Using *Nostoc commune* (purchased from the Freshwater Algae Culture Bank of the Chinese Academy of Sciences, code FACHB-1138) as raw material, this application provides a standardized and reproducible process for preparing an active extract. It should be noted that this application uses *Nostoc commune* from the cyanobacteria group as an example for extraction; those skilled in the art can also select other cyanobacteria, including but not limited to *Oscillatoria*. Oscillatoriales .

[0030] The specific extraction process is as follows: 1) Large-scale cell culture and harvesting: Liquid BG-11 medium was used; culture conditions: temperature 28 ± 5℃, light intensity 100 ± 10 μmol photons / m². - ² s - ², with an aeration rate of 0.5 vvm, continue culturing for 5-7 days until the late logarithmic growth stage; after the logarithmic growth stage, scale up the culture until it reaches the logarithmic growth stage in an outdoor greenhouse; collect the bacterial cells through a filter cloth, wash twice with water to obtain wet algae sludge. The yield of wet algae sludge at this stage is approximately 1.5-1.8 g / L of culture medium.

[0031] 2) Pretreatment and cell wall disruption: The wet algae sludge and deionized water were resuspended at a ratio of 1:5 (w / v) and homogenized in a high-speed homogenizer (8000~10000 rpm) for 2~3 min. Then, a gradient freeze-thaw-osmotic pressure shock combined cell wall disruption process was adopted: first in… Freeze at 20℃ for 6–8 h, then thaw in a 30℃ water bath. During thawing, add 0.2–0.4 mol / L NaCl solution to create an instantaneous osmotic pressure difference, causing structural rupture of the cell wall. Then, within 1 min, dilute with deionized water to restore an isotonic environment. Repeat this freeze-thaw-osmosis cycle 3–5 times. Before repeated freezing, administer low-power pulsed ultrasound (20–25 kHz, 300–400 W, 5 s / 5 s intervals, total time 8–10 min). After treatment, the cell wall disruption rate is >95% as determined by microscopy. 3) Extraction of active ingredients: Place the wet algae mud after cell wall disruption in a temperature-controlled water bath reactor, add deionized water at a solid-liquid ratio of 1:8~1:10 (w / v) for extraction; the extraction temperature is 38~45℃, the stirring speed is 200~300 rpm, and the extraction time is 2~4 h.

[0032] At the initial stage of extraction, 0.1-0.3% (w / v) cellulase was added for mild enzymatic hydrolysis for 30-60 min. Subsequently, the extraction system was subjected to low-intensity pulsed electric field treatment (electric field strength 1-3 kV / cm, pulse width 20-40 μs, treatment time 1-3 min) to promote the release of intracellular active substances. 4) Stabilization and extraction control: Add 0.01~0.05 mol / L phosphate buffer (pH 7.0~7.4) to the extraction system to maintain system stability. 10~15 min before the end of the extraction process, rapidly reduce the temperature of the extraction system from 38~45℃ to 10~15℃ and maintain this temperature for 3~5 min. Then restore the system temperature to 30~40℃. The above short-term low-temperature cycling treatment can be repeated 1~2 times. For example, continue to rapidly reduce the temperature to 10~15℃, maintain this temperature for 3~5 min, and then restore the system temperature to 30~40℃. 5) Separation and purification: Immediately after extraction, the temperature is lowered to 4℃. First, it is coarsely filtered through a 100 μm stainless steel screen to remove large particulate impurities, and then passed through a 0.45 μm microfiltration membrane to remove cell debris. Subsequently, the filtrate is sent to a fractionation membrane separation system for molecular weight screening: first, it is passed through a 50 kDa ultrafiltration membrane to remove large polysaccharides and protein aggregates; the permeate is then passed through a 10~30 kDa ultrafiltration membrane for fractionation and retention, thereby enriching cyanobacterial active substances in the range of 10~30 kDa. Based on membrane fractionation, the obtained solution is further separated by isoelectric point adjustment: the pH of the system is adjusted to 4.5~5.5, and the solution is allowed to stand at 4℃ for 30~60 min to allow some impurity proteins or pigments to selectively precipitate. The precipitate is then removed by low-temperature centrifugation (6000~8000 rpm, 10 min) to retain the dissolved target active component. Finally, the obtained target component solution was concentrated to 1 / 5 to 1 / 10 of its original volume using a vacuum rotary evaporator at 45°C and a water bath vacuum of -0.08 MPa to obtain the cyanobacterial extract.

[0033] Test Example 1: TLR4 / NF-κB Axis Verification Thermal damage destroys cells and releases endogenous danger signals (DAMPs). These DAMPs are recognized and activated by the cell membrane receptor TLR4, which in turn strongly initiates the downstream NF-κB transcription factor. After NF-κB enters the nucleus, it significantly upregulates the expression of pro-inflammatory cytokines such as TNF-α and IL-1β, driving the inflammatory response to clear necrotic tissue. However, overactivation can also lead to secondary tissue damage and delayed repair.

[0034] Experimental conditions: Cell models used were human keratinocytes (HaCaT cells) or macrophages (RAW264.7 cells); Thermal damage model: Cells were placed in a 45℃ water bath for 30 minutes to simulate photothermal damage; Extract treatment: Cyanobacterial extract was added immediately after thermal damage, with a 1% concentration group and a model group (without extract); Detection indicators: TLR4 protein expression, TLR4 protein level in cell lysates was detected by Western blotting; NF-κB activation: Nuclear translocation of the NF-κB p65 subunit was detected by immunofluorescence, or NF-κB level in cell supernatant was detected by ELISA.

[0035] Experimental results are as follows Figure 1 and Figure 2 As shown, TLR4 expression was significantly increased and NF-κB nuclear translocation was obvious in the model group (control group), indicating that thermal injury successfully activated the TLR4 / NF-κB inflammatory axis. Compared with the model group, TLR4 protein expression in the 1% cyanobacterial extract treatment group (sample group) was downregulated in a dose-dependent manner (p<0.05). NF-κB nuclear translocation was significantly reduced after extract treatment, showing that the extract can regulate the intensity and duration of NF-κB signaling.

[0036] Verification revealed that the cyanobacterial extract of this application can regulate TLR4, thereby mitigating the excessive NF-κB-driven inflammatory storm at its source by gently regulating the overactivation of TLR4 and preventing secondary tissue damage. It can also regulate NF-κB, precisely balancing its activity so that it can perform its necessary clearance function in the early stages of injury and then subside in a timely manner, thus preventing inflammation.

[0037] Verification revealed that the cyanobacterial extract of this application can mildly inhibit the overactivation of TLR4 receptors and achieve a dual regulatory effect of moderate activation in the early stage of inflammation to clear damaged tissue and timely resolution in the later stage to avoid secondary damage by regulating NF-κB signal transduction. This mechanism is different from the traditional "one-size-fits-all" anti-inflammatory strategy and is more in line with the physiological timing requirements of damage repair.

[0038] Test Example 2: HSP90 / PI3K-Akt Axis Verification Meanwhile, growth factors (such as EGF and FGF) in the damaged microenvironment activate another key repair pathway, PI3K-Akt. The heat shock protein HSP90 plays a "stabilizer" role in this process, protecting and stabilizing Akt protein, thereby enhancing this pro-survival signal. Activated Akt not only directly promotes cell proliferation and migration but also negatively regulates NF-κB activity through phosphorylation, thereby inhibiting excessive inflammatory responses and creating a favorable microenvironment for repair.

[0039] Experimental conditions: Human dermal fibroblasts (HDF cells) or HaCaT cells were used as cell models; Treatment: 42℃ water bath for 30 minutes; Extract treatment: Extracts (0.5%, 1%, 5%) were added immediately after heat treatment, and model groups and normal control groups were set up; Detection indicators: HSP90 mRNA expression was detected by qRT-PCR; HSP90 protein expression and localization: HSP90 fluorescence intensity and cell localization were detected by immunofluorescence.

[0040] Experimental results are as follows Figure 3 and Figure 4 As shown, after 30 min of heat treatment, compared with the model group, the extract of this application at test concentrations of 0.5%, 1%, and 5% significantly increased the gene expression level of HSP90, with upregulation rates of 21.42%, 51.34%, and 366.06%, respectively. After 30 min of heat treatment, compared with the model group, the cyanobacterial extract of this application at test concentrations of 0.5%, 1%, and 5% significantly increased the fluorescence intensity of HSP90, with upregulation rates of 27.08%, 44.64%, and 47.63%, respectively. It can be seen that after heat treatment, the expression of both HSP90 mRNA and protein in the model group increased, but the increase in each concentration group of the extract was significantly higher than that in the model group (p<0.05). Specifically, the 5% concentration group showed a 366.06% upregulation of HSP90 mRNA and a 47.63% upregulation of protein fluorescence intensity. Immunofluorescence showed that HSP90 in the extract group was more evenly distributed in the cytoplasm, suggesting that its molecular chaperone function is more stable.

[0041] Validation revealed that the cyanobacterial extract of this application significantly upregulates HSP90 expression and stabilizes its function, thereby enhancing the activity of the PI3K-Akt repair pathway. HSP90, as a key molecular chaperone, protects repair-related proteins such as Akt, promotes cell survival and proliferation, and naturally balances the inflammatory response through Akt's negative feedback regulation of NF-κB, achieving a synergistic effect of repair and anti-inflammation. The cyanobacterial extract of this application supports HSP90, assists in maintaining the molecular chaperone function of HSP90, and thus stabilizes key repair proteins, including Akt.

[0042] In summary, the results of this application demonstrate that the cyanobacterial extract, through multi-target intervention, synergistically balances the "TLR4-NF-κB" inflammation axis and the "HSP90-PI3K-Akt" repair axis, thereby effectively controlling inflammation, enhancing cell survival, and promoting orderly repair after heat injury. Specifically, the heat shock protein family enhances the cell's self-protection and repair capabilities, helping other proteins to fold, assemble, transport, stabilize, and degrade correctly, thus maintaining cellular "protein homeostasis," especially preventing protein misfolding and aggregation under stress conditions.

[0043] Test Example 3: Inhibition of FGF13 Expression During a thermal response, FGF13 in nerve cells becomes sensitive to heat stimulation. It enhances the function of sodium channels, enabling neurons to generate sustained action potentials in response to noxious heat stimulation, transmitting pain information to the central nervous system. FGF13 is only sensitive to heat stimulation and enhances the function of sodium channel Nav1.7, enabling neurons to generate sustained action potentials in response to noxious heat stimulation, transmitting pain information to the central nervous system. Therefore, inhibiting FGF13 expression can regulate heat pain.

[0044] Experimental conditions: Cell models used were mouse dorsal root ganglion neuron cell lines such as ND7 / 23 cells or human neuron models; Heat stimulation: 40℃ for 60 minutes; Extract treatment: 1% cyanobacterial extract was added after heat stimulation; Model group and normal control group were set up; Detection index: FGF13 mRNA expression was detected by qRT-PCR; Indirect assessment of Nav1.7 channel activity: Calcium imaging was used to detect changes in intraneuronal calcium ion flow.

[0045] Experimental results are as follows Figure 5 As shown, FGF13 mRNA expression was significantly upregulated in the model group after heat stimulation (p<0.01), consistent with increased heat pain sensitivity. FGF13 mRNA expression in the extract-treated group decreased in a dose-dependent manner (approximately 47% decrease in the 1% group, p<0.05).

[0046] In addition, calcium imaging showed that the calcium influx amplitude in the extract group neurons under thermal stimulation was significantly lower than that in the model group, suggesting that the activity of the Nav1.7 channel was inhibited.

[0047] FGF13 is a key regulator of thermal pain signal transduction; its upregulation enhances Nav1.7 channel function, leading to persistent pain signal transmission. The cyanobacterial extract in this application significantly inhibits FGF13 expression, thereby reducing neuronal sensitivity to thermal stimuli and achieving immediate relief and pain reduction. This provides a mechanistic basis for its effectiveness in relieving burning sensations after sun exposure and improving comfort after laser treatment.

[0048] Test Example 4: User Usage Results 4.1) Experimental method: First, the baseline skin values ​​of the blank control group and the sample group were measured. Then, the corresponding values ​​were tested at different times, such as immediately after injury, immediately after application of 1% of the blue algae extract of this application, 10 minutes, and 24 hours. The rate of change of the values ​​at different times compared with the baseline values ​​was calculated to measure the regulatory effect of the blue algae extract of this application.

[0049] The results are as follows Figure 6 and Figure 7 As shown, the cyanobacteria extract of this application can reduce water loss, with transdermal water loss decreasing by 14.25%; the cyanobacteria extract of this application can increase skin moisture, with skin moisture content increasing by 17.84% compared to the baseline value.

[0050] 4.2) After heat stimulation, the skin exhibited obvious inflammatory and sensitive reactions, such as redness. The skin sensitivity of the blank control group showed a relatively basic or unaffected state, indicating that skin sensitivity may remain at a relatively high level for a short period without specific intervention. Heat stimulation caused acute vasodilation, leading to increased secretion of inflammatory mediators, accelerated local blood flow, and increased hemoglobin content, resulting in increased skin redness. Skin color (a*) is used to represent skin color; a positive *a value indicates reddish skin, while a negative value indicates greenish skin. In this experiment, the baseline *a values ​​for the control group and the sample group were 7.4 and 7.94, respectively.

[0051] Experimental methods: First, the baseline skin values ​​of the blank control group and the sample group were measured. Then, the corresponding values ​​were tested at different times, such as immediately after injury, immediately after application of 1% of the blue algae extract of this application, 10 minutes, and 24 hours. The rate of change of the values ​​at different times compared with the baseline values ​​was calculated to measure the regulatory effect of the blue algae extract of this application.

[0052] The results are as follows Figure 8 and Figure 9 As shown, the blue algae extract of this application has a good immediate cooling effect and also shows significant efficacy in continuously regulating skin color.

[0053] In addition, tests were conducted on specific cases to assess changes in skin sensitivity, and the results were as follows: Figure 10As shown, compared with the control group, the redness area in the cyanobacteria extract group of this application was significantly reduced, and the overall skin condition was healthier. The soothing, anti-inflammatory and repairing components in the cyanobacteria extract of this application act on the skin and can reduce skin damage caused by heat stimulation.

[0054] In summary, this application provides a cyanobacterial extract that can synergistically regulate three key nodes in injury events: TLR4, NF-κB, and HSP90. It has broad prospects for cosmetic applications and has significant technical advantages compared to existing products.

[0055] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for preparing a cyanobacterial extract with photothermal damage repair effects, characterized in that, The preparation method includes the following steps: 1) Cultivate cyanobacterial strains to obtain wet algal sludge; 2) After resuspending the obtained wet algal mud in water, cell wall disruption was performed using a repeated freeze-thaw method; 3) Place the wet algae mud after cell wall disruption in a temperature-controlled water area and add cellulase for extraction; 4) After adding phosphate buffer to the extraction system, repeat the treatment with short-term low-temperature cycling; 5) After extraction, microfiltration and ultrafiltration membranes are used to remove cell residues and enrich cyanobacterial active substances in the range of 10-30 kDa. The resulting solution is then separated by isoelectric point adjustment and finally concentrated by evaporation to obtain the cyanobacterial extract.

2. The method for preparing the cyanobacterial extract with photothermal damage repair effect according to claim 1, characterized in that, Step 5) includes: cooling to 2-6°C after extraction, removing large particulate impurities through a sieve, and then removing cell debris through a 0.45μm microfiltration membrane; removing macromolecular polysaccharides and protein aggregates through a 40-50kDa ultrafiltration membrane; and then fractionating and retaining the permeate through a 10-30kDa ultrafiltration membrane to enrich cyanobacterial active substances in the range of 10-30 kDa. The pH of the system was adjusted to 4.5-5.5, and the mixture was allowed to stand at 4°C for 30-60 min to allow some impurities, proteins, or pigments to selectively precipitate. The precipitate was removed by centrifugation, and the resulting target component solution was concentrated using a vacuum rotary evaporator at 40-50°C to obtain the cyanobacterial extract.

3. The method for preparing the cyanobacterial extract with photothermal damage repair effect according to claim 1, characterized in that, Step 4) includes: adding 0.01~0.05 mol / L phosphate buffer to the extraction system, rapidly reducing the temperature of the extraction system from 38~45℃ to 10~15℃ 10~15 min before the end of the extraction process, maintaining this temperature for 3~5 min, and then restoring the system temperature to 30~40℃. The above short-term low-temperature cycling treatment can be repeated 1~2 times.

4. The method for preparing the cyanobacterial extract with photothermal damage repair effect according to claim 1, characterized in that, Step 3) includes: placing the broken-cell wall wet algae mud in a temperature-controlled water bath reactor, adding deionized water at a solid-liquid ratio of 1:(8~10) for extraction, with an extraction temperature of 38~45℃, a stirring speed of 200~300 rpm, and an extraction time of 2~4 h; wherein, at the initial stage of extraction, 0.1~0.3wt% cellulase is added for mild enzymatic hydrolysis for 30~60 min, and then the extraction system is subjected to low-intensity pulsed electric field treatment, with an electric field strength of 1~3 kV / cm, a pulse width of 20~40μs, and a treatment time of 1~3 min.

5. The method for preparing the cyanobacterial extract with photothermal damage repair effect according to claim 1, characterized in that, Step 2) includes: resuspending wet algae mud and deionized water at a ratio of 1:(3~10) and homogenizing it at 8000~10000 rpm for 2~3 min in a high-speed homogenizer; The freezer was then frozen at -15 to -25°C for 6 to 8 hours, and then thawed in a water bath at 30 to 35°C. During the thawing stage, 0.2 to 0.4 mol / L NaCl solution was added, and then the freezer was diluted with deionized water to restore the isotonic environment. This freeze-thaw-osmosis cycle was repeated 3 to 5 times.

6. The method for preparing the cyanobacterial extract with photothermal damage repair effect according to claim 5, characterized in that, Before repeated freezing, low-power pulsed ultrasound treatment is performed at a frequency of 20-25 kHz and a power of 300-400 W, with intervals of 3-10 seconds, for a total treatment time of 8-10 minutes.

7. A cyanobacterial extract with photothermal damage repair effects, characterized in that, It is prepared according to the preparation method of cyanobacterial extract with photothermal damage repair effect as described in any one of claims 1 to 6.

8. The application of the cyanobacterial extract with photothermal damage repair effect as described in claim 7 in the preparation of cosmetic products with heat damage repair function.

9. The application according to claim 8, characterized in that, The thermal damage repair is achieved by increasing the expression level of HSP90.

10. The application according to claim 9, characterized in that, The thermal damage repair is achieved by increasing the expression level of HSP90, thereby improving the stability of Akt protein.

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