CN103333270B - Collagen peptide grafted sodium alginate sulfuric ester, preparation method and its usage - Google Patents

Abstract

The invention belongs to the field of macromolecular chemical materials, and in particular relates to a collagen peptide grafted with sodium alginate sulfate, a preparation method and an application thereof. Collagen peptide is grafted with sodium alginate sulfate, which is obtained by activating the sodium carboxylate group of sodium alginate sulfate and then grafting reaction with collagen peptide at 25-65°C. The preparation process of the method is simple, and the prepared collagen peptide grafted with sodium alginate sulfate has good properties of removing hydrogen peroxide and promoting proliferation of fibroblasts, and can significantly promote wound healing.

Description

Collagen peptide grafted sodium alginate sulfate, preparation method and use thereof

technical field

The invention belongs to the field of macromolecular chemical materials, and in particular relates to a collagen peptide grafted with sodium alginate sulfate, a preparation method and an application thereof.

Background technique

There are many free hydroxyl groups and sodium carboxylate groups distributed on the skeleton of sodium alginate, which is an ideal chemical modification substance. Sodium alginate sulfate is the product of sulfonation modification of the hydroxyl group of sodium alginate. Compared with sodium alginate, sodium alginate sulfate not only retains the biocompatibility, non-toxic, non-immunogenic properties of sodium alginate, but also has good water solubility, hypoglycemic effect, anticoagulant, anti Oxidation, anti-tumor effect, anti-inflammatory effect and promotion of cell proliferation and other properties. Sodium alginate sulfate has a structure similar to heparin, has certain anticoagulant activity, and can promote wound healing in a manner similar to heparin. Due to the improvement of solubility, the application range of sodium alginate sulfate is wider, and it has many applications in food and medicine. Collagen peptide is a hydrolyzed product of collagen, which is easily absorbed by the human body and has excellent hydrophilicity, moisture absorption and moisturizing properties. If collagen peptides can be grafted onto sodium alginate sulfate, it will play a better role in wound healing. .

Contents of the invention

The technical problem to be solved by the present invention is to provide collagen peptide grafted with sodium alginate sulfate, a preparation method and use thereof. The preparation process of the method is simple, and the prepared collagen peptide grafted with sodium alginate sulfate has good properties of removing hydrogen peroxide and promoting proliferation of fibroblasts, and can significantly promote wound healing.

In order to solve the problems of the technologies described above, the technical solution provided by the invention is:

The collagen peptide grafted with sodium alginate sulfate is characterized in that it is obtained by activating the sodium carboxylate group of the sodium alginate sulfate and then grafting the collagen peptide at 25-65°C.

According to the above scheme, the substitution degree of the sodium carboxylate group in the collagen peptide grafted with sodium alginate sulfate is 0.062-0.469, preferably 0.124-0.469, more preferably 0.228-0.469.

The present invention also provides a preparation method of the collagen peptide grafted with sodium alginate sulfate.

The preparation method of collagen peptide grafted with sodium alginate sulfate is characterized in that: firstly, the sodium carboxylate group of sodium alginate sulfate is activated and reacted with carboxyl activator, and then it is mixed with collagen peptide at 25-65°C Grafting reaction and post-treatment are carried out.

According to the above scheme, the activation reaction is to react sodium alginate sulfate with carboxyl activator at 25-65°C for 12-28 hours under the condition of pH 5-7, and the carboxyl activator is 1- Mixture of (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

According to the above scheme, the sodium alginate sulfate in the activation reaction is firstly dissolved with a 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 5-7, and then a carboxyl activator is added. reaction.

According to the above scheme, the mass of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in the carboxyl activator The ratio is 3.3:1-6.7:1; the mass ratio of N-hydroxysuccinimide (NHS) to sodium alginate sulfate is 0.2:1-0.6:1.

According to the above scheme, the degree of substitution of sodium alginate sulfate (SO ₃ Na) is 1-1.8.

According to the above scheme, the mass ratio of the collagen peptide to sodium alginate sulfate is 0.4:1-2.0:1.

According to the above scheme, the reaction time for the grafting reaction after adding the collagen peptide is 10-30 minutes.

According to the above scheme, the post-treatment is dialysis, purification and drying after the completion of the reaction by adding collagen peptides.

According to the above scheme, the application of the collagen peptide grafted with sodium alginate sulfate in wound healing.

Beneficial effects of the present invention: the preparation process of the present invention is simple, low in cost, environment-friendly and remarkable in function; the prepared collagen peptide grafted with sodium alginate sulfate has good water solubility, can remove hydrogen peroxide and can promote fibroblast proliferation, Can significantly promote wound healing.

Description of drawings

Fig. 1 is the infrared spectrogram of the collagen peptide graft sodium alginate sulfate prepared in embodiment 1;

Fig. 2 is the scavenging rate of hydrogen peroxide to the collagen peptide graft sodium alginate sulfate prepared by embodiment 1-6;

Fig. 3 is the cell survival rate of the sodium alginate sulfate of the collagen peptide grafting sodium alginate sulfate prepared by embodiment 1,2,6 and ungrafted collagen peptide to fibroblast;

Fig. 4 is the observation of the effect of the collagen peptide grafting sodium alginate sulfate of embodiment 1 on burn wound healing;

5 is an optical microscope observation of the effect of collagen peptide grafted with sodium alginate sulfate in Example 2 on burn wound healing.

detailed description

The present invention will be further described below in conjunction with specific examples.

Example 1

Prepare 0.2mol/l 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, and adjust its pH value to 6.5 with 0.3mol/l sodium chloride solution. Measure 50ml of MES buffer solution into a three-neck flask, add 0.6g of sodium alginate sulfate to fully dissolve it. Subsequently, 0.40 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.12 g of N-hydroxysuccinimide (NHS) were sequentially added. After magnetic stirring at 55° C. for 20 hours, 0.48 g of collagen peptide was added, and magnetic stirring was continued for 10 min. The reacted solution was purified by dialysis for three days, and then freeze-dried to obtain collagen peptide-grafted sodium alginate sulfate. It was determined that the sodium carboxyl group in the collagen peptide-grafted sodium alginate sulfate was replaced by collagen peptide The degree of substitution was 0.436. Figure 1 shows the infrared spectrum of the collagen peptide grafted with sodium alginate sulfate characterized by infrared. In the figure: the absorption peaks at 1651cm ^-1 and 1555cm ^-1 are assigned to the amide I band and the amide II band respectively; the absorption peaks at 1245cm ^-1 and 875cm ^-1 are respectively assigned to the S=O bond Stretching vibration absorption peak and COS vibration absorption peak.

Example 2

Prepare 0.2mol/l 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, and adjust its pH value to 6.5 with 0.3mol/l sodium chloride solution. Measure 50ml of MES buffer solution into a three-neck flask, add 0.6g of sodium alginate sulfate to fully dissolve it. Subsequently, 0.60 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.18 g of N-hydroxysuccinimide (NHS) were added in sequence. After magnetic stirring at 45° C. for 20 hours, 1.2 g of collagen peptide was added, and magnetic stirring was continued for 10 min. The reacted solution was purified by dialysis for three days, and then freeze-dried to obtain collagen peptide-grafted sodium alginate sulfate. It was determined that the sodium carboxyl group in the collagen peptide-grafted sodium alginate sulfate was replaced by collagen peptide The degree of substitution was 0.310.

Example 3

Prepare 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution at pH 6.5, then measure an appropriate amount of the MES buffer solution into a three-necked flask, add 0.6g of sodium alginate sulfate to fully dissolve it. Subsequently, 1.19 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.36 g of N-hydroxysuccinimide (NHS) were sequentially added. After 12 hours of magnetic stirring at 35 °C, 0.48 g of collagen peptide was added, and the magnetic stirring reaction was continued. The solution after the reaction was dialyzed, purified, and dried to obtain collagen peptide grafted sodium alginate sulfate. After determination: the degree of substitution of the carboxyl sodium group in the collagen peptide grafted sodium alginate sulfate was replaced by collagen peptide is 0.062.

Example 4

Prepare 0.2mol/l 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, and adjust its pH value to 6.5 with 0.3mol/l sodium chloride solution. Measure the MES buffer solution into a three-necked flask, and add 0.6 g of sodium alginate sulfate to fully dissolve it. Subsequently, 0.40 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.12 g of N-hydroxysuccinimide (NHS) were sequentially added. After magnetic stirring at 35° C. for 24 hours, 0.48 g of collagen peptide was added, and magnetic stirring was continued for 30 min. The reacted solution was purified by dialysis for three days, and then freeze-dried to obtain collagen peptide-grafted sodium alginate sulfate. It was determined that the sodium carboxyl group in the collagen peptide-grafted sodium alginate sulfate was replaced by collagen peptide The degree of substitution was 0.359.

Example 5

Prepare a buffer solution of 2-(N-morpholino)ethanesulfonic acid (MES) at pH 5.5. Measure an appropriate amount of MES buffer solution into a three-necked flask, and add 0.6 g of sodium alginate sulfate to fully dissolve it. Subsequently, 0.80 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.12 g of N-hydroxysuccinimide (NHS) were sequentially added. After magnetic stirring at 35° C. for 28 hours, 0.24 g of collagen peptide was added, and magnetic stirring was continued for 20 min. The reacted solution was purified by dialysis for three days, and then freeze-dried to obtain collagen peptide-grafted sodium alginate sulfate. It was determined that the sodium carboxyl group in the collagen peptide-grafted sodium alginate sulfate was replaced by collagen peptide The degree of substitution was 0.228.

Example 6

Prepare 0.2mol/l 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, and adjust its pH value to 6.5 with 0.3mol/l sodium chloride solution. Measure 50ml of MES buffer solution into a three-neck flask, add 0.6g of sodium alginate sulfate to fully dissolve it. Subsequently, 1.19 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.36 g of N-hydroxysuccinimide (NHS) were sequentially added. After magnetic stirring at 35° C. for 20 hours, 0.24 g of collagen peptide was added, and magnetic stirring was continued for 10 min. The reacted solution was purified by dialysis for three days, and then freeze-dried to obtain collagen peptide-grafted sodium alginate sulfate. It was determined that the sodium carboxyl group in the collagen peptide-grafted sodium alginate sulfate was replaced by collagen peptide The degree of substitution was 0.124.

The collagen peptide grafted sodium alginate sulfate prepared by each of the above-mentioned embodiments is characterized as follows:

(1) The collagen peptides grafted with sodium alginate sulfate prepared in Examples 1-6 were respectively prepared into 1 mg/ml solutions as test samples, and then mixed with 6.0 ml of phosphate buffered saline solution (PBS, 0.1 mol/l , pH7.4) were mixed, and 1.0ml of hydrogen peroxide (H ₂ O ₂ , 40mmol/l) was added to the above mixed solution to obtain each test sample solution. After 10min, measure the absorbance of each test sample solution at 230nm with a UV spectrophotometer, calculate the hydrogen peroxide scavenging rate of the collagen peptide grafted sodium alginate sulfate prepared by each embodiment according to the following formula, the results are shown in Fig. 2, Wherein A _s is the absorbance of the test sample solution, A _b is the absorbance of the test sample solution without hydrogen peroxide, and A _c is the absorbance of the sample solution without the test sample.

It can be seen from Figure 2 that the hydrogen peroxide scavenging rate of sodium alginate sulfate increases after collagen peptide grafting modification, and increases with the increase of the degree of substitution. Proper scavenging of oxygen free radicals can improve vascular permeability, reduce exudate, reduce tissue edema and cell damage, thereby promoting accelerated wound healing, and hydrogen peroxide can be converted into oxygen free radicals in the body, so good peroxidation The hydrogen scavenging rate will be beneficial to the healing of the wound surface.

(2) The collagen peptide grafted with sodium alginate sulfate prepared in Examples 1, 2, and 6 and the sodium alginate sulfate of ungrafted collagen peptide were used to determine their promoting effect on fibroblasts, the experimental procedure as follows:

Under sterile conditions, rat skin fibroblasts were placed in culture flasks and cultured in DMEM medium containing 10% FBS in an incubator at 37°C and 5% CO ₂ . The fibroblasts after three passages were seeded in 96-well cell culture plates (6000 cells/well), and cultured with 200 μl DMEM medium containing 10% FBS for 24 hours. After adding the collagen peptide grafted sodium alginate sulfate (test sample) of each embodiment or the sodium alginate sulfate (comparative sample) without grafting collagen peptide and culturing for 48 hours, replace it with DMEM medium containing 10% FBS Subsequently, 20 μl of MTT solution was added and reacted in the dark for 4 hours. After aspirating the medium, add 150 μl dimethyl sulfoxide (DMSO) and react for 10 min to dissolve the MTT formazan crystals. The absorbance at 492nm was measured with a microplate reader, and the cell survival rate was calculated according to the formula. The results are shown in Figure 3, where A _s is the absorbance of the test sample or the control sample, and A _c is the blank control.

It can be seen from Figure 3 that the collagen peptide-grafted sodium alginate sulfate in each embodiment obtained after the modification of sodium alginate sulfate by collagen peptide grafting showed better cell viability, and was significantly higher. Sodium alginate sulfate in ungrafted collagen peptides, and with the increase of the degree of substitution, the cell viability tended to increase. The proliferation and differentiation of fibroblasts in the process of wound healing play an important role in promoting wound healing, the greater the survival rate of fibroblasts, the faster the wound healing.

(3) The collagen peptide grafted with sodium alginate sulfate in Example 1 was used for the healing of deep second-degree burn wounds.

SD rats were anesthetized by intraperitoneal injection of 0.8ml of 3% pentobarbital sodium. The back was depilated with 8% sodium sulfide solution. A constant temperature and constant voltage electric scald was used to scald the backs of SD rats for 15 seconds at a temperature of 75°C, resulting in deep second-degree burn wounds with an area of 3.14 cm ² . After the burn wound was sterilized with PVP-I with a volume ratio of 1:4 and povidone iodine diluted with normal saline, the wound was wrapped with oil gauze soaked in collagen peptide grafted with sodium alginate sulfate, fixed with sutures and needles, and wrapped eight times. A layer of sterile gauze, fixed with a self-adhesive elastic bandage. Change the dressing every other day. Then on the day of scalding, the 3rd day, the 7th day and the 14th day were observed respectively. The results of the wound healing photos on the day of scalding, the 3rd day, the 7th day and the 14th day are shown in Fig. The optical microscopy results of the day are shown in Figure 5.

It can be seen from Figure 4 that on the day of burn, the wound surface can be clearly distinguished from the normal area with the naked eye. The wound surface is round, the wound margin is overall, the boundary is clear, the wound surface is pale and lifeless, with poor luster and mild swelling; on the third day after injury, The area of the wound becomes smaller, the wound is dry and red, the skin is thin and the scab is close to the base layer, and a little healed skin can be seen locally; on the 7th day after injury, the wound shrinks significantly, the wound is dry and red, without exudation, the scab and the wound Separation and detachment, it can be seen that new skin grows, and the speed of skin climbing is fast; on the 14th day after injury, the wound grows well and has been completely covered by new epithelial tissue, no infection, no suture reaction, no redness, and the burn wound is completely heal.

It can be seen from Figure 5 that on the 7th day after injury, the number of capillaries, fibroblasts, fibroblasts, epidermal cells, and inflammatory cells increased significantly, and the wound was partially covered by epidermis; on the 14th day after injury, the wound was covered by epidermal cells, The epidermis healed completely, the collagen fibers were arranged neatly and parallel to the epidermis, and the connective tissue fibers were densely arranged. This shows that the collagen peptide grafted with sodium alginate sulfate of the present invention has a good healing effect on deep second-degree burn wounds.

Claims (6)

1. collagen peptide grafted sodium alginate sulfuric ester, is characterized in that: it is by the carboxyl sodium base of sodium alginate sulfuric esterAfter activating, group carries out with collagen peptide 25-65 DEG C that graft reaction obtains again, described collagen peptide grafting alginic acidThe substitution value that in sodium sulfuric ester, carboxyl sodium group is replaced by collagen peptide is 0.310-0.469; Described sodium alginate sulfuric esterSulfuric ester substitution value be 1-1.8; Collagen peptide grafted sodium alginate sulfuric ester is prepared by following methods: by alginic acidAfter the carboxyl sodium group of sodium sulfuric ester reacts with carboxyl activator is activated, then with collagen peptide 25-65 DEG C to carry out grafting anti-Should, post processing, in described priming reaction, sodium alginate sulfuric ester is to be first 5-7 with pH 2-(N-morpholino) ethyl sulfonic acid cushionsAfter solution dissolves, then add carboxyl activator to react, the mass ratio of described collagen peptide and sodium alginate sulfuric ester is0.4:1-2.0:1。

2. the preparation method of collagen peptide grafted sodium alginate sulfuric ester claimed in claim 1, is characterized in that: it is firstAfter the carboxyl sodium group of sodium alginate sulfuric ester is reacted with carboxyl activator is activated, then enter with collagen peptide 25-65 DEG CRow graft reaction, post processing, in described priming reaction, sodium alginate sulfuric ester is 2-(N-morpholino) second that is first 5-7 with pHAfter sulfonic acid cushioning liquid dissolves, then add carboxyl activator to react, described collagen peptide and sodium alginate sulfuric esterMass ratio is 0.4:1-2.0:1.

3. the preparation method of collagen peptide grafted sodium alginate sulfuric ester according to claim 2, is characterized in that: instituteUnder the condition that the priming reaction of stating is is 5-7 at pH, sodium alginate sulfuric ester is reacted to 12-with carboxyl activator at 25-65 DEG C28 hours, described carboxyl activator was 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxyl amberThe imido mixture of amber.

4. the preparation method of collagen peptide grafted sodium alginate sulfuric ester according to claim 3, is characterized in that: instituteState the matter of 1-in carboxyl activator (3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamideAmount is than being 3.3:1-6.7:1; The mass ratio of described N-hydroxy-succinamide and sodium alginate sulfuric ester is 0.2:1-0.6:1.

5. the preparation method of collagen peptide grafted sodium alginate sulfuric ester according to claim 2, is characterized in that: instituteThe reaction time of stating graft reaction is 10-30min.

6. collagen peptide grafted sodium alginate sulfuric ester according to claim 1 is preparing in wound surface healing medicineApplication.