Astragaloside-collagen sponge scaffold as well as preparation method and application thereof

Abstract

The invention relates to an astragaloside-collagen sponge scaffold as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The collagen scaffold in the astragaloside-collagen sponge scaffold is prepared by taking bovine achilles tendon as a raw material, and astragaloside is loaded on the collagen scaffold; the collagen scaffold is of a porous structure with highly interconnected inside, pores are tightly arranged, and the pore size is 40-200 [mu] m; the weight ratio of the astragaloside to the collagen sponge scaffold is (1-5): 200. The astragaloside-collagen scaffold disclosed by the invention not only can promote wound healing, but also can reduce hyperplasia of scar tissues.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an astragaloside IV-collagen sponge scaffold and a preparation method and application thereof. Background Art

[0002] In recent years, many natural medicines at home and abroad have achieved good clinical effects in promoting skin wound healing. With the rapid development of molecular biology technology and the cross-disciplinary reference, the research on wound healing at the molecular level has gradually become a hot topic. The mechanism of Chinese medicine promoting wound healing by regulating the action of wound-related cytokines has been discovered by more and more researchers.

[0003] The use of Chinese medicine for external treatment of wound healing has a long history in my country. Its research has gone from macroscopic to microscopic, from gross level to cellular level and even genetic molecular level, and has formed a certain theoretical system, but the corresponding mechanism is still in the exploratory stage. Overall, the research on external treatment of skin ulcers with Chinese medicine has indeed achieved gratifying results, but the overall research still lags behind other fields, and basic experiments are relatively scarce. Wound repair and healing is a very complex biological process, which requires us to devote ourselves to the regulatory mechanism of Chinese medicine on difficult-to-heal skin ulcers and strengthen the research on animal and cell models to conduct a large number of experiments and clinical work in order to make further progress.

[0004] Common treatment methods such as debridement, skin transplantation, and anti-infection therapy in modern medicine at home and abroad basically remain at the stage of waiting for the wound to heal itself, making many special wounds difficult to heal, and there is no good solution so far. Therefore, seeking a material that can accelerate and promote wound healing has become a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an astragaloside IV-collagen sponge scaffold capable of accelerating wound healing, and a preparation method and application thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In the first aspect, the present invention provides an astragaloside IV-collagen sponge scaffold, wherein the collagen scaffold is made of bovine Achilles tendon as raw material and is loaded with astragaloside IV; the collagen scaffold has a highly interconnected porous structure inside, the pores are closely arranged, and the pore size is 40-200 μm; the weight ratio of the astragaloside IV to the collagen sponge scaffold is (1-5):200.

[0008] Astragalus is known as the "holy medicine for sores". It has the functions of strengthening the body's defense system, treating qi and blood deficiency, enhancing the physiological metabolism of human cells, regulating immune function (significantly promoting the activity of natural killer cells (NK cells) in mice and stimulating the proliferation of NK cells), and plays an important role in anti-inflammatory and fibroblast proliferation in the process of wound healing. Unlike Western medicine for treating immune cells caused by trauma, external Chinese medicine can significantly increase the oxygen metabolism activity of these immune cells and enhance the role of fibronectin. Astragaloside IV is one of the main active ingredients of Astragalus. Due to its low content and good effect, it is known as "super Astragaloside polysaccharide". It is a cycloartine-type triterpenoid saponin compound with multiple pharmacological effects such as lowering blood sugar, protecting tissues and organs, regulating the body's immunity, anti-apoptosis, anti-inflammatory and anti-viral. It improves immunity and also has the function of promoting wound healing.

[0009] The present invention provides a collagen scaffold loaded with astragaloside IV, and the collagen scaffold is made of bovine Achilles tendon as a raw material, ensuring the porosity and loading capacity of the prepared collagen scaffold. After a series of treatments, not only the natural three-dimensional porous structure is retained, but also the collagen scaffold has a highly interconnected porous structure inside, the pores are closely arranged, and the pore size is 40-200μm. The microporous structure ensures the good capillary action of the collagen scaffold, so that blood can quickly and effectively penetrate through the collagen scaffold. The interconnected pore structure allows blood cells and proteins to enter, ensuring the nutrients required for effective wound healing. At the same time, as a carrier for loading astragaloside IV, it can continuously and effectively release the loaded astragaloside IV, achieving the effect of promoting wound repair and healing.

[0010] As a preferred implementation of the first aspect, the pore size is 40-100 μm.

[0011] As a preferred embodiment of the first aspect, the weight ratio of astragaloside IV to the collagen sponge scaffold is 1:200.

[0012] In a second aspect, the present invention provides a method for preparing the astragaloside IV-collagen sponge scaffold as described in the first aspect, comprising the following steps:

[0013] 1) Extract type I collagen from animal tissues and prepare collagen solution;

[0014] 2) mixing astragaloside IV with the collagen solution in step 1) to obtain a mixed solution;

[0015] 3) freeze-drying the mixed solution of step 3) to obtain astragaloside IV-collagen sponge scaffold.

[0016] As a preferred embodiment of the second aspect, the animal tissue in step 1) is bovine Achilles tendon.

[0017] As a preferred implementation of the second aspect, the step 1) is specifically as follows: using an ethanol solution to degrease and disinfect the bovine Achilles tendon, adding an acetic acid-pepsin solution for enzymatic extraction, collecting the supernatant by centrifugation, and using an isoelectric precipitation method to remove pepsin for purification to obtain a crude type I collagen extract; dissolving the crude extract in acetic acid for ultrafiltration purification column chromatography to obtain a purified type I collagen solution.

[0018] As a preferred implementation of the second aspect, the solid-to-liquid ratio of the bovine Achilles tendon to the acetic acid-pepsin solution is 1:60 (g / mL); the mass ratio of the bovine Achilles tendon to pepsin is 1:0.03.

[0019] As a preferred implementation of the second aspect, the acetic acid concentration in the acetic acid-pepsin solution is 0.5 mol / L; the relative molecular mass retained by the ultrafiltration purification column chromatography is 100 kDa, and the pressure during the ultrafiltration purification process is maintained below 0.8 MPa.

[0020] As a preferred embodiment of the second aspect, in the step 2), the weight ratio of astragaloside IV to the collagen sponge scaffold is (1-5):200.

[0021] In a third aspect, the present invention provides use of the astragaloside IV-collagen sponge scaffold as described in the first aspect in the preparation of a drug or preparation for treating wound healing.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention combines astragaloside IV with collagen to prepare an astragaloside IV-collagen sponge scaffold, which can promote wound healing. The interior of the astragaloside IV-collagen sponge scaffold of the present invention has a highly interconnected porous structure, the pores are closely arranged, and the pore size is 40-200 μm, which ensures the good capillary action of the collagen scaffold, so that blood can quickly pass through the collagen scaffold for effective penetration and astragaloside IV loading. The astragaloside IV-collagen scaffold of the present invention can not only promote wound healing but also reduce the proliferation of scar tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of extraction and purification of collagen solution;

[0025] Figure 2 This is the UV spectrum of high-purity collagen;

[0026] Figure 3 This is the infrared spectrum of the collagen sponge scaffold;

[0027] Figure 4 This is the SDS-PAGE gel electrophoresis image of the collagen sponge scaffold;

[0028] Figure 5 This is an electron microscopic image of the astragaloside IV-collagen sponge scaffold;

[0029] Figure 6 Schematic diagram of in vitro drug release from astragaloside IV-collagen sponge scaffold. DETAILED DESCRIPTION

[0030] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0031] In the following embodiments, bovine Achilles tendon and pepsin were provided by Zhejiang Chongshan Biological Products Co., Ltd.; glacial acetic acid, Coomassie Brilliant Blue R-250, copper sulfate and sodium hydroxide, AR, Tianjin Damao Chemical Reagent Factory; 75% ethanol solution by volume, Dezhou Anjie High-tech Disinfection Products Co., Ltd.; type I collagen, SLBQ7099V, Sigma, USA; GL-10MD large-capacity high-speed refrigerated centrifuge, Xiangyi Company; FD-1-50 vacuum freeze dryer, Beijing Bokang Company; UV-6000PC ultraviolet spectrophotometer, Shanghai Yuanxi Company; PowerPacTM universal electrophoresis instrument, Bio-Rad Company; HYP-3 intelligent digestion furnace, KDN-812 nitrogen determination instrument distillation device, Shanghai Fiber Inspection Company; WQF-510A Fourier transform infrared spectrometer, Beijing Duanli Analysis Company; Chirascan circular dichroism spectrometer, Applied Photophysics Company, UK; ultrafiltration purification column, with a relative molecular mass cutoff of 100kDa, Dongguan Fangtu Biotechnology Co., Ltd.

[0032] Example 1 Preparation method of astragaloside IV-collagen sponge scaffold

[0033] This embodiment provides a method for preparing an astragaloside IV-collagen sponge scaffold, and the specific steps are as follows:

[0034] 1. Prepare collagen solution ( Figure 1 ):

[0035] 500g of bovine Achilles tendon was frozen and sliced, soaked in 75% ethanol solution for 6 hours for degreasing and disinfection, rinsed with water, crushed, and dispersed in 0.5mol / L acetic acid-pepsin solution at a solid-liquid ratio of 1:60 (g / mL) [m (bovine Achilles tendon) : m (pepsin) = 1:0.03], and enzymatically extracted at below 10°C using a cold circulation device, with slow stirring during the extraction process. After 72 hours, the extract was centrifuged at 8000r / min at about 4°C for 5 minutes, the supernatant was collected, and entered the purification stage. The purification steps are as follows:

[0036] First, the isoelectric precipitation method is used to remove pepsin: slowly add 1 mol / L sodium hydroxide solution to the supernatant, stir slowly, adjust the pH of the solution to 5.8-7.0, and collagen precipitation can be observed. Let it stand for 2 minutes, filter, and quickly wash the collagen precipitate with pure water 3-4 times to obtain a crude extract of type I collagen. The crude extract is dissolved in 0.5 mol / L acetic acid to prepare a 1g / L collagen solution, and ultrafiltration purification column chromatography is performed. The relative molecular mass of the purification column is 100kDa, and the pressure is maintained below 0.8MPa during the ultrafiltration purification process. To ensure the stability of collagen, try to use a cold circulation device with a temperature of 4°C. During the purification process, purified water is continuously added until the pH of the purified collagen solution is ≈5, and a high-purity collagen solution is obtained.

[0037] 2. Preparation of Astragaloside IV-Collagen Sponge Scaffold

[0038] The concentrated collagen obtained after extraction and purification was diluted with 0.5 mol / L acetic acid to prepare 8 mg / ml collagen solution. 23.88 g of 8 mg / g collagen solution was mixed with 0.12 g of astragaloside IV and stirred thoroughly. The mixed collagen mixture was injected into a 12-well culture plate, 2 g per well. It was then placed in -20 ° C and frozen for 12 hours. After being taken out, it was immediately placed in a vacuum freeze dryer and vacuum dried for 24 hours to prepare an astragaloside IV-collagen sponge scaffold with a diameter of about 2 cm, and each scaffold contained about 10 mg of astragaloside IV. Using the same method, 24 g of 8 mg / g collagen solution was injected into a 12-well culture plate at 2 g per well and freeze-dried to prepare a collagen sponge scaffold without drug loading.

[0039] Example 2 Characterization of the High-Purity Collagen

[0040] 1. UV spectrometry

[0041] Method: The collagen solution prepared in Example 1 was diluted with 0.5 mol / L acetic acid to a concentration of 1 mg / ml, and the acetic acid solution was used as a blank control. A full wavelength scan was performed using a UV-6000PC ultraviolet spectrophotometer.

[0042] Results: Figure 2 As shown, the extracted collagen has ultraviolet absorption at a wavelength of 190nm to 300nm, and its maximum ultraviolet absorption peak is at 234nm, which is consistent with the ultraviolet absorption characteristic peak of type I collagen reported in the literature, indicating that the collagen extracted and purified in Example 1 is type I collagen.

[0043] 2. Infrared spectrum test

[0044] Method: The collagen sponge scaffold sample prepared in Example 1 was cut into pieces, and ground into fine powder with potassium bromide. The sample was infrared scanned by Fourier transform infrared spectrometer in the scanning range of 4000-500cm-1, and the results were analyzed by Origin8.0 software.

[0045] Results: The infrared spectrum of the collagen sponge scaffold is shown in Figure 3 As shown, at 3300-3400cm -1 3315cm in range -1 NH stretching vibration in the amide A band at 1656 cm -1 Nearby is the C=O stretching vibration peak in the amide I band, at 1540 cm -1 Nearby is the NH bending or CN stretching vibration in the amide II band, 1450 cm -1 -1230cm -1 The absorption peak near 1240cm indicates the integrity of the triple helix structure of collagen. -1 and 1450cm -1 The ratio of the peak intensities is very close to the characteristic value of collagen. Therefore, it can be seen that the collagen sponge scaffold prepared in Example 1 meets the characteristics of type I collagen, and the triple helical structure of the scaffold is relatively intact.

[0046] 3. SDS-PAGE gel electrophoresis

[0047] Method: The collagen solution prepared in Example 1 was prepared to a concentration of 1 mg / mL, and electrophoresis was performed using a 5% concentration gel and a 10% separation gel. Constant voltage electrophoresis was performed at a voltage of 80 V, and electrophoresis was stopped when bromophenol blue ran to the bottom of the gel. The separation gel was placed in a Coomassie Brilliant Blue R-250 solution for staining for 30 minutes, and decolorized with an acetic acid-ethanol decolorizing solution until the background color of the separation gel was transparent.

[0048] Results: Figure 4 As shown in the SDS-PAGE gel electrophoresis spectrum of the extracted collagen, there are two bands at a molecular weight of 130kDa, one band is due to the overlap of two α1 peptide chains, and the other band represents a single α2 peptide chain. There is a band at a molecular weight of 250kDa, which corresponds to the β chain in the collagen structure, which is a dimer of the α peptide chain. The band above 250kDa is the trimer γ chain of the α peptide chain. Since there are no other bands below the molecular weight of 100kDa, the results show that the structure of type I collagen is relatively intact, and it has not been decomposed into polypeptides or hydrolyzed into small molecule collagen. The extracted collagen has a high purity.

[0049] Example 3 Detection of the porosity of astragaloside IV-collagen sponge scaffold

[0050] Porosity of the scaffold: The porosity of the scaffold was determined by the ethanol penetration method. A centrifuge tube with a volume of 10 mL was filled with ethanol and weighed (denoted as W1). The scaffold was weighed (denoted as Ws) and then immersed in a centrifuge tube filled with ethanol. It was placed in an ultrasonic instrument for 30 minutes, and then filled with ethanol and weighed (denoted as W2). After taking out the sample that was fully absorbed with ethanol, the remaining ethanol and the centrifuge tube were weighed (denoted as W3). At the same time, the collagen sponge scaffold without drug loading was used as the control group, and the experiment was repeated 3 times to take the average value. Calculate the porosity:

[0051] Porosity = (W2-W3-Ws) / (W1-W3) x 100%.

[0052] Through electron microscope observation, the sponge scaffold is white, soft and loose ( Figure 5 ). Under an electron microscope, it can be observed that the interior has a highly interconnected porous structure, the pores are closely arranged, and the pore size ranges from 40-200μm.

[0053] Example 4 Detection of drug release from astragaloside IV-collagen sponge scaffold

[0054] In vitro drug release of the scaffold: The astragaloside IV-collagen sponge scaffold was completely immersed in 10 mL of phosphate buffer solution and placed in a 37°C constant temperature shaker. After being placed in the constant temperature shaker, the timing was started, and samples were taken and stored at 5 min, 10 min, 20 min, 30 min, 60 min, and 120 min. The drug content in the solution was detected by high performance liquid chromatography, and the cumulative release was calculated. The standard curve was obtained by the peak area of ​​the gradient concentration of astragaloside IV samples, and then the drug release at different times was calculated according to the standard curve, and the cumulative release was calculated after cumulative addition. Chromatographic conditions: The chromatographic column was Ultimate XB-C18 (4.6 mm × 250 mm, 5 μm); the mobile phase was water-methanol (85:15); the flow rate was 1.0 mL / min; the detection wavelength was 275 nm; the column temperature was 30°C; the injection volume was 5 μL. The experiment was repeated 3 times and the average was taken.

[0055] The results are as follows Figure 6 As shown in the figure, astragaloside IV in the astragaloside IV-collagen sponge scaffold can be rapidly released in vitro and is almost completely released within 30 minutes, which indicates that the collagen sponge scaffold does not affect the release of astragaloside IV.

[0056] Example 3 Animal Experiment

[0057] 1. Group processing

[0058] Fifteen SD rats were randomly divided into 5 groups, with 3 rats in each group. The first group of rats served as the negative control group, the second group of rats were given blank collagen sponge scaffolds, the third group of rats were given astragaloside IV-collagen scaffolds, the fourth group of rats were given simple astragaloside IV, and the fifth group served as the normal control group.

[0059] 2. Animal model preparation

[0060] 2.1 Preparation of immunosuppression model

[0061] Methods: The rats were weighed and recorded. Except for the normal group, the rats in the other groups were intraperitoneally injected with hydrocortisone (15 mg / kg) once a day for 3 consecutive days to establish the immunodeficient rat model.

[0062] 2.2 Wound modeling

[0063] SD rats were anesthetized (10% chloral hydrate, 3 ml / kg, intraperitoneal injection). The rats were placed in a prone position, the backs were shaved, and a circular skin with a diameter of 2 cm was excised on the midline of the back of the rats with a scalpel.

[0064] 2.3 Medication and postoperative care

[0065] Collagen sponge scaffolds, astragaloside IV-collagen sponge scaffolds, and pure astragaloside IV powder were implanted into the skin wounds of rats, and the wounds of rats were bandaged with medical tape. The wounds of the negative control group were only bandaged with gauze, without other treatment. After surgery, penicillin-streptomycin mixture (double antibiotic) was injected to prevent wound infection. The experimental mice were kept warm and raised in separate cages after they woke up. After completing all the above steps, the rats were moved into the cage for observation until they all woke up.

[0066] 3. Experimental results

[0067] 3.1 Healing rate test

[0068] On days 0, 7, 14, 21, and 28 after wound modeling, the wound area of ​​each group of rats was measured, and the average value of the group was taken. After attaching a translucent plastic film to the wound, the wound was traced and photographed along the edge, and the photos were scanned into the computer. The healing area was calculated using Image-ProPlus software, where the area before drug administration after successful modeling (day 0) was the original wound area. Healing rate (%) = (original wound area - current wound area) / original wound area × 100%, and the results are shown in Table 1 below:

[0069] Table 1: Healing status of different groups

[0070]

[0071] 3.2 Detection of thickness of new epithelium

[0072] On the 28th day after wound modeling, the thickness of the new epithelium of each group of rats was measured and the average value of the group was taken. Rats in each group were killed, and the full-thickness tissue including the entire wound surface and the surrounding 5mm was taken. The tissue blocks were used to prepare routine paraffin-embedded sections, and divided into 5 fields of view under a 40x optical microscope. The thickness of the new epithelium was measured once in each field of view and the average value was taken. The results are shown in Table 2:

[0073] Table 2: Thickness of new epithelium in different groups

[0074]

[0075] From the above animal experimental results, it can be seen that compared with the single collagen empty scaffold group and the astragaloside group, the astragaloside-collagen scaffold group of the present invention can promote wound healing. On the 28th day, its wound healing was 99.8%, while the other groups were all lower than 80%; at the same time, the astragaloside-collagen scaffold group of the present invention can also reduce the proliferation of scar tissue. On the 28th day, the thickness of the new epithelium in the astragaloside-collagen scaffold group was only 21.3μm, while the other groups were all above 30μm. It can be seen that the astragaloside-collagen scaffold of the present invention can not only promote wound healing but also reduce the proliferation of scar tissue.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An astragaloside IV-collagen sponge scaffold, characterized in that: The collagen scaffold is made of bovine Achilles tendon as raw material and is loaded with astragaloside IV; The collagen scaffold has a highly interconnected porous structure, the pores are closely arranged, and the pore size is 40-200 μm; The weight ratio of astragaloside IV to the collagen sponge scaffold is (1-5):

200.

2. The astragaloside IV-collagen sponge scaffold according to claim 1, characterized in that: The pore size is 40-100 μm.

3. The astragaloside IV-collagen sponge scaffold according to claim 1, characterized in that: The weight ratio of astragaloside IV to the collagen sponge scaffold is 1:

200.

4. A method for preparing the astragaloside IV-collagen sponge scaffold according to claim 1, characterized in that: The following steps are involved: 1) Extract type I collagen from animal tissues and prepare collagen solution; 2) mixing astragaloside IV with the collagen solution in step 1) to obtain a mixed solution; 3) freeze-drying the mixed solution of step 3) to obtain astragaloside IV-collagen sponge scaffold.

5. The preparation method according to claim 4, characterized in that: The animal tissue in step 1) is bovine Achilles tendon.

6. The preparation method according to claim 5, characterized in that: The step 1) specifically comprises: using an ethanol solution to degrease and disinfect the bovine Achilles tendon, adding an acetic acid-pepsin solution for enzymatic extraction, collecting the supernatant by centrifugation, and using an isoelectric precipitation method to remove pepsin for purification to obtain a crude type I collagen extract; dissolving the crude extract in acetic acid for ultrafiltration purification column chromatography to obtain a purified type I collagen solution.

7. The preparation method according to claim 6, characterized in that: The solid-liquid ratio of the bovine Achilles tendon to the acetic acid-pepsin solution is 1:60 (g / mL); the mass ratio of the bovine Achilles tendon to pepsin is 1:0.

03.

8. The preparation method according to claim 6, characterized in that: The acetic acid concentration in the acetic acid-pepsin solution is 0.5 mol / L; the relative molecular mass retained by the ultrafiltration purification column chromatography is 100 kDa, and the pressure during the ultrafiltration purification process is maintained below 0.8 MPa.

9. The preparation method according to claim 4, characterized in that: The weight ratio of astragaloside IV to the collagen sponge scaffold in step 2) is (1-5):

200.

10. Use of the astragaloside IV-collagen sponge scaffold according to claim 1 or 2 in the preparation of a medicine or preparation for treating wound healing.

Images