CN111235202A - Deer bone protein extract and preparation method and application thereof - Google Patents
Deer bone protein extract and preparation method and application thereof Download PDFInfo
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- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
- A61K38/014—Hydrolysed proteins; Derivatives thereof from animals from connective tissue peptides, e.g. gelatin, collagen
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
Abstract
The invention discloses a deer bone protein extract and a preparation method and application thereof, wherein the deer bone protein extract is extracted from red deer bones and sika deer bones by adopting an acid extraction and enzyme extraction method, and can play roles in repairing, resisting wrinkles and resisting skin aging from the following aspects of (1) improving the proliferation activity and the renewal capacity of skin keratinocytes at a cell level of ①, improving the proliferation activity of skin fibroblasts at ②, repairing the reduction of the proliferation capacity of the skin fibroblasts due to oxidative stress damage, and promoting the synthesis of endogenous collagen, and (2) supplementing the collagen lost by aged skin directly by enabling an exogenous deer bone protein extract to be absorbed into a dermis layer through skin penetration and integrated into collagen fibers of the deer bone protein extract.
Description
Technical Field
The invention relates to a deer bone protein extract and a preparation method and application thereof in the field of biomedicine.
Background
The skin, the largest organ of the body, comprises two distinct main components, a constantly renewed epidermis, consisting of keratinocytes, and a dermis, with an abundant matrix consisting of fibroblasts. Due to natural aging and accumulation of external environmental factors (especially ultraviolet rays), the cellular structure and function of skin tissues are changed, and the aging and damage of the skin such as relaxation and wrinkles are often caused. Skin aging is reflected at the cellular level as cellular aging, and the reduction of the proliferation capacity is the most direct manifestation of cellular aging.
Collagen is synthesized by fibroblasts in the dermal connective tissue and is the major structural protein in the extracellular matrix. Collagen exists in the form of collagen fibers in the skin, is a supporting structure of the skin, and plays a role in maintaining the elasticity of the skin, increasing the thickness of the skin and reducing the generation of wrinkles. The lack of collagen can cause skin aging phenomena such as dryness, roughness, and laxity.
It is known that collagen supplementation can repair damaged skin and delay skin aging. The collagen has great application prospect in the fields of cosmetics, foods, health products and medicines. China is a large country in deer industry, deer bones contain abundant collagen which accounts for about 57%, but the deer bones as a high-quality collagen resource are rarely researched.
Disclosure of Invention
The technical problem to be solved by the invention is how to obtain the deer bone protein extract which has no fishy smell and/or can improve the anti-oxidative damage activity of skin cells and/or improve the proliferation activity of the skin cells.
In order to solve the above technical problems, the present invention provides a method for preparing a deer bone protein extract, which is a method for preparing a deer bone protein extract by an acid extraction plus enzyme extraction method.
The method for preparing the deer bone protein extract provided by the invention comprises the following steps:
(1) acid extraction: extracting the decalcified deer bone powder with an acid solution, collecting an extracting solution, and naming the extracting solution as an acid-soluble collagen crude solution;
(2) enzyme extraction: adding pepsin into the crude acid-soluble collagen solution for enzymolysis, filtering after enzymolysis, collecting filtrate, and naming the filtrate as enzymatic acid-soluble collagen enzymatic hydrolysate;
(3) salting out: salting out the enzymatic acid-soluble collagen enzymatic hydrolysate, and collecting the precipitate;
(4) and (3) dialysis: dialyzing the precipitate to obtain deer bone protein extract.
In the above method, the method further comprises a step of drying the obtained dialysis product after dialyzing the precipitate to obtain a solid deer bone protein extract.
In the above method, the kind and concentration of the acid solution used in the acid extraction can be determined by a person skilled in the art through routine experiments, and for example, the acid solution can be acetic acid solution with acetic acid concentration of 0.5 mol/L.
In the above method, the pH value of the acid extraction system can be determined by a person skilled in the art through routine experiments, for example, the acid extraction is performed in a system with a pH value of 1.8.
In the above method, the ratio of the acid-soluble crude collagen solution and the pepsin in the enzymatic extraction can be determined by a person skilled in the art through routine experiments, and for example, the ratio of the acid-soluble crude collagen solution and the pepsin can be such that, per 1g of the crude acid-soluble collagen solution derived from the decalcified deer bone meal: 1000U of pepsin, wherein the acid-soluble collagen crude liquid is calculated by the mass of the decalcified deer bone powder from which the acid-soluble collagen crude liquid is derived.
In the above method, in the enzyme extraction, the temperature and time for the enzymolysis can be determined by a person skilled in the art through routine experiments, for example, the temperature for the enzymolysis can be 37 ℃, and the time for the enzymolysis can be 6 hours.
In the method, the decalcified deer bone powder is obtained by crushing decalcified deer bones.
The decalcified deer bone powder can be prepared by the following steps: decocting Os Cervi with water, collecting bone, soaking in hydrochloric acid solution until the bone is softened, washing with water until pH is neutral, taking out bone, oven drying, and pulverizing to obtain decalcified Os Cervi powder.
In the above method, the volume percentage of the hydrochloric acid solution may be 2% to 6%, for example, 4%.
In the above method, the decocting is carried out according to a method comprising the steps of: decocting Os Cervi in water twice, each for 1-3 hr.
In the above method, the cut-off for the dialysis may be 1000 Da.
In the above method, the drying may be freeze-drying.
In the above method, the freeze-drying may be performed according to a method comprising the steps of: freezing the dialyzate at-80 deg.C to obtain solid block, and freeze drying at-50 deg.C under 1.8Mpa in a freeze dryer to obtain dry powder.
The deer bone protein extract (i.e. deer bone acid extraction and enzyme extraction extract) extracted from the deer bone by the method also belongs to the protection scope of the invention.
In the above, the deer bone is red deer bone and/or sika deer bone, and the deer bone protein extract is red deer bone protein extract and/or sika deer bone protein extract.
Of the above deer bone protein extracts, 10mg/mL of aqueous solution of the deer bone protein extract has a pH of 4.7 at standard temperature (25 ℃) and standard atmospheric pressure (1atm), and 10mg/mL of aqueous solution of the deer bone protein extract has a pH of 5.5 at standard temperature (25 ℃) and standard atmospheric pressure (1 atm).
The deer bone protein extract has no fishy smell.
Any of the following applications also fall within the scope of the present invention:
u1. the application of the deer bone protein extract in enhancing the anti-oxidative damage activity of skin cells or preparing products for enhancing the anti-oxidative damage activity of skin cells;
u2. the application of the deer bone protein extract in resisting skin aging or preparing skin aging resisting products;
u3. the application of the deer bone protein extract in improving the proliferation activity of skin keratinocytes or preparing products for improving the proliferation activity of skin keratinocytes;
u4. the application of the deer bone protein extract in improving the proliferation activity of skin fibroblasts, or preparing products for improving the proliferation activity of skin fibroblasts;
u5. the application of the deer bone protein extract in repairing the decrease of the proliferation ability of skin fibroblasts caused by oxidative stress damage, or preparing products for repairing the decrease of the proliferation ability of skin fibroblasts caused by oxidative stress damage;
u6. the application of the deer bone protein extract in transdermal absorption into dermis and integration into collagen fiber, or preparing products capable of transdermal absorption into dermis and integration into collagen fiber;
u7. the application of the deer bone protein extract in supplementing collagen lost from aged skin or preparing products for supplementing collagen lost from aged skin.
As above, the skin cells may be skin keratinocytes and/or skin fibroblasts.
Wherein the product can be cosmetics, foods, health products or medicines.
As used herein, the skin cell can be a human skin cell, the skin keratinocyte can be a human skin keratinocyte, and the skin fibroblast can be a human skin fibroblast.
The deer bone protein extract (deer bone acid extraction and enzyme extraction extract) extracted from the deer bone by adopting the acid extraction and enzyme extraction method is obviously superior to the deer bone acid extraction extract, the deer bone enzyme extraction extract, a commercial recombinant human collagen product and a commercial deer bone protein extract product in the aspect of enhancing the anti-oxidative stress injury activity of skin cells.
The deer bone protein extract extracted from the red deer bone and the sika deer bone by adopting an acid extraction and enzyme extraction method can play roles of repairing, resisting wrinkles and resisting skin aging from the aspects of (1) improving the proliferation activity and the renewal capacity of skin keratinocytes at a cell level of ①, improving the proliferation activity of skin fibroblasts at ②, repairing the reduction of the proliferation capacity of the skin fibroblasts caused by oxidative stress damage, and promoting the synthesis of endogenous collagen, and (2) supplementing the collagen lost by skin aging directly by enabling the exogenous deer bone protein extract to be absorbed into a dermis layer through skin penetration and being integrated into collagen fibers of the deer bone protein extract.
Drawings
FIG. 1 shows the effect of deer bone protein extract on the proliferation of HaCaT cell line. Wherein P <0.05 and P <0.01, compared to control.
FIG. 2 shows the effect of sika deer bone protein extract on the proliferation of HaCaT cell line. Wherein P <0.05, P <0.01, P <0.001, compared to control.
FIG. 3 shows the effect of deer bone protein extract on the proliferation of HFF-1 cell line. Wherein P <0.05 and P <0.01, compared to control.
FIG. 4 shows the effect of sika deer bone protein extract on the proliferation of HFF-1 cell line. Wherein P <0.01 compared to control.
FIG. 5 shows different concentrations H2O2Induce the cell survival rate of HFF-1 cells with oxidative damage.
FIG. 6 shows the pairs of deer bone protein extracts H2O2Effects of HFF-1 cell proliferation that induce oxidative damage. Wherein P is compared with control group<0.01，***P<0.001。
FIG. 7 shows the pair of protein extracts of sika deer bone H2O2Effects of HFF-1 cell proliferation that induce oxidative damage. Wherein P is compared with control group<0.01，***P<0.001。
FIG. 8 is an SDS-PAGE electrophoresis band of the fluorescence-labeled deer bone protein extract. In the figure, A: bright field; b: 365 nm. 1 is a red deer bone protein extract without marked fluorescence, 2 is a red deer bone protein extract without marked fluorescence, 3 is a Marker (strips from large to small are 250kDa, 130kDa, 100kDa, 70kDa, 55kDa, 35kDa and 25kDa in sequence), 4 is a red deer bone protein extract marked by FITC, and 5 is a red deer bone protein extract marked by FITC.
FIG. 9 shows second harmonic and two-photon imaging dynamic observation of skin penetration of deer bone protein extract.
Fig. 10 shows the second harmonic wave combined with two-photon imaging in vitro observation of the percutaneous absorption of the deer bone protein extract.
FIG. 11 is a SDS-PAGE electrophoresis pattern of the deer bone extract obtained by hot water extraction, acid extraction and enzyme extraction and the red deer bone protein extract and the sika deer bone protein extract of example 1. In the figure, 1. protein marker; 2. extracting sika deer bone with hot water; 3. extracting with Cervus Nippon Temminck bone enzyme; 4. extracting with enzyme (such as the protein extract of the bone of Cervus Nippon Temminck of example 1); 5. extracting sika deer bone with acid; 6. extracting Os Cervi with hot water; 7. extracting with enzyme to obtain extract; 8. a red deer bone acid extraction enzyme extraction extract (i.e., the red deer bone protein extract of example 1); 9. the extract of red deer bone by acid extraction.
FIG. 12 is a graph showing the effect of various products on the proliferative activity of HaCaT cells. In the figure, P <0.05, P <0.01, P <0.001, compared to the control group.
FIG. 13 is a graph showing the effect of various products on the proliferative activity of HFF-1 cells. In the figure, P <0.05, P <0.01, P <0.001, compared to the control group.
FIG. 14 shows various product pairs H2O2Effects of HFF-1 cell proliferation that induce oxidative damage. In the figure, P is compared with the model group<0.05，**P<0.01，***P<0.001。
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The solutions in the following examples are water solutions as solvents unless otherwise specified.
Reagent: leg bones of deer (from cultured red deer and sika deer) are from agricultural academy of sciences of Jilin province; the BCA protein quantitative kit is from Shanghai Biyuntian biotechnology limited; acetic acid is from national pharmaceutical group chemical agents, ltd; the pepsin is from chemical reagent of national medicine group, the product number is 64007137, and the enzyme activity is more than or equal to 1200.0U/g; DMEM high-glucose medium, fetal bovine serum, sodium pyruvate were obtained from Gibco, USA; the trypsin and streptomycin double antibody is from Sigma company in the United states; the CCK-8 reagent is a product of the Japan college chemical research institute; 3% H2O2Solution of Beijing seaDe run pharmaceutical products.
The instrument comprises the following steps: cell-grade ultrafine pulverizer, denda micro mechanical ltd; a digital display constant temperature water bath kettle and a JintanCity crystal wave experimental instrument are long; lyophilizers, Beijing Bo Yi kang laboratory instruments, Inc.; a dryer, Shanghai sperm macro laboratory equipments Ltd; carbon dioxide incubator Thermo; desktop centrifuge, Eppendorf; super clean bench, suzhou sujing; microplate reader, Bio-Rad; two-photon confocal microscopy, OLYMPUS; multifunctional extraction tank, model TQ-3m3Purchased from Tujiang south county Liou petrochemical equipment Co., Ltd.
Cell: the human normal skin immortalized keratinocyte strain HaCaT cell strain is offered by the dermatology of Beijing medical hospital, and is cultured by a DMEM high-sugar culture medium in a conventional way; the human skin fibroblast strain HFF-1 is from a stem cell bank of Chinese academy of sciences and is cultured by a culture medium containing 84% of DMEM, 15% of fetal calf serum and 1% of sodium pyruvate.
Experimental animals: BALB/c nude mouse, male, 6-8 weeks old, Beijing Witonglihua laboratory animal technology Co., Ltd., license number: SCXK (Kyoto) 2016-.
Example 1 acid extraction with enzyme extraction method protein extract from deer bone (Cervus Elaphus L., Cervus Nippon Temminck)
Preparation of deer bone protein extract
1. Pretreatment of
Weighing appropriate amount of leg bone of Cervus Elaphus L, cleaning, removing skin, muscle and periosteum to obtain bone, cutting off spongy bone at two ends of the bone, removing bone marrow from the bone, washing the bone with running water until there is no oil slick in water, air drying, pulverizing into bone pieces (2cm × 2cm), and cleaning to remove oil slick in bone. The bone pieces were dried (55-65 ℃) to dryness to give bone, which was weighed.
2. Decocting and decocting
Decocting dried bone in water in a multifunctional extraction tank for 2 times (0.1Mpa, 100 deg.C) for 2.5 hr each time, filtering (100 mesh), collecting residue (bone), and oven drying (60-65 deg.C).
3. Decalcification of calcium
Placing the filter residue (bone) in a soaking pool, adding 4% (v/v) HCl solution, soaking for 4 days until the bone is softened, filtering, and washing with water until pH is neutral; taking out bone, oven drying at 60 deg.C, and pulverizing into coarse bone powder, which is decalcified deer bone powder.
4. Acid extraction
Adding 10 times of water into the decalcified red deer bone powder, adding acetic acid to adjust the concentration of the acetic acid to 0.5mol/L, adding hydrochloric acid to adjust the pH value to 1.8, soaking for 3 days at 4 ℃, and stirring once every 2h during soaking; filtering (100 mesh), and collecting filtrate to obtain crude solution of acid soluble collagen.
5. Enzyme extraction
Adding pepsin into the crude acid-soluble collagen solution, placing the mixture into a digital display water bath constant temperature oscillator, carrying out enzymatic reaction at 37 ℃ for 6 hours, stirring the mixture once every 1 hour, filtering the mixture by using filter cloth, and collecting filtrate to obtain enzymatic acid-soluble collagen enzymatic hydrolysate.
Wherein, the adding amount of the pepsin meets the requirement that the acid soluble collagen crude liquid is calculated by the mass of the decalcified red deer bone powder from the acid soluble collagen crude liquid, and 1000U of the pepsin is added into every 1g of the decalcified red deer bone powder.
6. Salting out
Adding NaCl solid into the enzymatic acid-soluble collagenase hydrolysate while stirring to enable the concentration of NaCl to reach 2mol/L, and standing for 24 hours at normal temperature (24 ℃); centrifuging (8000rmp, 20min) after salting out, and collecting precipitate; re-dissolving the precipitate with appropriate amount of 0.5mol/L acetic acid solution, and repeating the above steps to perform secondary salting-out to obtain precipitate.
7. Dialysis
Adding 0.5mol/L acetic acid solution into the precipitate until dissolving, injecting the dissolved solution into a dialysis bag (specification of the dialysis bag: 77mm in width, available in length; molecular weight cut-off: 1000Da), clamping the dialysis bag in two sections with clamps, soaking in deionized water, repeatedly dialyzing, and replacing water every 4h until the pH of the solution outside the dialysis bag is neutral (dialysis reference time: 24 h).
8. Freeze drying
Taking out the dialyzate, freezing at-80 deg.C to obtain solid, and freeze drying at-50 deg.C under 1.8Mpa in a freeze dryer to obtain dry powder, which is the protein extract of Os Cervi (enzyme extraction method of acid extraction of Os Cervi).
Preparation of sika deer bone protein extract
The operation is completely the same as the first step except that the red deer leg bone in the first step is replaced by the sika deer leg bone, and the sika deer bone protein extract (namely the sika deer bone acid extraction enzyme extraction extract) is obtained.
Identification of deer bone protein extract
1. Determination of Properties and pH
Weighing 20mg of the red deer bone protein extract obtained in the step one, dissolving the red deer bone protein extract in 2mL of PBS solution with the pH value of 7.2, and preparing the red deer bone protein extract PBS solution with the concentration of 10 mg/mL.
Weighing 20mg of the sika deer bone protein extract obtained in the second step, dissolving the sika deer bone protein extract in 2mL of PBS solution with the pH value of 7.2, and preparing the sika deer bone protein extract PBS solution with the concentration of the sika deer bone protein extract of 10 mg/mL.
The pH of the solutions of the deer bone protein extract PBS and the sika deer bone protein extract PBS were measured at standard temperature (25 ℃) and standard atmospheric pressure (1atm) using a pH meter. The result is expressed to one decimal place.
Weighing the deer bone protein extract obtained in the step one, 20mg of the deer bone protein extract is dissolved in 2mL of ddH with pH of 6.42And O solution, preparing into a red deer bone protein extract water solution with the concentration of the red deer bone protein extract of 10 mg/mL.
Weighing the sika deer bone protein extract obtained in the second step, wherein 20mg of the sika deer bone protein extract is dissolved in 2mL of ddH with pH value of 6.42And preparing a sika deer bone protein extract aqueous solution with the concentration of the sika deer bone protein extract of 10mg/mL in the O solution.
The pH of the aqueous solutions of the deer bone protein extract and the sika deer bone protein extract were measured at standard temperature (25 ℃) and standard atmospheric pressure (1atm) using a pH meter. The result is expressed to one decimal place.
The results show that: the red deer bone protein extract obtained in the step one and the sika deer bone protein extract obtained in the step two are white flocculent, fine and smooth, have no taste, have better solubility in water and PBS, and have no particle deposition. The pH of 10mg/mL of the PBS solution of the Cervus elaphus Linnaeus bone protein extract at standard temperature (25 ℃) and standard atmospheric pressure (1atm) is 7.0, the pH of 10mg/mL of the PBS solution of the Cervus elaphus Linnaeus bone protein extract at standard temperature (25 ℃) and standard atmospheric pressure (1atm) is 7.2, the pH of 10mg/mL of the aqueous solution of the Cervus elaphus Linnaeus bone protein extract at standard temperature (25 ℃) and standard atmospheric pressure (1atm) is 4.7, and the pH of 10mg/mL of the aqueous solution of the Cervus elaphus Linnaeus bone protein extract at standard temperature (25 ℃) and standard atmospheric pressure (1atm) is 5.5.
2. Protein content determination of deer bone protein extract
And (2) operating according to the requirements of the BCA method protein content determination kit, diluting a standard Bovine Serum Albumin (BSA) (Biyuntian, P0009) into 0-2000 mu g/mL, respectively adding 20 mu L of BSA standard and the red deer bone protein extract obtained in the first step or the red deer bone protein extract obtained in the second step into a 96-well plate, adding 200 mu L of BCA working solution into each well, and incubating for 30min at 37 ℃. And (4) measuring under an enzyme-labeling instrument A595nm, and measuring the protein concentration of the sample by taking the OD value as an abscissa and the sample concentration as an ordinate.
The result shows that 190.28g of decalcified red deer bone powder can obtain 2.643g of red deer bone protein extract, the protein content in the red deer bone protein extract is 77.00 percent, which indicates that 1g of decalcified red deer bone powder can obtain 10.70mg of protein; 147.63g of decalcified sika deer bone powder can obtain 0.537g of sika deer bone protein extract, the protein content in the sika deer bone protein extract is 72.47%, which indicates that 1g of decalcified sika deer bone powder can obtain 2.64mg of protein.
Pharmacodynamic detection of deer bone protein extract
1. In vitro cell assay
The model of the human normal skin immortalized keratinocyte cell line (HaCaT cell) has a human normal skin epithelial structure, can simulate human skin cells and is applied to the research of cosmetics on skin anti-aging. The HFF-1 cell strain is derived from the foreskin tissue of foreign newborn boys and has the shape and characteristics of normal skin fibroblasts. This study used HaCaT cells and HFF-1 cells as in vitro models to study the anti-skin aging activity of deer bone protein extracts.
1. Deer bone protein extract for promoting proliferation of skin keratinocyte and skin fibroblast
1.1 Effect of Cervus elaphus bone protein extract on the proliferation of HaCaT cell line
The experiment was performed in triplicate, each replicate as follows: HaCaT was added at 5X 104Inoculating each well into a 96-well plate, culturing for 12h at 37 ℃ by using a DMEM high-sugar complete culture medium, randomly dividing into an administration group and a control group, setting 5 treatments in the administration group, wherein each treatment comprises 5 wells, each well is respectively added with 100 mu L of red deer bone protein extract solution (the solute is the red deer bone protein extract obtained in the step one, and the solvent is serum-free DMEM high-sugar culture medium), and the content of the red deer bone protein extract in the 5 treatments is respectively 0.004, 0.016, 0.063, 0.250 and 1.000 mg/mL. The control group is provided with 5 multiple wells, and each well is added with an equal volume of serum-free DMEM high-sugar medium. Culturing in an incubator at 37 ℃ for 48h, detecting the proliferation activity of HaCaT cells by using a CCK-8 reagent, measuring the absorbance at 450nm by using an enzyme-labeling instrument, and calculating the cell survival rate (%). All data were processed statistically using an independent sample t-test of the SPSS12.0(spssinc., USA) statistical software.
The result shows that the red deer bone protein extract can promote the proliferation of HaCaT cells, and no cytotoxicity is seen when the concentration is as high as 1mg/mL, wherein the red deer bone protein extract solution of 0.063-0.250 mg/mL can promote the proliferation of HaCaT cells in a dose-dependent manner, the red deer bone protein extract solution of 0.063mg/mL has a significant difference (P <0.05) compared with a control group, and the red deer bone protein extract solution of 0.250mg/mL has a very significant difference (P <0.01) compared with the control group (figure 1).
1.2 Effect of sika deer bone protein extract on HaCaT cell line proliferation
The procedure was exactly the same as 1.2 except that the red deer bone protein extract of 1.1 was replaced with the red deer bone protein extract obtained in step two.
The result shows that the sika deer bone protein extract can promote the proliferation of HaCaT cells, and no cytotoxicity is seen when the concentration is as high as 1mg/mL, wherein the sika deer bone protein extract solution of 0.004-0.250 mg/mL can obviously promote the proliferation of HaCaT cells, the sika deer bone protein extract solution of 0.004mg/mL has very significant difference (P <0.01) compared with a control group, the sika deer bone protein extract solution of 0.016-0.250 mg/mL has very significant difference (P <0.001) compared with the control group, and the sika deer bone protein extract solution of 0.063mg/mL has the most significant proliferation promoting effect (figure 2).
1.3 Effect of Cervus elaphus Linnaeus bone protein extract on HFF-1 cell proliferation
The experiment was performed in triplicate, each replicate as follows: HFF-1 was added at 5X 104Inoculating each well into a 96-well plate, culturing for 12h at 37 ℃ by using a DMEM + FBS + sodium pyruvate culture medium (consisting of 84% DMEM, 15% fetal calf serum and 1% sodium pyruvate aqueous solution), randomly dividing the culture medium into an administration group and a control group, and setting 5 treatments in each administration group, wherein each treatment comprises 5 multiple wells, each well is respectively added with 100 mu L of a red deer bone protein extract solution (the solute is the red deer bone protein extract obtained in the step one, and the solvent is serum-free DMEM high-sugar medium), and the content of the red deer bone protein extract in the 5 treatments is respectively 0.004, 0.016, 0.063, 0.250 and 1.000 mg/mL. The control group is provided with 5 multiple wells, and each well is added with an equal volume of serum-free DMEM high-sugar medium. Culturing in an incubator at 37 ℃ for 48h, detecting the proliferation activity of the HFF-1 cells by using a CCK-8 reagent, measuring the absorbance at 450nm by using an enzyme-labeling instrument, and calculating the cell survival rate (%). All data were processed statistically using independent sample t-tests of SPSS12.0(SPSS inc., USA) statistical software.
The results show that the deer bone protein extract obtained in the first step can promote the proliferation of HFF-1 cells, compared with the control group, the deer bone protein extract solution of 0.25mg/mL can obviously promote the proliferation of normal HFF-1 cells (P <0.05), and the deer bone protein extract solution of 1.00mg/mL can very obviously promote the proliferation of normal HFF-1 cells (P <0.01) (FIG. 3).
1.4 Effect of Cervus Nippon Temminck bone protein extract on HFF-1 cell proliferation
The procedure was exactly the same as 1.3 except that the red deer bone protein extract of 1.3 was replaced with the red deer bone protein extract obtained in step two.
The results show that the sika deer bone protein extract obtained in the second step can promote the proliferation of HFF-1 cells, and compared with the control group, the sika deer bone protein extract solutions of 0.25mg/mL and 1.00mg/mL can greatly promote the proliferation of normal HFF-1 cells (P <0.01) (FIG. 4).
2. Antioxidant activity of deer bone protein extract
2.1 establishing H2O2Model for inducing HFF-1 cell oxidative damage
HFF-1 cells were cultured at 5X 104Inoculating each well into 96-well plate, culturing at 37 deg.C for 12 hr with DMEM + FBS + sodium pyruvate medium, randomly dividing into model group and control group, setting 4 treatments in each model group, and adding 2 μ L of 40mM H in each treatment well, wherein each treatment well comprises 5 multiple wells2O2Solution (solute is H)2O2Solvent is serum-free DMEM high-sugar medium), H in the 4 treatments2O2The contents of (A) are 200, 400, 800 and 1600 μ M, respectively. Control group set 5 multiple wells, each well added with an equal volume of serum-free DMEM high-glucose medium). Culturing at 37 deg.C for 24 hr. Detecting cell proliferation by CCK-8 reagent, calculating cell survival rate (%), and determining H2O2Induction of HFF-1 cell oxidative damage model.
The results are shown in FIG. 5, H2O2Cell viability was 86.38%, 77.42%, 9.71%, 5.84% at concentrations of 200, 400, 800, 1600 μ M, respectively, and correlation analysis of r-0.9051 indicated H2O2Concentration is inversely related to% cell viability, i.e. H2O2The higher the concentration, the lower the cell viability. R20.8192, the independent variable can explain the model of the dependent variable 81.92% variation, indicating that the model fits well as a whole; p<0.05, indicating that the regression equation is remarkably true.
2.2 determination of Activity of deer bone protein extract against oxidative Damage
2.2.1 HFF-1 cells at 5X 104Inoculating each well into 96-well plate, culturing at 37 deg.C with DMEM + FBS + sodium pyruvate culture medium for 12 hr, randomly dividing into control group, model group, and administration group, wherein the control group and model group are respectively provided with 1 treatment, and the administration group is provided with 5 treatments, each treatmentAnd 5 multiple holes. Model group and administration group 2. mu.L of 40mM H was added to each well of each treatment2O2Solution (solute is H)2O2Solvent is serum-free DMEM high-sugar medium), H in each well2O2The content of (A) is 400 mu M, and the HFF-1 cells are induced to generate oxidative damage. 50 μ L of Cervus elaphus Linnaeus bone protein extract solution (solute is the Cervus elaphus Linnaeus bone protein extract obtained in step one, and solvent is serum-free DMEM high-sugar medium) is added into each well of 5 treatments of administration group, wherein the content of the Cervus elaphus Linnaeus bone protein extract in the 5 treatments is 0.004, 0.016, 0.063, 0.250 and 1.000mg/mL respectively. And respectively adding 50 mu L of serum-free DMEM high-sugar medium into each hole in the model group. Each well of the control group was added with 52. mu.L of serum-free DMEM high-sugar medium. Culturing in an incubator at 37 ℃ for 24h, detecting cell proliferation by using a CCK-8 reagent, and calculating the cell survival rate (%). All data were processed statistically using independent sample t-tests of SPSS12.0(SPSS inc., USA) statistical software.
2.2.2 the deer bone protein extract in the step 2.2.1 is replaced by the deer bone protein extract obtained in the step two, and the other operations are completely the same as the operations in the step 2.2.1.
As shown in FIGS. 6 and 7, the results of the 0.063-1.000 mg/mL red deer bone protein extract solution and the 0.016-1.000 mg/mL sika deer bone protein extract solution in H2O2Induced oxidative damage of HFF-1 cells with very significant protection (. about.P)<0.01，***P<0.001) and the effect is dose-dependent, the result shows that the red deer bone protein extract (0.063-1.000 mg/mL) and the sika deer bone protein extract (0.016-1.000 mg/mL) can reduce H2O2Has antioxidant injury activity on the injury of the proliferation activity of skin fibroblasts, and has the most obvious antioxidant injury effect when the concentration is 1.000 mg/mL.
3. Whole animal experiments
(1) Fluorescent-labeled deer bone protein extract
And (3) adding 9mL of PBS into 500mg of the red deer bone protein extract obtained in the first step or the sika deer bone protein extract obtained in the second step, adding 1mL of carbonate buffer solution, and fully stirring on a magnetic stirrer to obtain a deer bone protein extract solution with the concentration of 50 mg/mL. 10mg of FITC was added to 12.5mL of PBS and mixed, 2.5mL of carbonate buffer was added, vortexed, and mixed thoroughly. Slowly adding FITC solution into the prepared deer bone protein extract solution with 200 μ L pipette, placing the magnetic stirrer into a 4 deg.C refrigerator, and stirring for 24 hr to fully mark. Taking out the protein after fluorescent labeling, putting the protein into a 1kD dialysis bag, putting the protein into a big beaker which is protected from light and contains ultrapure water, changing water every 4h, and stopping dialysis when no fluorescence is detected in the ultrapure water. And (4) taking out the fluorescent protein, and freeze-drying the fluorescent protein into dry powder for later use.
(2) SDS-PAGE detection of fluorescence labelling
The loading amount of the fluorescence labeling deer bone protein extract is 100 mu g, the loading volume is 5 mu L, and the constant pressure of 100V is 150min by adopting 5 percent concentrated glue and 10 percent separation glue. And after the electrophoresis is finished, observing a fluorescent band at 365nm, then observing a protein band under white light after Coomassie brilliant blue dyeing, and determining whether the protein is marked by the fluorescent probe according to the relative positions of the fluorescent band and the protein band.
The results are shown in FIG. 8, where A is Coomassie brilliant blue gel imaging and B is FITC-labeled deer bone protein extract fluorescence image. As can be seen from FIG. 8, deer bone protein has more distinct protein bands around 130kD and 40 kD.
(3) Second harmonic and two-photon imaging of skin tissue
Taking 8 Babl/c nude mice (6-8 weeks old, SPF grade, Beijing Wittingle laboratory animal technology limited company, license number SCXK (Jing) 2012-doped 0001), uniformly coating the fluorescence-labeled deer bone protein extract on back skin tissues, keeping out of the sun, fixing four limbs on an object stage after the skin naturally absorbs for 0h, 1h, 2h, 3h, 4h, 24h and 48h under the room temperature condition, and collecting living images under a two-photon confocal microscope. After the collection, the whole layer of skin tissue of 1cm multiplied by 0.5cm is taken down at each time point, and is tiled on a glass slide for image collection again. Signals are collected from the back in the second harmonic imaging and the two-photon fluorescence imaging, the second harmonic excitation wavelength is 950nm, the two-photon fluorescence excitation wavelength is 925nm, the exciting light is focused on a sample through an objective lens, and the generated back second harmonic signals and the fluorescence signals are collected through a 25-time water-immersed microscope objective lens with the numerical aperture of 1.05. A high-pass filter is used in the two-photon fluorescence channel, and the wavelength range is 495-540 nm; and the second harmonic adopts a narrow-pass filter with the wavelength range of 465-485 nm. And (3) carrying out full-layer Z-axis scanning on the tissue, stepping by 1 mu m, acquiring an image by 512 x 512 pixels at an image acquisition speed of 2 mu m/pixel, and carrying out Imaris 8.4.2 image rendering processing on the obtained image.
1mg/mL FITC marked deer bone protein extract is uniformly smeared on the back skin of a nude mouse, and the percutaneous absorption condition of the nude mouse along with time is observed by combining a two-photon confocal microscope and a second harmonic imaging method, so that the result shows that the percutaneous absorption depth of the deer bone protein extract is increased along with time, the second harmonic signal is gradually enhanced, and the peak value is reached after 48 hours of smearing, which shows that the collagen fibers of the dermis are orderly increased (figure 9).
The skin tissue coated with the FITC labeled deer bone protein extract is taken down according to time points and then is statically observed for the transdermal absorption condition, and the result shows that the transdermal absorption depth of the deer bone protein extract is still increased along with the increase of time without the influence of respiration, and the second harmonic signal intensity reaches the peak after being coated for 48 hours. The tissues were cryosectioned and longitudinal observations also showed that the depth of transdermal absorption increased with time, accumulation at the follicular site and increased the alignment of collagen fibers in the dermal layer (fig. 10).
In conclusion, the deer bone protein extract extracted from the deer bone can play the roles of repairing, resisting wrinkles and resisting skin aging in the aspects of (1) improving the proliferation activity and the renewal capacity of skin keratinocytes at a cell level, improving the proliferation activity of skin fibroblasts at ②, repairing the reduction of the proliferation capacity of the skin fibroblasts due to oxidative stress damage, and promoting the synthesis of endogenous collagen, and (2) supplementing the collagen lost by aged skin directly by absorbing the exogenous deer bone protein extract into a dermis layer through the skin and integrating the exogenous deer bone protein extract into collagen fibers of the deer bone protein extract.
Comparative examples 1,
The acid extraction, hot water extraction, acid extraction and enzyme extraction of example 1 were performed simultaneously in parallel experiments.
1. Hot water extraction process
The method comprises decocting 1370g of the decalcified red deer bone powder of example 1 and 876g of the decalcified red deer bone powder of example 1 in water of 0.1Mpa twice, each for 2.5 hr, adding 13.70L of water at a ratio of 1:10 for the first time, adding 8.22L of water at a ratio of 1:6 for the second time, and mixing the bone soup for 22L. Concentrating the filtrate for 7 hr to obtain 1.5L concentrated solution, freezing the filtrate at-80 deg.C for 2 hr, freeze-drying at low temperature for 65 hr, and collecting dry powder to obtain 14.73g of hot water extraction method extract of red deer bone and 13.87g of hot water extraction method extract of sika deer bone.
Although the yield of the product obtained by the hot water extraction method is high (1.075 percent of red deer bone and 1.583 percent of red deer bone), the SDS-PAGE protein electrophoresis result shows that no obvious protein band is found in the red deer bone hot water extraction method extract and the red deer bone hot water extraction method extract, and obvious protein bands are found in the red deer bone protein extract obtained in example 1 (red deer bone acid extraction and enzyme extraction extract) and the red deer bone protein extract obtained in example 1 (red deer bone acid extraction and enzyme extraction extract) (figure 11).
2. Acid extraction method
Adding 10 times of water by mass into the decalcified red deer bone powder in example 1 or the decalcified red deer bone powder in example 1, adding acetic acid to adjust the acetic acid concentration to 0.5mol/L, adding hydrochloric acid to adjust the pH to 1.8, soaking at 4 ℃ for 3 days, and stirring once every 2h during soaking; filtering (100 mesh), and collecting filtrate to obtain crude solution of acid soluble collagen. Salting out, dialyzing and drying were carried out according to step one, 6 to 8, of example 1 to obtain a red deer bone acid extract and a red deer bone acid extract.
192g of the decalcified red deer bone powder obtained in example 1 to obtain 2.657g of red deer bone extract by acid extraction, the yield is 1.384%, and the protein content is 70.45%; 138.5g of the decalcified sika deer bone powder of example 1 to obtain 0.518g of the sika deer bone acid extraction extract, the yield is 0.374%, and the protein content is 68.04%.
Although the yield of the acid extraction method is similar to that of the acid extraction enzyme extraction method in example 1 (in example 1, 2.643g of the red deer bone protein extract can be obtained from 190.28g of the decalcified red deer bone powder, the yield is 1.389%, and 0.304g of the red deer bone protein extract can be obtained from 138.5g of the decalcified red deer bone powder, the yield is 0.364%), the protein contents of the red deer bone extract and the red deer bone extract obtained by the acid extraction method are lower than those of the acid extraction enzyme extraction method in example 1 (in example 1, the protein content of the red deer bone extract is 77%, and the protein content of the red deer bone extract is 72.47%), and the acid extraction enzyme extraction method is superior to the acid extraction method.
3. And (3) enzyme extraction method:
190g of the decalcified red deer bone powder in the embodiment 1 and 142.11g of the decalcified red deer bone powder in the embodiment 1 are respectively ground by a traditional Chinese medicine grinder. Adding 10 times of water by mass, adding acetic acid to adjust the concentration of the acetic acid to 0.5mol/L, adding hydrochloric acid to adjust the pH to 1.8, adding pepsin, placing in a digital display water bath constant-temperature oscillator, stirring once every 1h at the enzymatic reaction temperature of 37 ℃ for the enzymatic reaction time of 6h, and filtering through filter cloth to obtain the enzymatic acid-soluble collagen enzymatic hydrolysate. Salting out, dialyzing and drying were carried out according to step one, 6 to 8, of example 1 to obtain a red deer bone enzyme extract and a red deer bone enzyme extract.
Wherein, the adding amount of the pepsin meets the requirement that 1000U of the pepsin is added into every 1g of the decalcified deer bone powder by the mass of the decalcified deer bone powder.
190g of the decalcified red deer bone powder of example 1 gave 1.367g of the red deer bone enzyme extract, and 142.11g of the decalcified red deer bone powder of example 1 gave 0.304g of the red deer bone enzyme extract.
Taking the extract yield as an examination index, the red deer bone protein extract yield of the acid-extraction enzyme-extraction method of example 1 was 1.389% (190.28g of decalcified red deer bone powder could obtain 2.643g of red deer bone protein extract), the red deer bone protein extract yield of the acid-extraction enzyme-extraction method of example 1 was 0.364% (147.63g of decalcified red deer bone powder could obtain 0.537g of red deer bone protein extract), the enzyme-extraction method yields were 0.719% (190g of decalcified red deer bone powder could obtain 1.367g of red deer bone protein extract), and 0.214% (142.11g of decalcified red deer bone powder could obtain 0.518g of red deer bone protein extract), respectively. It can be seen that the acid-addition enzyme extraction method of example 1 all gave better yields than the enzyme extraction method.
The protein contents of the acid-extraction enzyme extraction method of example 1 were 77% (red deer bone protein extract) and 72.47% (red deer bone protein extract), respectively, and the protein contents of the enzyme extraction method were 95.82% (red deer bone protein extract) and 61.32% (red deer bone protein extract), respectively, using the protein content as an index for the examination. Although the protein content of the red deer bone extract obtained by the enzyme extraction method is higher than that of the acid extraction enzyme extraction method, the protein content of the red deer bone extract obtained by the enzyme extraction method is lower than that of the acid extraction enzyme extraction method. And from the comparison of color and odor of the finished product obtained after freeze drying, the sample obtained by the acid-added enzyme extraction method of example 1 is white sponge-like solid, has no peculiar smell and no fishy smell, and the sample obtained by the enzyme extraction method is yellow powder and has more obvious fishy smell (Table 1).
Therefore, the method for extracting deer bone protein extract is preferably the acid-extraction enzyme extraction method of example 1, compared in terms of color, smell, yield and protein content of the obtained product.
TABLE 1 comparison of the properties of the respective products
Comparative examples 2,
The red deer bone acid extraction extract, the red deer bone enzyme extraction extract and the red deer bone enzyme extraction extract obtained in comparative example 1, the red deer bone acid extraction enzyme extraction extract and the red deer bone acid extraction enzyme extraction extract obtained in example 1, and a commercially available deer bone protein extract product (deer brand deer bone collagen capsule, harbin cargilt bioengineering ltd, lot No. 20170602) and a commercially available recombinant human collagen product (human collagen lyophilized powder, Tianjin Yiyan biotechnology ltd, lot No. 20180101) were simultaneously subjected to parallel experiments to determine the effects of the products on the proliferation activity of HaCaT cells and HFF-1 cells and the antioxidant activity of the products. The experimental procedures were the same as in example 1.
The results show that:
(1) compared with the extracts obtained by an acid extraction method and an enzyme extraction method,the red deer bone protein extract and the sika deer bone protein extract (red deer bone acid extraction enzyme extraction extract and sika deer bone acid extraction enzyme extraction extract) of example 1 had the strongest proliferative activity on HaCaT cells and HFF-1 cells and the strongest antioxidant stress injury activity on HFF-1 cells (fig. 12, 13, 14). The method comprises the following specific steps: the highest survival rate of HaCaT cells under the action of the red deer bone protein extract in example 1 is 111.6% (0.25mg/mL), the highest survival rate of acid extraction red deer bone extract is 107.7% (0.25mg/mL), and the highest survival rate of enzyme extraction red deer bone extract is 110.2% (0.25 mg/mL); the highest survival rate of HaCaT cells under the action of the sika deer bone protein extract in example 1 is 114.1% (0.063mg/mL), the highest survival rate of acid extraction sika deer bone extract is 109.4% (0.063mg/mL), and the highest survival rate of enzyme extraction sika deer bone extract is 110.1% (0.063 mg/mL). The highest survival rate of HFF-1 cells under the action of the red deer bone protein extract in example 1 is 119.1% (1mg/mL), the highest survival rate of acid extraction red deer bone extract is 110.0% (1mg/mL), and the highest survival rate of enzyme extraction red deer bone extract is 112.8% (1 mg/mL); the highest survival rate of HFF-1 cells under the action of the sika deer bone protein extract in example 1 is 120.3% (1mg/mL), the acid extraction method of the sika deer bone extract is 112.6% (1mg/mL), and the enzyme extraction method of the sika deer bone extract is 115.8% (1 mg/mL). Cervus elaphus Linnaeus bone protein extract of example 1 under the action of H2O2The survival rate of HFF-1 cells damaged by the induced oxidative stress is 110.7 percent (1mg/mL) at most, the survival rate of the red deer bone extract by acid extraction is 94.87 percent (1mg/mL), and the survival rate of the red deer bone extract by enzyme extraction is 93.94 percent (1 mg/mL); the highest survival rate of HFF-1 cells under the action of the sika deer bone protein extract in example 1 is 114.9% (1mg/mL), the highest survival rate of acid-extracted sika deer bone extract is 96.06% (1mg/mL), and the highest survival rate of enzyme-extracted sika deer bone extract is 97.28% (1 mg/mL). The red deer bone protein extract and the sika deer bone protein extract (red deer bone acid extraction enzyme extraction extract and sika deer bone acid extraction enzyme extraction extract) of example 1 are resistant to H2O2The concentration of HFF-1 cell oxidative stress injury is lower, as shown in FIG. 14, 0.063mg/mL red deer bone protein extract and 0.016mg/mL sika deer bone protein extract can obviously repair H2O2Induced reduction of proliferative activity of HFF-1 cells (P)<0.01), indicating examples1 deer bone protein extract pair H2O2The induced HFF-1 cell oxidative stress injury has obvious protective effect, and the protective effect is shown only in the concentration of 0.25mg/mL of the acid extraction method red deer bone extract, the enzyme extraction method red deer bone extract, the acid extraction method red deer bone extract and the enzyme extraction method red deer bone extract; it is shown that the red deer bone protein extract and the sika deer bone protein extract (red deer bone acid extraction enzyme extraction extract and sika deer bone acid extraction enzyme extraction extract) of example 1 have better antioxidant stress injury activity on HFF-1 cells than acid extraction red deer bone extract, enzyme extraction red deer bone extract, acid extraction sika deer bone extract and enzyme extraction sika deer bone extract.
(2) The commercial deer bone protein extract products have certain proliferation promoting effect on HaCaT cells and HFF-1 cells (FIGS. 12 and 13), but the antioxidant injury activity is inferior to that of the deer bone protein extracts (Cervus Elaphus L., Cervus Nippon Temminck) of example 1, and the deer bone protein extracts (Cervus Elaphus L. acid extraction enzyme extraction extract and Cervus Nippon Temminck. acid extraction enzyme extraction extract) of example 1 for resisting H2O2The concentration of HFF-1 cell oxidative stress injury is lower, as shown in FIG. 14, 0.063mg/mL red deer bone protein extract and 0.016mg/mL sika deer bone protein extract can obviously repair H2O2Induced reduction of proliferative activity of HFF-1 cells (P)<0.01), indicating that the deer bone protein extract of example 1 is on H2O2The induced HFF-1 cell oxidative stress injury has obvious protection, and the protection is shown only at the concentration of 0.25mg/mL of a commercial deer bone protein extract product. Therefore, the deer bone protein extract (red deer, sika deer) of example 1 has better activity of resisting oxidative stress injury on HFF-1 cells than the commercially available deer bone protein extract product.
(3) The commercial recombinant human collagen product also has a certain proliferation promoting effect on HaCaT cells and HFF-1 cells (FIGS. 12 and 13), but has no effect of resisting oxidative damage (FIG. 14). As shown in FIG. 14, the extract of the deer bone protein of example 1at 0.063-1 mg/ml and the deer bone protein of example 1at 0.016-1 mg/mlThe protein extracts can obviously enhance H2O2Cell survival (P) of HFF-1 cells induced oxidative stress injury<0.05), and the commercial recombinant human collagen product does not show obvious effect of resisting oxidative stress damage (P is more than 0.05), which shows that the red deer bone protein extract and the sika deer bone protein extract (the red deer bone acid extraction enzyme extraction extract and the sika deer bone acid extraction enzyme extraction extract) in example 1 have obviously better activity of resisting oxidative stress damage to HFF-1 cells than the commercial recombinant human collagen product.
In conclusion, the red deer bone protein extract and the sika deer bone protein extract (red deer bone acid extraction enzyme extraction extract and sika deer bone acid extraction enzyme extraction extract) of example 1 have the proliferation promoting activity on HaCaT cells and HFF-1 cells, and on H2O2The induced HFF-1 cells have antioxidant stress injury activity. The red deer bone protein extract and the sika deer bone protein extract (red deer bone acid extraction enzyme extraction extract and sika deer bone acid extraction enzyme extraction extract) in example 1 have higher proliferation promoting capability on HaCaT cells and HFF-1 cells than the red deer bone extract, enzyme extraction red deer bone extract, acid extraction sika deer bone extract and enzyme extraction sika deer bone extract in the acid extraction method, are similar to the commercial sika deer bone protein extract products and the commercial recombinant human collagen products, but have obviously better antioxidant injury effect on HFF-1 cells than the red deer bone extract, enzyme extraction red deer bone extract, acid extraction sika deer bone extract, enzyme extraction sika deer bone extract, commercial sika deer bone protein extract products and commercial recombinant human collagen products.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. A method for preparing deer bone protein extract comprises the following steps:
(1) acid extraction: extracting the decalcified deer bone powder with an acid solution, collecting an extracting solution, and naming the extracting solution as an acid-soluble collagen crude solution;
(2) enzyme extraction: adding pepsin into the crude acid-soluble collagen solution for enzymolysis, filtering after enzymolysis, collecting filtrate, and naming the filtrate as enzymatic acid-soluble collagen enzymatic hydrolysate;
(3) salting out: salting out the enzymatic acid-soluble collagen enzymatic hydrolysate, and collecting the precipitate;
(4) and (3) dialysis: dialyzing the precipitate to obtain deer bone protein extract.
2. The method of claim 1, wherein: the method further comprises the step of drying the obtained dialysis product after dialyzing the precipitate to obtain a deer bone protein extract in a solid state.
3. The method according to claim 1 or 2, characterized in that: the acid solution is acetic acid solution with acetic acid concentration of 0.5 mol/L.
4. A method according to any one of claims 1-3, characterized in that: the acid extraction was carried out in a system with a pH of 1.8.
5. The method according to any one of claims 1-4, wherein: in the enzyme extraction, the enzymolysis temperature is 37 ℃, and the enzymolysis time is 6 h.
6. The method according to any one of claims 1-5, wherein: the decalcified deer bone powder is prepared by the following steps: decocting Os Cervi with water, collecting bone, soaking in hydrochloric acid solution until the bone is softened, washing with water until pH is neutral, taking out bone, oven drying, and pulverizing to obtain decalcified Os Cervi powder.
7. The method according to any one of claims 1-6, wherein: the cut-off molecular weight of the dialysis was 1000 Da.
8. The method according to any one of claims 1-7, wherein: the deer bone is red deer bone and/or sika deer bone, and the deer bone protein extract is red deer bone protein extract and/or sika deer bone protein extract.
9. Deer bone protein extract obtained by extraction from deer bone using the method of any one of claims 1-8.
10. Any of the following applications:
u1. use of the deer bone protein extract of claim 9 in enhancing the anti-oxidative damage activity of skin cells or preparing products for enhancing the anti-oxidative damage activity of skin cells;
u2. use of the deer bone protein extract of claim 9 in anti-skin aging or preparing anti-skin aging products;
u3. use of the deer bone protein extract of claim 9 for increasing the proliferation activity of skin keratinocytes or for preparing a product for increasing the proliferation activity of skin keratinocytes;
u4. use of the deer bone protein extract of claim 9 for increasing the proliferation activity of skin fibroblasts or for preparing a product for increasing the proliferation activity of skin fibroblasts;
u5. use of the deer bone protein extract of claim 9 in repairing the decrease of the proliferation ability of skin fibroblasts caused by oxidative stress damage or preparing a product for repairing the decrease of the proliferation ability of skin fibroblasts caused by oxidative stress damage;
u6. use of the deer bone protein extract of claim 9 in transdermal absorption into the dermis layer and integration into autologous collagen fibers, or in the preparation of a product capable of transdermal absorption into the dermis layer and integration into autologous collagen fibers;
u7. use of the deer bone protein extract of claim 9 in supplementing collagen lost from aged skin or in preparing a product for supplementing collagen lost from aged skin.
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