A method for preparing calf blood extract and application of the calf blood extract

Abstract

The application is applied to the field of skin care products and relates to a method for preparing calf blood extract and application of the calf blood extract. Specifically, the application provides a method for preparing calf blood extract. Meanwhile, the application provides calf blood extract and application thereof in preventing and repairing ultraviolet light damage of cells. The calf blood extract prepared by the method provided by the application transports oxygen to the face and skin of human body, the transported oxygen activates human fibroblasts, and the activity of the fibroblasts is improved, which is beneficial to the anti-aging of human skin at the cell level; the application utilizes the abnormally high efficiency of hemoglobin in combination with CO gas, fully combines calf blood extract with CO gas to form carbonylated calf blood extract, so as to continuously support the increase of human fibroblasts and simultaneously play the anti-photoaging effect of preventing light damage and repairing after damage on human fibroblasts.

Description

Technical Field

[0001] This invention relates to the field of skincare products, specifically a method for preparing calf blood extract and the application of the calf blood extract. Background Technology

[0002] Population aging refers to the dynamic process by which the proportion of elderly people in the total population increases due to a decrease in the number of young people and an increase in the number of older people, resulting from declining birth rates and increased life expectancy. The definition of population aging is relatively broad. Internationally, it is generally considered that a country or region is considered an aging society when the population aged 60 and above accounts for 10% of the total population, or the population aged 65 and above accounts for 7%. Data released by the National Bureau of Statistics in 2025 shows that in 2024, the population aged 60 and above was approximately 310 million, accounting for 22.0% of the national population, of which the population aged 65 and above was approximately 220 million, accounting for 15.6% of the national population. my country has entered a moderately aging society. With the accelerating global aging process, skin aging has become one of the key factors affecting the quality of life of middle-aged and elderly people. As the largest and outermost organ of the human body, the skin not only undertakes the physiological functions of protecting the body, maintaining water balance, and metabolic circulation, but also directly affects an individual's appearance and mental health. Under the combined effects of natural aging and the external environment, skin problems such as decreased elasticity, increased wrinkles, dryness, and sagging have become a core issue that urgently needs to be addressed in the skincare field.

[0003] Human skin is structured into the epidermis, dermis, and subcutaneous tissue. The epidermis, from the outside in, consists of the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. The epidermis acts as a barrier between the human body and the environment, preventing transepidermal water loss, participating in the formation of an acidic protective film, and preventing microbial invasion. The dermis, located below the epidermis, is the largest part of the skin and includes the superficial and deep (reticular) layers. Human fibroblasts and collagen fibers reside in this layer, playing a crucial role in providing tensile strength and mechanical support.

[0004] The mechanisms of skin aging are complex, involving multiple factors and levels of biological processes. The main mechanisms include: 1) Internal aging mechanisms: With age, the regenerative capacity of skin cells weakens, the number of fibroblasts in the dermis decreases, and their function declines, leading to insufficient synthesis of collagen, elastin, and matrix molecules, and a decrease in the structural support of the dermis. The basement membrane and hemidesmosomes gradually degenerate, weakening the connection between the epidermis and dermis, reducing the efficiency of substance exchange and signal transduction, and exacerbating wrinkle formation and skin sagging. 2) External environmental factors: Ultraviolet radiation is a major cause of exogenous aging. It not only directly damages collagen and elastin fibers but also induces the massive generation of free radicals, triggering oxidative stress damage. Environmental pollution, extreme temperatures, and other factors further exacerbate the decline in skin barrier function and moisture loss, resulting in multiple signs of aging such as age spots, roughness, and wrinkles.

[0005] Anti-aging skin products on the market exhibit different technological characteristics: First, they achieve synergistic effects on multiple targets through the combination of various ingredients; second, they utilize liposomes, nanocarriers, and microencapsulation technologies to improve the stability of active ingredients and achieve slow release, prolonging the duration of action and reducing irritation; third, they utilize bio-fermentation and synthetic biology to produce high-purity, high-activity ingredients (such as various peptides, ectoine, and ergothioneine), ensuring quality and sustainability, and potentially generating new, highly effective molecules.

[0006] In general, the current field of anti-aging cosmetics has evolved from passive defense (such as sun protection and moisturizing) and post-event remedies (such as anti-oxidation) to active intervention (such as promoting collagen regeneration) and root-cause regulation (such as autophagy and epigenetics).

[0007] As is well known, human skin fibroblasts are key cells in maintaining skin structural stability and youthful function, playing important roles in synthesizing collagen, elastic fibers, and matrix molecules, as well as participating in wound healing and immune regulation. Their activity and quantity directly affect skin elasticity, firmness, and repair capacity. Therefore, enhancing fibroblast activity and promoting collagen regeneration have become key directions in anti-aging research. Traditional skincare ingredients have limited efficiency in directly and continuously activating fibroblasts, making it difficult to achieve structural anti-aging at the cellular level.

[0008] According to reports, calf blood extract has effects such as promoting cell metabolism, repairing tissue damage, and improving microcirculation. However, current research and application of calf blood extract are still relatively limited. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a method for preparing calf blood extract and the application of the calf blood extract. The calf blood extract can significantly enhance fibroblast activity and promote collagen regeneration, thereby enhancing the skin's self-repair and regeneration capabilities at the cellular level. This addresses the growing demand for skin rejuvenation in the context of an aging population and meets consumers' expectations for highly effective, continuous, and multi-dimensional anti-aging products.

[0010] The present invention is achieved through the following technical solution: Specifically, in a first aspect, the present invention provides a method for preparing calf blood extract, which includes the following preparation steps: 1) Calf blood separation and extraction: The calf blood was centrifuged and ultrafiltered; the calf blood was lysed using 7.2% NaCl solution and purified water and then ultrafiltered; the supernatant after centrifugation was mixed with the product after the lysis and ultrafiltration steps to obtain the calf blood extract; 2) Inactivation: N-acetylcysteine ​​was added to the calf blood extract, deoxygenated, filtered, heated, and centrifuged to obtain the supernatant; 3) Carbonylation: CO is passed through the supernatant. The process is stopped when the carbonylated protein reaches a certain proportion, and carbonylated calf blood extract is obtained. 4) Freeze-drying: Carbonylated calf blood extract is repackaged and freeze-dried; The method has at least one of the following characteristics: During the separation and extraction of calf blood, the ratio of lysed calf blood, purified water, and 7.2% NaCl solution is 1:3.5-4.5:0.45-0.95. During the inactivation process, the heating temperature is 71-80 °C; During the inactivation process, the amount of N-acetylcysteine ​​added was 0.015-0.045 wt.%. During the carbonylation process, the proportion of carbonylated protein is 91-99%; During the freeze-drying process, the freeze-drying procedure is as follows: freeze-drying at -40°C for 2.5-5.5 h, freeze-drying at -35°C and pressure <5 Pa for 5-15 h, and freeze-drying at -25°C and pressure <5 Pa for 16-24 h.

[0011] In some implementations, during the separation and extraction of calf blood, the ratio of calf blood, purified water, and 7.2% NaCl solution is 1:3.7-4.3:0.5-0.7.

[0012] In some implementations, the heating temperature during the inactivation process is 74-76 °C.

[0013] In some embodiments, the amount of N-acetylcysteine ​​added during the inactivation process is 0.017-0.03 wt.%.

[0014] In some implementations, the carbonylation process involves a carbonylation protein ratio of 93-97%.

[0015] In some implementations, the freeze-drying process includes freeze-drying at -40 °C for 3-5 h, freeze-drying at -35 °C and pressure <5 Pa for 7-12 h, and freeze-drying at -25 °C and pressure <5 Pa for 17-22 h.

[0016] In some implementations, the method has at least one of the following features: 1) In the process of separating and extracting calf blood, the ratio of calf blood, purified water, and 7.2% NaCl solution is 1:4:0.5; 2) During the inactivation process, the heating temperature is 75 °C; 3) During the inactivation process, the amount of N-acetylcysteine ​​added was 0.02 wt.%; 4) During the carbonylation process, the proportion of carbonylated protein is 95%; 5) During the freeze-drying process, the freeze-drying procedure is as follows: freeze-drying at -40°C for 4 hours, freeze-drying at -35°C and pressure <5 Pa for 10 hours, and freeze-drying at -25°C and pressure <5 Pa for 20 hours.

[0017] In some implementations, the method has at least one of the following features: 1) During the separation and extraction of calf blood, the centrifugal force is 10000 g-14000 g, preferably 12000 g, the centrifugation time is 10-20 min, preferably 15 min, the centrifugation temperature is 2-6 ℃, preferably 4 ℃, the number of centrifugations is 1-3 times, preferably 2 times, and after centrifugation, it is preferred to perform ultrafiltration through a 0.65 μm filter membrane; 2) During the separation and extraction of calf blood, the preferred method for ultrafiltration during lysis is to use a 0.22 μm membrane pack and a 100 KD hollow fiber column; 3) During the inactivation process, the heating time is 5-20 h, preferably 10 h; 4) During the inactivation process, the centrifugal force is 10,000 g-20,000 g, preferably 15,500 g, and the centrifugation time is 10-30 min, preferably 20 min; 5) Pre-freezing may be optionally used during the freeze-drying process.

[0018] In a second aspect, the present invention provides a calf blood extract prepared according to the method described in the first aspect.

[0019] In a third aspect, the present invention provides the use of calf blood extract as described in the second aspect in the prevention and repair of ultraviolet light damage to cells.

[0020] The beneficial effects of this invention are: 1. The substances extracted from calf blood include both nutrients that promote growth and development, such as amino acids, and hemoglobin. Through hemoglobin, the calf blood extract prepared by the method provided in this invention transports oxygen to the human face and skin. The transported oxygen activates human fibroblasts, and the increased activity of fibroblasts is beneficial for the human skin to fight aging at the cellular level. 2. This invention utilizes the exceptionally high binding efficiency of hemoglobin with CO gas to fully combine calf blood extract with CO gas to form carbonylated calf blood extract, thereby continuously supporting the increase of human fibroblasts and providing anti-photoaging effects such as preventing photodamage and repairing damage to human fibroblasts. Attached Figure Description

[0021] Figure 1 Comparison of HFF-1 fibroblast survival rates in vitro, where control represents the control group; Model represents the model group; CO-Hb represents calf blood extract containing carbonylated hemoglobin; GF represents calf blood extract without carbonylated hemoglobin. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0023] Example 1 – Preparation of Calf Blood Extract

[0024] Step 1: Calf blood separation and extraction: First, centrifugation and ultrafiltration: 500 mL of collected bovine blood was centrifuged at 12000 g for 15 min at 4°C. The supernatant (feed solution) was collected, balanced, and then centrifuged a second time using the same parameters as the first centrifugation. The supernatant was collected and weighed, and stored at 4°C. The solution containing blood cells was diluted with 0.9% NaCl solution to a protein concentration of 10 g / dL. The resulting protein solution was then ultrafiltered using a 0.65 μm membrane with 0.9% NaCl solution, with an ultrafiltration factor of 5 times.

[0025] Second, lysis and ultrafiltration: Blood cells in the solution were lysed using a static mixer with 7.2% NaCl solution. The ratio of bovine blood, purified water, and 7.2% NaCl solution during lysis was 1:4:0.5. The lysate was then ultrafiltered through a 0.22 μm membrane and a 100 KD hollow fiber column. The permeate volume after ultrafiltration was 60% of the supernatant volume after two centrifugations, and the protein concentration was maintained at 2.9 g / dL. The hemoglobin yield, endotoxin, and immunoglobulin (IgG) content after lysis were measured. The hemoglobin yield after lysis was calculated based on the product of the hemoglobin concentration and volume before and after lysis. Method for calculating the yield of hemoglobin after lysis: Hemoglobin yield after lysis = 100% Third, mixing: The supernatant after the above two centrifugations is mixed with the product after the lysis and ultrafiltration step to obtain calf blood extract.

[0026] Step 2 Inactivation: 0.02 wt% N-acetylcysteine ​​was added to the calf blood extract, and deoxygenation was performed using a degassing membrane. After deoxygenation, the methemoglobin (FMetHb) content was 4%. The deoxygenated product was filtered through a 0.45 μm filter membrane into a sterile Schott flask, which was then heated in a 75°C water bath for 10 h. The sample was then centrifuged at 15500 g for 20 min, filtered, and the supernatant was retained for later use. The hemoglobin yield after inactivation was calculated.

[0027] Method for calculating hemoglobin yield after inactivation: Hemoglobin yield after inactivation = 100% Step 3 Carbonylation: The inactivated product is passed through CO gas. During the gas passage, the liquid is kept stirred at a speed ≥300 r / min (too high a speed will generate more bubbles, while too low a speed will make it difficult to eliminate bubbles). Approximately 1 mL is extracted during the gas passage and analyzed for carbonylated hemoglobin using a blood gas analyzer. When the proportion of CO-bound protein (carbonylated protein) (i.e., carbonylation rate) reaches 95%, the gas passage is stopped, yielding the carbonylated calf blood extract.

[0028] Step 4: Freeze-drying: Dispense the product from the carbonylation step into vials and pre-freeze the vials in a -80°C freezer for 18 hours. Then, place the pre-frozen product in a freezer for vacuum freeze-drying. The freeze-drying program is as follows: freeze-dry at -40°C for 4 hours, freeze-dry at -35°C and pressure <5Pa for 10 hours, and freeze-dry at -25°C and pressure <5Pa for 20 hours. The product is then obtained, and its water content is measured.

[0029] Comparative Example 1 – Changing the pyrolysis parameter 1

[0030] Calf blood separation and extraction: First, centrifugation and ultrafiltration: 500 mL of collected bovine blood was centrifuged at 12000 g for 15 min at 4°C. The supernatant was collected, balanced, and then centrifuged a second time using the same parameters as the first centrifugation. The supernatant was collected and weighed, and stored at 4°C. The solution containing blood cells after centrifugation was diluted with 0.9% NaCl solution to a protein concentration of 10 g / dL. The resulting protein solution was then ultrafiltered 5 times using a 0.65 μm membrane with 0.9% NaCl solution.

[0031] Second, lysis and ultrafiltration: Blood cells in the solution were lysed using a static mixer with 7.2% NaCl solution. The ratio of bovine blood, purified water, and 7.2% NaCl solution during lysis was 1:5:1. The lysate was then ultrafiltered through a 0.22 μm membrane and a 100 KD hollow fiber column. The permeate volume after ultrafiltration was 60% of the supernatant volume after two centrifugations, and the protein concentration was maintained at 2.9 g / dL. The hemoglobin yield, endotoxin, and immunoglobulin (IgG) content after lysis were measured.

[0032] Third, mixing: The supernatant after the above two centrifugations is mixed with the product after the pyrolysis and ultrafiltration step to obtain the final product.

[0033] Comparative Example 2 – Changing the pyrolysis parameters 2

[0034] Calf blood separation and extraction: First, centrifugation and ultrafiltration: 500 mL of collected bovine blood was centrifuged at 12000 g for 15 min at 4°C. The supernatant was collected, balanced, and then centrifuged a second time using the same parameters as the first centrifugation. The supernatant was collected and weighed, and stored at 4°C. The solution containing blood cells after centrifugation was diluted with 0.9% NaCl solution to a protein concentration of 10 g / dL. The resulting protein solution was then ultrafiltered 5 times using a 0.65 μm membrane with 0.9% NaCl solution.

[0035] Second, lysis and ultrafiltration: Blood cells in the solution were lysed using a static mixer with 7.2% NaCl solution. The ratio of bovine blood, purified water, and 7.2% NaCl solution during lysis was 1:3:0.4. The lysate was then ultrafiltered through a 0.22 μm membrane and a 100 KD hollow fiber column. The permeate volume after ultrafiltration was 60% of the supernatant volume after two centrifugations, and the protein concentration was maintained at 2.9 g / dL. The hemoglobin yield, endotoxin, and immunoglobulin (IgG) content after lysis were measured.

[0036] Third, mixing: The supernatant after the above two centrifugations is mixed with the product after the pyrolysis and ultrafiltration step to obtain the final product.

[0037] Verification Example 1 – Effect of Different Pyrolysis Parameters on Products

[0038] Bovine blood (intraerythrocyte fluid) has an osmotic pressure of approximately 280–300 mOsm / kg. Purified water has an osmotic pressure of 0. When added, it creates a hypotonic environment, driving water to rapidly enter the red blood cells, causing the cells to swell and rupture (hemolysis), releasing intracellular substances such as hemoglobin, enzymes, lipids, and nucleic acids.

[0039] Medium-ratio purified water (blood:water = 1:1 to 1:5) was used for deproteinized calf blood extract, while high-ratio purified water (blood:water = 1:5 to 1:20) was used for large-scale extraction and preparation of hemoglobin. The effects of different ratios of bovine blood, purified water, and 7.2% NaCl solution on hemoglobin yield and other impurities during lysis were compared. The lysis and ultrafiltration products were collected, and their hemoglobin content was detected using a blood gas analyzer. The hemoglobin yield after lysis was calculated based on the product of hemoglobin concentration and volume before and after lysis.

[0040] Leukocytes can also be lysed under hypotonic conditions. When the purified water content in the lysate is high, leukocytes are overly lysed, resulting in the production of endotoxins and immunoglobulins (IgG) that are harmful to the human body. The endotoxin content in the lysate can be detected by an endotoxin analyzer, and the content of the lysed immunoglobulin IgG can be detected by an enzyme-linked immunosorbent assay (ELISA) reader.

[0041] Table 1 below shows a comparison of bovine blood, purified water, and 7.2% sodium chloride solution at different proportions.

[0042] Table 1 Effect of different pyrolysis conditions on the products

[0043] Note: Other preparation conditions remain unchanged.

[0044] Depending on the application scenario, the control of endotoxins and IgG levels for hemoglobin varies. When applied to areas in contact with human skin, IgG should be controlled at ≤0.5%, and endotoxins at ≤5 EU / mL. Based on the above lysis comparison, different mixture ratios significantly affect the lysis products. Although the lysis conditions of Comparative Example 1 meet the control requirements for IgG and endotoxins, the hemoglobin yield in the product is far below the normal 90% yield level, thus lacking advantage. The IgG and endotoxin levels in the product of Comparative Example 2 significantly exceed the general control levels, suggesting that the ratio of bovine blood, purified water, and 7.2% sodium chloride solution is not up to standard. Therefore, considering all factors, a lysis parameter of 1:4:0.5 for bovine blood, purified water, and 7.2% NaCl solution was selected.

[0045] Verification Example 2 – Inactivation Effect at Different Heating Temperatures

[0046] The product prepared in step 1 of Example 1 was divided into three portions, according to YY / T 0771.3.

[0047] -2009 Animal-derived Medical Devices Part 3: Validation methods for the removal and inactivation of viruses and transmissible spongiform encephalopathy (TSE) factors were commissioned to a third party for validation. The indicator viruses included were poliovirus Sabin 1 (PV1, non-enveloped RNA virus), bovine viral diarrhea virus (BVDV, enveloped RNA virus), pseudorabies virus (PRV, enveloped DNA virus), and porcine parvovirus (PPV, non-enveloped DNA virus).

[0048] After inactivation of the virus, a heat inactivation step was performed. The heating temperatures were set at 60℃, 70℃, and 75℃, and the heating time was 10 h. The products heated at 60℃ and 70℃ were used as Comparative Examples 3 and 4. The experimental results are shown in Table 2 below.

[0049] Table 2 Comparison of virus inactivation effects at different heating temperatures

[0050] After virus inactivation, a higher virus titer reduction coefficient indicates a better inactivation effect. When the virus titer reduction coefficient is ≤4 logs, it indicates poor virus inactivation. Based on the results above, increasing the heating temperature from 60℃ to 75℃ significantly improves the virus inactivation effect. Heating at 60℃ and 70℃ for 10 hours did not achieve the minimum required PPV virus titer reduction coefficient, indicating poor inactivation effect for this type of virus. Furthermore, the PV1 virus titer reduction coefficient at this heating temperature was smaller than that at 75℃ for 10 hours. Therefore, heating at 75℃ for 10 hours was selected as the optimal parameter for virus inactivation.

[0051] Comparative Example 5 – Effect of Different NAC Addition Amounts on Product Yield 1

[0052] The preparation methods for steps 1, 3, and 4 are the same as in Example 1. Step 2 involves inactivation: 0.01 wt% N-acetylcysteine ​​is added to the calf blood extract, and deoxygenation is performed using a degassing membrane. The deoxygenated product is filtered through a 0.45 μm filter into a sterile Schott flask, which is then heated in a 70°C water bath for 10 h. The sample is then centrifuged at 15500 g for 20 min, filtered, and the supernatant is retained for later use. The yield is calculated.

[0053] Comparative Example 6 – Effect of Different NAC Addition Amounts on Product Yield 2

[0054] The preparation methods for steps 1, 3, and 4 are the same as in Example 1. Step 2 involves inactivation: 0.05 wt% N-acetylcysteine ​​is added to the calf blood extract, and deoxygenation is performed using a degassing membrane. The deoxygenated product is filtered through a 0.45 μm filter into a sterile Schott flask, which is then heated in a 70°C water bath for 10 h. The sample is then centrifuged at 15500 g for 20 min, filtered, and the supernatant is retained for later use. The yield is calculated.

[0055] Verification Example 3 – Effect of NAC Addition Amount on Product Yield

[0056] To verify the effect of NAC addition on the product yield after virus inactivation, the product yield under different NAC addition amounts was investigated. NAC addition was calculated as a percentage of the initial feed volume by mass. The results are shown in Table 3 below.

[0057] Table 3 Product yield at different NAC addition levels

[0058] The results show that the product yield generally increases with the increase of the NAC addition ratio. The product yield after heating at 75℃ for 10 hours without NAC addition is significantly lower than that with NAC addition. The product yield when the NAC addition is 0.02% is close to that when the NAC addition is 0.05%. Considering all factors, a NAC addition of 0.02% was selected.

[0059] Comparative Example 7 – Effect of Different Carbonylation Rates on the Product 1

[0060] The preparation methods for steps 1, 2, and 4 are the same as in Example 1. Step 3, carbonylation: The inactivated product is passed through CO gas. During the gas passage, the liquid is kept under stirring at a speed ≥300 r / min (too high a speed easily generates more bubbles, while too low a speed makes it difficult to eliminate bubbles). Approximately 1 mL is extracted during the gas passage and analyzed for carbonylated hemoglobin using a blood gas analyzer. When the proportion of CO-bound protein (carbonylated protein) reaches 70%, the gas passage is stopped, yielding the carbonylated calf blood extract.

[0061] Comparative Example 8 – Effect of Different Carbonylation Rates on the Product 2

[0062] The preparation methods for steps 1, 2, and 4 are the same as in Example 1. Step 3, carbonylation: The inactivated product is passed through CO gas, and the liquid is kept stirred during the gas introduction process at a speed ≥300 r / min (too high a speed will generate more bubbles, while too low a speed will make it difficult to eliminate bubbles). During the gas introduction process, approximately 1 mL is extracted and analyzed for carbonylated hemoglobin using a blood gas analyzer. When the proportion of CO-bound protein (carbonylated protein) reaches 90%, the gas introduction is stopped, yielding the carbonylated calf blood extract.

[0063] Verification Example 4 – Effect of Different Carbonylation Rates on the Product

[0064] The calf blood extract solutions (before freeze-drying) prepared in Examples 1, 7, and 8 were placed in air for 24 hours. 1 mL of each solution was then taken and the protein concentration was measured using a blood gas analyzer. The results are shown in the table below. Based on the results, higher carbonylation levels resulted in better stability after 24 hours, reflected in a lower proportion of methemoglobin. Lower methemoglobin levels are beneficial for the oxygen-carrying function of the calf blood extract.

[0065] Table 4. Comparison of stability of products with different carbonylation rates

[0066] Comparative Example 9 – Freeze-drying parameters

[0067] The preparation methods for steps 1-3 are the same as in Example 1. Step 4: freeze drying: The product from the carbonylation step is dispensed into vials, and the vials are pre-frozen in a -80°C freezer for 18 hours. The pre-frozen product is then placed in a freezer for vacuum freeze drying. The freeze drying program is as follows: freeze drying at -40°C for 2 hours, freeze drying at -35°C and pressure <5Pa for 10 hours, and freeze drying at -25°C and pressure <5Pa for 20 hours. The water content is then measured.

[0068] Comparative Example 10 – Freeze-drying parameters

[0069] The preparation methods for steps 1-3 are the same as in Example 1. Step 4: freeze drying: The product from the carbonylation step is dispensed into vials, and the vials are pre-frozen in a -80°C freezer for 18 hours. The pre-frozen product is then placed in a freezer for vacuum freeze drying. The freeze drying program is as follows: freeze drying at -40°C for 6 hours, freeze drying at -35°C and pressure <5Pa for 10 hours, and freeze drying at -25°C and pressure <5Pa for 20 hours. The water content is then measured.

[0070] Comparative Example 11 – Freeze-drying parameters

[0071] The preparation methods for steps 1-3 are the same as in Example 1. Step 4: freeze drying: The product from the carbonylation step is dispensed into vials, and the vials are pre-frozen in a -80°C freezer for 18 hours. The pre-frozen product is then placed in a freezer for vacuum freeze drying. The freeze drying program is as follows: freeze drying at -40°C for 4 hours, freeze drying at -35°C and pressure <5Pa for 10 hours, and freeze drying at -25°C and pressure <5Pa for 15 hours. The water content is then measured.

[0072] Comparative Example 12 – Freeze-drying parameters

[0073] The preparation methods for steps 1-3 are the same as in Example 1. Step 4: freeze drying: The product from the carbonylation step is dispensed into vials, and the vials are pre-frozen in a -80°C freezer for 18 hours. The pre-frozen product is then placed in a freezer for vacuum freeze drying. The freeze drying program is as follows: freeze drying at -40°C for 4 hours, freeze drying at -35°C and pressure <5Pa for 10 hours, and freeze drying at -25°C and pressure <5Pa for 10 hours. The water content is then measured.

[0074] Comparative Example 13 – Freeze-drying parameters

[0075] The preparation methods for steps 1-3 are the same as in Example 1. Step 4: freeze drying: The product from the carbonylation step is dispensed into vials, and the vials are pre-frozen in a -80°C freezer for 18 hours. The pre-frozen product is then placed in a freezer for vacuum freeze drying. The freeze drying program is as follows: freeze drying at -40°C for 4 hours, freeze drying at -35°C and pressure <5Pa for 10 hours, and freeze drying at -25°C and pressure <5Pa for 25 hours. The water content is then measured.

[0076] Verification Example 5 – Comparison of product moisture content under different freeze-drying conditions

[0077] The freeze-drying conditions have a significant impact on the water content of calf blood extract. The water content of the freeze-dried product was determined according to the Karl Fischer method as specified in the Chinese Pharmacopoeia (2025 edition), and the results are shown in Table 5 below.

[0078] Table 5. Effects of different freeze-drying parameters on the product

[0079] Note: The pre-freeze-drying conditions were the same, all involving placement in a -80℃ freezer for 18 hours.

[0080] The results showed that the water content of the calf blood extract freeze-dried powder prepared according to the freeze-drying procedure of Example 1 was significantly lower than that of the freeze-dried powder prepared under the freeze-drying conditions of Comparative Examples 11 and 12. When the freeze-drying conditions of the second and third stages remained unchanged, extending the freeze-drying time of the first stage to 6 hours did not change the water content of the product compared to the product with a freeze-drying time of 4 hours. Shortening the freeze-drying time in this stage significantly increased the water content. When the freeze-drying conditions of the first and second stages remained unchanged, extending the freeze-drying time of the third stage did not significantly change the water content compared to Example 1. Based on industry-standard criteria (water content controlled at ≤3%) and considering both freeze-drying time and cost, the freeze-drying procedure parameters of Example 1 were selected as the freeze-drying parameter combination.

[0081] Verification Example 6 – In Vitro Cell Experiment

[0082] 1. Research Methods

[0083] HFF-1 cells (a type of human fibroblast) were irradiated with UVB lamps to induce phototoxic reactions, simulating skin damage and aging. Subsequently, different formulations of calf blood extract were added to observe whether the different test products could exert anti-photoaging effects on HFF-1 cells, such as preventing photodamage and repairing damage.

[0084] 2. Grouping

[0085] The in vitro cell experiments were grouped as shown in Table 6 below.

[0086] Table 6 Grouping of In Vitro Cell Experiments

[0087] Note: The concentration of the extract was determined using a blood gas analyzer, model ABL90 FLEX.

[0088] 3. Experimental Procedure

[0089] 3.1 HFF-1 Cell Culture

[0090] HFF-1 cell lines were seeded in DMEM growth medium containing 15% fetal bovine serum (FBS) and supplemented with 1% of two antibiotics (penicillin and streptomycin) and cultured in a humid environment at 37°C and 5% CO2.

[0091] 3.2 Cell Pretreatment

[0092] HFF-1 cells were seeded in 96-well cell culture plates at a density of 0.5 x 10⁴ cells / well. Cells were cultured at 37°C in a 5% CO₂ incubator for 24 h. After 24 h of cell adhesion, the old culture medium was removed. Working solutions were prepared using complete medium containing 15% FBS to a final concentration of 1000 ppm for both carbonylated hemoglobin (CO-Hb) calf blood extract and carbonylated hemoglobin-free calf blood extract (GF). The experiment consisted of a control group, a model group, and test sample groups (CO-Hb and GF), with six replicates per group. 100 µL of complete medium containing 1000 ppm of the corresponding test sample was added to the test sample groups, while the control and model groups received an equal volume of complete medium without the test sample. All plates were pre-incubated at 37°C in a 5% CO₂ incubator for 24 h.

[0093] 3.3 Establishment of UVB photodamage model

[0094] After 24 h of pretreatment with the test sample, the culture medium in the wells was aspirated, and the cells were gently washed once with preheated DPBS (37°C). Then, 50 µL of preheated DPBS was added to each well to cover the cells and prevent drying. The cell culture plate was placed in a UV irradiation chamber (model: XEPU-1235L, XEPU (Shanghai) Scientific Instruments Co., Ltd.), with a vertical irradiation distance of 15 cm between the light source and the cells. The model group and the test sample group received UVB irradiation at a dose of 60 mJ / cm²; the control group cells were completely wrapped in aluminum foil under the same conditions to avoid light and were not irradiated. After the irradiation program, the DPBS in the wells was immediately aspirated. 100 µL of complete culture medium containing 1000 ppm of the corresponding test sample was added to the test sample group, while 100 µL of complete culture medium without the test sample was added to the control and model groups. The cell culture plate was returned to the incubator and incubated for another 24 h under the same conditions.

[0095] 3.4 CCK-8 cell viability assay

[0096] After UVB irradiation and incubation for 24 h, cell viability was assessed using the Cell Counting Kit-8 (CCK-8) method. The culture medium in each well was aspirated, and the cells were washed once with pre-warmed DPBS. A working solution was prepared at a 1:10 ratio (CCK-8 stock solution: complete culture medium), with 100 µL added to each well. The culture plate was incubated at 37°C in a 5% CO2 incubator for 2 h in the dark. After incubation, the optical density (OD) of each well was measured at 450 nm using a microplate reader to calculate the relative cell viability.

[0097] 3.5 Statistical Methods

[0098] Data are expressed as mean ± standard deviation (Mean ± SD). Differences between groups were assessed using one-way ANOVA with Tukey's multiple comparison test (post-hoc test) on GraphPad Prism software (Version 10.2.3, San Diego, CA, USA). A p-value < 0.05 was considered statistically significant.

[0099] 4. Experimental Results

[0100] Figure 1These are the statistical results of HFF-1 cell survival rate. The comparison of HFF-1 cell survival rates between the model group and the control group shows successful model establishment. Compared with the model group, the results show that the CO-Hb group significantly improved the HFF-1 cell survival rate, with a statistically significant difference. However, no improvement in HFF-1 cell survival rate was observed in the GF group.

[0101] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope defined in the claims.

Claims

1. A method for preparing calf blood extract, comprising the following preparation steps: 1) Calf blood separation and extraction: The calf blood was centrifuged and ultrafiltered; the calf blood was lysed using 7.2% NaCl solution and purified water and then ultrafiltered; the supernatant after centrifugation was mixed with the product after the lysis and ultrafiltration steps to obtain the calf blood extract; 2) Inactivation: N-acetylcysteine ​​was added to the calf blood extract, deoxygenated, filtered, heated, and centrifuged to obtain the supernatant; 3) Carbonylation: CO is passed through the supernatant. The process is stopped when the carbonylated protein reaches a certain proportion, and carbonylated calf blood extract is obtained. 4) Freeze-drying: Carbonylated calf blood extract is repackaged and freeze-dried; The method has at least one of the following characteristics: During the separation and extraction of calf blood, the ratio of lysed calf blood, purified water, and 7.2% NaCl solution is 1:3.5-4.5:0.45-0.

95. During the inactivation process, the heating temperature is 71-80 °C; During the inactivation process, the amount of N-acetylcysteine ​​added was 0.015-0.045 wt.%. During the carbonylation process, the proportion of carbonylated protein is 91-99%; During the freeze-drying process, the freeze-drying procedure is as follows: freeze-drying at -40°C for 2.5-5.5 h, freeze-drying at -35°C and pressure <5 Pa for 5-15 h, and freeze-drying at -25°C and pressure <5 Pa for 16-24 h.

2. The method for preparing calf blood extract as described in claim 1, wherein during the separation and extraction of calf blood, the ratio of calf blood, purified water, and 7.2% NaCl solution is 1:3.7-4.3:0.5-0.

7.

3. The method for preparing calf blood extract as described in claim 1, wherein the heating temperature during the inactivation process is 74-76 °C.

4. The method for preparing calf blood extract as described in claim 1, wherein the amount of N-acetylcysteine ​​added during the inactivation process is 0.017-0.03 wt.%.

5. The method for preparing calf blood extract as described in claim 1, wherein the carbonylation process involves carbonylated protein at a ratio of 93-97%.

6. The method for preparing calf blood extract as described in claim 1, wherein the freeze-drying process comprises freeze-drying at -40 °C for 3-5 h, freeze-drying at -35 °C and pressure <5 Pa for 7-12 h, and freeze-drying at -25 °C and pressure <5 Pa for 17-22 h.

7. The method for preparing calf blood extract according to any one of claims 1-6, wherein the method has at least one of the following characteristics: 1) In the process of separating and extracting calf blood, the ratio of calf blood, purified water, and 7.2% NaCl solution is 1:4:0.5; 2) During the inactivation process, the heating temperature is 75 °C; 3) During the inactivation process, the amount of N-acetylcysteine ​​added was 0.02 wt.%; 4) During the carbonylation process, the proportion of carbonylated protein is 95%; 5) During the freeze-drying process, the freeze-drying program is as follows: freeze-drying at -40°C for 4 hours, freeze-drying at -35°C and pressure <5 Pa for 10 hours, and freeze-drying at -25°C and pressure <5 Pa for 20 hours.

8. The method for preparing calf blood extract according to any one of claims 1-7, wherein the method has at least one of the following characteristics: 1) During the separation and extraction of calf blood, the centrifugal force is 10000 g-14000 g, preferably 12000 g, the centrifugation time is 10-20 min, preferably 15 min, the centrifugation temperature is 2-6 ℃, preferably 4 ℃, the number of centrifugations is 1-3 times, preferably 2 times, and after centrifugation, it is preferred to perform ultrafiltration through a 0.65 μm filter membrane; 2) During the separation and extraction of calf blood, the preferred method for ultrafiltration during lysis is to use a 0.22 μm membrane pack and a 100 KD hollow fiber column; 3) During the inactivation process, the heating time is 5-20 h, preferably 10 h; 4) During the inactivation process, the centrifugal force is 10,000 g-20,000 g, preferably 15,500 g, and the centrifugation time is 10-30 min, preferably 20 min; 5) Pre-freezing may be optionally used during the freeze-drying process.

9. A calf blood extract, prepared according to any one of claims 1-8.

10. The use of the calf blood extract as described in claim 8 in preventing and repairing ultraviolet light damage to cells.

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