阅读说明：本技术 天然胶原材料及天然胶原材料的制备方法、用途 (Natural collagen material, preparation method and application thereof ) 是由 景伟 王华生 赵博 魏鹏飞 李红阳 胡苗苗 于 2021-09-06 设计创作，主要内容包括：本公开属于生物医用材料领域,具体来说,本公开涉及一种天然胶原材料,天然胶原材料的制备方法、应用,和皮下填充剂。本公开中的天然胶原材料中III型胶原蛋白的含量显著提高,具有优异的弹性和皮肤修复功能,可有效恢复皮肤弹性、紧致肌肤,增加皮肤水润度,实现受损皮肤修复、抗衰老等功效,适合作为受损、衰老皮肤的皮下填充材料。(The disclosure belongs to the field of biomedical materials, and particularly relates to a natural collagen material, a preparation method and application of the natural collagen material, and a subcutaneous filler. The content of the type III collagen in the natural collagen material is remarkably improved, the natural collagen material has excellent elasticity and skin repairing function, can effectively recover the elasticity and tighten the skin of the skin, increases the skin moisture, realizes the effects of repairing the damaged skin, resisting aging and the like, and is suitable for being used as a subcutaneous filling material for the damaged and aged skin.)

1. A natural collagen material, wherein said natural collagen material comprises type I collagen and type III collagen; based on the total mass of the natural collagen material, the content of the type I collagen is 30-35%, and the content of the type III collagen is 65-70%;

the natural collagen material is derived from small intestine submucosa, preferably pig small intestine submucosa.

2. The natural collagen material according to claim 1, wherein said natural collagen material has a residual amount of DNA of less than 4ng/mg and a residual amount of α -Gal of less than 1.0U/L.

3. A preparation method of a natural collagen material is characterized by comprising the following steps:

taking a small intestine submucosa material, and sequentially carrying out virus inactivation treatment, degreasing treatment, cell removal treatment and enzymolysis treatment to obtain a collagen crude extract;

purifying and drying the crude collagen extraction solution to obtain a natural collagen material;

preferably, the native collagen material is the native collagen material of any one of claims 1-2.

4. The method according to claim 3, wherein the virus inactivation treatment comprises: cleaning a small intestine submucosa material, soaking the small intestine submucosa material in a virus inactivation solution, and performing virus inactivation treatment under the oscillation condition; preferably, the volume ratio of the small intestine submucosa material to the virus inactivation solution is (0.5-5): 30; preferably, the virus inactivation solution is an aqueous solution comprising (0.5-5)% (v/v) peroxyacetic acid and (20-25)% (v/v) ethanol; preferably, the time of the virus inactivation treatment is (1-5) h.

5. The production method according to claim 3 or 4, wherein the step of degreasing comprises: soaking the virus-inactivated small intestine submucosa material in a degreasing solution, and performing degreasing treatment under the ultrasonic condition; preferably, the volume ratio of the small intestine submucosa material to the degreasing solution is (0.1-1): 2; preferably, the degreasing solution is 1-2 wt% alkaline lipase solution; preferably, the degreasing solution has a pH of 7-10; preferably, the small intestine submucosa material is soaked in the degreasing fluid, the degreasing fluid is replaced after ultrasonic treatment for 30-60min, and the operation is repeated for more than 3 times.

6. The method for preparing a drug according to any one of claims 3 to 5, wherein the step of the decellularization treatment comprises: soaking the degreased small intestine submucosa material in a cell removing solution, and performing cell removing treatment under the ultrasonic condition; preferably, the volume ratio of the small intestine submucosa material to the decellularization solution is (0.5-5): 30; preferably, the decellularization solution is a phosphate buffer comprising 0.02-0.05 wt% trypsin and 0.1-0.4 wt% EDTA; preferably, the time of the decellularization treatment is 30min, and then the decellularized small intestine submucosa material is obtained by washing and drying.

7. The method according to any one of claims 3 to 6, wherein the step of subjecting to enzymatic treatment comprises:

taking a small intestine submucosa material without cells, crushing the small intestine submucosa material, and soaking the small intestine submucosa material in an acid solution;

crushing and stirring the soaked small intestine submucosa material by adopting crushing equipment, adding pepsin, performing enzymolysis treatment, centrifuging, and removing precipitates to obtain a collagen crude extract;

preferably, the volume ratio of the small intestine submucosa material to the acidic solution is (0.5-5): 30; preferably, the acid solution is (0.1-1) mol/L acetic acid solution, the time for soaking the small intestine submucosa material in the acid solution is 12h, and the temperature equipment is 4-10 ℃; preferably, the mass ratio of the small intestine submucosa material to the pepsin is (8-12) to 1; preferably, the enzymolysis treatment is carried out for 48h to 96h under the stirring condition.

8. The method of any one of claims 3-7, wherein the step of purifying comprises:

filtering the crude collagen extraction solution for one time, and then performing salting-out treatment to obtain precipitate particles; dissolving the precipitate particles in an acidic solution, washing and dissolving for the second time, and performing membrane separation, filtration and concentration to obtain a collagen concentrated solution containing natural collagen materials.

9. Use of a native collagen material according to any one of claims 1 to 2, or a native collagen material prepared according to the method of any one of claims 3 to 8, for the preparation of a medical cosmetic material or skin care product; preferably, the medical cosmetic material is a subcutaneous filler; preferably, the subcutaneous filler is an injection.

10. A subcutaneous filler, wherein the subcutaneous filler comprises the native collagen material of any one of claims 1-2, or the native collagen material produced by the method of any one of claims 3-8.

Technical Field

The disclosure belongs to the field of biomedical materials, and particularly relates to a natural collagen material, a preparation method and application of the natural collagen material, and a subcutaneous filler.

Background

Collagen (collagen) is an important structural protein of an animal extracellular matrix and accounts for about 25-30% of the total protein content. Collagen supports the integrity, elasticity and strength of connective tissue, thereby maintaining the morphology and function of skin, cartilage and bone. The skin is affected by natural aging and external environmental factors, collagen of skin tissues is lost, and various skin problems such as skin looseness, wrinkles, color spots and the like are caused. How to repair skin damage and delay skin aging is an important problem which is generally concerned by people at present.

There are 28 kinds of collagen contained in the human body, and mainly type I collagen and type III collagen are distributed in the skin. The I type collagen fibers are thick in diameter, are arranged tightly, are in a compact strip bundle shape, are criss-cross and are very suitable for forming a firm fiber network, and are relatively hard collagen. The type III collagen fiber is a fine fiber in a loose net shape, and the higher the content of the type III collagen fiber, the finer the fiber bundle, and the higher the elasticity. The ratio of type I/III collagen content in the skin of infants is about 1. With age, type III collagen decreases and type I collagen increases. The ratio of type I/III collagen in adult skin is about 2.5, and the type III collagen content decreases in a cliff-like manner. With the lapse of type III collagen, the dermal tissue undergoes phase collapse, fiber rupture, and lipoatrophy, which results in loss of elasticity and luster of the skin, and the appearance of spots and wrinkles. The III type collagen has excellent elasticity and repair function, so that the supplementation of the exogenous III type collagen is an important way for delaying senility and repairing skin injury.

At present, the sources of collagen mainly include natural collagen extracted from tissues and artificial collagen obtained by techniques such as genetic engineering. At present, the collagen is widely used and mainly obtained naturally, and is generally extracted from tendons, corium and tails of animals such as cows, pigs, rats, fishes and the like. However, the natural collagen extracted by the conventional method is mainly type I collagen, and the content of type III collagen is low. Further, the removal of the impurity components (nucleic acid fragments, cell fragments) from collagen extracted by an acid method, an alkaline method, a salt method, an enzymatic method, or the like requires a complicated purification process.

The high-purity III collagen can be prepared by adopting a genetic engineering means, and the problems of complex preparation process, high cost, long period and the like exist. On the other hand, currently, a prokaryotic expression system of escherichia coli is mostly adopted for preparing the collagen, and certain endotoxin possibly generated in bacterial cells such as escherichia coli is mixed into the collagen, so that the application of the collagen in the biomedical field is limited.

Disclosure of Invention

Problems to be solved by the invention

In view of the problems in the prior art. For example, the natural collagen extracted by the traditional method has low content of type III collagen and high content of impurities; the method for preparing the type III collagen by the genetic engineering has the problems of high cost, complex preparation process and certain biological potential safety hazard. Therefore, the natural collagen material has high content of type III collagen obtained by taking small intestine submucosa as a raw material, low immunogenicity and high biosafety, has excellent functions of repairing skin and recovering skin elasticity, and is suitable for being used as a subcutaneous filling material for damaged and aged skin.

Means for solving the problems

In a first aspect, the present disclosure provides a native collagen material, wherein the native collagen material comprises type I collagen and type III collagen; based on the total mass of the natural collagen material, the content of the type I collagen is 30-35%, and the content of the type III collagen is 65-70%;

the natural collagen material is derived from small intestine submucosa, preferably pig small intestine submucosa.

In some embodiments, the native collagen material of the present disclosure, wherein the native collagen material has a residual amount of DNA less than 4ng/mg and a residual amount of α -Gal less than 1.0U/L.

In a second aspect, the present disclosure provides a method for preparing a natural collagen material, comprising the following steps:

taking a small intestine submucosa material, and sequentially carrying out virus inactivation treatment, degreasing treatment, cell removal treatment and enzymolysis treatment to obtain a collagen crude extract;

purifying and drying the crude collagen extraction solution to obtain a natural collagen material;

preferably, the native collagen material is a native collagen material according to the first aspect.

In some embodiments, the method of manufacturing according to the present disclosure, wherein the step of virus inactivation treatment comprises: cleaning a small intestine submucosa material, soaking the small intestine submucosa material in a virus inactivation solution, and performing virus inactivation treatment under the oscillation condition; preferably, the volume ratio of the small intestine submucosa material to the virus inactivation solution is (0.5-5): 30; preferably, the virus inactivation solution is an aqueous solution comprising (0.5-5)% (v/v) peroxyacetic acid and (20-25)% (v/v) ethanol; preferably, the time of the virus inactivation treatment is (1-5) h.

In some embodiments, the method of manufacturing according to the present disclosure, wherein the step of degreasing comprises: soaking the virus-inactivated small intestine submucosa material in a degreasing solution, and performing degreasing treatment under the ultrasonic condition; preferably, the volume ratio of the small intestine submucosa material to the degreasing solution is (0.1-1): 2; preferably, the degreasing solution is 1-2 wt% alkaline lipase solution; preferably, the degreasing solution has a pH of 7-10; preferably, the small intestine submucosa material is soaked in the degreasing fluid, the degreasing fluid is replaced after ultrasonic treatment for 30-60min, and the operation is repeated for more than 3 times.

In some embodiments, the method of manufacturing according to the present disclosure, wherein the step of decellularizing comprises: soaking the degreased small intestine submucosa material in a cell removing solution, and performing cell removing treatment under the ultrasonic condition; preferably, the volume ratio of the small intestine submucosa material to the decellularization solution is (0.5-5): 30; preferably, the decellularization solution is a phosphate buffer comprising 0.02-0.05 wt% trypsin and 0.1-0.4 wt% EDTA; preferably, the time of the decellularization treatment is 30min, and then the decellularized small intestine submucosa material is obtained by washing and drying.

In some embodiments, the preparation method according to the present disclosure, wherein the step of performing enzymatic treatment comprises:

taking a small intestine submucosa material without cells, crushing the small intestine submucosa material, and soaking the small intestine submucosa material in an acid solution;

crushing and stirring the soaked small intestine submucosa material by adopting crushing equipment, adding pepsin, performing enzymolysis treatment, centrifuging, and removing precipitates to obtain a collagen crude extract;

preferably, the volume ratio of the small intestine submucosa material to the acidic solution is (0.5-5): 30; preferably, the acid solution is (0.1-1) mol/L acetic acid solution, the time for soaking the small intestine submucosa material in the acid solution is 12h, and the temperature equipment is 4-10 ℃; preferably, the mass ratio of the small intestine submucosa material to the pepsin is (8-12) to 1; preferably, the enzymolysis treatment is carried out for 48h to 96h under the stirring condition.

In some embodiments, the method of manufacturing according to the present disclosure, wherein the step of purifying comprises:

filtering the crude collagen extraction solution for one time, and then performing salting-out treatment to obtain precipitate particles; dissolving the precipitate particles in an acidic solution, washing and dissolving for the second time, and performing membrane separation, filtration and concentration to obtain a collagen concentrated solution containing natural collagen materials.

In a third aspect, the present disclosure provides the use of a native collagen material according to the first aspect, or a native collagen material prepared according to the method of the second aspect, in the preparation of a medical cosmetic material or skin care product; preferably, the medical cosmetic material is a subcutaneous filler; preferably, the subcutaneous filler is an injection.

In a fourth aspect, the present disclosure provides a subcutaneous filler, wherein the subcutaneous filler comprises the native collagen material according to the first aspect, or the native collagen material prepared according to the method of the second aspect.

ADVANTAGEOUS EFFECTS OF INVENTION

In some embodiments, the natural collagen material provided by the present disclosure is derived from small intestine submucosa, and compared with collagen extracted from achilles tendon, dermis, tail, and the like, the content of type III collagen in the natural collagen material provided by the present disclosure is significantly increased, and the natural collagen material has excellent elasticity and skin repair function, can effectively restore skin elasticity, tighten skin, increase skin moisture, and achieve the effects of damaged skin repair, aging resistance, and the like.

In some embodiments, the method for preparing a natural collagen material provided by the present invention can remove intracellular components (such as DNA fragments, cell membranes, and cell nucleus fragments) in the upper layer tissue of the small intestine mucosa through degreasing and decellularization, reduce residues of immunogenic substances such as nucleic acids, improve the biological safety of the natural collagen material, simplify the purification process of the natural collagen material, and reduce the preparation difficulty and the preparation cost.

In some embodiments, the subcutaneous fillers provided by the present disclosure, including the natural collagen materials provided by the present disclosure, are suitable for subcutaneous tissue filling, effectively tightening skin, improving wrinkles and damage of skin, and restoring elasticity, luster and water wettability of skin. In addition, the natural collagen material has a longer retention time and a longer lasting effect than fillers such as hyaluronic acid.

Drawings

FIG. 1 shows SEM pictures of natural collagen material, wherein A, B, C, D in FIG. 1 is scanning electron microscope pictures under a field of view of 1.00mm, 500 μm and 100 μm in sequence.

Figure 2 shows a fourier transform infrared spectrum of a native collagen material.

FIG. 3 shows a UV-VIS absorption spectrum of a native collagen material.

Figure 4 shows a circular dichroism plot of native collagen material.

Detailed Description

Various exemplary embodiments, features and aspects of the invention will be described in detail below. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps which are well known to those skilled in the art have not been described in detail so as not to obscure the invention.

All units used in the specification are international standard units unless otherwise stated, and numerical values and numerical ranges appearing in the present invention should be understood to include systematic errors inevitable in industrial production.

In the present specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

In the present specification, reference to "some particular/preferred embodiments," "other particular/preferred embodiments," "embodiments," and the like, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

In the present specification, the numerical range represented by "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In the present specification, "v/v" represents a volume percentage content, and "wt%" represents a mass percentage content.

In addition, in the present specification, the "water" includes any feasible water that can be used, such as deionized water, distilled water, ion-exchanged water, double distilled water, high purity water, and purified water.

In the present specification, when "normal temperature" or "room temperature" is used, the temperature may be 10 to 40 ℃.

Natural collagen material

A first aspect of the present disclosure provides a native collagen material comprising type I collagen and type III collagen; based on the total mass of the natural collagen material, the content of the type I collagen is 30-35%, and the content of the type III collagen is 65-70%;

the natural collagen material is derived from small intestine submucosa, preferably pig small intestine submucosa.

The natural collagen material disclosed by the invention is obtained by taking Small Intestinal Submucosa (SIS) as a raw material, the content of III-type collagen in the extracted collagen is higher, a natural, safe and low-immunogenicity collagen source is provided for supplementing the loss of collagen, and the natural collagen material is suitable for being used as a skin collagen supplementing material. After being applied to damaged or aged skin, the natural collagen material can exert the effects of tightening the skin and recovering the elasticity of the skin, so that the skin is delicate and glossy, the state of the damaged skin is effectively improved, and the skin aging is delayed.

In some preferred embodiments, the native collagen material is derived from porcine small intestine submucosa, which contains high levels of type III collagen, and is suitable as a subcutaneous filler material for repairing skin elasticity.

In some embodiments, the natural collagen material provided by the present disclosure has a low content of immunogenic substances, wherein the residual amount of DNA is less than 4ng/mg, and the detection of α -galactosidase (α -Gal) by an enzyme-linked immunosorbent assay method has a residual amount of less than 1.0U/L of the detection limit of the α -galactosidase detection kit. The natural collagen material has low content of immune source substances and high biological safety, is suitable for being implanted into subcutaneous tissues, and can be used as a filling material for repairing damaged and aged skin and recovering skin elasticity.

Preparation method of natural collagen material

A second aspect of the present disclosure provides a method for preparing a native collagen material, comprising the steps of:

taking a small intestine submucosa material, and sequentially carrying out virus inactivation treatment, degreasing treatment, cell removal treatment and enzymolysis treatment to obtain a collagen crude extract;

and purifying and drying the crude collagen extraction solution to obtain a natural collagen material.

The preparation method disclosed by the invention can effectively remove intracellular components such as nucleic acid fragments, cell membranes, cell nucleus fragments and the like and reduce the residues of immune source substances through degreasing and decellularization treatment of the small intestine submucosa material. Compared with the traditional tissue collagen extraction method, the preparation method disclosed by the invention can effectively simplify the purification treatment steps of collagen, and can be used for preparing the natural collagen material with high type III collagen content and high biological safety. Compared with the method for preparing the type III collagen by genetic engineering, the preparation method disclosed by the invention has the advantages of low cost, simple steps, easiness in operation and the like.

In some embodiments, the natural collagen material with the content of type I collagen of 30-35% and the content of type III collagen of 65-70% by total mass can be prepared by applying the preparation method disclosed by the present disclosure, and the natural collagen material with the reasonable ratio of type III collagen to type I collagen is provided for skin repair and anti-aging.

In some embodiments, the small intestine submucosa material is the intermediate small intestine submucosa material that is left after the mucosal and fascia layers of the small intestine have been removed. The small intestine submucosa material is washed with clear water and used for subsequent treatment.

When the volume of the small intestine submucosa material is measured, the cleaned material is placed on a stainless steel net for 1-5min and then is put into a measuring cylinder for measurement.

In some embodiments, the step of virus inactivation treatment comprises: the small intestine submucosa material is cleaned and then soaked in a virus inactivation solution, and virus inactivation treatment is carried out under the oscillation condition.

For the virus inactivation solution, it is an aqueous solution comprising (0.5-5)% (v/v) peroxyacetic acid and (20-25)% (v/v) ethanol. Illustratively, the amount of peroxyacetic acid in the virus inactivation solution is 0.8% (v/v), 1% (v/v), 1.5% (v/v), 2% (v/v), 2.5% (v/v), 3% (v/v), 3.5% (v/v), 4% (v/v), 4.5% (v/v), etc.; the content of ethanol in the virus-inactivating solution is 21% (v/v), 22% (v/v), 23% (v/v), 24% (v/v), or the like. In some preferred embodiments, the virus inactivation solution is an aqueous solution comprising 1% (v/v) peroxyacetic acid and 24% (v/v) ethanol.

According to the volume ratio of the small intestine submucosa material to the virus inactivation solution (0.5-5) to 30, for example, 0.8:30, 1:30, 2:30, 3:30, 4:30, and the like. In some preferred embodiments, the volume ratio of small intestine submucosa material to virus inactivation solution is 1: 30. Soaking the small intestine submucosa material in a virus inactivation solution, and soaking under the oscillation condition. The small intestine submucosa material is soaked in the virus inactivating solution for 1-5h, preferably for 2h, and the shaking speed is 40 r/min. The soaked small intestine submucosa material is washed with water. The oscillation can be achieved by means of a shaker, magnetic stirrer, or the like.

By the virus inactivation treatment, virus components in the small intestine submucosa material can be effectively inactivated, the content of bacterial endotoxin can be further reduced, and the biological safety of the natural collagen material is ensured.

In some embodiments, the step of degreasing comprises: and (3) soaking the virus-inactivated small intestine submucosa material in a degreasing solution, and performing degreasing treatment under the ultrasonic condition.

Further, the degreasing solution is 1-2 wt% alkaline lipase solution. Illustratively, the concentration of the alkaline lipase solution is 1 wt%, 1.2 wt%, 1.4 wt%, 1.5 wt%, 1.7 wt%, 2 wt%, etc. In some preferred embodiments, the pH of the degreasing solution is 7-10, e.g., pH 7, 8, 9, 10, etc. Exemplary small intestine submucosa material to degreasing solution volume ratios are 0.1:2, 0.2:2, 0.5:2, 0.8:2, 1:2, and so forth, in terms of a small intestine submucosa material to degreasing solution volume ratio of (0.1-1): 2. Preferably, the volume ratio of the small intestine submucosa material to the degreasing solution is 1:10, the small intestine submucosa material is soaked in the degreasing solution, and the degreasing solution is replaced after degreasing treatment is carried out for 30-60min under the ultrasonic condition; the procedure was repeated 3 more times and then the small intestine submucosa material was washed with water.

Through degreasing treatment, non-collagen impurity components mixed in the crude collagen extracting solution can be effectively reduced, so that the subsequent purification treatment process is simplified, and the loss of collagen is avoided. Compared with the existing treatment mode of placing tissues in water for ultrasonic cleaning, the alkaline lipase solution with the concentration of 2 wt% is selected in the method, so that the unwanted impurity components such as lipid can be more effectively removed, and the loss of collagen components in the extraction process is reduced.

In some embodiments, the step of decellularizing comprises: and (3) soaking the degreased small intestine submucosa material in a cell removing solution, and performing cell removing treatment under the ultrasonic condition.

For the decellularization solution, a phosphate buffer containing 0.02-0.05 wt% trypsin and 0.1-0.4 wt% EDTA. Illustratively, the content of trypsin in the phosphate buffer is 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt%, etc., and the content of EDTA is 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, etc. In some preferred embodiments, the phosphate buffer contains 0.02 wt% trypsin and 0.1 wt% EDTA. Soaking the small intestine submucosa material in the cell removing solution according to the volume ratio of the small intestine submucosa material to the cell removing solution of (0.5-5) to 30, and soaking for 30min under the ultrasonic condition. Further, the small intestine submucosa material and the cell removing solution are soaked in an ultrasonic cleaning machine for 30min according to the volume ratio of 1: 30.

After the ultrasound treatment, the small intestine submucosa material is washed and dried. In some more specific embodiments, the drying is carried out under conditions in which the small intestine submucosa material is dried at a temperature of 37 ℃ for 24 hours.

The conditions of the cell removing treatment in the disclosure are mild, so that the immune components such as nucleic acid, cell membranes, nuclear fragments and the like can be sufficiently removed, the immunogenicity of the natural collagen material is reduced, the natural collagen material is suitable for being implanted into subcutaneous tissues, and the loss of the collagen components is avoided.

In the conventional collagen extraction process, after a crude collagen extraction solution is obtained, it is necessary to repeat a purification operation of salting out and dissolving to remove impurity components such as nucleic acid mixed in collagen. This process results in loss of collagen and, due to the use of large amounts of neutral salts, requires washing or dialysis at a later stage, is time consuming and complicated to operate. In the method, the immune source substances such as fat, nucleic acid, alpha-Gal and the like can be reduced to the minimum by combining degreasing and decellularization treatment, so that the purification process of the crude collagen solution can be completed by only once salting out and microfiltration membrane filtration.

In some embodiments, the step of enzymatically treating comprises: taking a small intestine submucosa material without cells, crushing the small intestine submucosa material, and soaking the small intestine submucosa material in an acid solution; and (3) crushing and stirring the soaked small intestine submucosa material by adopting crushing equipment, adding pepsin, performing enzymolysis treatment, centrifuging, and removing precipitates to obtain a collagen crude extract solution.

Further, the acidic solution is an acetic acid solution of 0.1 to 1mol/L, and illustratively, the concentration of the acetic acid solution is 0.2mol/L, 0.3mol/L, 0.5mol/L, 0.7mol/L, 0.8mol/L, or the like. Preferably, the concentration of the acetic acid solution is 0.5 mol/L. The dried small intestine submucosa material is cut into pieces and then mixed with the acidic solution at a volume ratio of small intestine submucosa material to acidic solution of (0.5-5):30, e.g., 0.8:30, 1:30, 2:30, 3:30, 4:30, etc. In some preferred embodiments, the small intestine submucosa material is soaked in an acidic solution in a volume ratio of 1: 30. In some preferred embodiments, the small intestine submucosa material is soaked in an acidic solution for 12 hours at a low temperature of 4-10 ℃ to facilitate the solubilization of collagen.

For the enzymolysis treatment, the soaked small intestine submucosa material is pulverized and stirred at low temperature for 5min by a pulverizer, then pepsin is added into the acidic solution according to the mass ratio of 1 (8-12) of the pepsin to the small intestine submucosa material, and the mixture is mechanically stirred at low temperature (4-10 ℃) for 48-96h, so that the collagen in the small intestine submucosa material is fully dissolved, and the extraction efficiency of type III collagen and type I collagen is improved. In some preferred embodiments, the mass ratio of pepsin to small intestine submucosa material is 1: 10; after adding pepsin into the acidic solution, the time of low-temperature mechanical stirring is 72 h.

After the enzymolysis treatment, the mixed solution is centrifuged, and the obtained supernatant is the collagen crude extraction solution dissolved with the collagen.

In some embodiments, the step of purifying comprises: filtering the crude collagen extraction solution for one time, and then performing salting-out treatment to obtain precipitate particles; dissolving the precipitate particles in an acidic solution, washing and dissolving for the second time, and performing membrane separation, filtration and concentration to obtain a collagen concentrated solution containing natural collagen materials.

Further, the acidic solution is an acetic acid solution of 0.5mol/L to sufficiently dissolve the native collagen material in the precipitated particles.

For filtration, the solution is filtered using a microfiltration membrane to filter out particles in the solution. Wherein, the micro-filtration membrane aperture of the first filtration is 0.2-0.4 μm, and insoluble particles in the collagen crude extract solution can be removed by the first filtration. The aperture of the filter membrane filtered after the secondary cleaning is 0.05-0.1 μm, and the redundant micromolecular impurities can be removed through the filtering after the secondary cleaning, so that the purity of the collagen concentrated solution is improved. In the purification step in the method, only pepsin and residual non-collagen components in the material need to be removed, so that the purification of the crude collagen extraction solution can be completed only by once salting out and microporous membrane filtration, and the method has the advantages of simple steps and less loss of collagen.

In some embodiments, the native collagen material is obtained by freeze-drying a collagen concentrate.

According to the preparation method provided by the disclosure, through interaction of each step and optimization of treatment conditions of each step, the purification process can be simplified, the efficient extraction of collagen in the small intestine submucosa material is realized, and the natural collagen material containing 65-70% of type III collagen and 30-35% of type I collagen is obtained. The proportion of the type III collagen in the natural collagen material is high, the content of the immune source substance is low, the biosafety is high, and the collagen material is in a loose and porous structure and is suitable for the adhesion and growth of cells; the natural collagen material maintains the triple-helix conformation of collagen, has high collagen activity, and is suitable for being used as a subcutaneous filling material for repairing damaged and aged skin.

Subcutaneous filler

A third aspect of the present disclosure provides a subcutaneous filler comprising a native collagen material according to the first aspect, or a native collagen material prepared according to the method of the second aspect.

Use of natural collagen material

A fourth aspect of the present disclosure provides the use of a native collagen material, in some embodiments, as or in the preparation of a medical cosmetic material. In some embodiments, the medical cosmetic material is a subcutaneous filler. The natural collagen material may be mixed with one or more excipients and formulated into any administrable dosage form. Exemplary adjuvants include solvents, thickeners, solubilizing agents, emulsifiers, binders, disintegrants, lubricants, wetting agents, tonicity adjusting agents, pH adjusting agents, stabilizers, surfactants, and the like. In some preferred embodiments, the subcutaneous filler is an injection.

In other embodiments, the native collagen material is used to prepare a skin care product. The skin care product can be patch type, water preparation type, gel type, emulsion type or cream type.

The natural collagen material disclosed by the invention has the advantages that the proportion of the type III collagen and the type I collagen is close to that of the skin in the infancy period, the high-content type III collagen is obtained, the natural collagen material is suitable for subcutaneous filling, the elasticity of the skin can be effectively recovered, the skin is tightened, and the moisture and the glossiness of the skin are increased.

Examples

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

This example provides a method for preparing a native collagen material, comprising the steps of:

(1) cleaning pretreatment: at room temperature, the mucosal layer and fascia layer of the small intestine are scraped, the middle small intestine submucosa is left, and the material is washed by purified water for standby.

(2) Virus inactivation: preparing a virus inactivation water solution (1% (v/v) peroxyacetic acid + 24% (v/v) ethanol), soaking the cleaned small intestine submucosa material in the virus inactivation solution, wherein the ratio of the small intestine submucosa material to the solution is 1:30, placing the small intestine submucosa material on a shaking table, and soaking the small intestine submucosa material for 2 hours at room temperature, wherein the speed of the shaking table is 40 r/min. And cleaning with purified water after soaking.

(3) Degreasing: preparing 2 wt% alkaline lipase solution, soaking the virus-killed small intestine submucosa material in a degreasing solution at a ratio of 1:10(v: v), ultrasonically soaking in an ultrasonic cleaning machine for 30min, replacing the degreasing solution, and repeating the operation for 3 times. Purified water washes the small intestine submucosa material.

(4) And (3) cell removal: preparing a decellularized phosphate buffer solution (0.025 wt% of trypsin and 0.19 wt% of EDTA), transferring the small intestine submucosa material into a decellularized solution, ultrasonically soaking the small intestine submucosa material and the decellularized solution in an ultrasonic cleaning machine for 30min at a ratio of 1:30(v: v), cleaning the small intestine submucosa material by purified water after ultrasonic treatment, and drying the small intestine submucosa material in an oven at 37 ℃ for 24 h.

(5) Acid soaking: cutting the dried small intestine submucosa material into about 1cm²The small intestine submucosa material after being decellularized is soaked for 12 hours by 0.5mol/L acetic acid solution at 4 ℃, and the ratio of the small intestine submucosa material to the decellularized solution is 1:30(v: v).

(6) Enzymolysis: pulverizing the soaked small intestine submucosa material with a pulverizer at low temperature, stirring for 5min, adding pepsin (the mass ratio of pepsin to small intestine submucosa material is 1:10) into the solution, and mechanically stirring at low temperature for 72 h. And after enzymolysis, centrifuging the mixed solution at 8000r/min for 10min, and taking the supernatant as a crude collagen solution.

(7) And (3) purification: the crude collagen solution is treated by a microfiltration membrane, and insoluble particles are filtered by a filter membrane with the aperture of 0.2-0.4 micron. Salting out by using neutral salt sodium chloride or ammonium sulfate, centrifuging after salting out to obtain precipitated particles, washing by using distilled water, dissolving by using 0.5mol/L acetic acid, and concentrating by using an ultrafiltration membrane with the pore diameter of 0.05-0.1 micrometer to remove redundant micromolecular impurities to obtain the high-purity collagen concentrated solution.

(8) And (3) drying: and (4) freeze-drying the high-purity collagen concentrated solution to obtain the natural collagen material. Placing the collagen concentrated solution into a freeze-drying machine (FD-1A-50 of Beijing Bo Yi kang laboratory instruments Co., Ltd.) cold trap for pre-cooling for 60-120min, starting a vacuum pump after pre-cooling, and vacuum drying, wherein the temperature of the cold trap is set to-35 deg.C to-45 deg.C, such as-37 deg.C, -38 deg.C, -40 deg.C, -42 deg.C, and-44 deg.C. Preferably, the temperature of freeze-drying is-40 ℃. The natural collagen material is obtained by a freeze-drying step.

The residual quantity of DNA in the natural collagen material is lower than 4ng/mg, the alpha-galactosidase (alpha-Gal) is detected by adopting an enzyme-linked immunosorbent assay method, and the residual quantity is lower than the detection limit of the alpha-galactosidase detection kit by 1.0U/L.

Performance testing

The natural collagen material prepared in example 1 was tested as follows:

1. SEM analysis

Shearing a proper amount of natural collagen material with proper size, adhering the natural collagen material on a sample table, observing the surface appearance of the collagen sponge by using a scanning electron microscope, and setting the accelerating voltage to be 10 kV.

The microstructure and morphology of the lyophilized native collagen material was studied with SEM. As shown in FIG. 1, SEM shows a highly interconnected porous structure, the natural collagen material has a loose and porous structure as a whole, and it can be seen that the existing pore size of the partial fibrosis structure is in the range of 150-500 μm, which is suitable for the adhesion and growth of cells.

2. Fourier transform infrared spectroscopy detection

Weighing about 2mg of natural collagen material, freezing, grinding and crushing by liquid nitrogen, uniformly mixing with 200mg of potassium bromide, pressing into a sheet under 15MPa after complete grinding, and preparing a sample by using an infrared spectrometer at 4000-400 cm^-1Performing infrared spectrum scanning 64 times with resolution of 4cm^-1。

Figure 2 shows a fourier transform infrared spectrum of a native collagen material. Collagen infrared spectrum at 3293.53cm^-1Near is the absorption peak of the stretching vibration of N-H, 1634.48cm^-1Near the C ═ O stretching vibration absorption peak of the amide I band, 1545.88cm^-1The vicinity is the N-H bending vibration absorption peak of the amide II band. 1450 and 1230cm^-1The ratio of the nearby absorption peaks is more than 0.5, which indicates the integrity of the triple-helical structure of the collagen. Collagen protein is 1455.99 and 1236.05cm^-1The ratio of peak intensities was 1.02, which is a characteristic value of collagen.

3. Ultraviolet-visible absorption spectroscopy

Dissolving the freeze-dried collagen in 0.5mol/L acetic acid solution to enable the concentration of the collagen solution to reach 0.5mg/mL, comparing with 0.5mol/L HAC solution, and scanning an ultraviolet absorption spectrum of the collagen solution within a wavelength range of 200-400 nm by using an ultraviolet spectrophotometer.

FIG. 3 shows a UV-VIS absorption spectrum of a native collagen material. Greater light absorption occurs at a wavelength around 235 nm. The ultraviolet spectrogram of the collagen is actually the result of the addition of various ultraviolet chromogenic groups of protein molecules, and the maximum ultraviolet absorption peak of the collagen I is at 235nm due to the hydroxyproline and proline containing a benzene ring structure, which is consistent with the ultraviolet absorption characteristic peak of the collagen I reported in the literature. The absorption peak occurs mainly due to the n → pi transition of the carbonyl group in the peptide chain.

4. Circular Dichroism (CD)

Preparing a collagen solution with the concentration of 20 mug/mL, and scanning by using a circular dichroism spectroscopy instrument within the range of 180-260 nm. Setting parameters: the thickness of the liquid pool is 1mm, the scanning speed is 10nm/min, the scanning temperature is 19 ℃, and the width of the slit is 1.5 nm.

In the extreme ultraviolet region, the CD spectrum of collagen has a maximum positive peak at 222nm, a maximum negative peak at 197nm, and a crossover point at about 213 nm. The ratio of positive to negative peaks (Rpn) is unique to triple helix conformation and can be used to identify triple helix conformations.

The range of Rpn of the collagen with triple-helix structure is 0.09-0.15. Figure 4 shows a circular dichroism plot of native collagen material, the chromatogram corresponding to the characteristics of collagen, with an Rpn value of 0.112, indicating the integrity of the collagen triple helix structure. The results show that the purification process does not compromise the triple helix conformation of collagen.

5. Analysis of collagen composition

According to the experiment, a high performance liquid chromatography-mass spectrometry (HPLC-MS) combined technology is adopted, collagen in a sample is specifically degraded by trypsin, characteristic polypeptides of I, III type collagen are searched and identified by the HPLC-MS combined technology, quantitative analysis is carried out on the characteristic polypeptides, different types of collagen in materials can be identified, and accurate quantification is carried out. And (3) respectively denaturing the standard substance and the sample, performing enzymolysis, and detecting an zymolyte by adopting HPLC-MS. Wherein the lyophilized natural collagen material is pulverized, and 10mg is weighed, and NH is added₄HCO₃The buffer (50mM, pH 8.0) was prepared as a 1mg/mL solution. Denaturing at 100 ℃ for 10min, cooling to room temperature, adding trypsin according to the mass ratio of 50:1 of collagen to trypsin, carrying out enzymolysis at 37 ℃ for 16h, and directly detecting the collagen component in the enzymolysis product by HPLC-MS. Through analysis aiming at the detection result, the natural collagen material comprises 65 to 70 mass percent of type III collagen and 30 to 3 mass percent of type III collagen5% type I collagen.

The above examples of the present disclosure are merely examples provided for clearly illustrating the present disclosure and are not intended to limit the embodiments of the present disclosure. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present disclosure should be included in the protection scope of the claims of the present disclosure.