CN110623860A - Giant salamander active peptide and hyaluronic acid composition capable of effectively promoting fibroblast proliferation - Google Patents
Giant salamander active peptide and hyaluronic acid composition capable of effectively promoting fibroblast proliferation Download PDFInfo
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Abstract
The invention discloses a giant salamander active peptide and hyaluronic acid composition capable of effectively promoting fibroblast proliferation, which is prepared by mixing a giant salamander active peptide solution with the mass volume concentration of 1 ~ 10% and the pH value of 5 ~ 7 with hyaluronic acid according to the mass ratio of 1 ~ 8: 1, wherein the molecular weight of the giant salamander active peptide is less than 2000Da, the mole percentages of lysine and arginine are respectively 7.2% and 9.7%, the molecular weight of the hyaluronic acid is 42.5 ~ 145kDa, the giant salamander active peptide can be naturally crosslinked with hyaluronic acid rich in a plurality of hydroxyl groups through hydrogen bonds, the natural affinity of the hyaluronic acid and human skin cells is utilized, the effect of promoting the proliferation of the skin fibroblasts can be effectively improved, and the composition has a wide application prospect in the fields of medicines and cosmetics.
Description
Technical Field
The invention belongs to the technical field of skin care products, and particularly relates to a giant salamander active peptide and hyaluronic acid composition capable of effectively promoting fibroblast proliferation.
Background
The bioactive peptide is mainly a product obtained by carrying out enzymolysis on protein by using enzyme, and the protein from the same source is subjected to enzymolysis by using different enzymes and enzymolysis methods, and the enzymolysis product also has different biological activities due to different structures. The peptide yield of 5h of giant salamander meat enzymolysis by 5 enzymes such as alkaline protease, neutral protease, Flavourzyme, Protamex and Alcalase is researched by Xiujing, and the peptide yield obtained by the Protamex enzymolysis of the giant salamander meat is found to be the highest and 13.75%, and the optimal conditions of the enzyme enzymolysis of the giant salamander meat are that the feed-liquid ratio is 1: 35. the temperature is 60 ℃ and the enzymolysis time is 7h (Bijing, Chengding, Caomina. study of the preparation process of the giant salamander polypeptide [ J ]. food science and technology, 2012, 37 (2): 66-68, 72). Liuxin and the like research on enzymolysis of giant salamander meat by alkaline protease and papain (1: 3 (g/g)), and the optimal conditions are found to be that the feed-liquid ratio is 1: 7. the temperature is 55 ℃, the pH value is 7.5 and the enzymolysis time is 5 hours, the peptide yield reaches 36.18 percent (Liuxin, Quchaoxia, Huangshixi, and the like. the study on the proteolysis process of feeding giant salamanders [ J ] food science and technology, 2012, 37 (2): 82-88). Wangheli et al performed detailed single-factor and orthogonal experimental studies on the enzymatic hydrolysis of giant salamander meat using Aspergillus sp. acidic protease (wangheli, zhanwei, shinning, et al. preparation of giant salamander meat enzymatic hydrolysate and its antioxidant properties study [ J ]. north river fishery, 2012, (9): 1-4). It can be seen that the giant salamander peptide is giant salamander meat, and the yield, structure, molecular weight and activity of the giant salamander peptide obtained by different enzymes and enzymolysis methods are different.
Chinese invention patent number ZL201610314751.X discloses a giant salamander active peptide and application, wherein the giant salamander active peptide is prepared according to the following steps:
homogenizing giant salamander meat, adding 1 ~ 3-time volume of 0.01 ~ 0.1.1M phosphate buffer solution with the pH value of 7 ~ 8 and 0.1 ~ 1% of complex enzyme with the weight ratio of the homogenate to carry out enzymolysis for 12 ~ 48 h, wherein the complex enzyme is marine alkaline protease and papain which are mixed according to the activity unit ratio of 7: 3;
centrifuging to obtain supernatant, separating with trypsin fixed ultrafiltration membrane separator with molecular weight cutoff below 4000 Da, and collecting liquid penetrating through the ultrafiltration membrane;
collecting chromatographic products by Sephadex LH-20 molecular sieve chromatographic column according to 1ml per tube, and freeze-drying the 30 th 30 ~ 36 th tube chromatographic product;
d. c, preparing the freeze-dried product obtained in the step c into a deionized water solution of 10 ~ 50mg/ml, centrifuging for 10 min at 8000 rpm and 4 ℃, and filtering by using a 0.22 mu m water film to obtain a supernatant;
e, preparing a 5% methanol buffer solution by using ddH2O, performing suction filtration through a water film with the aperture of 0.45 mu m, performing ultrasonic degassing to obtain a mobile phase A of the high performance liquid chromatography column, preparing a 95% methanol buffer solution by using ddH2O, performing suction filtration through an oil film with the aperture of 0.45 mu m, and performing ultrasonic degassing to obtain a mobile phase B of the high performance liquid chromatography column, performing gradient elution by using the mobile phase A and the mobile phase B under the conditions that the flow rate is 1ml/min, the elution gradient distribution is 0 ~ 20min, the mobile phase A is 100%, the mobile phase B is 20.5 ~ 40min, the mobile phase A is 50%, the mobile phase B is 50%, the mobile phase B is 40.5 ~ 60min, and the mobile phase B is 100%, injecting a supernatant into a sample inlet of a C18 column high performance liquid chromatograph, and collecting an absorption peak with;
and f, volatilizing the methanol in the collected matter, and carrying out vacuum freeze drying to obtain the giant salamander peptide product.
The prepared giant salamander bioactive peptide has the effect of promoting skin fibroblast proliferation.
Hyaluronic acid has become an important skin-friendly cosmetic raw material due to its good water-retaining effect, and is widely added to masks, lotions, creams, and lotions. Meanwhile, various compositions of hyaluronic acid have been developed. Chinese patent No. ZL201380080735.3 discloses a "biomaterial substance reinforced with rubber properties by natural crosslinking of collagen and hyaluronic acid, and a method for manufacturing and using the same", which is a novel substance exhibiting rubber properties by mixing collagen and hyaluronic acid and providing an optimum mixing ratio considering molecular structural characteristics of collagen and hyaluronic acid and natural crosslinking conditions of non-chemical or physical crosslinking, thereby greatly improving quality and reliability of products. The Chinese patent ZL201410718100.8 discloses a composition of stem cells and hyaluronic acid and an application thereof, which is a composition of mesenchymal stem cells and hyaluronic acid and has the advantages of quick response of hyaluronic acid and long duration of stem cells.
However, there is no report on the combination of giant salamander active peptide solution and hyaluronic acid to improve the fibroblast proliferation promoting effect.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides a giant salamander active peptide and hyaluronic acid composition for effectively promoting fibroblast proliferation.
The technical scheme includes that the giant salamander active peptide and hyaluronic acid composition capable of effectively promoting fibroblast proliferation is characterized in that the giant salamander active peptide and hyaluronic acid composition is prepared by mixing a giant salamander active peptide solution with the mass volume concentration of 1 ~ 10% and the pH value of 5 ~ 7 with hyaluronic acid according to the mass ratio of 1 ~ 8: 1, the molecular weight of the giant salamander active peptide is less than 2000Da, the mole percentages of lysine and arginine are 7.2% and 9.7%, respectively, and the molecular weight of the hyaluronic acid is 42.5 ~ 145 kDa.
The giant salamander active peptide is prepared by the following steps:
a. homogenizing giant salamander meat, adding 0.01 ~ 0.1.1M phosphate buffer solution with the volume being 1 ~ 4 times and the volume being pH 4 ~ 5 and 0.5 ~ 10% of complex enzyme into the homogenate, and carrying out enzymolysis for 1 ~ 12h at the ultrasonic frequency of 15 ~ 25 kHz and the temperature of 30 ~ 60 ℃ under the condition that the ultrasonic frequency is 15, 25 kHz and the temperature is 30, ~ ℃, wherein the complex enzyme is prepared by mixing marine acid protease, pepsin, flavourzyme and papain according to the activity unit ratio of 4: 3: 2: 1;
b. centrifuging the enzymolysis solution to obtain supernatant, separating with ultrafiltration membrane separator with cut-off molecular weight of 2000Da, and collecting liquid passing through ultrafiltration membrane;
c. adding the obtained liquid into macroporous adsorption resin DA201 according to the proportion of 1 g: 10mL, adjusting the pH value to be 5 ~ 8, oscillating at the room temperature of 150r/min for 6 ~ 12h, adding ethanol with the volume concentration of 60 ~ 95%, and oscillating at the room temperature of 150r/min for 6 ~ 24h for analysis to obtain an analysis liquid;
d. dialyzing the resolution solution, and freeze-drying to obtain the giant salamander active peptide.
The invention takes giant salamander meat as raw material and adopts marine acid proteaseAspergillus sp.The molecular weight of the giant salamander active peptide is less than 2000Da, the giant salamander active peptide has higher content of lysine and arginine residues with positive charges, can be naturally crosslinked with hyaluronic acid rich in a plurality of hydroxyl groups through hydrogen bonds, utilizes the natural affinity of the hyaluronic acid and human skin cells, can effectively improve the effect of promoting the proliferation of skin fibroblasts, and has wide application prospect in the fields of medicines and cosmetics.
Drawings
FIG. 1 is an electron micrograph of a composition according to an embodiment of the present invention.
FIG. 2 is a graph of an infrared spectrum of a composition of an example of the present invention.
FIG. 3 is a photomicrograph of a composition of an example of the invention at 200. mu.g/mL, co-cultured with fibroblasts for 36 h.
FIG. 4 is a photomicrograph of fibroblasts cultured alone for 36h in accordance with an embodiment of the present invention.
Detailed Description
The giant salamander active peptide and hyaluronic acid composition for effectively promoting fibroblast proliferation is prepared by mixing a giant salamander active peptide solution with the mass volume concentration of 8% and the pH value of 6 and hyaluronic acid according to the mass ratio of 5: 1, the molecular weight of the giant salamander active peptide is less than 2000Da, the mole percentages of lysine and arginine are respectively 7.2% and 9.7%, and the molecular weight of hyaluronic acid is 80 kDa. The giant salamander active peptide solution with the mass volume concentration of 8% and the pH value of 6 is prepared by adding water into giant salamander active peptide to prepare a giant salamander active peptide solution with the mass volume of 8% (w/v), and adjusting the pH value to 6 by using dilute hydrochloric acid.
The giant salamander active peptide is prepared by the following steps:
a. homogenizing giant salamander meat, adding 2 times volume of 0.05M phosphate buffer solution with pH 4 and composite enzyme with mass ratio of 5% to homogenate, and performing enzymolysis at ultrasonic frequency of 20kHz and temperature of 50 deg.C for 8 hr; the complex enzyme is 3500u/mg marine acid proteaseAspergillus sp.6000u/mg pepsin, 6000u/mg flavourzyme and 6000u/mg papain in a unit ratio of activity of 4: 3: 2: 1, mixing; marine acid proteaseAspergillus sp.Pepsin, flavourzyme and papain are all purchased from outsourcing.
b. Centrifuging the enzymolysis solution to obtain supernatant, separating with ultrafiltration membrane separator with cut-off molecular weight of 2000Da, and collecting liquid passing through ultrafiltration membrane;
c. the resulting liquid was measured as 1 g: adding 10mL of the solution into treated macroporous adsorption resin DA201, adjusting the pH to 6, shaking at 150r/min at room temperature for 8h, adding 85% ethanol, and shaking at 150r/min at room temperature for 20h to obtain a solution;
d. dialyzing the resolution solution, and freeze-drying to obtain the giant salamander active peptide.
The obtained giant salamander active peptide is detected according to the method of the prior art, the molecular weight of the giant salamander active peptide is less than 2000Da, and the mole percentages of lysine and arginine are respectively 7.2% and 9.7%. The embodiment of the invention can be freeze-dried and then powdered to prepare powder and spray for preparing products for promoting the proliferation of skin fibroblasts.
An electron micrograph and an infrared spectrum of the powder prepared from the composition of the example of the present invention are shown in fig. 1 and 2, respectively, and it can be seen from fig. 1 that the composition of the example shows a structure formed by natural crosslinking. FIG. 2 shows that the IR spectrum of the composition of the present invention has changed significantly from the IR spectrum of typical hyaluronic acid.
Experiment:
the composition of the embodiment of the invention promotes the proliferation of fibroblast cells in the skin of mice
Fibroblasts from Balb/c mice skin were suspended (5X 10)3Each cell/well) is inoculated in a 96-well culture plate, wherein part of wells are added with the composition (composition of giant salamander active peptide and hyaluronic acid) freeze-dried powder solution, the giant salamander active peptide freeze-dried powder solution and the hyaluronic acid solution of the embodiment of the invention, so that the final concentration is respectively 2, 20 and 200 mug/ml. 5% CO at 37 ℃2After 36h incubation in the incubator, 10. mu.l of tetrazole solution (cytometry kit-8) was added to each well, mixed well, and incubated in the incubator for 2h to measure the absorbance at 450 nm.
The composition freeze-dried powder solution of the embodiment of the invention is prepared by preparing the composition of the embodiment of the invention into freeze-dried powder and then dissolving the freeze-dried powder into serum-free culture medium to prepare different concentrations of 2 mug/ml, 20 mug/ml and 200 mug/ml.
The giant salamander active peptide freeze-dried powder solution is prepared by dissolving giant salamander active peptide freeze-dried powder in a serum-free culture medium to prepare different concentrations of 2 microgram/ml, 20 microgram/ml and 200 microgram/ml.
The hyaluronic acid solution is prepared by dissolving hyaluronic acid in serum-free culture medium to obtain different concentrations of 2 mug/ml, 20 mug/ml and 200 mug/ml.
The results are shown in Table 1. The results show that the fibroblast of the skin of the mouse increases along with the increase of the concentration of the composition, the giant salamander active peptide and the hyaluronic acid solution, but the composition provided by the embodiment of the invention has the effect of remarkably promoting the proliferation of the fibroblast of the skin of the mouse compared with the giant salamander active peptide or the hyaluronic acid solution with the same concentration (table 1).
TABLE 1
A photomicrograph of the composition of the present example, 200. mu.g/mL, co-cultured with fibroblasts for 36h, is shown in FIG. 3. The photomicrograph of fibroblasts cultured alone for 36 hours in the examples of the present invention is shown in FIG. 4. By contrast, it can also be seen that the composition of the embodiment of the present invention has the effect of significantly promoting the proliferation of fibroblasts in the skin of mice.
Claims (2)
1. The composition of giant salamander active peptide and hyaluronic acid for effectively promoting fibroblast proliferation is characterized by being prepared by mixing giant salamander active peptide solution with the mass volume concentration of 1 ~ 10% and the pH value of 5 ~ 7 with hyaluronic acid according to the mass ratio of 1 ~ 8: 1, wherein the molecular weight of the giant salamander active peptide is less than 2000Da, the mole percentages of lysine and arginine are 7.2% and 9.7%, respectively, and the molecular weight of the hyaluronic acid is 42.5 ~ 145 kDa.
2. The composition of giant salamander active peptide and hyaluronic acid effective in promoting fibroblast proliferation according to claim 1, wherein the giant salamander active peptide is prepared by the following steps:
a. homogenizing giant salamander meat, adding 0.01 ~ 0.1.1M phosphate buffer solution with the volume being 1 ~ 4 times and the volume being pH 4 ~ 5 and 0.5 ~ 10% of complex enzyme into the homogenate, and carrying out enzymolysis for 1 ~ 12h at the ultrasonic frequency of 15 ~ 25 kHz and the temperature of 30 ~ 60 ℃ under the condition that the ultrasonic frequency is 15, 25 kHz and the temperature is 30, ~ ℃, wherein the complex enzyme is prepared by mixing marine acid protease, pepsin, flavourzyme and papain according to the activity unit ratio of 4: 3: 2: 1;
b. centrifuging the enzymolysis solution to obtain supernatant, separating with ultrafiltration membrane separator with cut-off molecular weight of 2000Da, and collecting liquid passing through ultrafiltration membrane;
c. adding the obtained liquid into macroporous adsorption resin DA201 according to the proportion of 1 g: 10mL, adjusting the pH value to 5 ~ 8, oscillating for 6 ~ 12h at the room temperature of 150r/min, adding ethanol with the volume concentration of 60 ~ 95%, and oscillating for 6 ~ 24h at the room temperature of 150r/min for analysis to obtain an analysis liquid;
d. dialyzing the resolution solution, and freeze-drying to obtain the giant salamander active peptide.
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Cited By (8)
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| CN111758967A (en) * | 2020-07-15 | 2020-10-13 | 张兆熙 | Giant salamander collagen peptide composition and application thereof |
| CN111772195A (en) * | 2020-07-15 | 2020-10-16 | 张兆熙 | Giant salamander collagen peptide, preparation method and preparation thereof |
| CN112353748A (en) * | 2020-12-07 | 2021-02-12 | 成都高新区九州华昀医疗美容门诊部有限公司 | Cosmetic composition based on platelet-rich plasma and giant salamander active peptide |
| CN113249420A (en) * | 2021-05-12 | 2021-08-13 | 四川原石科技有限公司 | Method for improving bioactivity of giant salamander peptide, giant salamander peptide glycosylation product and application |
| CN113384480A (en) * | 2021-06-17 | 2021-09-14 | 张家界金驰天问农业科技有限公司 | Giant salamander peptide-fullerene compound and preparation method and application thereof |
| CN113398004A (en) * | 2021-07-27 | 2021-09-17 | 四川原石科技有限公司 | Giant salamander peptide-hyaluronic acid mixture and preparation method and application thereof |
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| CN114949169A (en) * | 2022-06-29 | 2022-08-30 | 九江高科制药技术有限公司 | Giant salamander polypeptide/carboxymethyl chitosan composite cream and preparation method thereof |
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| CN111772195B (en) * | 2020-07-15 | 2022-05-20 | 张兆熙 | Giant salamander collagen peptide, preparation method and preparation thereof |
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| CN113249420B (en) * | 2021-05-12 | 2022-08-26 | 四川原石科技有限公司 | Method for improving bioactivity of giant salamander peptide, giant salamander peptide glycosylation product and application |
| CN113249420A (en) * | 2021-05-12 | 2021-08-13 | 四川原石科技有限公司 | Method for improving bioactivity of giant salamander peptide, giant salamander peptide glycosylation product and application |
| CN113384480A (en) * | 2021-06-17 | 2021-09-14 | 张家界金驰天问农业科技有限公司 | Giant salamander peptide-fullerene compound and preparation method and application thereof |
| CN113398004B (en) * | 2021-07-27 | 2022-04-26 | 四川原石科技有限公司 | Giant salamander peptide-hyaluronic acid mixture and preparation method and application thereof |
| CN113398004A (en) * | 2021-07-27 | 2021-09-17 | 四川原石科技有限公司 | Giant salamander peptide-hyaluronic acid mixture and preparation method and application thereof |
| CN113730306A (en) * | 2021-09-18 | 2021-12-03 | 武汉海思普莱生命科技有限公司 | Anti-aging composition, anti-aging vesicle and preparation method and application of anti-aging vesicle |
| CN114949169A (en) * | 2022-06-29 | 2022-08-30 | 九江高科制药技术有限公司 | Giant salamander polypeptide/carboxymethyl chitosan composite cream and preparation method thereof |
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