CN103739734A - Carboxymethyl carrageenan-collagen peptide, and preparation method and use thereof - Google Patents

Abstract

The invention belongs to the field of medical polymer material science, and particularly relates to carboxymethyl carrageenan-collagen peptide, and a preparation method and use thereof. The carboxymethyl carrageenan-collagen peptide is prepared from a carboxyl sodium group of carboxymethyl carrageenan subjected to grafting reaction together with collagen peptide at the temperature of 25-35 DEG C after being activated. The amide substitution degree of the carboxymethyl carrageenan-collagen peptide is 0.043-0.442. The carboxymethyl carrageenan-collagen peptide has good ability of removing hydroxyl radical (.OH) and DPPH. radical, and can promote growth of fibroblasts and accelerate healing of a wound.

Description

Carboxymethyl carrageenin-collagen peptide, preparation method and its usage

Technical field

The present invention relates to carboxymethyl carrageenin-collagen peptide, preparation method and its usage, belong to medical bio polymer material science field.

Background technology

Carrageenin is a kind of natural seaweed sulfated polysaccharide of structure uniqueness, has very strong electronegativity, and this is that it can present multiple bioactive key, and these biological activated energies strengthen along with electronegative increase.Can while Han You – SO after its carboxymethylation₄^2-he – COOH, structure and heparin are similar, and compared with the heparin surrogate synthetic with other, the carrageenin after carboxymethylation can not only be avoided the shortcoming of synthetic sulfated polysaccharides, also has that technique is simple, cost advantage lower, free from environmental pollution.

Collagen peptide has good biological activity and physical properties, makes it can be used as bio-medical material, participates in the differentiation and migration of cell, makes skin, bone and bone tendon have certain physical strength, plays the part of sheath in zooblast.In collagen peptide, also there is aminoacid sequence (Gly-X-Y), be rich in-NH₂, hydrophilic radicals such as-OH and have good moisture retention, can supply with the needed moisture of skin.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of carboxymethyl carrageenin-collagen peptide, preparation method and application thereof.This carboxymethyl carrageenin-collagen peptide has good removing hydroxy radical qiao (OH) and DPPH free radical ability, can promote fibroblastic growth, accelerates the healing of wound surface.

The present invention is that the technical scheme of its technical problem employing of solution is as follows:

Carboxymethyl carrageenin-collagen peptide, it is to carry out graft reaction and obtain with collagen peptide 25-35 ℃ again after the carboxyl sodium group of carboxymethyl carrageenin is activated.

Press such scheme, the acid amides substitution value (number that the carboxyl sodium group on average each carboxymethyl carrageenin is replaced by the amino group on collagen peptide) of described carboxymethyl carrageenin-collagen peptide is 0.043-0.442.

The preparation method of above-mentioned carboxymethyl carrageenin-collagen peptide, is characterized in that: it is after carboxymethyl carrageenin and carboxyl activator activation, to carry out graft reaction, aftertreatment again with collagen peptide 25-35 ℃.

Press such scheme, described priming reaction is that carboxymethyl carrageenin is dissolved in to MES(2-(N-morpholine) ethyl sulfonic acid solution) in damping fluid, the pH of regulation system is 6.0-7.0, then in system, add carboxyl activator to react 10-30 hour at 25-35 ℃, described carboxyl activator is the mixture of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS).

Press such scheme, the mass ratio of 1-in described carboxyl activator (3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS) is 1.5:1-5.6:1; Described N-hydroxy-succinamide (NHS) is 0.1:1-0.5:1 with the mass ratio of carboxymethyl carrageenin.

Press such scheme, the mass ratio of described carboxymethyl carrageenin and collagen peptide is 1:1-1:3.

Press such scheme, described in to add the reaction times of carrying out graft reaction after collagen peptide be 10min-30min.

Press such scheme, described aftertreatment is the solution distill water dialysis after reaction is finished, cooling drying.

The application of above-mentioned carboxymethyl carrageenin-collagen peptide aspect Promote cell's growth.

The application of above-mentioned carboxymethyl carrageenin-collagen peptide in medical wound dressing.

Beneficial effect of the present invention:

Carboxymethyl carrageenin collagen peptide of the present invention has good removing hydroxy radical qiao (OH) and DPPH free radical ability, can promote fibroblastic growth; As medical wound dressing, can accelerate surface of a wound hemostasis, accelerating wound, successful; Good biocompatibility, life-time service can not cause the ill symptomses such as the skin sensitivity of wound site or anaphylaxis.

Accompanying drawing explanation

The infared spectrum of carboxymethyl carrageenin-collagen peptide that Fig. 1 obtains for the embodiment of the present invention 1;

Carboxymethyl carrageenin-collagen peptide that Fig. 2 obtains for the embodiment of the present invention 1 is at 1d, 2d, 3d and add MTT reagent after impact on fibroblastic growth;

Carboxymethyl carrageenin-collagen peptide that Fig. 3 obtains for the embodiment of the present invention 1 when scalding, scald after the wound healing picture of 3d, 7d and 14d;

Carboxymethyl carrageenin-collagen peptide that Fig. 4 obtains for the embodiment of the present invention 1 when scalding, the pathological section photo of 3d, 7d and 14d after scalding.

Embodiment

In order to understand better the present invention, below in conjunction with embodiment, further illustrate content of the present invention, but content of the present invention is not only confined to the following examples.

Embodiment 1

Getting 0.6g carboxymethyl carrageenin (CKC) and be dissolved in 0.2mol/L2-(N-morpholino) ethyl sulfonic acid (MES) damping fluid, is 6.5 by adding 0.3mol/L NaCl solution 50ml to regulate the pH of MES damping fluid, stirs.Then add 0.32gEDC and 0.09g NHS pressed powder, keep 23 ℃ constant, continuously stirring 20h.In solution, add 0.6g collagen peptide again, stir 24min.After reaction finishes, by mixing solutions distillwater dialysis 3d, gained finished product obtains the carboxymethyl carrageenin-collagen peptide (CKC-CP) of purifying with freeze drier freeze-drying.The CKC-CP substitution value obtaining is 0.25.Carboxymethyl carrageenin and carboxymethyl carrageenin-collagen peptide are shown in Fig. 1 through the infared spectrum of Infrared Characterization.As seen from the figure: the amino on collagen peptide carries out acid amides with the carboxyl sodium group of carboxymethyl carrageenin and reacts, and collagen peptide has successfully been introduced in carboxymethyl carrageenin sugar unit structure.

Embodiment 2

Get 0.6g carboxymethyl carrageenin (CKC) and be dissolved in 2-(N-morpholino) ethyl sulfonic acid (MES) damping fluid, adding the pH of NaCl solution regulation system is 6.0, stirs.Then in system, add 0.36g EDC and 0.17g NHS, 27 ℃ of temperature maintenances are constant, continuously stirring 15h.In solution, add 0.8g collagen peptide solid again, stir 18min.After reaction finishes, by mixing solutions distill water dialysis, gained finished product obtains the carboxymethyl carrageenin-collagen peptide (CKC-CP) of purifying with freeze drier freeze-drying, and the CKC-CP substitution value obtaining is 0.12.

Embodiment 3

Get 0.4g carboxymethyl carrageenin (CKC) and be dissolved in 0.2mol/L2-(N-morpholino) ethyl sulfonic acid (MES) damping fluid, the pH of regulation system is 7.0, stirs.Then in system, add 0.25g EDC and 0.19g NHS, keep 23 ℃ of constant continuously stirring 25h of temperature.In solution, add 1.0g collagen peptide solid again, stir 28min.After reaction finishes, by mixing solutions distillwater dialysis 3d, gained finished product obtains the carboxymethyl carrageenin-collagen peptide (CKC-CP) of purifying with freeze drier freeze-drying, and the CKC-CP substitution value obtaining is 0.40.

Embodiment 4

Getting 0.5g carboxymethyl carrageenin (CKC) and be dissolved in 0.2mol/L2-(N-morpholino) ethyl sulfonic acid (MES) damping fluid, is 6.5 by the pH that adds 0.3mol/L NaCl solution 50ml to carry out regulation system, stirs.Then in system, add 0.40gEDC and 0.14g NHS pressed powder, keep 20 ℃ constant, continuously stirring 23h.In solution, add 1.2g collagen peptide solid again, stir 26min.After reaction finishes, by mixing solutions distillwater dialysis 3d, gained finished product obtains the carboxymethyl carrageenin-collagen peptide (CKC-CP) of purifying with freeze drier freeze-drying, and the CKC-CP substitution value obtaining is 0.32.

Embodiment 5

Getting 0.8g carboxymethyl carrageenin (CKC) and be dissolved in 0.2mol/L2-(N-morpholino) ethyl sulfonic acid (MES) damping fluid, is 6.0 by the pH that adds 0.3mol/L NaCl solution 50ml to carry out regulation system, stirs.Then add wherein 0.24gEDC and 0.18g NHS, keep 30 ℃ of temperature constant, continuously stirring 21h.In solution, add 1.4g collagen peptide solid again, stir 18min.After reaction finishes, by mixing solutions distillwater dialysis 3d, gained finished product obtains the carboxymethyl carrageenin-collagen peptide (CKC-CP) of purifying with freeze drier freeze-drying, and the CKC-CP substitution value obtaining is 0.44.

Embodiment 6

Getting 0.6g carboxymethyl carrageenin (CKC) and be dissolved in 2-(N-morpholino) ethyl sulfonic acid (MES) damping fluid, is 7.0 by the pH that adds 0.3mol/L NaCl solution 50ml to come in regulation system, stirs.Then in system, add 0.33g EDC and 0.08g NHS pressed powder, keep 20 ℃ of temperature constant, continuously stirring 15h.In solution, add 0.8g collagen peptide solid again, stir 22min.After reaction finishes, by mixing solutions distillwater dialysis 3d, gained finished product obtains the carboxymethyl carrageenin-collagen peptide (CKC-CP) of purifying with freeze drier freeze-drying, and the CKC-CP substitution value obtaining is 0.18.

Carboxymethyl carrageenin-collagen peptide prepared by above-described embodiment carries out following performance characterization:

(1) carboxymethyl carrageenin-collagen peptide of being prepared by embodiment 1-6 carries out soluble test, and result shows: carboxymethyl carrageenin-collagen peptide is water-soluble good, and all test samples all can fully dissolve in water.

(2) radical scavenging activity test

Radical scavenging activity, also referred to as resistance of oxidation, is removed unnecessary free radical in body, can alleviate their damages to body.Free radical is to be in vivo unbound state, with molecule, atom or the ion of unpaired electronics, comprises hydroxy radical qiao (OH) and DPPH free radical etc.

Hydroxy radical qiao is one of chemical substance that offensiveness is the strongest, almost can dissimilar reaction occur with all biomacromolecules, and have very high rate constant.We adopt o-phenanthroline at this: in test tube, add 0.5ml0.75mol/l phenanthroline ethanol solution, then add successively PBS damping fluid 1ml and the carboxymethyl carrageenin-collagen peptide sample solution 0.5ml of 0.2mmol/l (pH7.4), after fully mixing, add 0.5ml0.75mmol/l copperas solution and 0.5ml0.01wt%H₂o₂.This solution is water-bath 1h at 37 ℃, measures absorbancy at 536nm place.Wherein As is the absorbancy of test sample solution, and Ab is the absorbancy that does not add the test sample solution of hydrogen peroxide, and Ap is the absorbancy that does not add the sample solution of testing sample.

Scavenging action to hydroxyl free radical (%)=(As-Ap)/(Ab-Ap) × 100%

The removing of DPPH free radical is widely used in evaluating the resistance of oxidation of antioxidant.Concrete operation method is as follows: by 2.8ml1.0 × 10⁴the DPPH-ethanol solution of mol/l and 1.0ml carboxymethyl carrageenin-collagen peptide sample solution mix, at the room temperature 20min that keeps in Dark Place, and under 4000r/min centrifugal 10min, get supernatant liquor and measure absorbancy under 515nm.Wherein Ai is the absorbancy of test sample, and Aj is the absorbancy that dehydrated alcohol substitutes test sample, and A0 is the absorbancy that distilled water substitutes sample.

DPPH free radical scavenging activity (%)=[1-(Ai-Aj)]/A₀× 100%

Result is as shown in table 1.

Table 1

Antioxidant property	Embodiment 1	Embodiment 2	Embodiment 3
				Scavenging action to hydroxyl free radical (OH)/%	32	29	31
DPPH free radical scavenging activity/%	13	16	17

(2) promote chick embryo fibroblast increment test:

Chick embryo fibroblast with tryptic digestion in logarithmic phase, by cell harvesting to containing in the DMEM substratum of serum, and the carboxymethyl carrageenin-collagen peptide in embodiment 1 is mixed with respectively to certain density carboxymethyl carrageenin-collagen protein peptide solution, join in 96 well culture plates, then to culture plate, add aforementioned DMEM substratum.Afterwards, culture plate is put in to 5%CO₂in incubator, under 37 ℃ of conditions, cultivate after 24h, take out culture plate, in culture plate, add MTT reagent, on enzyme-linked immunosorbent assay instrument, read the OD value (absorbance) in each hole, 570nm wavelength place.Every group of experiment repeats 6 times, and result represents with X ± S, and X is the average of 6 times, and S is degree of fluctuating.And according to above-mentioned experiment condition, compare test with carboxymethyl carrageenin.The results are shown in following table 1.

Table 1 carboxymethyl carrageenin-collagen peptide is the impact on chick embryo fibroblast growth under different concns (ppm)

$(\stackrel{\‾}{X}\±s,n=6)$

?	10ppm	50ppm	100ppm	500ppm	1000ppm
						CKC	0.804±0.004	0.812±0.008	0.828±0.012	0.802±0.007	0.762±0.005
Embodiment 1	0.819±0.003	0.824±0.004	0.842±0.003	0.821±0.004	0.783±0.008
						Embodiment 2	0.811±0.008	0.817±0.011	0.839±0.005	0.814±0.008	0.777±0.003
Embodiment 3	0.821±0.016	0.843±0.002	0.857±0.007	0.835±0.008	0.790±0.002

Table 1 can be found out thus: under identical concentration conditions, the absorbancy of carboxymethyl carrageenin-collagen peptide of the present invention (OD value) is obviously greater than carboxymethyl carrageenin.And carboxymethyl carrageenin-collagen peptide of the present invention absorbancy (OD) value of (10-1000ppm) under different concns shows that it all has promoter action to the growth of chick embryo fibroblast.When concentration is 100ppm, the viable cell quantity of gained is maximum, and now carboxymethyl carrageenin-collagen peptide promotes that chick embryo fibroblast proliferation function is the strongest.When concentration is greater than 100ppm, the short chick embryo fibroblast energy for growth of carboxymethyl carrageenin-collagen peptide all can diminish gradually, and chick embryo fibroblast also has de-wall phenomenon to occur.The CKC-CP sample substitution value that 1-3 obtains in conjunction with the embodiments in addition can find out, along with the increase of substitution value, absorbancy (OD) value strengthens to some extent, the CKC-CP that shows high substitution value promote aspect chick embryo fibroblast propagation more obvious.

Another passing through set different incubation times (1d, 2d, 3d), will cultivate 1d, 2d, and the culture plate after 3d is put and is examined under a microscope and scanning respectively, then aftercultivation 3d, adds MTT reagent to show also microscopic examination of reaction, as shown in Figure 2.By Fig. 2, found out: first day chick embryo fibroblast has just joined in substratum, and under microscope, cell is less; Within second day, chick embryo fibroblast has growth compared with first day; Within the 3rd day, increase more; Add after MTT reagent, inoblast has color reaction, and cell is examined under a microscope more obvious.Illustrate that carboxymethyl carrageenin-collagen peptide is along with the growth of incubation time can promote the growth of chick embryo fibroblast quantity.

(3) healing for burn wound by carboxymethyl carrageenin-collagen peptide of embodiment 1:

Experimental group: with ether by rat anesthesia, reject the hair of rat, with 90 ℃ of boiling hot 10s of scald apparatus, a circular deep ii degree burn wound is burnt in the back of rat, carboxymethyl carrageenin-collagen peptide of embodiment 1 is configured to the solution that mass concentration is 5wt%, be coated on mouse wound, then at the 3rd day, within the 7th day and the 14th day, observe, according to healing wound area, calculate wound healing rate

Wound healing rate=(healing wound area during original wound area-observation)/original wound area × 100%;

Control group: adopt the carboxymethyl carrageenin aqueous solution that mass concentration is 5wt%.

Table 2 wound healing rate

Number of days	Experimental group	Control group
			3	9.97±2.58	7.87±3.64
7	38.47±2.51	20.76±2.16
			14	89.4±2.43	51.94±3.61

And scalding the same day, the 3rd day, within the 7th day and the 14th day, to observe respectively, wound healing photo the results are shown in Figure 3, and scald same day, the 3rd day, the pathological section figure of the 7th day and the 14th day was shown in Fig. 4.From Fig. 3 and Fig. 4 associative list 2: to deep ii degree burn, wound healing has promoter action to carboxymethyl carrageenin-collagen peptide of the present invention, in 14d left and right, can substantially heal with the wound of carboxymethyl carrageenin-collagen peptide group processing, healing rate reaches 89%, and healing time is significantly shorter than carboxymethyl carrageenin control group; Can be used as the healing of dressing for burn wound.Results of animal conforms to cell experiment result.This is mainly because carboxymethyl carrageenin-collagen peptide is water-soluble strong, can enter cell and tissue space, promote cell, intercellular substance and organize interphase interaction, and there is good removing hydroxy radical qiao (OH) and DPPH free radical ability, thereby can promote fibroblastic growth accelerated wound healing process.

Claims (10)

Translated fromChinese

1.羧甲基卡拉胶-胶原蛋白肽，其特征在于：它是将羧甲基卡拉胶的羧基钠基团进行活化后再与胶原蛋白肽25-35℃进行接枝反应得到的。1. Carboxymethyl carrageenan-collagen peptide, characterized in that: it is obtained by activating the sodium carboxylate group of carboxymethyl carrageenan and then grafting reaction with collagen peptide at 25-35°C.2.根据权利要求1所述的羧甲基卡拉胶-胶原蛋白肽，其特征在于：所述羧甲基卡拉胶-胶原蛋白肽的酰胺取代度为0.043-0.442。2. The carboxymethyl carrageenan-collagen peptide according to claim 1, characterized in that: the amide substitution degree of the carboxymethyl carrageenan-collagen peptide is 0.043-0.442.3.权利要求1所述的羧甲基卡拉胶-胶原蛋白肽的制备方法，其特征在于：它是将羧甲基卡拉胶与羧基活化剂活化后再与胶原蛋白肽25-35℃进行接枝反应、后处理。3. the preparation method of carboxymethyl carrageenan-collagen protein peptide described in claim 1 is characterized in that: it is to connect with collagen protein peptide 25-35 ℃ after carboxymethyl carrageenan and carboxyl activator activation Branch reaction, post-processing.4.根据权利要求3所述的羧甲基卡拉胶-胶原蛋白肽的制备方法，其特征在于：所述的活化反应是将羧甲基卡拉胶溶于MES缓冲液中，调节体系的pH为6.0-7.0，然后在体系中加入羧基活化剂在25-35℃下反应10-30小时，所述的羧基活化剂为1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的混合物。4. the preparation method of carboxymethyl carrageenan-collagen peptide according to claim 3 is characterized in that: described activation reaction is that carboxymethyl carrageenan is dissolved in MES damping fluid, and the pH of adjustment system is 6.0-7.0, then add a carboxyl activator in the system to react for 10-30 hours at 25-35°C, the carboxyl activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodi Mixture of imine hydrochloride and N-hydroxysuccinimide.5.根据权利要求3所述的羧甲基卡拉胶-胶原蛋白肽的制备方法，其特征在于：所述的羧基活化剂中1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的质量比为1.5:1-5.6:1；所述N-羟基琥珀酰亚胺与羧甲基卡拉胶的质量比为0.1:1-0.5:1。5. the preparation method of carboxymethyl carrageenan-collagen peptide according to claim 3 is characterized in that: 1-(3-dimethylaminopropyl)-3-ethyl in the described carboxyl activator The mass ratio of carbodiimide hydrochloride and N-hydroxysuccinimide is 1.5:1-5.6:1; the mass ratio of described N-hydroxysuccinimide and carboxymethyl carrageenan is 0.1:1- 0.5:1.6.根据权利要求3所述的羧甲基卡拉胶-胶原蛋白肽的制备方法，其特征在于：所述羧甲基卡拉胶与胶原蛋白肽的质量比为1:1-1:3。6. the preparation method of carboxymethyl carrageenan-collagen peptide according to claim 3, is characterized in that: the mass ratio of described carboxymethyl carrageenan and collagen peptide is 1:1-1:3.7.根据权利要求3所述的羧甲基卡拉胶-胶原蛋白肽的制备方法，其特征在于：所述加入胶原蛋白肽后进行接枝反应的反应时间为10min-30min。7. The preparation method of carboxymethyl carrageenan-collagen peptide according to claim 3, characterized in that: the reaction time for grafting reaction after adding the collagen peptide is 10min-30min.8.根据权利要求3所述的羧甲基卡拉胶-胶原蛋白肽的制备方法，其特征在于：所述的后处理为将反应结束后的溶液用蒸馏水透析，冷却干燥。8. The preparation method of carboxymethyl carrageenan-collagen peptide according to claim 3, characterized in that: the post-treatment is to dialyze the solution after the reaction with distilled water, and cool and dry.9.权利要求1-8中任一项所述的羧甲基卡拉胶-胶原蛋白肽在促进细胞生长方面的应用。9. The application of the carboxymethyl carrageenan-collagen peptide described in any one of claims 1-8 in promoting cell growth.10.根据权利要求1-8任一项所述的羧甲基卡拉胶-胶原蛋白肽在医用伤口敷料中的应用。10. The application of carboxymethyl carrageenan-collagen peptide according to any one of claims 1-8 in medical wound dressing.

Images