阅读说明：本技术 一种活性木质素及其制备方法和应用 (Active lignin and preparation method and application thereof ) 是由 黄曹兴 裴雯慧 郑力铭 王许才 勇强 于 2021-08-12 设计创作，主要内容包括：本发明公开了一种活性木质素及其制备方法和应用。所述的应用为活性木质素在制备用于促进软骨缺损修复的药物中的应用。本发明以漆酶催化阿拉伯糖、木糖脱氢聚合合成的活性木质素,将其用于软骨缺损模型中进行修复试验,发现所制备的活性木质素可以促进软骨缺损修复。本发明通过生物法合成的活性木质素在促进软骨缺损修复方面效果显著,具有巨大的应用潜力和经济效益。(The invention discloses active lignin, a preparation method and application thereof. The application is the application of active lignin in preparing a medicament for promoting cartilage defect repair. According to the invention, laccase is used for catalyzing active lignin synthesized by arabinose and xylose dehydrogenation polymerization, and the active lignin is used for a repair test in a cartilage defect model, so that the prepared active lignin can promote cartilage defect repair. The active lignin synthesized by the biological method has obvious effect on promoting the repair of cartilage defect, and has huge application potential and economic benefit.)

1. Application of active lignin in preparing medicine for promoting repair of cartilage defect is provided.

2. The use of claim 1, wherein the active lignin promotes chondrogenic differentiation of mesenchymal stem cells.

3. The activated lignin according to claim 1, wherein the activated lignin is obtained by laccase catalyzed reaction of arabinose or xylose with isoeugenol.

4. A method for producing the activated lignin according to claim 1 or 3, characterized by the following steps:

1) mixing arabinose or xylose with isoeugenol uniformly, adding acetic acid/sodium acetate buffer solution and ethanol under aseptic condition, and dissolving completely;

2) adding laccase, mixing, performing enzymolysis reaction under controlled temperature, centrifuging, retaining solid substance to obtain crude product, and freeze drying;

3) washing the crude product with dichloroethane/ethanol, collecting liquid part, and performing rotary evaporation to obtain solid, namely the active lignin.

5. The method for preparing active lignin according to claim 4, wherein the step 1) is specifically: preparing an acetic acid/sodium acetate buffer solution with the pH value of 5, and mixing the acetic acid/sodium acetate buffer solution with ethanol in the same volume; then 5g of arabinose or 5g of xylose is taken to be evenly mixed with 5g of isoeugenol, and the prepared acetic acid/sodium acetate and ethanol buffer mixed liquid is added under the aseptic condition for full dissolution.

6. The method for preparing active lignin according to claim 4, wherein the step 2) is specifically: adding 1mL of laccase with enzyme activity of 1000IU/mL, uniformly mixing, then placing the system in a water bath kettle at 30 ℃ for reacting for 24 hours, adding 1mL of laccase, and simultaneously adding absolute ethyl alcohol; after 5 days of reaction under aseptic conditions, the crude product was washed, centrifuged to retain the precipitated fraction and freeze-dried.

7. The method for preparing active lignin according to claim 4, wherein step 3) is specifically: adding the freeze-dried crude product into a dichloroethane/ethanol mixed solution with the volume ratio of 2: 1, reacting for 6 hours, centrifuging, collecting liquid, and performing rotary evaporation to obtain the active lignin.

Technical Field

The invention belongs to the technical field of new biological materials, and particularly relates to active lignin as well as a preparation method and application thereof.

Background

Articular cartilage damage is one of the most common diseases in clinic, and once damaged, the articular cartilage is difficult to repair spontaneously due to the avascular structure and the poor migration capability of chondrocytes, and can cause secondary joint degeneration, and if reasonable treatment is lacked, joint pain can be caused finally, even osteoarthritis can be developed, so that great pain is brought to patients. Hydrogel materials, which have been shown to significantly promote cartilage defect repair. Although cartilage tissue engineering techniques are widely used in the field of cartilage repair, integration between the implanted hydrogel scaffold and the host tissue is crucial for efficient tissue regeneration, particularly for cartilage, which may facilitate the healing process and restoration of mechanical function. Poor binding of implanted biomaterial to native cartilage often leads to tissue fibrosis, resulting in inefficient mechanical load transfer, failure of the binding of new cartilage to native cartilage, and ultimately failure of cartilage regeneration. However, good and stable bonding between implanted biomaterials and native cartilage remains a great challenge. For cartilage regeneration, the existing adhesive hydrogel is far from ideal, has biocompatibility, is convenient to use and high in cost benefit, and can effectively promote cartilage regeneration.

Lignin is the most abundant natural polyphenol and a by-product of the wood hydrolysis and pulping industry. However, these lignin by-products are often incinerated as low grade fuels and even discarded as waste. With the development and progress of society, people pay more and more attention to the environment and sustainable development. Lignin resources that have not been fully utilized have been extensively studied. Due to the unique characteristics of other natural polymers, such as oxidation resistance, anti-inflammation, good antibacterial activity and the like. In the biomedical field, it is a common approach to develop biomedical materials using natural polymers, and lignin can be used as a bioactive compound to supplement common biomaterials. However, the lignin component extracted from industrial lignin is very complex, the purification process is complicated, the content of functional groups is low, the extraction process and the pretreatment method can affect the physical and chemical properties of lignin, the structure is not adjustable, and even if the lignin comes from the same material and the pulping process, different extraction conditions can affect the properties of the lignin, so the use requirement cannot be met.

Disclosure of Invention

Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide the active lignin with high purity and rich content of phenolic hydroxyl functional groups. The invention also aims to provide a preparation method of the active lignin. The invention also aims to solve the technical problem of providing the application of the active lignin in preparing the medicine for promoting the repair of the cartilage defect.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

application of active lignin in preparing medicine for promoting repair of cartilage defect is provided.

The active lignin promotes the chondrogenic differentiation of the mesenchymal stem cells.

The active lignin is obtained by catalyzing the reaction of arabinose or xylose and isoeugenol by laccase.

A method for preparing the active lignin comprises the following steps:

1) mixing arabinose or xylose with isoeugenol uniformly, adding acetic acid/sodium acetate buffer solution and ethanol under aseptic condition, and dissolving completely;

2) adding laccase, mixing, performing enzymolysis reaction under controlled temperature, centrifuging, retaining solid substance to obtain crude product, and freeze drying;

3) washing the crude product with dichloroethane/ethanol, collecting liquid part, and performing rotary evaporation to obtain solid, namely the active lignin.

The step 1) is specifically as follows: preparing an acetic acid/sodium acetate buffer solution with the pH value of 5, and mixing the acetic acid/sodium acetate buffer solution with ethanol in the same volume; then 5g of arabinose or 5g of xylose is taken to be evenly mixed with 5g of isoeugenol, and the prepared acetic acid/sodium acetate and ethanol buffer mixed liquid is added under the aseptic condition for full dissolution.

The step 2) is specifically as follows: adding 1mL of laccase with enzyme activity of 1000IU/mL, uniformly mixing, then placing the system in a water bath kettle at 30 ℃ for reacting for 24 hours, adding 1mL of laccase, and simultaneously adding absolute ethyl alcohol; after 5 days of reaction under aseptic conditions, the crude product was washed, centrifuged to retain the precipitated fraction and freeze-dried.

The step 3) is specifically as follows: adding the freeze-dried crude product into a dichloroethane/ethanol mixed solution with the volume ratio of 2: 1, reacting for 6 hours, centrifuging, collecting liquid, and performing rotary evaporation to obtain active lignin solid. Wherein, the reaction of arabinose and isoeugenol as raw materials obtains arabinose-derived active lignin (DHP-A), and the reaction of xylose and isoeugenol as raw materials obtains xylose-derived active lignin (DHP-X).

Has the advantages that: compared with the prior art, the invention has the advantages that: the active lignin provided by the invention has high purity (98%), definite structure and good biocompatibility and oxidation resistance. In vitro experiments show that the active lignin can promote mesenchymal stem cells to promote chondrogenic differentiation. In vivo experiments show that the active lignin can promote the repair of rat cartilage defect. The active lignin provided by the invention can effectively promote the repair of cartilage defects, and can be widely applied to the preparation of medicines for promoting the repair of cartilage defects. In addition, the active lignin is prepared by adopting an enzyme catalysis synthesis method, and the obtained lignin has high purity, has rich phenolic hydroxyl functional group content (more than 3.5mmol/g), is an ideal antioxidant and has good practicability.

Drawings

FIG. 1 is ｃA DHP-A and DHP-X2D HSQC NMR spectrum;

FIG. 2 is a graph showing the results of cell viability data 24h after DHP treatment;

FIG. 3 is a graph showing the effect of different DHP concentrations on DPPH and superoxide anion radical scavenging;

FIG. 4 is ROS scavenging flow data and fluorescence plot; vs IL-1 β control group, P < 0.0001;

FIG. 5 is a graph of gross observations and immunohistochemical staining of Alisin blue, type II collagen on differently treated adult bone marrow stem cell chondrocytes; scale Bar: 200 mu m;

FIG. 6 is a diagram of a cartilage defect model.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1

And (3) synthesis of active lignin: an acetic acid/sodium acetate buffer of pH 5 was prepared and mixed with an equal volume of ethanol. Then 5g of arabinose and 5g of isoeugenol (test 1), 5g of xylose and 5g of isoeugenol (test 2) are respectively weighed and evenly mixed, and 70mL of prepared acetic acid/sodium acetate and ethanol buffer mixed liquid is respectively added under aseptic condition for full dissolution. Adding 1mL of laccase with enzyme activity of 1000IU/mL respectively, uniformly mixing, placing the system in a water bath kettle at 30 ℃ for reaction for 24 hours, adding 1mL of laccase, and adding 30mL of absolute ethyl alcohol simultaneously. After aseptic reaction for 5 days, washing with distilled water, centrifuging, retaining precipitate portion to obtain crude product, and freeze drying. Adding 100mL of dichloroethane/ethanol mixed solution with the volume ratio of 2: 1 into the freeze-dried crude product, reacting for 6h, centrifuging, collecting the liquid part, and performing rotary evaporation to obtain the active lignin DHP. The DHP from run 1 and run 2 were named DHP-A and DHP-X, respectively, with yields of 81.2% and 83.5%, and purities of 98.1% and 98.6%, respectively.

Adopts a 2D-HSQC nuclear magnetic resonance spectroscopy method,³¹The results of characterization and determination of the structure, functional group content and molecular weight of the active lignin by P quantitative nuclear magnetic resonance spectroscopy and gel permeation chromatography are shown in Table 1.

TABLE 1 content of different DHP functional groups and molecular weights

As can be seen from Table 1, the weight average molecular weight (1890g/mol) and the number average molecular weight (1310g/mol) of DHP-A are both smaller than the weight average molecular weight (2560g/mol) and the number average molecular weight (1620g/mol) of DHP-X. The polydispersity of DHP-A was 1.44, which is lower than that of DHP-X (1.58), and the molecular weight distributions of both lignin samples were relatively narrow, with the ratio of weight average molecular weight to number average molecular weight being less than 3.

³¹The content of phenolic hydroxyl group (3.5mmol/g) of DHP-A is lower than that of DHP-X (4.3mmol/g) by ｃA P quantitative method nuclear magnetic resonance spectroscopy, and the content of alcoholic hydroxyl group is very close to that of DHP-X and is respectively 0.8 mmol/g and 0.6 mmol/g. Wherein, DHP-A and DHP-X both have rich phenolic hydroxyl content, which shows that the DHP-A and DHP-X have good antioxidant function.

From the 2D-HSQC NMR spectrum (FIG. 1), it can be seen that the side chain region shows beta-O-4 aryl ether bond 5 structure (A, A'), resinol beta-beta structure (B), and phenyl coumaran beta-5 structure (C). The signal with the chemical shift of 86.0/4.11ppm of the beta position of the beta-O-4 ether linkage structure connected with the ligneous syringyl (S) (A) the related signal peaks at the alpha and beta positions of the resin alcohol beta-beta structure (B) can be identified at the positions of 84.9/4.69 and 53.7/3.05ppm respectively. Meanwhile, the spectrum can clearly see the signals of the beta-5 (C) structure, the chemical shift of alpha is 86.8/5.49ppm, and the chemical shift of beta is 53.1/3.49 ppm. The guaiacyl (G) building block signals are clearly visible in the aromatic ring region. The related signals of the 2, 5 and 6 positions are respectively 111.0/7.01, 114.4/6.73-115.3/6.98ppm and 119.0/6.82 ppm. Meanwhile, the phenomenon that the side chain position of the G-type lignin is oxidized into a ketone group (G') is found, and the signal of the 2-position is 112.5/7.32 ppm. In addition, the cinnamyl alcohol end group (I) structure is found in both lignins, and the related signal peaks of alpha positions and beta positions of the structure are respectively 128.5/6.47 and 128.5/6.25 ppm. From these chemical shifts, it can be seen that lignin biosynthesized using different sugars has the basic structure of native lignin.

Example 2 determination of cytotoxicity and antioxidant Properties of active Lignin

(1) Cytotoxicity assays

Toxicity of active lignin to human bone marrow mesenchymal stem cells was tested by co-culturing cells with different concentrations (0.1ug/mL, 1ug/mL, 10ug/mL, 50ug/mL) of DHP-X and DHP-A for 24h, and cell viability was determined by cck-8.

FIG. 2 is data on cell viability. At low concentrations (0.1-10. mu.g/mL), DHP concentration was substantially non-toxic to BMSCs cells at 24h, and no cell death occurred. When the concentration of DHP is 1 mu g/mL, DHP-X has no obvious effect of promoting cell proliferation, and DHP-A has ｃA certain effect of promoting osteoblast proliferation, which is probably because DHP-A has higher hydroxyl content and thus shows certain physiological activity. DHP-X at 50. mu.g/mL had a more pronounced effect on cell proliferation. However, when the DHP-A concentration is 50. mu.g/mL, no significant effect on cell proliferation is obtained. Based on the significance of the DHP on the promotion of the proliferation of the BMSCs cells, 1 mug/mL is finally selected as the optimal concentration for subsequent experiments.

(2) Measurement of antioxidant Property

In vitro antioxidant performance was determined by measuring the difference in the scavenging capacity of the samples for DPPH radicals and superoxide anion radicals. Respectively taking 2mL of DHP solution with different mass concentrations (1ug/mL, 10ug/mL, 100ug/mL, 500ug/mL, 800ug/mL and 1000ug/mL) into a 10mL centrifuge tube, adding 2mL of DPPH solution (dissolved in absolute ethyl alcohol and with the concentration of 0.2mmol/L), uniformly mixing, and reacting at room temperature in a dark place for 30 min. After completion, the absorbance (A) was measured at 517nm₁). Respectively taking 2mL of DHP solution (dissolved in dimethyl sulfoxide) and 2mL of absolute ethyl alcohol with different mass concentrations (1ug/mL, 10ug/mL, 100ug/mL, 500ug/mL, 800ug/mL and 1000ug/mL) to be mixed and react to be recorded as a control group, taking 2mL of absolute ethyl alcohol and 2mL of DPPH solution (dissolved in absolute ethyl alcohol and having a concentration of 0.2mmol/L) to be mixed and react to be recorded as a blank group, and respectively recording the light absorption values as A₂And A₀The test was repeated twice for each sample. DPPH radical clearance (Scavenging activity) was calculated according to the following equation:

Scavenging activity＝(1-(A₁-A₂)/A₀)×100％

in the formula: the Scavenging activity is DPPH free radical clearance rate; a. the₁The absorbance of the mixed solution of DHP and DPPH is obtained; a. the₂The absorbance of the mixed solution of DHP and absolute ethyl alcohol is obtained; a. the₀The absorbance of the mixed solution of absolute ethyl alcohol and DPPH is shown.

The superoxide anion radical scavenging capacity of DHP solutions of different mass concentrations (1ug/mL, 10ug/mL, 100ug/mL, 500ug/mL, 800ug/mL, 1000ug/mL) was determined using the Solebao superoxide anion radical scavenging kit.

As can be seen from FIG. 3, DHP-A has ｃA higher DPPH radical scavenging capacity than DHP-X. When the mass concentration is 0.5g/L, the scavenging capacity of DHP-A on DPPH is 70%, and the scavenging capacity of DHP-X is 55%. For DHP-A, DPPH radical scavenging effect is enhanced with increasing sample concentration. While DHP-A gave the best scavenging effect at 0.5g/L for DPPH radicals. When the DHP- ｃA concentration is above peak, DPPH clearance decreases to ｃA final value with increasing concentration. Superoxide anion radical clearance increased with increasing DHP concentration, eventually reaching nearly 100%, with no difference between the two DHPs.

In vivo antioxidant properties the intracellular levels of reactive oxygen species were known by flow cytometry through the detection of cellular fluorescence signals. The fluorescence intensity indirectly represents the level of peroxide in the cell.

Flow cytometry detection results show that after cells are stimulated by IL-1 betｃA, high-concentration active oxygen is generated, the generation of the active oxygen is inhibited by DHP, and the inhibiting effect of the DHP-X is better than that of the DHP-A. Then cell staining was performed using the oxidation sensitive stain DCFH-DA. The results of the experiment are shown in fig. 4, the IL-I beta stimulated control group shows stronger fluorescence than the blank group, which indicates that the cells of the control group generate more active oxygen; the fluorescence intensity of the treatment group added with DHP in advance is obviously lower than that of the control group and the blank group, which shows that the treatment group obviously reduces the generation of active oxygen in cells. This is consistent with flow cytometry results. The above results indicate that DHP inhibits oxidative stress in chondrocytes, and that DHP-X has ｃA stronger effect of inhibiting oxidative stress than DHP-A.

Example 3 Activity Lignin accelerated cartilage differentiation assay

BMSCs were first induced to chondrogenic differentiation by a commercial kit (adult mesenchymal stem cell chondrogenic differentiation induction medium, Setaria, HUXMA-90041), 1ug/mL DHP-A, DHP-X was added during the induction for 18 days, and after the induction, the chondrocytes were taken out and observed under a stereomicroscope and photographed. Frozen sections were immunohistochemically stained for alisertine blue and type ii collagen. The gross observation and staining pattern of the chondrocytes is shown in figure 5.

As can be seen from FIG. 5, the induction of cartilage differentiation of adult bone marrow stem cells was successful. Gross size analysis of the formed chondrocytes revealed that the control group had ｃA size similar to that of DHP- ｃA induced chondrocytes, and that lignin had ｃA significantly smaller size than that of DHP-X induced chondrocytes. DHP-A induced chondrocytes have ｃA smoother surface and are more spherical, probably because DHP-A induces BMSCs to form chondrocytes with ｃA better effect.

Alisin Blue (AB) is a water-soluble multivalent basic dye with copper present in the structure. When the pH is 2.5, the dye forms a salt bond with acidic proteoglycan of sulfuric acid, carboxylic acid and salts thereof, and becomes blue. The depth of the coloration can reflect the synthesis condition of the cartilage extracellular matrix. After 19 days of chondrogenic induction medium induction culture, staining with alisnew blue was performed. The experimental result shows that the BMSCs are successfully chondrogenic induction and differentiation. Compared with a control group, the staining area of the chondrosasphere section formed by the lignin group is smaller, but the internal structure is more compact; the DHP-A chondrocyte section has ｃA larger staining areｃA and is more aggregated inside; the stained area of the chondrocyte pellet section in DHP-X group was smaller, but the inside thereof was formed into a more dense morphology. The results indicate that the induced differentiation extracellular matrix glycosaminoglycan content increases and ｃA more aggregated morphology is formed after DHP- ｃA treatment.

The cartilage matrix is composed primarily of collagen and proteoglycans. The research finds that the collagen is the main component and accounts for 50 to 80 percent. The collagen fiber mainly comprises type II collagen, and forms fibrous skeleton of articular cartilage matrix. To evaluate the effect of active lignin on BMSCs chondrogenesis, Col2 α 1 immunohistochemical staining assays were performed after 19d culture. Immunohistochemical staining analysis shows that compared with ｃA control group, staining of ｃA cartilage marker gene Col2 ｃA1 in ｃA cartilage ball section after DHP-A treatment is obviously enhanced and is consistent with an Alisin blue staining result.

Example 4: experiment for promoting cartilage defect repair by using active lignin in vivo

All the operations in this example strictly adhere to the relevant guidelines of the U.S. public health agency on caring and using the experimental animals, and all the raising, operation, euthanasia and the like of the experimental animals are strictly performed according to the guidelines of the ethics committee of the experimental animals in the affiliated drum hospital of the medical college of Nanjing university.

Animals were divided into five groups: normal group, defective group, pure HA group, 0.1% DHP group, and 1% DHP group.

A total of 30 rats (male, 12 weeks old) were selected for this experiment. The treatment method comprises five treatment modes, including a normal group (Sham) and a damage-free group (Control); hyaluronic acid group 0.1% DHP addition amount hydrogel treatment group (m-HA + 0.1% DHP); hyaluronic acid-based 1% DHP addition amount hydrogel treatment group (m-HA + 1% DHP); hyaluronic acid only hydrogel treated group (m-HA). For the m-HA + 0.1% DHP and m-HA + 1% DHP treated groups, the corresponding hydrogels were injected into the cartilage defect using a syringe. For the m-HA treated group, a hyaluronic acid hydrogel was injected into the defect. Rat anesthesia mode: aeroanesthesia (isoflurane), after anesthesia, rats are in supine position. After conventional disinfection, an inner side bone bypass is adopted, the bone is dislocated towards the outer side, the inner side of the femur is exposed, and a bone cartilage transplantation device with corresponding size is used for manufacturing a bone cartilage defect of a full-layer cartilage defect with the diameter of 2mm and the depth of 3 mm. The tissue debris and blood clots are removed by flushing with normal saline, the patella is repositioned, and the muscle layer, fascia layer and skin incision are closed layer by layer. Disinfecting the skin incision with iodophor; the intramuscular injection of penicillin after operation prevents infection, and the rat can move freely after operation.

Experimental rats were euthanized 3 weeks after surgery. And acquiring the knee joint specimen, and taking a picture of the whole body. FIG. 6 is a cartilage defect model; the samples of each group were approximately observed after 3 weeks of filling with HA hydrogel at different DHP addition levels.

As can be analyzed from fig. 6, compared with the HA group specimens, the HA group specimens at 3 weeks after the operation can see the cartilage abrasion traces of the condyle part of the femur, the cartilage defect is obvious, the defect part is dark red, and a little white scar-like fibrous tissue grows into the partial defect. The 0.1% DHP group can be seen to have smooth articular surface and no obvious cartilage defect, the repaired tissue and the normal cartilage tissue are difficult to distinguish visually under the visual observation, and the surface of the tissue repaired by the experimental group is found to be smooth; compared with the 0.1% DHP group, the defect area of the 1% DHP group is filled with a large amount of new chondrocytes, similar to surrounding normal cartilage, and the boundary is not clear. Whereas in the 0.01% DHP group, the articular surface was not continuous and there was a significant cartilage defect in the scraped area.

Gross observation showed that the cartilage defects in the treated groups were almost completely filled, with significant tissue recess at the defect at 3 weeks post-implantation, compared to the untreated group. The high concentration DHP addition group showed a smoother, more intact morphology with no apparent boundaries at certain sites.

The experimental result shows that the HA group HAs poor effect of repairing cartilage defect under general observation; the 0.01% DHP group has a general repairing effect, and the 0.1% DHP group and the 1% DHP group have better repairing effects.