Application of umbilical cord extract in improving effects of preventing hair loss and increasing hair density and hair diameter of cacumen biotae

Abstract

The invention discloses an application of an umbilical cord extract in improving the effects of preventing hair loss and increasing hair density and hair diameter of cacumen biotae. The preparation method of the umbilical cord extract comprises the following steps: washing an animal umbilical cord, cutting the animal umbilical cord into pieces, digesting the animal umbilical cord with pancreatin, adding a serum-free mesenchymal stem cell culture medium, blowing and beating to disperse tissues, filtering, performing low-energy red light irradiation on the filtered tissue fluid, incubating for 10-12 hours, adding the serum-free mesenchymal stem cell culture medium with the same volume, continuously incubating for 48-72 hours, and performing freeze drying to obtain the umbilical cord extract. And adding an isometric serum-free mesenchymal stem cell culture medium, adding mir-135b at the same time, treating for 48-72 hours, taking out tissue fluid, performing freeze thawing, ultrasonic treatment and filtration, and diluting filtrate with sterile water to obtain the serum-free mesenchymal stem cell culture medium. The umbilical cord extract can remarkably improve the effects of preventing alopecia and increasing the hair density and the hair diameter of the cacumen biotae, and can be used for preparing cosmetics and medicines with the effects of preventing alopecia and increasing the hair density and the hair diameter after being mixed with the cacumen biotae extract.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the use of umbilical cord extract in improving the efficacy of Platycladus orientalis leaves in preventing hair loss and increasing hair density and hair diameter. Background Art

[0002] Hair is produced by hair follicles, and the development of human hair follicles is completed during the embryonic period. After birth, hair follicles generally cannot regenerate, but present a cyclical cycle, including the growth phase, regression phase and resting phase. During the growth phase, hair grows rapidly; during the regression phase: hair stops growing; during the resting phase: hair falls off easily. There are many causes of hair loss, of which nearly 90% are androgenic alopecia (AGA), also known as male pattern hair loss (MPHL) or female pattern hair loss (FPHL). Androgenic alopecia is the most common clinical non-scarring hair loss, which often occurs during adolescence and late adolescence, and is characterized by the progressive miniaturization of scalp hair follicles. The disease is mainly manifested in the receding hairline of the forehead and (or) the progressive reduction and thinning of the hair on the top of the head in men, while in women it is mainly manifested in the progressive reduction and thinning of the hair on the top of the head. At present, the prevention and treatment of hair loss is not only an important issue in the medical field, but also an important area of ​​concern in daily necessities, and various anti-hair loss products are emerging in an endless stream.

[0003] In recent years, there have been many studies on the anti-hair loss field of Platycladus orientalis leaves. According to the ancient book "Compendium of Materia Medica", Platycladus orientalis leaves have a significant effect on the treatment of hair loss. Platycladus orientalis leaves contain flavonoids, phenylpropanoids, terpenes and volatile oils. Modern research has confirmed that the main active ingredient of Platycladus orientalis leaves in the treatment of hair loss is flavonoid extracts. Studies have found that Platycladus orientalis leaf volatile oils and Platycladus orientalis leaf water extracts can induce the expression of β-catenin protein in mouse hair follicle cells, thereby promoting hair follicle regeneration and hair growth, while Platycladus orientalis leaf alcohol extracts, isoparinetol and rosin lactone in Platycladus orientalis leaves can enhance the vitality of hair follicles, maintain the normal growth of hair follicles, and thus promote hair growth. Other studies have found that flavonoid compounds in Platycladus orientalis leaves can treat oxidative damage in UV-induced androgenic alopecia. Some researchers have used Lactobacillus plantarum CCFM1348 to improve the biotransformation of Platycladus orientalis leaf extracts and thus improve the effect of Platycladus orientalis leaves on mouse hair growth. Therefore, how to improve the anti-hair loss effect of Platycladus orientalis leaves is a major direction of current research.

[0004] Animal umbilical cord is an animal-derived cosmetic raw material that is easy to obtain and has relatively low immunogenicity. The biologically active substances obtained from animal raw materials vary greatly due to the different extraction processes, extraction methods and treatment methods. The present invention provides a method for extracting animal umbilical cord. The animal umbilical cord extract extracted by the method is used in combination with a Platycladus orientalis leaf extract to significantly improve the anti-hair loss effect of Platycladus orientalis leaves and increase hair density and hair diameter. Summary of the invention

[0005] The first object of the present invention is to provide a use of an umbilical cord extract in improving the efficacy of Platycladus orientalis leaves in preventing hair loss and increasing hair density and hair diameter. The preparation steps of the umbilical cord extract are: washing and chopping a fresh animal umbilical cord, digesting it with trypsin, adding a serum-free mesenchymal stem cell culture medium, blowing and dispersing the tissue, and then filtering it, irradiating the filtered tissue fluid with low-energy red light and incubating it for 10 to 12 hours, adding an equal volume of serum-free mesenchymal stem cell culture medium, and continuing to incubate for 48 to 72 hours, adding an equal volume of serum-free mesenchymal stem cell culture medium, and adding mir-135b at the same time, treating it for 48 to 72 hours, taking out the tissue fluid, performing freeze-thaw treatment, then ultrasonically treating it, filtering it, and diluting the filtrate with sterile water to obtain the umbilical cord extract.

[0006] Preferably, the low-energy red light irradiation is irradiation with 620 nm low-energy red light for 10-15 min with an energy density of 0.5-1 J / cm 2 .

[0007] Preferably, the dosage of mir-135b is calculated based on a final concentration of 1 nmol / L.

[0008] Preferably, the incubation is carried out in a 37°C incubator.

[0009] Preferably, the freeze-thaw treatment is specifically as follows: quick freezing at -80°C for 1 h, and thawing in warm water at 37°C; quick freezing at -80°C for 1 h, and thawing in warm water at 37°C.

[0010] Preferably, the ultrasonic treatment is specifically: ultrasonic treatment at 4° C. for 4-10 min, with an operating frequency of 80 kHz.

[0011] Preferably, the first filtration is performed using a 100-300 μm filter, and the second filtration is performed using a 10-40 μm filter.

[0012] Preferably, the trypsin digestion treatment is specifically: using 0.25% trypsin at 4°C for 8-12 h.

[0013] Preferably, the dilution with sterile water is specifically diluted to a concentration of 10% by mass.

[0014] The second object of the present invention is to provide a composition comprising Platycladus orientalis leaf extract and umbilical cord extract, wherein the preparation steps of the umbilical cord extract are as described above.

[0015] Preferably, in the composition, the mass ratio of the Platycladus orientalis leaf extract to the umbilical cord extract is 1:3.

[0016] The third object of the present invention is to provide a use of the above composition in preparing a product having the effects of preventing hair loss and increasing hair density and hair diameter.

[0017] Preferably, the products include cosmetics and medicines.

[0018] The fourth object of the present invention is to provide a product having the effects of preventing hair loss and increasing hair density and hair diameter, which contains the above composition as an active ingredient.

[0019] The present invention has discovered a method for treating umbilical cords, and the extract obtained by extraction can significantly improve the efficacy of Platycladus orientalis leaves in preventing hair loss and increasing hair density and hair diameter. The umbilical cord extract is mixed with Platycladus orientalis leaf extract, and can be used as a new type of raw material for preparing cosmetics and medicines with the functions of preventing hair loss and increasing hair density and hair diameter, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Take partial hair pictures for the hair mirror. DETAILED DESCRIPTION

[0021] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0022] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0023] Example 1 Preparation of umbilical cord extract: In the clean bench, take fresh pig umbilical cord and wash it in PBS containing penicillin (100 U / mL) and streptomycin (0.1 mg / mL) for 3 times, 5 min each time; then use sharp scissors to cut the tissue into 1 mm 2 About 100 μm, wash twice with calcium-free and magnesium-free PBS, 5 min each time; then add an equal volume of 0.25% trypsin and shake at 4°C for 12 h. Add an equal volume of serum-free mesenchymal stem cell culture medium (serum-free culture medium produced by Ecosai Biotechnology (Taicang) Co., Ltd., with a catalog number of ME000-N023), blow and disperse the tissue for 2 h, and filter the tissue liquid with a 100 μm filter; the filtered tissue liquid is placed on an open plate and irradiated with 620 nm low-energy red light for 10 min, with an energy density of 0.5-1 J / cm 2Then, the tissue fluid was placed in a 37°C incubator with 5% CO2 for 12 h, an equal volume of serum-free mesenchymal stem cell culture medium was added, and the incubation continued for 72 h. An equal volume of serum-free mesenchymal stem cell culture medium was added, and mir-135b (final concentration 1 nmol / L) was added for 72 h. The tissue fluid was taken out, bottled, 10 mL / bottle, and placed at -80°C for 3 h until it became solid; thawed in warm water at 37°C; frozen at -80°C for 3 h until it became solid, and thawed in warm water at 37°C; then ultrasonically treated at 4°C, 500W ultrasonic cell disruptor, working frequency was 80 kHz, duration was 4 min; the obtained tissue fluid was filtered using a 40 μm filter; then the filtered tissue fluid was diluted with sterile water to a concentration of 10% by mass, and the obtained liquid was the umbilical cord extract, which was stored at -80°C.

[0024] Example 2 Umbilical cord extract enhances the efficacy of Platycladus orientalis in preventing hair loss, increasing hair density and hair diameter: Female hair loss patients with hair loss grade 2-4 were selected and randomly divided into 5 groups, 4 people in each group; The first group, 2 mL of 0.3% Thuja orientalis leaf extract solution (Shanghai Zhina Biotechnology Co., Ltd., FM133 Thuja orientalis leaf extract, prepared with sterile water, filtered and sterilized at 0.22 μm) was applied to the entire scalp 3 days a week for a total of 12 weeks; The second group, 2 mL of 0.9% animal umbilical cord extract 1 (prepared according to the preparation method of Example 1 of ZL202311368155.6) solution was applied to the entire scalp 3 days a week for a total of 12 weeks; The third group, smeared 2 mL of 0.9% animal umbilical cord extract 2 (prepared in Example 1 of the present invention) solution on the entire scalp 3 days a week for a total of 12 weeks; The fourth group, 2 mL of a solution containing 0.3% by mass of Platycladus orientalis leaf extract and 0.9% by mass of animal umbilical cord extract 1 was applied to the entire scalp 3 days a week for a total of 12 weeks; The fifth group, 2 mL of a solution containing 0.3% orientalis leaf extract and 0.9% animal umbilical cord extract 2 was applied to the entire scalp 3 days a week for a total of 12 weeks; Data Collection: On the first and last day, the subjects' hair was combed 60 times and the lost hair was counted. The results are shown in Table 1, where the change rate = ((the amount of hair loss in the first week - the amount of hair loss in the 12th week) / the amount of hair loss in the first week) × 100%, p < 0.05 * , p<0.01 ** , p<0.005 *** .

[0025] On the first and last day, the same position (with the marked point) was used to take photos using the hair mirror of the Hongxin Medical Skin Image Processing System. The results are shown in Figure 1 The hair density and average hair diameter per unit area were analyzed. The results are shown in Tables 2 and 3. The rate of change of hair density per unit area = ((hair density at week 12 - hair density at week 1) / hair density at week 1) × 100%, p < 0.05 * , p<0.01 ** , p<0.005 *** The change rate of average hair diameter = ((average hair diameter at week 12 - average hair diameter at week 1) / average hair diameter at week 1) × 100%, p < 0.01 ** , p<0.005 *** .

[0026] Table 1. Statistical analysis of hair loss counts after using different samples (roots)

[0027] Table 2. Statistical analysis of hair density counts after using different samples (roots / cm 2 )

[0028] Table 3. Statistical analysis of average hair diameter after using different samples (μm)

[0029] Depend on Figure 1 It can be seen from Table 1, Table 2 and Table 3 that the umbilical cord extract provided by the present invention can significantly enhance the effect of Platycladus orientalis leaves in preventing hair loss, increasing hair density and hair diameter, and its effect is better than the umbilical cord extract of ZL202311368155.6.

[0030] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. The use of umbilical cord extract in improving the efficacy of Platycladus orientalis leaves in preventing hair loss, increasing hair density and hair diameter, characterized in that: The preparation steps of the umbilical cord extract are as follows: washing and chopping the fresh animal umbilical cord, digesting it with trypsin, adding serum-free mesenchymal stem cell culture medium, blowing and dispersing the tissue, and then filtering it, irradiating the filtered tissue fluid with low-energy red light and incubating it for 10 to 12 hours, adding an equal volume of serum-free mesenchymal stem cell culture medium, continuing to incubate for 48 to 72 hours, adding an equal volume of serum-free mesenchymal stem cell culture medium, and adding mir-135b at the same time, treating it for 48 to 72 hours, taking out the tissue fluid, performing freeze-thaw treatment, and then ultrasonically treating it, filtering it, and diluting the filtrate with sterile water to obtain the umbilical cord extract.

2. The use according to claim 1, characterized in that The low-energy red light irradiation is to use 620 nm low-energy red light irradiation for 10-15 min, with an energy density of 0.5-1 J / cm 2 .

3. The use according to claim 1, characterized in that The final concentration of mir-135b is 1 nmol / L.

4. The use according to claim 1, characterized in that The incubation is carried out in a 37°C incubator.

5. The use according to claim 1, characterized in that: The freeze-thaw treatment is specifically as follows: quick freezing at -80°C for 1 hour, thawing in warm water at 37°C; quick freezing at -80°C for 1 hour, thawing in warm water at 37°C; the ultrasonic treatment is specifically as follows: ultrasonic treatment at 4°C for 4-10 minutes, with an operating frequency of 80 kHz.

6. A composition, characterized in that The invention comprises Platycladus orientalis leaf extract and umbilical cord extract, and the preparation steps of the umbilical cord extract are as described in claim 1.

7. The composition according to claim 6, characterized in that The mass ratio of the Platycladus orientalis leaf extract to the umbilical cord extract is 1:

3.

8. Use of the composition according to claim 6 or 7 in preparing a product having the effects of preventing hair loss and increasing hair density and hair diameter.

9. The use according to claim 8, characterized in that The products include cosmetics and medicines.

10. A product having the effects of preventing hair loss and increasing hair density and hair diameter, characterized in that: Contains the composition according to claim 6 or 7 as an active ingredient.

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