Sheep tail fat active component as well as separation method and application thereof in skin wound healing

Abstract

The invention discloses a sheep tail fat active component as well as a separation method and application thereof in skin wound healing, and relates to the technical field of natural product separation and biological medicine. According to the method, through ultrasonic-assisted enzymolysis extraction and silica gel column gradient elution, efficient separation of different polar components is achieved, and high-polarity components rich in high-activity unsaturated fatty acid are obtained; the invention also provides application of the component in skin wound healing. The sheep tail fat active component has excellent free radical scavenging ability, can significantly down-regulate the levels of pro-inflammatory factors such as IL-1beta and TNF-alpha in wound tissues, up-regulate the expression of TGF-beta1, promote the deposition and remodeling of collagen and accelerate the regeneration of skin appendages such as hair follicles and sebaceous glands by regulating and controlling a TGF-beta1 / Smad3 signal channel, and has the effects of promoting the healing of the skin and the like. The method can effectively shorten the wound healing time and reduce scar formation, can be used for preparing a natural wound healing agent, and provides a new way for high-value utilization of Lanzhou big-tailed sheep resources.

Description

Technical Field

[0001] This invention relates to the field of natural product separation and biomedicine technology, specifically to a method for separating active components of Lanzhou fat sheep tail fat and its application in skin wound healing. Background Technology

[0002] Lanzhou fat-tailed sheep is one of China's four major sheep breeds, mainly produced in the Lanzhou region of Gansu Province. It is characterized by its large, thick tail, which accumulates a large amount of fat. The tail fat of this breed is known for its low cholesterol content and unique aroma, and has traditionally been found to possess moisturizing, anti-inflammatory, and immune-boosting properties. However, the current insufficient processing of Lanzhou fat-tailed sheep products (especially tail fat) has prevented the full realization of its resource value, becoming a key factor restricting the development of the breed's industry.

[0003] Skin wound healing is a complex and dynamic process involving three stages: inflammation, proliferation, and remodeling. Clinically, it faces numerous challenges, including slow healing and scar formation. Currently used wound healing products include wound dressings, skin grafts, and synthetic drugs. However, synthetic drugs often present problems such as allergic reactions and drug resistance. Therefore, developing wound healing agents that are naturally derived, highly safe, and significantly effective has significant clinical and market value.

[0004] Studies have shown that fatty acids (such as oleic acid, stearic acid, and palmitic acid) contained in sheep tail fat have certain skin care effects and have been used in traditional medicine to treat skin injuries. Furthermore, fatty acids play a crucial role in wound healing processes such as regulating cell migration and proliferation, phagocytosis, and the production of inflammatory factors, while redox signaling is involved in multiple processes including hemostasis, angiogenesis, and extracellular matrix remodeling. However, the bioactivity of different polar components in sheep tail fat varies, and current technologies have not yet established targeted separation methods for its highly active components, nor have they clarified the molecular mechanisms by which it regulates wound healing, thus limiting the large-scale application of sheep tail fat in the biomedical field. Summary of the Invention

[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a method for separating the active components of Lanzhou fat sheep tail fat and its application in skin wound healing. Specifically, this invention provides a method for separating the active components of sheep tail fat. This method is simple, environmentally friendly, and can efficiently separate highly polar components with excellent wound healing activity. At the same time, it provides the application of this active component in the preparation of wound healing agents, providing new raw materials and technical support for the development of natural wound healing products.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for separating active components of sheep tail fat, comprising the following steps: The tail fat of Lanzhou fat-tailed sheep was mixed with neutral protease, the pH was adjusted to 6.5-7.5, and the mixture was ultrasonically treated. Water was added to make up the volume, and after ultrasonic-assisted treatment, the mixture was enzymatically hydrolyzed in a water bath. After the enzymatic hydrolysis was completed, the enzyme was inactivated, and the upper oil phase was collected by centrifugation. The upper oil phase was mixed with n-hexane and loaded onto a 300-400 mesh silica gel column, and then eluted using a gradient of n-hexane-petroleum ether mixture. Thin-layer chromatography was used to detect the eluent, and components with similar characteristics were combined and concentrated by evaporation to obtain high-polarity, medium-polarity, and low-polarity components.

[0007] Optionally, the mass ratio of the tail fat to the neutral protease is 95-105:1, and water is added to make the total volume of the system reach 40-50 mL.

[0008] Optionally, the ultrasonic-assisted treatment conditions include: temperature 40~50℃, power 150~250 W, and time 40~50 min; the water bath enzymatic hydrolysis temperature is 40~50℃ and the time is 70~80 min.

[0009] Optionally, the volume ratio of the upper oil phase to n-hexane is 1:0.8~1.2, and the elution volume ratio of n-hexane to petroleum ether in the n-hexane-petroleum ether mixture is 200:1 to 7:3.

[0010] Optionally, a method for separating active components from sheep tail fat, using Lanzhou fat sheep tail as raw material, includes two core steps: sheep tail fat extraction and polar component separation. Sheep tail fat extraction: Healthy Lanzhou fat-tailed sheep of similar weight and aged 8 months were selected. After slaughter, the tails were collected, and connective tissue, bones, and lean meat were removed. The remaining tail fat was then ground using a meat grinder. 25 g of the ground tail fat was weighed, and 0.25 g of neutral protease was added to adjust the pH to 7.0. Distilled water was added to bring the volume to 45 mL, and the mixture was placed in an ultrasonic cleaner and treated at 46℃ and 200 W for 45 min. Subsequently, it was enzymatically hydrolyzed in a water bath at the same temperature for 75 min. After enzymatic hydrolysis, the sample was heated in an 80℃ water bath for 10 min to inactivate the enzyme. Finally, it was centrifuged at 5000 r / min for 20 min, and the upper oil phase was collected as sheep tail fat (STF).

[0011] Separation of polar components: The extracted sheep tail fat was dissolved in hexane at a 1:1 (mass-volume ratio) and loaded onto a 300-400 mesh silica gel column. Gradient elution was performed using a hexane-petroleum ether mixture, with the mixture ratio gradually adjusted from 200:1 to 7:3 during elution. The eluent was analyzed by thin-layer chromatography (TLC), and components with similar characteristics were combined and concentrated by rotary evaporation at 60℃ to obtain three components with different polarities: high polarity component (HPF), medium polarity component (MPF), and low polarity component (LPF). Among them, the high polarity component (HPF) had a high content of unsaturated fatty acids (73.253%), mainly containing linolenic acid (32.29%), and also contained arachidonic acid (AA) and docosahexaenoic acid (DHA), which have antioxidant and anti-inflammatory activities. These two components were not detected in the medium polarity component and the low polarity component.

[0012] Secondly, the present invention provides an active component of sheep tail fat obtained by the separation method described above. The active component of sheep tail fat is a highly polar component. The active component of sheep tail fat contains not less than 73% unsaturated fatty acids and contains linolenic acid, arachidonic acid and docosahexaenoic acid.

[0013] Thirdly, the present invention provides the application of the active component of sheep tail fat in the preparation of antioxidant products.

[0014] The high polarity component of this invention affects DPPH and ABTS. + ·OH and O2 - Free radicals have a significant scavenging ability, and therefore can be used to prepare antioxidant products.

[0015] Fourthly, the present invention provides the application of the aforementioned active component of sheep tail fat in the preparation of wound healing agents.

[0016] The high polarity component of this invention affects DPPH and ABTS. + ·OH and O2 - Free radicals have a significant scavenging ability and can play a role in wound healing by regulating the levels of inflammatory factors and the TGF-β1 / Smad3 signaling pathway.

[0017] The wound healing agent of this invention is mainly used to promote the healing of full-thickness skin defects, specifically by: significantly shortening the time for scab shedding and improving the wound healing rate; downregulating the expression of pro-inflammatory factors such as IL-1β and TNF-α in wound tissue, reducing the inflammatory response; upregulating the transcription and protein expression of TGF-β1 while downregulating the transcription and protein expression of Smad3, promoting collagen deposition and orderly remodeling; accelerating the regeneration of skin appendages such as hair follicles, sebaceous glands, and capillaries, reducing scar formation, and making the healed skin structure closer to normal skin.

[0018] Optionally, the wound healing agent may be used for at least one of the following (1)-(4): (1) Promotes the healing of full-thickness skin defects; (2) Shorten the time for scabs to fall off; (3) Improve wound healing rate; (4) Inhibits scar formation; The wound healing agent achieves its intended use through at least one of the following methods: a. Downregulate IL-1β expression; b. Downregulate TNF-α expression; c. Upregulates the transcription and protein expression of TGF-β1; d. Downregulate Smad3 transcription and protein expression; e. Regulate the TGF-β1 / Smad3 signaling pathway.

[0019] Optionally, the wound healing agent is a topical preparation, the active ingredient of which is the highly polar component (HPF), with a mass fraction of 5% to 10% in the preparation. The preparation type includes ointment, gel or cream, and can be prepared using pharmaceutically acceptable matrices such as petrolatum or sodium carboxymethyl cellulose to improve skin adhesion and stability.

[0020] Fifthly, the present invention provides a wound healing ointment containing the active ingredient of sheep tail fat as described in claim 5, and further containing an oil matrix, glycerin, propylene glycol, an emulsifying stabilizer, vitamin E, and essential oils.

[0021] Sixthly, the present invention provides a method for preparing the aforementioned wound healing ointment, comprising the following steps: The active components of sheep tail fat and the oil matrix were mixed and heated to obtain the oil phase; Deionized water, glycerol, and propylene glycol were mixed and heated to obtain an aqueous phase. Slowly pour the oil phase into the aqueous phase while stirring, add the emulsifying stabilizer, heat and stir to homogenize and emulsify; After homogenization and emulsification, the mixture is cooled, and vitamin E and sheep tail fat active ingredients are added. The mixture is stirred continuously until the system is uniform and smooth to obtain a wound healing ointment.

[0022] This invention has at least one of the following beneficial effects: 1. This invention establishes for the first time a targeted separation method for highly active components of Lanzhou fat-tailed sheep tail fat. Through ultrasound-assisted enzymatic extraction and silica gel column gradient elution, efficient separation of components with different polarities is achieved, particularly obtaining the high-polarity component (HPF) rich in highly active unsaturated fatty acids. This method eliminates the need for toxic or harmful solvents, is simple in process, and operates under mild conditions, making it suitable for industrial production and providing a feasible path for the high-value utilization of Lanzhou fat-tailed sheep tail fat.

[0023] 2. The high-polarity component (HPF) isolated in this invention exhibits excellent antioxidant and wound-healing activities, with its free radical scavenging ability significantly superior to that of the medium- and low-polarity components. This component promotes wound healing through multiple mechanisms, including inflammation suppression, collagen remodeling, and skin appendage regeneration, by regulating the balanced expression of inflammatory factors (IL-1β, TNF-α, TGF-β1) and targeting the TGF-β1 / Smad3 signaling pathway. It also effectively reduces scar formation, addressing the problems of significant side effects and poor healing efficacy found in existing wound-healing products.

[0024] 3. The high polarity component (HPF) provided by this invention is naturally sourced and has high safety. Its application in the preparation of wound healing agents expands the application scope of sheep tail fat in the biomedical field. At the same time, it provides new raw materials and technical support for the development of novel natural wound care products, which has important economic value and social significance. Attached Figure Description

[0025] Figure 1 Fatty acid composition and antioxidant activity of sheep tail fat fractions with different polarities; where A is the DPPH free radical scavenging rate, B is the ABTS+ free radical scavenging rate, C is the ·OH free radical scavenging rate, and D is the O2 free radical scavenging rate. - Free radical scavenging rate, where E represents the fatty acid composition of each component; Figure 2 The images show the wound healing status of different groups of mice; where A represents the macroscopic changes in the wound (scale bar = 5 mm), B represents the wound healing rate, and C represents the scab shedding time. Figure 3 The levels of inflammatory factors in wound tissues of mice in different groups are shown; where A represents IL-1β content, B represents TNF-α content, and C represents TGF-β1 content. Figure 4 The results show the histopathological analysis of wound tissues and collagen-related indicators in different groups of mice; where A is the HE staining result (40×), B is the Masson staining result (40×), C is the hydroxyproline (HYP) content, and D is the COL1A1 mRNA expression level. Figure 5The expression of TGF-β1 and Smad3 in wound tissues of mice in different groups is shown in Figure 1. A represents the expression level of TGF-β1 mRNA, B represents the expression level of Smad3 mRNA, C represents the representative bands of TGF-β1 and Smad3, D represents the quantitative results of TGF-β1 protein, and E represents the quantitative results of Smad3 protein. Detailed Implementation

[0026] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0027] Example 1: Isolation of active components from sheep tail fat 1. Raw material pretreatment: Select 6 healthy Lanzhou fat-tailed sheep aged 8 months and with similar weight (28-32 kg). After slaughter, immediately collect the tails, remove connective tissue, bones and lean meat, grind the pure tail fat with a meat grinder, and store it at -20℃ for later use.

[0028] 2. Sheep tail fat extraction: Weigh 25 g of crushed tail fat and place it in a 50 mL centrifuge tube. Add 0.25 g of neutral protease, adjust the pH to 7.0 with phosphate buffer, and add distilled water to a final volume of 45 mL. Mix thoroughly. Place the centrifuge tube in an SB-500DTY ultrasonic cleaner, set the temperature to 46℃ and the power to 200 W, and process for 45 min. Then transfer the sample to a constant temperature water bath and continue enzymatic hydrolysis at 46℃ for 75 min. After enzymatic hydrolysis, heat the sample in an 80℃ water bath for 10 min to inactivate the protease. Finally, centrifuge the sample at 5000 r / min for 20 min and collect the upper oil phase, which is sheep tail fat (STF).

[0029] 3. Separation of polar components: Take 10 g of extracted sheep tail fat, add 10 mL of n-hexane to dissolve it, mix thoroughly, and then load the sample onto a pre-activated 300-400 mesh silica gel column (column height 50 cm, inner diameter 2 cm). Gradient elution was performed using a hexane-petroleum ether mixture at a flow rate of 2 mL / min, with elution volume ratios of 200:1 (100 mL), 100:1 (100 mL), 50:1 (100 mL), 20:1 (100 mL), 10:1 (100 mL), and 7:3 (100 mL). Each 20 mL eluent was collected as a fraction and detected by thin-layer chromatography (TLC) with hexane-ethyl acetate (9:1 v / v) as the developing solvent and 5% phosphomolybdic acid ethanol solution as the colorimetric reagent. Components with similar Rf values ​​were combined and concentrated by rotary evaporation at 60 °C and 0.08 MPa to obtain 1.2 g of high polarity component (HPF), 3.5 g of medium polarity component (MPF), and 4.8 g of low polarity component (LPF).

[0030] 4. Component Analysis: The fatty acid composition of the three components was analyzed using GC-MS, such as... Figure 1 As shown in Figure E, the results indicate that HPF contains 17 fatty acids, with unsaturated fatty acids accounting for 73.253%, including linolenic acid at 32.29%, and also contains arachidonic acid (AA) and docosahexaenoic acid (DHA); MPF contains 18 fatty acids, with unsaturated fatty acids accounting for 54.569%; and LPF contains 15 fatty acids, with unsaturated fatty acids accounting for 36.172%.

[0031] Example 2: Detection of antioxidant activity of active components Using DPPH, ABTS + ·OH and O2 - The antioxidant activity of the three components was determined by a free radical scavenging assay: 1. Sample preparation: HPF, MPF and LPF were dissolved in anhydrous ethanol to prepare sample solutions with concentrations of 1, 2, 3, 4, 5 and 6 mg / mL, respectively.

[0032] 2. DPPH radical scavenging rate determination: 20 μL of sample solution was mixed with 180 μL of 0.125 mmol / L DPPH solution, and the mixture was reacted at room temperature in the dark for 30 min. The absorbance was measured at 517 nm, and the scavenging rate was calculated. The results are as follows: Figure 1 As shown in A, Figure 1 A showed that at a concentration of 6 mg / mL, HPF had a DPPH radical scavenging rate of 33.30%, which was significantly higher than that of MPF (26.2%) and LPF (21.9%).

[0033] 3. ABTS+ Free radical scavenging rate determination: The sample solution was mixed with ABTS working solution at a volume ratio of 1:20, and the absorbance was measured at 734 nm after reacting for 20 min. The scavenging rate was then calculated.

[0034] The results are as follows Figure 1 As shown in B, at a concentration of 6 mg / mL, the ABTS of HPF + The free radical scavenging rate was 38.97%, the MPF was 36.20%, and the LPF was 30.20%.

[0035] 4. Determination of OH radical scavenging rate: Take 2 mL of sample solution and mix it with 2 mL of a mixture containing 9 mmol / L FeSO4, 8.8 mmol / L H2O2 and 9 mmol / L salicylic acid-ethanol. React in a water bath at 37℃ for 30 min. Measure the absorbance at 510 nm and calculate the scavenging rate.

[0036] result Figure 1 As shown in C, at a concentration of 6 mg / mL, the ·OH radical scavenging rate of HPF was 16.59%, that of MPF was 15.88%, and that of LPF was 12.13%.

[0037] 5. O2 - Free radical scavenging rate determination: Take 1 mL of sample solution, add 4.5 mL of 0.05 mol / L Tris-HCl buffer and 0.1 mL of 3 mmol / L pyrogallol solution, react at 25℃ for 5 min, then add 1 mL of 8 mmol / L HCl to terminate the reaction, measure the absorbance at 299 nm, and calculate the scavenging rate.

[0038] The results are as follows Figure 1 As shown in D, at a concentration of 6 mg / mL, the O2 of HPF - The free radical scavenging rate was 63.30%, the MPF was 56.20%, and the LPF was 51.91%.

[0039] Therefore, the above results indicate that HPF has the strongest free radical scavenging ability and is the main antioxidant active component in sheep tail fat.

[0040] Example 3: Effect of active components on healing of full-thickness skin defects in mice 1. Animal model establishment: 100 SPF-grade Balb / c female mice weighing 28-32 g at 8 weeks of age were selected. After 7 days of acclimatization, they were anesthetized with 10% chloral hydrate (4 mL / kg) in the abdominal cavity, the skin on the back was disinfected with 75% alcohol, the hair was shaved and the residual hair was removed, and a full-thickness skin defect was created on the back with an 8 mm diameter sterile circular punch.

[0041] 2. Grouping and Administration: Mice were randomly divided into 5 groups of 20 mice each: blank control group (NT), positive control group (PC, mupirocin ointment applied topically), HPF group, MPF group, and LPF group. The administration was 0.3 g once daily for 21 consecutive days.

[0042] 3. Wound healing index testing: (1) Scab shedding time: The time it takes for the scab to completely fall off the mouse wound is observed and recorded daily.

[0043] like Figure 2 A and Figure 2 As shown in Figure C, the results indicate that the average scab shedding time was 17 days in the NT group, 13.3 days in the PC group, 14 days in the LPF group, 13.3 days in the MPF group, and 11.7 days in the HPF group. Therefore, the scab shedding time in the HPF group was significantly shorter than that in the other groups.

[0044] (2) Wound healing rate: The wound was photographed every 2 days, and the wound area was calculated using ImageJ software to calculate the healing rate.

[0045] like Figure 2 A and Figure 2 As shown in Figure B, the results indicated that on day 7 of administration, the wound healing rate in the HPF group was 50.0%, which was superior to that in the PC group (48.4%), MPF group (39.7%), LPF group (46.1%), and NT group (35.4%). On day 15 of administration, the wound healing rate in the HPF group was 97.9%, compared to 94.5% in the PC group, 96.5% in the MPF group, 92.6% in the LPF group, and 92.4% in the NT group. On day 21 of administration, the wound healing rate in the HPF group reached 100%, with smooth wound surfaces, complete hair regrowth, and no scar formation, while all other groups showed varying degrees of scarring.

[0046] (3) Measurement of inflammatory factor levels: On days 3, 5, 7, 14 and 21 after administration, three mice in each group were sacrificed and wound tissues were collected. The levels of IL-1β, TNF-α and TGF-β1 were detected by ELISA kit.

[0047] like Figure 3 A, Figure 3 B and Figure 3 As shown in Figure C, the results showed that the levels of IL-1β and TNF-α in the HPF group were significantly lower than those in other groups at each time point, while the level of TGF-β1 was significantly higher in the HPF group during the inflammatory period (3-7 days), indicating that HPF can effectively inhibit the expression of pro-inflammatory factors and promote the secretion of growth factors.

[0048] (4) Histopathological analysis: On days 7, 14 and 21 after administration, three mice in each group were sacrificed and wound tissues were taken for HE staining and Masson staining.

[0049] like Figure 4 As shown in Figure A, HE staining results indicated that the HPF group completed epidermal re-epithelialization by day 7, exhibited significant angiogenesis and sebaceous gland and hair follicle regeneration by day 14, and restored normal skin structure by day 21; Figure 4 As shown in Figure B, Masson staining results indicate that collagen deposition in the HPF group was orderly, forming a network structure, and scar formation was minimal, while collagen arrangement in other groups was disordered, with varying degrees of scar tissue.

[0050] (5) HYP analysis: Three mice were randomly selected from each group on days 7, 14 and 21 after modeling and drug administration. After sacrifice, the wound and surrounding skin were taken to determine the hydroxyproline content, and the hydroxyproline content in normal skin (NS) was used as a control.

[0051] like Figure 4 As shown in Figure C, the results indicated that the hydroxyproline content in the wound tissue of each group increased with time. On days 7 and 14 after modeling and drug administration, there were no significant differences between the PC group and the HPF group. On day 21 after modeling, the HPF group had the highest HYP content in the wound, with no significant difference between the PC group and the HPF group. The MPF and LPF groups showed no significant difference compared to the NT group, indicating that the PC and HPF groups can promote collagen deposition in the wound.

[0052] (6) Molecular mechanism detection: qRT-PCR and Western blot were used to detect the mRNA and protein expression levels of TGF-β1, Smad3 and COL1A1 in wound tissue.

[0053] like Figure 5 A- Figure 5 E and Figure 4 As shown in Figure D, the results showed that the HPF group significantly upregulated the mRNA and protein expression of TGF-β1 and COL1A1 on days 3 and 7, while significantly downregulated the mRNA and protein expression of Smad3, indicating that HPF promotes collagen remodeling and wound healing by regulating the TGF-β1 / Smad3 signaling pathway.

[0054] Example 4: Preparation and application of wound healing ointment 1. Raw material formula The formula uses the high polarity component (HPF) of Lanzhou fat sheep tail fat as the core active ingredient, combined with natural excipients and functional ingredients, as follows (total 100 g): Key active ingredient: HPF 8 g; Oil matrix: Lanzhou fat tail fat (conventional extraction, reference: Zhang Yuxuan, Zhou Tianyu, Chen Ying, et al. Optimization of process for extraction of Lanzhou fat tail fat by ultrasound-assisted enzymatic hydrolysis [J]. China Oils and Fats, 2025, 50(10): 1-7+30.) 5g, jojoba oil 6.165g; Moisturizing ingredients: 1.638g glycerin, 2g propylene glycol; Emulsion stabilizers: Tween 80 2 g, glyceryl monostearate 2 g, xanthan gum 1 g, carbomer 940 0.3 g; Functional excipient: Vitamin E 150 mg (antioxidant, protects active ingredients); Soothing ingredients: 0.15 mL of anti-inflammatory essential oil (such as lavender essential oil, to reduce skin irritation); Deionized water: Add to 100 g.

[0055] 2. Preparation process Raw material pretreatment: Lanzhou fat sheep tail was used as raw material, and Lanzhou fat sheep tail fat was obtained by ultrasonic-assisted enzymatic hydrolysis. HPF was separated and purified by silica gel column chromatography (300~400 mesh silica gel column, n-hexane-petroleum ether gradient elution) (the specific separation method of HPF is the same as in Example 1), and frozen for later use.

[0056] Oil phase preparation: Weigh 6 g of HPF, Lanzhou big-tailed sheep tail fat, and jojoba oil, place them in a beaker, stir and heat to 80℃, keep warm for 20 min until completely melted and mixed.

[0057] Aqueous phase preparation: In a separate beaker, add deionized water, glycerol, and propylene glycol, heat in a water bath to 80°C, and stir until homogeneous and transparent.

[0058] Emulsification and homogenization: Slowly pour the oil phase into the aqueous phase while stirring. Add Tween 80, glyceryl monostearate, xanthan gum, and carbomer 940. Maintain an environment of 65°C and homogenize and emulsify at 1000 r / min for 20 min.

[0059] Post-processing: Cool to below 40℃, add vitamin E and the remaining 2 g HPF, stir continuously until the system is uniform and smooth, cool to 20±5℃, filter, and fill.

[0060] 3. Applicable Scenarios It is suitable for acute full-thickness skin defects, including traumatic wounds, surgical incisions, and skin abrasions, and is especially suitable for skin repair scenarios where scar reduction is required.

[0061] 4. How to use Wound pretreatment: Clean the wound surface with saline solution to remove dirt and necrotic tissue, and dry the skin around the wound.

[0062] Application method: Apply an appropriate amount of ointment evenly to the wound surface and the surrounding 0.5 cm of skin, with a thickness of about 1 mm, to ensure complete coverage of the wound.

[0063] Frequency of use: Once a day, continue until the wound scabs fall off and epithelialization is complete; during the scab formation period, the frequency can be adjusted to once every 12 hours depending on the moisture level of the wound.

[0064] Precautions: Avoid rubbing vigorously when applying. If the wound becomes infected (redness, swelling, pus, etc.), anti-infection treatment should be performed before use. Avoid contact between the wound and water or contaminants during use. If contact is necessary, cover with sterile gauze.

[0065] 5. Application Advantages Significant healing effect: HPF can downregulate the levels of pro-inflammatory factors such as IL-1β and TNF-α, upregulate the expression of TGF-β1, promote the orderly deposition of collagen and the regeneration of skin appendages (hair follicles and sebaceous glands) through the TGF-β1 / Smad3 signaling pathway, shorten the scab shedding time by more than 30%, and improve the wound healing rate by 25% compared with conventional care.

[0066] High safety: The raw materials are all from natural sources and have passed skin sensitivity tests without causing allergic reactions such as erythema or edema. The pH value is stable at around 5.4, which matches the acid-base environment of human skin. The hygiene indicators such as heavy metals and microorganisms meet the relevant standards for cosmetics and topical preparations.

[0067] High stability: The product has passed heat resistance (70℃) and cold resistance (-20℃) tests without delamination, discoloration, or odor change. It has good light stability, and the aluminum sealed packaging can extend the shelf life to 18 months, effectively protecting the bioactivity of HPF.

[0068] It nourishes and repairs simultaneously: The HPF in the formula works synergistically with natural oils to promote wound healing while providing long-lasting hydration to the skin, improving dryness and roughness around the wound, and achieving both repair and maintenance effects.

[0069] 6. Taboos and Precautions Contraindications: This product is contraindicated for individuals with allergies to lanolin, jojoba oil, or any of the ingredients in the formula.

[0070] Special circumstances: Infected wounds, diabetic foot ulcers, and other chronic wounds should be used under the guidance of a doctor; pregnant women and infants should undergo a skin sensitivity test before use.

[0071] Storage conditions: Store in a cool, dry place (5~25℃), away from direct sunlight and high temperatures. It is recommended to use within 3 months after opening.

[0072] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for isolating the active components of lanolin characterized by, The method comprises the following steps: The tail fat of Lanzhou big-tailed sheep is mixed with neutral protease, the pH is adjusted to 6.5-7.5, ultrasonic treatment is performed, water is added to constant volume, after ultrasonic assisted treatment, water bath enzymolysis is performed, after the enzymolysis is completed, inactivation is performed, and the upper oil phase is collected by centrifugation; The upper oil phase is mixed with n-hexane, and is loaded onto a 300-400 mesh silica gel column, and gradient elution is performed by using a n-hexane-petroleum ether mixture; The eluate is detected by thin layer chromatography, components with similar characteristics are combined, and evaporation and concentration are performed to obtain high-polarity components, medium-polarity components and low-polarity components.

2. The separation method of claim 1, wherein, The mass ratio of the tail fat to the neutral protease is 95-105:1, and water is added to make the total volume of the system reach 40-50 mL.

3. The separation method of claim 1, wherein, The ultrasonic assisted treatment conditions include a temperature of 40-50 DEG C, a power of 150-250 W, and a time of 40-50 min; and the water bath enzymolysis is performed at a temperature of 40-50 DEG C for 70-80 min.

4. The separation method of claim 1, wherein, The volume ratio of the upper oil phase to n-hexane is 1:0.8-1.2, and the elution volume ratio of n-hexane to petroleum ether in the n-hexane-petroleum ether mixture is 200:1 to 7:

3.

5. The active ingredient of lanolin sheep tail fat obtained by the separation method according to any one of claims 1 to 4, characterized in that, The active component of the sheep tail fat is the high-polarity component, the content of unsaturated fatty acids in the active component of the sheep tail fat is not less than 73%, and the active component of the sheep tail fat contains linolenic acid, arachidonic acid and docosahexaenoic acid.

6. The application of the active component of the sheep tail fat in claim 5 in the preparation of an antioxidant product.

7. The application of the active component of the sheep tail fat in claim 5 in the preparation of a wound healing agent.

8. The application in claim 7, characterized in that, The use of the wound healing agent is at least one of the following (1)-(4): (1) promoting the healing of full-thickness skin defect wounds; (2) shortening the scab shedding time; (3) improving the wound healing rate; (4) inhibiting scar formation; The wound healing agent realizes the use by at least one of the following ways: a. down-regulating the expression of IL-1β; b. down-regulating the expression of TNF-α; c. up-regulating the transcription and protein expression of TGF-β1; d. down-regulating the transcription and protein expression of Smad3; e. regulating the TGF-β1 / Smad3 signaling pathway.

9. A wound healing ointment characterized in that, The wound healing ointment contains the active component of the sheep tail fat in claim 5, and further contains a fat base, glycerol, propylene glycol, an emulsifying stabilizer, vitamin E and essential oil.

10. The method of making the wound healing salve of claim 9, characterized in that, The method comprises the following steps: The active component of the sheep tail fat and the fat base are mixed and heated to obtain an oil phase; Deionized water, glycerol and propylene glycol are mixed and heated to obtain an aqueous phase; The oil phase is slowly poured into the aqueous phase while stirring, and an emulsifying stabilizer is added, and the system is homogenized and emulsified by heating and stirring; After the homogenization and emulsification are completed, the system is cooled, vitamin E and the active component of the sheep tail fat are added, and the system is continuously stirred until it is uniform and delicate to obtain a wound healing ointment.

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