Cubilose enzymolysis product and application thereof

Abstract

The invention relates to the technical field of biological products, in particular to a cubilose enzymolysis product and application thereof.By means of the cubilose enzymolysis product, a good environment can be provided for growth and periodic development of hair follicle cells by reducing the oxidative stress pressure and inflammatory response of the skin hair follicle microenvironment and promoting blood flow microcirculation, and therefore the hair follicle microenvironment can be effectively protected. Therefore, the hair follicle cycle is improved and maintained, and hair growth is promoted.

Description

Technical Field

[0001] This invention belongs to the field of bioproduct technology, and specifically relates to an enzymatic hydrolysis product of bird's nest and its application. Background Technology

[0002] Androgenic alopecia (AGA), also known as seborrheic alopecia, is a hair loss disorder that primarily occurs during and after puberty. Research has found that its main cause is an increase in dihydrotestosterone (DHT), a precursor to testosterone, leading to a hormonal imbalance in the hair follicles and consequently damaging them. Pathologically, AGA is characterized by a shortened anagen phase, smaller hair follicles, and inhibited hair growth (Li Linlin. Establishment and Preliminary Study of Androgenic Alopecia Mouse Model. Master's Thesis, Southern Medical University, 2021).

[0003] While AGA (Alopecia Areata Gay) does not cause obvious pain or physical inconvenience, it significantly impacts appearance and negatively affects daily life and social interactions, causing considerable psychological stress and burden for sufferers. Therefore, researching effective prevention and treatment methods for hair loss is of great importance.

[0004] Currently, the main treatments for AGA include medication, hair transplantation, and low-energy laser therapy. For medication, commonly used drugs include finasteride and minoxidil. Finasteride is a specific inhibitor of androgen 5α-reductase, which can effectively lower dihydrotestosterone levels, but its side effects are also significant, such as erectile dysfunction, decreased libido, and gynecomastia. Minoxidil is a potassium channel opener approved by the FDA for clinical treatment of hair loss. It can dilate local blood vessels in hair follicles and promote hair growth, but it requires long-term use, and symptoms are prone to relapse after discontinuation, and it can also cause contact dermatitis. Hair transplantation involves separating hair follicles from the occipital scalp and transplanting them to the affected area. It is also one of the important methods for treating hair loss, but medication is needed to stabilize hair loss before surgery, and medication must be continued after surgery for maintenance. Hair transplantation surgery requires a high level of skill. There are also some hair care products on the market that are believed to promote hair growth and health, including various shampoos, conditioners and head massage agents, which may alleviate hair loss to some extent. However, the effect varies greatly depending on the individual. The active ingredient carriers, surfactants or preservatives contained in them may also have a certain degree of irritation and may cause discomfort to the user (Gu Zhijing et al. A massage cream composition for strengthening hair roots CN105106042 A).

[0005] Due to the limited availability of drugs for AGA and the fact that existing drugs also face more or less toxic side effects, there is an urgent need to explore its new pathological mechanisms and actively seek to develop new preventive and therapeutic drugs to provide more new treatment options for patients with hormone-induced alopecia.

[0006] In my country, bird's nest has long been considered a traditional tonic, rich in nutrients. Classical Chinese medical texts, such as the *Compendium of Materia Medica*, record its effects as nourishing yin and moistening the lungs, replenishing qi and strengthening the spleen and stomach, and relieving cough and phlegm. Modern research shows that bird's nest can inhibit influenza virus activity and promote cell growth. Its components, such as epidermal growth factor and glycoproteins, can promote the regeneration of human tissues and cells, improve cellular immune function, enhance the body's resistance to viruses, and regulate blood pressure and metabolic rate, among other benefits.

[0007] Currently, bird's nest is mainly consumed orally after stewing. However, due to the poor solubility of bird's nest protein, only about 5% of the protein is extracted through stewing, and a significant portion of the protein structure remains intact after stewing. Furthermore, as people age, the amount of proteases secreted by the body gradually decreases, leading to difficulties in digestion and low bioavailability of bird's nest protein, thus limiting its nutritional benefits.

[0008] Therefore, developing a new application for sialic acid is of great significance. Summary of the Invention

[0009] In view of the above, it is necessary to provide an enzymatic hydrolysate of bird's nest and its application. The enzymatic hydrolysate of bird's nest can improve and maintain the hair follicle cycle and promote hair growth.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] A method for preparing an enzymatic hydrolysate of bird's nest includes the following steps:

[0012] (1) Extraction of sialic acid from bird's nest: bird's nest is crushed to obtain bird's nest powder. Water is added to the bird's nest powder to make it swell and then a compound enzyme is added. Enzymatic hydrolysis is carried out under ultrasonic conditions. After the enzyme is inactivated, the supernatant is collected by centrifugation. The supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3kDa. Then it is purified and freeze-dried to obtain the sialic acid from bird's nest.

[0013] (2) Preparation of bird's nest protein oligopeptides: The enzymatic hydrolysis product retained by ultrafiltration in step (1) is subjected to combined enzymatic hydrolysis by trypsin, pepsin and flavor protease in a reaction vessel to obtain bird's nest protein oligopeptides.

[0014] (3) Preparation of enzymatic hydrolysate of bird’s nest: The enzymatic hydrolysate of bird’s nest is obtained by mixing sialic acid and oligopeptides obtained by enzymatic hydrolysis of bird’s nest.

[0015] In this invention, further, in step (1), the ratio of bird's nest powder to added water is 1g:20mL.

[0016] In this invention, the complex enzyme in step (1) is papain and neuraminidase in a mass ratio of 9-10:1.

[0017] In this invention, the ultrasonic conditions in step (1) are: ultrasonic power 400W, time 30 minutes; the enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature 50-60℃, enzymatic hydrolysis time 1-2 hours, and enzyme-to-material ratio 1:100.

[0018] In this invention, the enzymatic hydrolysis conditions of step (2) are as follows: pH value 7.5, trypsin 10000U / g, pepsin 10000U / g, flavor protease 5000U / g, enzymatic hydrolysis temperature 55-65℃, and enzymatic hydrolysis time 8-12 hours.

[0019] The present invention also provides the application of the above-mentioned enzymatic hydrolysis products of bird's nest, specifically in the preparation of a hair loss prevention preparation.

[0020] The present invention also provides a method for using enzymatic hydrolysate of bird's nest, the method comprising using enzymatic hydrolysate of bird's nest in the preparation of an anti-seborrheic alopecia preparation.

[0021] When using, apply the anti-seborrheic alopecia preparation to the affected area, or combine topical application with oral administration for prevention and treatment.

[0022] The present invention has the following beneficial effects:

[0023] This invention proposes an enzymatic hydrolysis product of bird's nest. First, the enzymatic hydrolysis product of bird's nest is separated and extracted from bird's nest by ultrasonic enzymatic hydrolysis. Ultrasonic assistance is used to promote the dissociation of sialic acid from bird's nest under the action of compound enzymatic hydrolysis, thereby significantly improving the yield of enzymatic hydrolysis product of bird's nest. Then, bird's nest oligopeptides are prepared by pepsin, trypsin and flavor protease. Then, sialic acid and enzymatic hydrolysis oligopeptides of bird's nest are mixed to obtain a mixture of bird's nest oligopeptides and bird's nest sialic acid, that is, the enzymatic hydrolysis product of bird's nest.

[0024] Next, mice were administered enzymatic hydrolysates of bird's nest via gavage and / or topical application. The hair growth effect and potential mechanism of action of the enzymatic hydrolysates were evaluated by detecting the effects of the enzymatic hydrolysates on hair regeneration, as well as the expression levels of oxidative stress, inflammatory response, and angiogenesis-related factors in mouse skin tissue. The results showed that androgen treatment in mice reduced the number of hair follicles and inhibited hair growth. Treatment with enzymatic hydrolysates of bird's nest improved this hair growth inhibition to some extent. Biochemical analysis revealed a decrease in the level of the oxidative stress marker MDA and an increase in the level of the antioxidant enzyme GSH-Px in the mouse skin, indicating that enzymatic hydrolysates of bird's nest can improve oxidative stress in the skin of androgenic alopecia mice. Meanwhile, the enzymatic hydrolysate of bird's nest also reduced the expression levels of some inflammatory factors in skin tissue, such as IL-1β, IL-6, and TNFα, indicating that the enzymatic hydrolysate of bird's nest has an anti-inflammatory effect. In addition, the enzymatic hydrolysate of bird's nest effectively increased the expression of VEGF, a key factor in vascular development, and inhibited the expression of TGFβ, suggesting that the enzymatic hydrolysate of bird's nest may participate in improving microcirculation in the skin and enhancing hair follicle cell activity. Furthermore, the enzymatic hydrolysate of bird's nest also increased the expression of key members of the Wnt pathway, such as β-Catenin and Frizzled7, and improved the expression level of IGF-IR.

[0025] The comprehensive test results show that enzymatic hydrolysates of bird's nest can improve and maintain the hair follicle cycle and promote hair growth by reducing oxidative stress and inflammatory response in the skin's hair follicle microenvironment and promoting blood microcirculation. Attached Figure Description

[0026] Figure 1 The DPPH scavenging rate of enzymatically hydrolyzed oligopeptides from bird's nest obtained at different enzymatic hydrolysis times;

[0027] Figure 2 This shows the hair growth of mice during the experiment;

[0028] Figure 3 The length of newly grown hair in mice and the weight of newly grown hair per unit area;

[0029] Figure 4 The distribution and number of hair follicles in mouse skin tissue were statistically analyzed.

[0030] Figure 5 The expression of oxidative stress-related factors MDA and GSH-Px in mouse skin tissue;

[0031] Figure 6 The expression of immune-related factors IL-1β, IL-6 and TNFα in mouse skin tissue;

[0032] Figure 7The expression of growth factors Vegf and Tgfb1 at the mRNA level in mouse skin tissue;

[0033] Figure 8 The effect of enzymatic hydrolysates of bird's nest on the expression levels of Wnt pathway-related proteins in the skin;

[0034] Figure 9 The effect of enzymatic hydrolysates of bird's nest on the expression level of IGF1Rβ in the skin. Detailed Implementation

[0035] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0036] Example 1:

[0037] This embodiment provides an enzymatic hydrolysate of bird's nest, the preparation method of which includes the following steps:

[0038] (1) Extraction of sialic acid from bird's nest: Bird's nest was crushed to obtain bird's nest powder. Water was added to the bird's nest powder to dissolve it and set aside for use. The ratio of bird's nest powder to water was 1g:20mL. Then, a compound enzyme was added. The compound enzyme was papain and neuraminidase with a mass ratio of 9:1. Enzymatic hydrolysis was carried out under ultrasonic conditions. The ultrasonic conditions were: ultrasonic power 400W, time 30 minutes; the enzymatic hydrolysis conditions were: enzymatic hydrolysis temperature 50℃, enzymatic hydrolysis time 2 hours, enzyme-to-material ratio 1:100; after enzyme inactivation, the supernatant was collected by centrifugation. The supernatant was ultrafiltered at room temperature using an ultrafiltration membrane with a molecular weight cutoff of 3kDa and an ultrafiltration pressure of 40psi. Then, a second separation and purification was carried out by ion exchange column chromatography. D301 type anion exchange resin was used as the ion exchange resin. The loading concentration was 8.0mg / mL, the loading volume was 8.5BV, and the loading speed was 1mL / min. After separation and purification by ultrafiltration and dynamic column chromatography, the bird's nest sialic acid was obtained by freeze drying.

[0039] (2) Preparation of bird's nest protein oligopeptides: The enzymatic hydrolysis product retained by ultrafiltration in step (1) is subjected to combined enzymatic hydrolysis by trypsin, pepsin and flavor protease in a reaction vessel to obtain bird's nest protein oligopeptides; the enzymatic hydrolysis conditions are: pH value 7.5, trypsin 10000U / g, pepsin 10000U / g, flavor protease 5000U / g, enzymatic hydrolysis temperature 55℃, and enzymatic hydrolysis time 10 hours;

[0040] (3) Preparation of enzymatic hydrolysate of bird’s nest: The enzymatic hydrolysate of bird’s nest is obtained by mixing sialic acid and oligopeptides obtained by enzymatic hydrolysis of bird’s nest.

[0041] Example 2:

[0042] This embodiment provides an enzymatic hydrolysate of bird's nest, the preparation method of which includes the following steps:

[0043] (1) Extraction of sialic acid from bird's nest: Bird's nest was crushed to obtain bird's nest powder. Water was added to the bird's nest powder to dissolve it and set aside for use. The ratio of bird's nest powder to water was 1g:20mL. Then, a compound enzyme was added. The compound enzyme was papain and neuraminidase with a mass ratio of 10:1. Enzymatic hydrolysis was carried out under ultrasonic conditions. The ultrasonic conditions were: ultrasonic power 400W, time 30 minutes; the enzymatic hydrolysis conditions were: enzymatic hydrolysis temperature 55℃, enzymatic hydrolysis time 2 hours, enzyme-to-material ratio 1:100; after enzyme inactivation, the supernatant was collected by centrifugation. The supernatant was ultrafiltered at room temperature using an ultrafiltration membrane with a molecular weight cutoff of 3kDa and an ultrafiltration pressure of 40psi. Then, a second separation and purification was carried out by ion exchange column chromatography. D301 type anion exchange resin was used as the ion exchange resin. The loading concentration was 8.0mg / mL, the loading volume was 8.5BV, and the loading speed was 1mL / min. After separation and purification by ultrafiltration and dynamic column chromatography, the bird's nest sialic acid was obtained by freeze drying.

[0044] (2) Preparation of bird's nest protein oligopeptides: The enzymatic hydrolysis product retained by ultrafiltration in step (1) is subjected to combined enzymatic hydrolysis by trypsin, pepsin and flavor protease in a reaction vessel to obtain bird's nest protein oligopeptides; the enzymatic hydrolysis conditions are: pH value 7.5, trypsin 10000U / g, pepsin 10000U / g, flavor protease 5000U / g, enzymatic hydrolysis temperature 60℃, and enzymatic hydrolysis time 8 hours;

[0045] (3) Preparation of enzymatic hydrolysate of bird’s nest: The enzymatic hydrolysate of bird’s nest is obtained by mixing sialic acid and oligopeptides obtained by enzymatic hydrolysis of bird’s nest.

[0046] Experimental example:

[0047] To illustrate the effectiveness of this application, the applicant conducted the following experiment:

[0048] Experiment 1:

[0049] This experiment compared the extraction yield of sialic acid from bird's nest using different extraction methods, including group 1: water extraction, group 2: enzymatic hydrolysis, group 3: ultrasonic mixed single enzyme method, and group 4: ultrasonic mixed enzymatic hydrolysis. In the water extraction method, the compound enzyme was replaced with deionized water; in the enzymatic hydrolysis method, the ultrasonic treatment was omitted; in the ultrasonic mixed single enzyme method, papain hydrolysis was combined with ultrasonic treatment; and in the ultrasonic mixed enzymatic hydrolysis method, the enzymatic hydrolysis method described in Example 2 of this application was used. Except for the emphasized differences, the other treatment methods were the same. The extraction rate and purity under each treatment method were recorded, as shown in Table 1.

[0050] Table 1 Comparison of sialic acid yield and purity using different extraction methods

[0051] Extraction rate (%) purity(%) Group 1 7.28％ 85.3％ Group 2 8.30％ 90.4％ Group 3 9.12％ 88.5％ Group 4 10.21％ 92.8％

[0052] As shown in Table 1, the enzymatic hydrolysis method using ultrasonic mixing of papain and neuraminidase described in this application can improve the extraction yield of sialic acid, and its purity is as high as 92.8%.

[0053] Experiment 2:

[0054] This experiment tested the in vitro antioxidant activity of the bird's nest oligopeptides prepared by the method described in this application. DPPH was weighed and dissolved in 95% ethanol, then diluted to a 500mL volumetric flask to a concentration of 0.20mmol / L. Then, 2mL of the DPPH solution and 2mL of each concentration sample solution were taken and placed in 15mL test tubes. This value was designated as test tube A. i Take 2 mL of 95% ethanol solution and 2 mL of each concentration of sample solution into a 15 mL test tube, mix well, and this is control tube A. j Take 2 mL of DPPH solution and 2 mL of 95% ethanol solution in a 15 mL test tube, mix well, and record the value as blank tube A. c After preparing the solutions in each tube, incubate them at room temperature in the dark for 30 minutes to allow the reaction to proceed. Use 95% ethanol solution for the zeroing tube and measure the absorbance of each tube at a wavelength of 517 nm. Calculate the DPPH scavenging rate of different concentrations of enzymatic hydrolysis products using the following formula 3-1.

[0055]

[0056] Where: A0—the absorbance value of the reference tube at 517nm, A x —Absorbance value of the experimental tube at 517nm, A xo —Absorbance value of the blank tube at 517nm.

[0057] Test results are as follows Figure 1 As shown in the results of the activity analysis, the bird's nest oligopeptides have good antioxidant activity in vitro, and the DPPH scavenging rate can reach about 40% when the enzymatic hydrolysis time is 8 hours.

[0058] Experiment 3:

[0059] This experiment tested the effect of enzymatic hydrolysates of bird's nest on improving hair growth in mice with hair loss:

[0060] 1. Establishment of a mouse hair loss model: After one week of acclimatization, mice were injected intraperitoneally with the anesthetic sodium pentobarbital. The hair on the back was shaved short with a hair clipper (the shaved area was about 1.5cm × 2cm). An appropriate amount of hair removal cream (VeeTine) was applied. After 5 minutes, the hair was gently wiped off with a wet tissue to expose the skin on the back. Then, the residual hair removal cream was completely wiped off with a wet tissue.

[0061] 2. Grouping and processing of experimental animals:

[0062] After hair removal, the mice were randomly divided into the following groups:

[0063] (1) Blank control group: Apply 0.2ml of distilled water to the back and administer 0.2ml of distilled water by gavage every day.

[0064] (2) Model group: Apply 0.2ml of distilled water to the back and administer 0.2ml of distilled water by gavage every day.

[0065] (3) Positive control group: Apply 0.2 ml of 5% minoxidil to the back daily and administer 0.2 ml of distilled water by gavage.

[0066] (4) Back application group of enzymatic hydrolysate of bird's nest: Apply 0.2ml of enzymatic hydrolysate of bird's nest (150mg / kg body weight) to the back daily and administer 0.2ml of distilled water by gavage.

[0067] (5) Bird's nest enzymatic hydrolysis product gavage group: Apply 0.2ml of distilled water to the back of the patient daily and administer 0.2ml of bird's nest enzymatic hydrolysis product (150mg / kg body weight) by gavage.

[0068] (6) Back application of enzymatic hydrolysate of bird's nest + gavage group: 0.2ml of enzymatic hydrolysate of bird's nest (150mg / kg body weight) was applied to the back daily, and 0.2ml of enzymatic hydrolysate of bird's nest (150mg / kg body weight) was administered by gavage.

[0069] Except for the blank control group, all other groups of mice were given subcutaneous injections of testosterone propionate 0.5 mg / kg body weight, once every two days. Mice had free access to water and food during the remaining time. Mice were housed in an SPF environment with suitable temperature and humidity, accompanied by 12-hour / 12-hour light / dark cycles.

[0070] Hair growth in mice during the experiment was as follows: Figure 2As shown, the skin of mice is pink during the resting phase of hair follicles. After hair removal, the hair follicles are activated, then enter the growth phase and turn black, eventually becoming gray. In our experiment, we found that the skin of mice in all groups immediately after hair removal was distinctly pink. At 7 days, the skin of the normal control group turned black, indicating that the hair follicles had entered the growth phase. The skin of the positive control group mice was partially grayish-black, indicating that some hair follicles had also entered an activated state after treatment with the positive control drug minoxidil. However, no significant activation of hair follicles was observed in the mice treated with enzymatic hydrolysates of bird's nest, whether applied topically or administered orally.

[0071] After 14 days of drug administration, all mice in the normal control group grew new hair, and mice in the positive control group had partial areas of skin covered by new hair. Mice in the other groups treated with bird's nest enzymatic hydrolysate showed little or no hair growth, and the hair was short and sparse. The area of ​​activated hair follicles in the skin of mice treated with topical application (and topical application + gavage) was larger than that in the gavage group. After 21 days of drug administration, the previously bald areas in the normal control group were completely covered by hair. Compared to the model group, the positive drug group also had hair covering most of the bald areas. Although hair had grown in the model group and the gavage group, the growth was uneven, exhibiting a "patchy" alopecia area. The hair growth in the bird's nest enzymatic hydrolysate topical treatment group was relatively uniform, and the relative hair coverage area was significantly higher than that in the model group.

[0072] 3. Measurement of mouse hair length and weight:

[0073] To further evaluate the effects of enzymatic hydrolysates of bird's nest on hair growth in mice, we conducted a statistical analysis of the length of newly grown hair and the weight of hair per unit area in mice, as follows:

[0074] (1) Hair length measurement

[0075] On day 21 of the experiment, five hairs were taken from the front, middle and back regions of the back of each group of mice, and their lengths were measured and recorded using calipers.

[0076] (2) Hair weight measurement

[0077] On day 21 of the experiment, a 2cm × 2cm area was marked in the middle of the back of each group of mice. The hair in the area was carefully cut off, weighed on a balance, and recorded.

[0078] The results are as follows Figure 3 As shown in the figure, under the influence of androgens, hair growth in mice was significantly inhibited, with both hair length and quantity being significantly lower than those in the normal control group. Oral administration alone had no significant effect on improving hair growth, while combined treatment with topical application of enzymatic hydrolysate of bird's nest significantly promoted hair growth, resulting in increased hair length and weight per unit area.

[0079] 4. Mouse skin tissue sections:

[0080] Hair growth is related to the development of hair follicles. Therefore, we further examined the growth of hair follicles in the skin under the action of enzymatic hydrolysate of bird's nest. On day 21 of the experiment, the back of the mice was simply shaved, and the skin tissue was taken. After being washed with physiological saline, it was fixed in tissue fixative, then dehydrated by gradient alcohol and xylene, and then embedded in paraffin using a paraffin embedding machine.

[0081] After the embedded tissue is serially sectioned, it is placed on a detachable slide and dewaxed in stages. The main procedures are as follows:

[0082] Xylene I → Xylene II → 100% anhydrous ethanol → 90% anhydrous ethanol → 80% anhydrous ethanol → 70% anhydrous ethanol

[0083] →50% anhydrous ethanol → distilled water, 15 min for each reagent.

[0084] After staining with hematoxylin and eosin, the sections were dehydrated, mounted, and allowed to dry completely before being observed and photographed under a microscope. The number of hair follicles was observed and recorded at 100× field of view after staining. Compared with the model group, *p<0.05, ***p<0.001, N≥5.

[0085] like Figure 4 As shown, under normal circumstances, hair follicles are evenly distributed and neatly arranged. When androgens are excessive, the normal hair follicle cycle is disrupted, leading to follicle cell apoptosis and ultimately a reduction in the number of hair follicles. Gavage treatment with enzymatically hydrolyzed bird's nest did not significantly improve the reduction in hair follicle count caused by increased androgens. Compared to gavage, applying enzymatically hydrolyzed bird's nest to the back increased the number of hair follicles in mice, indicating that applying enzymatically hydrolyzed bird's nest to the back can improve hormone-induced hair loss.

[0086] 5. Mechanism of hair growth promotion by enzymatic hydrolysates of bird's nest:

[0087] Hair follicle development is a cyclical process influenced by multiple factors that collectively maintain the microenvironment for hair follicle development. The stability of the hair follicle microenvironment is crucial for normal hair follicle development. Oxidative stress, immune inflammation, and abnormalities in blood flow and nutrient supply can all affect the homeostasis of the hair follicle microenvironment. Therefore, we examined the expression levels of cellular and biochemical factors affecting hair follicle development, including oxidative stress, immune factors, and angiogenesis-related factors, in mouse skin tissue.

[0088] Figure 5 The expression of oxidative stress-related factors MDA and GSH-Px in mouse skin tissue was compared with that in the model group. *p<0.05, **p<0.01, N≥5;

[0089] Figure 6 The expression of immune-related factors IL-1β, IL-6 and TNFα in mouse skin tissue was compared with that in the model group. *p<0.05, **p<0.01, ***p<0.001, N≥5;

[0090] Depend on Figure 5 and 6 The results showed that, compared with normal control mice, the oxidative stress in the skin tissue of the model group mice under androgen treatment increased, manifested as an increase in the level of lipid peroxidation product MDA and a decrease in the level of antioxidant enzyme GSH-Px, and varying degrees of immune response. Applying enzymatic hydrolysate of bird's nest to the skin could alleviate oxidative stress and inflammatory response to some extent.

[0091] In addition, hair follicle development depends on normal blood circulation to supply nutrients, and normal microcirculation around the hair follicle is particularly crucial. VEGF is a key regulator in the process of vascular development. Figure 7 The expression levels of growth factors Vegf and Tgfb1 in mouse skin tissue at the mRNA level were determined by... Figure 7 As can be seen, the enzymatic hydrolysate of bird's nest can promote the expression of Vegf in the skin, suggesting that the enzymatic hydrolysate of bird's nest may improve the microcirculation of blood around hair follicles by promoting the development of vascular network in the scalp, thereby providing sufficient nutrients for hair follicle metabolism and improving the developmental microenvironment.

[0092] TGF-β is a transforming growth factor. Previous studies have found that TGF-β can induce apoptosis in hair follicle cells. Furthermore, treatment with enzymatic hydrolysate of bird's nest via gavage combined with topical application significantly inhibited the expression of TGF-β1. Figure 7 This suggests that enzymatic hydrolysates from bird's nest may help enhance hair follicle cell activity.

[0093] The classic Wnt / β-catenin pathway plays a crucial regulatory role in hair follicle formation and normal developmental cycles. Moderate stimulation of the Wnt pathway promotes hair growth. For example, studies have shown that astragaloside can significantly increase the expression of Wnt pathway members such as Wnt3a, Wnt5a, and Frizzled 7 in reconstructed hair follicles of mice, thus activating the Wnt pathway. In this study, Western blot analysis was used to detect the expression of Wnt pathway-related proteins. The results showed that enzymatic hydrolysates from bird's nest could increase β-catenin expression and inhibit the expression level of the Wnt pathway negative regulator GSK3β (e.g.,...). Figure 8 As shown in the figure, topical administration is more effective than gavage.

[0094] During cell growth and differentiation, some growth factors, such as IGF, FGF, EGF, and their receptors, participate in a series of complex signaling network regulation processes. These growth factors activate downstream signaling pathways by binding to their receptors, thereby regulating physiological processes such as cell proliferation and differentiation. Western blot analysis revealed that enzymatic hydrolysates from bird's nest can increase the expression level of insulin-like growth factor receptor I (IGF-I) in the skin. Figure 9 As shown in the figure, the enzymatic hydrolysate of bird's nest may enhance the IGF1R-mediated signaling cascade. Previous studies have shown that after IGF-IR binds to its ligands, its self-activation induces the activation of downstream Ras-MAPK and PI3K-Akt pathways, which are of great significance for normal cell growth. This suggests that the enzymatic hydrolysate of bird's nest may promote cell proliferation by enhancing the activity of the IGF-IR pathway.

[0095] The above embodiments are merely examples of several implementations of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention.

Claims

1. A bird's nest enzymatic hydrolysis product, characterized in that, The preparation method of the enzymatic hydrolysate of bird's nest includes the following steps: (1) Extraction of sialic acid from bird's nest: bird's nest is crushed to obtain bird's nest powder. Water is added to the bird's nest powder to make it swell and then a compound enzyme is added. Enzymatic hydrolysis is carried out under ultrasonic conditions. After the enzyme is inactivated, the supernatant is collected by centrifugation. The supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3kDa. Then it is purified and freeze-dried to obtain the sialic acid from bird's nest. (2) Preparation of bird's nest protein oligopeptides: The enzymatic hydrolysis product retained by ultrafiltration in step (1) is subjected to combined enzymatic hydrolysis by trypsin, pepsin and flavor protease in a reaction vessel to obtain bird's nest protein oligopeptides. (3) Preparation of enzymatic hydrolysate of bird’s nest: The enzymatic hydrolysate of bird’s nest is obtained by mixing sialic acid and oligopeptides obtained by enzymatic hydrolysis of bird’s nest.

2. The enzymatic hydrolysate of bird's nest according to claim 1, characterized in that, In step (1), the ratio of bird's nest powder to added water is 1g:20mL.

3. The enzymatic hydrolysate of bird's nest according to claim 1, characterized in that, The complex enzyme in step (1) is papain and neuraminidase in a mass ratio of 9-10:

1.

4. The enzymatic hydrolysate of bird's nest according to claim 1, characterized in that, The ultrasonic conditions in step (1) are: ultrasonic power 400W, time 30 minutes; the enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature 50-60℃, enzymatic hydrolysis time 1-2 hours, enzyme-to-material ratio 1:

100.

5. The enzymatic hydrolysate of bird's nest according to claim 1, characterized in that, The enzymatic hydrolysis conditions in step (2) are as follows: pH value 7.5, trypsin 10000U / g, pepsin 10000U / g, flavor protease 5000U / g, hydrolysis temperature 55-65℃, and hydrolysis time 8-12 hours.

6. The application of the enzymatic hydrolysate of bird's nest according to any one of claims 1-5, characterized in that, The enzymatic hydrolysate of bird's nest is used to prepare a preparation for preventing seborrheic alopecia.

7. The application according to claim 6, characterized in that, The application includes applying the prepared anti-seborrheic alopecia preparation to the affected area.

8. The application according to claim 6, characterized in that, The application includes applying the prepared anti-seborrheic alopecia preparation to the affected area and taking it orally.

9. A method for using the enzymatic hydrolysis product of bird's nest as described in any one of claims 1-5, characterized in that, The method includes preparing an anti-seborrheic alopecia preparation using enzymatic hydrolysates of bird's nest.

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