CN116036024B

CN116036024B - Plant polyphenol microsphere and preparation method and application thereof

Info

Publication number: CN116036024B
Application number: CN202211430244.4A
Authority: CN
Inventors: 李娜; 昝兴杰; 魏绍银; 孙林啸
Original assignee: Wenzhou Research Institute Of Guoke Wenzhou Institute Of Biomaterials And Engineering
Current assignee: Wenzhou Research Institute Of Guoke Wenzhou Institute Of Biomaterials And Engineering
Priority date: 2022-11-15
Filing date: 2022-11-15
Publication date: 2024-03-08
Anticipated expiration: 2042-11-15
Also published as: CN116036024A

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to a plant polyphenol microsphere, and a preparation method and application thereof. The invention provides a plant polyphenol microsphere, wherein the components of the plant polyphenol microsphere comprise plant polyphenol and calcium ions distributed in the plant polyphenol; the plant polyphenol microsphere has a porous structure. The plant polyphenol microsphere provided by the invention is a metal-plant polyphenol assembly, and the plant polyphenol is self-maintained in a microsphere structure, so that the plant polyphenol can be ensured to have a natural conformation for maintaining biological activity. Meanwhile, the porous three-dimensional microsphere structure can release plant polyphenol to a certain extent, so that the stability of the plant polyphenol is improved, the biocompatibility and the bioavailability are improved, and the porous three-dimensional microsphere structure has excellent antioxidant and anti-inflammatory effects and has good research and application prospects in the field of biomedical materials.

Description

Plant polyphenol microsphere and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a plant polyphenol microsphere, and a preparation method and application thereof.

Background

Plant polyphenols are a class of secondary metabolites with a polyphenol structure that are widely present in plants. The ortho-position phenolic hydroxyl in the phenolic hydroxyl structure is easy to oxidize, consumes oxygen in the environment, has strong capturing capability on free radicals such as active oxygen and the like, and ensures that the polyphenol has excellent oxidation resistance and free radical scavenging capability. Therefore, the plant polyphenol is a naturally occurring active oxygen nitrogen molecule (RONS) scavenger, and can be widely applied to the treatment or prevention of various diseases related to oxidative stress in daily life. However, the disadvantages of poor bioavailability, poor stability, etc. limit the application of plant polyphenols in antioxidant and anti-inflammatory aspects.

To date, various micro-nanoparticles have been used to reduce the generation of excessive RONS and to defend against oxidative stress. E.Marin et al synthesized a supported MnO ₂ PSS/PAH polymer vesicles of (2) can well reduce H in an oxidative stress model ₂ O ₂ Is contained in the composition; antonPopov et al CeO ₂ The nano particles are doped into the microvesicles, so that CeO can be maintained ₂ The oxidation resistance of the nanoparticles; chinese patent publication No. CN106565964a discloses a method for preparing metal polyphenol vesicle material with micro/nano multilayer composite structure, wherein the template is metal organic framework compound, polyphenol is introduced into the system in etching mode, the etching time is long, and it takes 0.5-4 h. In these microparticles, other substances are required as carriers for the active ingredient to function, and the natural conformation and biological activity of the active ingredient are affected to some extent.

Disclosure of Invention

The invention aims to provide a plant polyphenol microsphere, a preparation method and application thereof, and the plant polyphenol microsphere provided by the invention can keep the natural conformation of plant polyphenol and has good antioxidant and anti-inflammatory effects.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a plant polyphenol microsphere, wherein the components of the plant polyphenol microsphere comprise plant polyphenol and calcium ions distributed in the plant polyphenol;

The plant polyphenol microsphere has a porous structure.

Preferably, the plant polyphenol microsphere has a diameter of 1-500 μm.

Preferably, the plant polyphenol comprises one or more of phenolic acid plant polyphenol, acid ester plant polyphenol, flavonoid plant polyphenol, lignan plant polyphenol, and procyanidin.

The invention provides a preparation method of the plant polyphenol microsphere, which comprises the following steps:

firstly mixing a plant polyphenol solution, a carbonate solution and a calcium salt solution, and standing to obtain plant polyphenol doped calcium carbonate microspheres;

and (3) carrying out secondary mixing and etching on the plant polyphenol doped calcium carbonate microspheres and acid liquor to obtain the plant polyphenol microspheres.

Preferably, the molar concentration of the carbonate solution is 0.1-5 mol/L;

the molar concentration of the calcium salt solution is 0.1-5 mol/L;

the concentration of the plant polyphenol solution is 0.1-300 mg/mL.

Preferably, the first mixing comprises: mixing plant polyphenol solution and carbonate solution, and adding calcium salt solution;

or mixing plant polyphenol solution and calcium salt solution, and adding carbonate solution;

adding the calcium salt solution or the carbonate solution under stirring or ultrasonic conditions;

The stirring speed is 200-5000 rpm;

the frequency of the ultrasonic wave is 10-90 kHz.

Preferably, the acid solution comprises one or more of hydrochloric acid solution, nitric acid solution and acetic acid solution;

the molar concentration of the acid liquor is 0.1-5 mol/L.

The invention provides the plant polyphenol microsphere according to the technical scheme or the application of the plant polyphenol microsphere obtained by the preparation method according to the technical scheme in preparation of antioxidant preparations and anti-inflammatory preparations.

Preferably, the antioxidant formulation comprises a hydrogen peroxide scavenging formulation, a superoxide anion radical scavenging formulation, a DPPH radical scavenging formulation or an ABTS radical scavenging formulation.

Preferably, the anti-inflammatory agent comprises an anti-osteoarthritis agent, an anti-acute pancreatitis agent, an anti-chronic obstructive pulmonary disease agent, an anti-acute wound inflammation agent, or an anti-chronic wound inflammation agent.

The invention provides a plant polyphenol microsphere, wherein the components of the plant polyphenol microsphere comprise plant polyphenol and calcium ions distributed in the plant polyphenol; the plant polyphenol microsphere has a porous structure. The plant polyphenol microsphere provided by the invention is a metal-plant polyphenol assembly, and the plant polyphenol is self-maintained in a microsphere structure without being carried by a carrier, so that the plant polyphenol can be ensured to have a natural conformation for maintaining biological activity. Meanwhile, the porous three-dimensional microsphere structure can release plant polyphenol to a certain extent, so that the stability of the plant polyphenol is improved, the biocompatibility and the bioavailability are improved, and the porous three-dimensional microsphere structure has excellent antioxidant and anti-inflammatory effects and has good research and application prospects in the field of biomedical materials.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is an SEM image of tannic acid microspheres obtained in example 1;

FIG. 2 is an SEM image of gallic acid microspheres obtained in example 2;

FIG. 3 is an SEM image of catechin microspheres obtained in example 3;

FIG. 4 is an SEM image of procyanidin-doped calcium carbonate microspheres obtained in example 6;

FIG. 5 is an SEM image of procyanidin microspheres obtained in example 6;

FIG. 6 is a TEM image of procyanidin microspheres obtained in example 6;

FIG. 7 is a graph showing the total antioxidant capacity of procyanidin (OPC) microspheres and procyanidins (OPC);

FIG. 8 is a graph showing the hydrogen peroxide scavenging ability of procyanidin (OPC) microspheres and procyanidins (OPC);

FIG. 9 is a graph showing the detection of the superoxide anion radical scavenging capacity of procyanidin (OPC) microspheres and procyanidins (OPC);

FIG. 10 is a graph showing DPPH radical scavenging ability test of procyanidin (OPC) microspheres and procyanidins (OPC);

FIG. 11 is a graph showing the detection of the ability of procyanidin (OPC) microspheres to scavenge ABTS free radicals from procyanidins (OPC);

FIG. 12 is a graph showing the viability of procyanidin (OPC) microspheres and mouse monocytes macrophages (RAW 264.7) and mouse dendritic cells (DC 2.4) under treatment with OPC;

FIG. 13 is a graph showing endocytic behavior of RAW264.7, DC2.4, and NIH3T3 cells On Procyanidin (OPC) microspheres;

FIG. 14 is a graph showing the change in cell viability following hydrogen peroxide treatment of RAW264.7 cells and DC2.4 cells;

FIG. 15 is a CLSM graph of DCFH-DA fluorescent probe for detecting reactive oxygen species levels in RAW264.7 cells and DC2.4 cells;

FIG. 16 is a white light image taken by a fluorescence microscope of DC2.4 cell migration;

FIG. 17 is a graph showing inflammatory factor IL-6 and TNF- α levels in supernatants of different groups of RAW264.7 cells after LPS induction;

FIG. 18 is a graph of H & E staining of an osteoarthritis model, and graphs of IL-6, IL-1β, TNF- α, and MMP-13 expression levels in an osteoarthritis model;

FIG. 19 is a graph of H & E staining of an acute pancreatitis model;

FIG. 20 is a graph of H & E staining of a model of chronic obstructive pulmonary disease;

FIG. 21 is a white light image of the skin wound surface and a H & E staining image of the wound surface of the acute wound model control group, the L-microsphere group and the H-microsphere group;

FIG. 22 is a white light image of the skin wound surface and a H & E staining image of the wound surface of the chronic wound model control group, the L-microsphere group and the H-microsphere group.

Detailed Description

the plant polyphenol microsphere has a porous structure.

In the present invention, the plant polyphenol microsphere preferably has a diameter of 1 to 500. Mu.m, more preferably 5 to 100. Mu.m, and most preferably 10 to 50. Mu.m.

In the present invention, the plant polyphenol preferably includes one or more of phenolic acid plant polyphenol, acid ester plant polyphenol, flavonoid plant polyphenol, lignan plant polyphenol, and procyanidin, more preferably includes one or more of phenolic acid plant polyphenol, flavonoid plant polyphenol, lignan plant polyphenol, and procyanidin, and most preferably one or more of procyanidin, phenolic acid plant polyphenol, and flavonoid plant polyphenol; the phenolic plant polyphenols preferably comprise one or more of tannins, gallic acid, epigallocatechin gallate, tea polyphenols, grape polyphenols, olive polyphenols, cocoa polyphenols and apple polyphenols, more preferably comprise one or more of tannins, gallic acid, epigallocatechin gallate, tea polyphenols, cocoa polyphenols and apple polyphenols, most preferably comprise one or more of tannins, gallic acid and epigallocatechin gallate; the flavonoid plant polyphenol preferably comprises one or more of flavonols, anthocyanins, flavonoids and catechins, more preferably comprises one or more of anthocyanins, flavonoids and catechins, most preferably comprises flavonoids and/or catechins; the lignan plant polyphenol preferably comprises one or more of resveratrol, forsythin and arbutin, more preferably comprises resveratrol and/or arbutin, and most preferably comprises resveratrol; the lignan plant polyphenol preferably includes one or more of curcumin and pinoresinol, and more preferably includes curcumin. When the plant polyphenol is two or more of the above specific choices, the invention does not have any special limitation on the ratio of the above specific substances, and the plant polyphenol is mixed according to any ratio. In an embodiment of the present invention, the plant polyphenol is specifically tannic acid, gallic acid, catechin, epigallocatechin gallate, curcumin or procyanidins.

The plant polyphenol microsphere provided by the invention is a metal-plant polyphenol assembly, and the plant polyphenol is self-maintained in a microsphere structure without being carried by a carrier, so that the plant polyphenol can be ensured to have a natural conformation for maintaining biological activity. Meanwhile, the porous three-dimensional microsphere structure can release plant polyphenol to a certain extent, so that the stability of the plant polyphenol is improved, the biocompatibility and the bioavailability are improved, and the porous three-dimensional microsphere structure has excellent antioxidant and anti-inflammatory effects and has good research and application prospects in the field of biomedical materials.

In the present invention, the temperature of all steps is preferably 1 to 50 ℃, more preferably 2 to 40 ℃, and most preferably 4 to 30 ℃ unless otherwise specified.

In the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise.

The invention mixes the plant polyphenol solution, the carbonate solution and the calcium salt solution for the first time, and stands the mixture to obtain the plant polyphenol doped calcium carbonate microsphere.

In the present invention, the concentration of the plant polyphenol solution is preferably 0.1 to 300mg/mL, more preferably 0.5 to 100mg/mL, and most preferably 1 to 50mg/mL.

In the present invention, the molar concentration of the carbonate solution is preferably 0.1 to 5mol/L, more preferably 0.2 to 3mol/L, and most preferably 0.3 to 1mol/L. In the present invention, the carbonate in the carbonate solution preferably includes one or more of ammonium bicarbonate, ammonium carbonate, sodium carbonate, and potassium carbonate, more preferably includes one or more of ammonium carbonate, sodium carbonate, and potassium carbonate; when the carbonate is two or more of the above specific choices, the present invention does not have any particular limitation on the ratio of the above specific substances, and the above specific substances may be mixed in any ratio. In an embodiment of the invention, the carbonate is in particular ammonium carbonate, sodium carbonate or potassium carbonate.

In the present invention, the molar concentration of the calcium salt solution is preferably 0.1 to 5mol/L, more preferably 0.2 to 3mol/L, and most preferably 0.3 to 1mol/L. In the present invention, the calcium salt in the calcium salt solution preferably comprises calcium chloride and/or calcium nitrate; when the calcium salt is calcium chloride and calcium nitrate, the invention does not limit the proportion of the specific substances, and the specific substances are mixed according to any proportion. In an embodiment of the invention, the calcium salt is specifically calcium chloride or calcium nitrate.

In the present invention, the volume ratio of the plant polyphenol solution to the carbonate solution is preferably 1:0.5 to 10, more preferably 1:0.8 to 5, most preferably 1:1 to 3; the volume ratio of the plant polyphenol solution to the calcium salt solution is preferably 1:0.5 to 10, more preferably 1:0.8 to 5, most preferably 1:1 to 3.

In the present invention, the first mixing preferably includes: mixing plant polyphenol solution and carbonate solution, adding calcium salt solution, or mixing plant polyphenol solution and calcium salt solution, and adding carbonate solution. In an embodiment of the present invention, the first mixing is specifically adding the calcium salt solution after mixing the plant polyphenol solution and the carbonate solution. In an embodiment of the present invention, the calcium salt solution or the carbonate solution is specifically added by 1 to 5mL of calcium chloride/calcium nitrate solution within 3 seconds. The addition of the calcium salt solution or carbonate solution is preferably carried out under stirring or ultrasound; the stirring speed is preferably 200 to 5000rpm, more preferably 500 to 3000rpm, and most preferably 1000 to 2000rpm; the frequency of the ultrasound is preferably 10 to 90kHz, more preferably 20 to 80kHz, and most preferably 30 to 70kHz. After the addition of the calcium salt or carbonate, the present invention also preferably includes continuing stirring or sonication; the time for continuing stirring or ultrasonic treatment is preferably 10 to 100 seconds, more preferably 20 to 80 seconds, and most preferably 30 to 60 seconds.

In the present invention, the time for the standing is preferably 5 to 60 minutes, more preferably 10 to 40 minutes, and most preferably 15 to 30 minutes.

After the standing is finished, the invention also preferably comprises centrifugation and washing which are sequentially carried out; the rotational speed of the centrifugation is preferably 500 to 8000rpm, more preferably 1000 to 6000rpm, and most preferably 2000 to 5000rpm; the time is preferably 2 to 30 minutes, more preferably 3 to 20 minutes, and most preferably 4 to 10 minutes. In the present invention, the washing liquid used for the washing is preferably ultrapure water; the washing conditions are not particularly limited in the present invention, and may be carried out by a process well known to those skilled in the art. In the present invention, the number of times of centrifugation is preferably 1 to 10 times, more preferably 2 to 8 times, and most preferably 3 to 5 times; the number of times of washing is preferably 1 to 10 times, more preferably 2 to 7 times, and most preferably 3 to 4 times.

The invention adopts a coprecipitation method to uniformly dope plant polyphenol into calcium carbonate microspheres to obtain the plant polyphenol doped calcium carbonate microspheres.

After the plant polyphenol doped calcium carbonate microsphere is obtained, the plant polyphenol doped calcium carbonate microsphere is etched by second mixing with acid liquor, so that the plant polyphenol microsphere is obtained.

In the present invention, the molar concentration of the acid solution is preferably 0.1 to 5mol/L, more preferably 0.2 to 3mol/L, and most preferably 0.3 to 1mol/L. In the present invention, the acid solution preferably includes one or more of a hydrochloric acid solution, a nitric acid solution and an acetic acid solution, more preferably a hydrochloric acid solution and/or a nitric acid solution; when the acid liquid is two or more of the above specific choices, the invention does not have any special limitation on the ratio of the above specific substances, and the acid liquid is mixed according to any ratio. In an embodiment of the present invention, the acid solution is specifically a hydrochloric acid solution, a nitric acid solution or an acetic acid solution.

In the present invention, the volume ratio of the acid solution to the carbonate solution is preferably 1 to 50:1, more preferably 3 to 40:1, most preferably 5 to 30:1.

in the present invention, the second mixing is preferably adding an acid solution to the plant polyphenol doped calcium carbonate microspheres. In the present invention, the second mixing is preferably performed under vortex conditions; the rotational speed of the vortex is preferably 200 to 5000rpm, more preferably 500 to 4000rpm, and most preferably 2000 to 3000rpm; the time is preferably 1 to 60 seconds, more preferably 10 to 50 seconds, and most preferably 20 to 40 seconds. After the second mixing is completed, the present invention also preferably includes sequentially performing centrifugation, washing, and drying. In the present invention, the rotational speed of the centrifugation is preferably 500 to 8000rpm, more preferably 1000 to 6000rpm, and most preferably 2000 to 5000rpm; the time is preferably 2 to 30 minutes, more preferably 3 to 20 minutes, and most preferably 4 to 10 minutes. In the present invention, the washing liquid for washing is preferably ultrapure water; the washing conditions are not particularly limited in the present invention, and may be carried out by a process well known to those skilled in the art. In the present invention, the number of times of centrifugation and washing is preferably 1 to 10 times, more preferably 2 to 8 times, and most preferably 3 to 5 times. In the present invention, the drying is preferably freeze-drying; the temperature of the freeze drying is preferably-80 to-10 ℃, more preferably-70 to-20 ℃, and most preferably-60 to-30 ℃; the time is preferably 30 to 500 minutes, more preferably 60 to 400 minutes, and most preferably 100 to 300 minutes.

According to the invention, the acid liquor is adopted to etch the plant polyphenol doped calcium carbonate microspheres, so that carbonate templates in the microspheres are removed, the microsphere structure of the plant polyphenol is maintained, and a porous morphology is generated; meanwhile, the plant polyphenol can capture and complex calcium ions in the solution, namely cation-pi interaction occurs, so that the metal ion-plant polyphenol composite plant polyphenol microsphere is obtained.

The preparation method of the plant polyphenol microsphere provided by the invention has the advantages of simple steps, mild conditions, uniform size, good dispersibility, better cell biocompatibility, excellent anti-inflammatory and antioxidant capabilities, and good research and application prospects in the field of biomedical materials.

The invention provides the application of the plant polyphenol microsphere or the plant polyphenol microsphere obtained by the preparation method in the technical scheme in preparation of antioxidant and anti-inflammatory preparations.

In the present invention, the antioxidation preferably includes hydrogen peroxide scavenging, superoxide anion radical scavenging, DPPH radical scavenging or ABTS radical scavenging, i.e., the antioxidation preparation preferably includes hydrogen peroxide scavenging preparation, superoxide anion radical scavenging preparation, DPPH radical scavenging preparation or ABTS radical scavenging preparation; the anti-inflammatory agent preferably comprises osteoarthritis, acute pancreatitis, chronic obstructive pulmonary disease, acute wound inflammation, or chronic wound inflammation, i.e., the anti-inflammatory agent preferably comprises an anti-osteoarthritis agent, an anti-acute pancreatitis agent, an anti-chronic obstructive pulmonary disease agent, an anti-acute wound inflammation agent, or an anti-chronic wound inflammation agent.

The method of the present invention is not particularly limited, and may be performed by a process well known to those skilled in the art.

In order to further illustrate the present invention, the following description of the present invention provides a plant polyphenol microsphere, its preparation method and application with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.

Example 1

Preparing tannic acid microspheres:

at 25 ℃, 1mL of tannic acid solution with the concentration of 5mg/mL and 1mL of potassium carbonate solution with the concentration of 1mol/L are mixed, placed on a magnetic stirrer, the stirring rotation speed is adjusted to 1200rpm, 3mL of calcium chloride solution with the concentration of 0.33mol/L is added in 3s, stirring is continued for 40s, standing is carried out for 20min, centrifugation is carried out for 5min under the condition of 1000rmp, washing is carried out by adopting ultrapure water, centrifugation is carried out for 4 times, and washing is carried out for 3 times. At this time, the tannic acid doped calcium carbonate microspheres with good dispersion and uniform size can be observed under a microscope.

5mL of 1mol/L hydrochloric acid solution is added into the tannic acid-doped calcium carbonate microsphere, vortex is carried out while acid liquor is added, centrifugal washing is carried out sequentially for 10s, the rotating speed is 3500rpm, the time is 4min, ultrapure water is adopted for washing, the centrifugation is carried out for 4 times, and the washing is carried out for 3 times. Freeze-drying at-40deg.C for 60min to obtain tannic acid microsphere shown in figure 1.

As can be seen from FIG. 1, the prepared tannic acid microsphere has a particle size of 1-5 μm, uniform size, complete morphology and good dispersibility.

Example 2

Preparing gallic acid microspheres:

after 1mL of gallic acid solution with the concentration of 10mg/mL and 1mL of ammonium carbonate solution with the concentration of 0.5mol/L are mixed at the temperature of 40 ℃, the mixture is placed on a magnetic stirrer, the stirring rotation speed is adjusted to 1500rpm, 2mL of calcium chloride solution with the concentration of 0.25mol/L is added in 3s, stirring is continued for 30s, the mixture is kept stand for 20min, the mixture is centrifuged for 5min under the condition of 2000rmp, the mixture is washed by ultrapure water, the centrifugation is carried out for 4 times, and the washing is carried out for 3 times. At this time, the gallic acid-doped calcium carbonate microspheres with good dispersion and uniform size can be observed under a microscope.

To the gallic acid-doped calcium carbonate microspheres, 10mL of a nitric acid solution with a concentration of 0.5mol/L was added, vortexing was performed while adding an acid solution for 10s, centrifugal washing was performed sequentially at a rotation speed of 3500rpm for 4min, washing was performed with ultrapure water, centrifugation was performed 6 times, and washing was performed 5 times. Freeze drying at-30deg.C for 120min to obtain gallic acid microsphere shown in figure 2.

As can be seen from FIG. 2, the prepared gallic acid microsphere has a particle size of 1-5 μm, uniform size, complete morphology and good dispersibility.

Example 3

Preparation of catechin microspheres:

at 20 ℃, 1mL of catechin solution with the concentration of 20mg/mL and 1mL of sodium carbonate solution with the concentration of 1mol/L are mixed, placed on a magnetic stirrer, the stirring speed is adjusted to 1200rpm, 3mL of calcium nitrate solution with the concentration of 0.33mol/L is added in 3s, stirring is continued for 40s, standing is carried out for 15min, centrifugation is carried out for 4min under 1500rmp, washing is carried out by adopting ultrapure water, centrifugation is carried out for 4 times, and washing is carried out for 3 times. At this time, catechin-doped calcium carbonate microspheres with good dispersion and uniform size were observed under a microscope.

Adding 20mL of hydrochloric acid solution with the concentration of 0.25mol/L into a proper amount of catechin-doped calcium carbonate microspheres, adding the acid solution, swirling the solution for 30s while, sequentially performing centrifugal washing at the rotating speed of 4000rpm for 4min, washing by adopting ultrapure water, centrifuging for 7 times, and washing for 6 times. Freeze drying at-50deg.C for 360min to obtain catechin microsphere shown in figure 3.

As can be seen from FIG. 3, the prepared catechin microsphere has a particle size of 1-5 μm, a uniform size and good dispersibility.

Example 4

Preparation of epigallocatechin gallate microspheres:

Mixing 1mL of epigallocatechin gallate solution with the concentration of 40mg/mL and 1mL of sodium carbonate solution with the concentration of 1mol/L at the temperature of 10 ℃, placing on a magnetic stirrer, regulating the stirring rotation speed to 1600rpm, adding 3mL of calcium chloride solution with the concentration of 0.33mol/L in 3s, continuously stirring for 30s, standing for 20min, centrifuging for 8min at 3000rmp, washing with ultrapure water, centrifuging for 4 times, and washing for 3 times. At this time, the epigallocatechin gallate-doped calcium carbonate microspheres with good dispersion and uniform size can be observed under a microscope.

10mL of nitric acid solution with the concentration of 0.5mol/L is added into a proper amount of the epigallocatechin gallate-doped calcium carbonate microsphere, vortex is carried out while acid liquor is added for 20s, centrifugal washing is sequentially carried out at the rotating speed of 4500rpm for 4min, the centrifugation is carried out for 7 times, and the washing is carried out for 6 times. Freeze drying at-45deg.C for 60min to obtain epigallocatechin gallate microsphere.

Example 5

Preparation of curcumin microspheres:

at 40 ℃, 1mL of curcumin solution with the concentration of 30mg/mL and 1mL of potassium carbonate solution with the concentration of 1mol/L are mixed, placed on a magnetic stirrer, the stirring speed is adjusted to 2000rpm, 5mL of calcium chloride solution with the concentration of 0.2mol/L is added in 3s, stirring is continued for 40s, standing is carried out for 10min, centrifugation is carried out for 10min under 2500rmp, washing is carried out by adopting ultrapure water, centrifugation is carried out for 4 times, and washing is carried out for 3 times. At this time, curcumin-doped calcium carbonate microspheres with good dispersion and uniform size can be observed under a microscope.

And adding 25mL of hydrochloric acid solution with the concentration of 0.2mol/L into a proper amount of curcumin-doped calcium carbonate microspheres, swirling the mixture while adding the acid solution for 40s, sequentially performing centrifugal washing at the rotation speed of 5000rpm for 4min, performing centrifugation for 10 times and washing for 9 times. Freeze drying at-30deg.C for 400min to obtain curcumin microsphere.

Example 6

Preparation of procyanidine (OPC) microspheres:

mixing 1mL of procyanidine solution with the concentration of 60mg/mL and 1mL of sodium carbonate solution with the concentration of 1mol/L at 25 ℃, placing on a magnetic stirrer, regulating the stirring rotation speed to 1200rpm, adding 3mL of calcium chloride solution with the concentration of 0.33mol/L in 3s, continuing stirring for 40s, standing for 15min, centrifuging for 5min under 1000rmp, washing with ultrapure water, centrifuging for 4 times, and washing for 3 times. The procyanidine-doped calcium carbonate microspheres shown in fig. 4 were obtained.

And 5mL of hydrochloric acid solution with the concentration of 1mol/L is added into a proper amount of the procyanidine-doped calcium carbonate microsphere, vortex is carried out while acid solution is added, the time is 10s, centrifugal washing is sequentially carried out, the rotating speed is 3500rpm, the time is 4min, the centrifugal washing is carried out for 8 times, and the washing is carried out for 7 times. Freeze drying at-45deg.C for 240min to obtain procyanidin microsphere shown in figure 5 and figure 6.

As can be seen from fig. 4, the procyanidine-doped calcium carbonate microsphere formed by coprecipitation of procyanidine and calcium carbonate has complete appearance, shows a loose and porous structure, has a particle size of 1-5 μm, and is uniform in size and good in dispersibility.

As can be seen from FIGS. 5 and 6, the prepared gallic acid microsphere has a particle size of 1-5 μm, a uniform size, a complete morphology and good dispersibility.

Example 7

Preparation of procyanidine (OPC) microspheres:

mixing 1mL of procyanidine solution with the concentration of 60mg/mL and 2mL of sodium carbonate solution with the concentration of 0.5mol/L at 25 ℃, placing on a magnetic stirrer, regulating the stirring rotation speed to 1200rpm, adding 1mL of calcium chloride solution with the concentration of 1mol/L in 3s, continuing stirring for 40s, standing for 15min, centrifuging for 4min under 1000rmp, washing with ultrapure water, centrifuging for 4 times, and washing for 3 times. At this time, the procyanidine-doped calcium carbonate microspheres with good dispersion and uniform size can be observed under a microscope.

And 5mL of hydrochloric acid solution with the concentration of 1mol/L is added into a proper amount of the procyanidine-doped calcium carbonate microsphere, vortex is carried out while acid solution is added, the time is 10s, centrifugal washing is sequentially carried out, the rotating speed is 3500rpm, the time is 4min, the centrifugal washing is carried out for 4 times, and the washing is carried out for 4 times. Freeze drying at-45deg.C for 300min to obtain procyanidin microsphere.

Example 8

Preparation of procyanidine (OPC) microspheres:

mixing 1mL of pure water, 1mL of procyanidine solution with the concentration of 5mg/mL and 1mL of potassium carbonate solution with the concentration of 1mol/L at 25 ℃, placing on a magnetic stirrer, regulating the stirring rotation speed to 1200rpm, adding 3mL of calcium chloride solution with the concentration of 0.33mol/L in 3s, continuously stirring for 30s, standing for 10min, centrifuging for 4min under 1000rmp, washing with ultrapure water, centrifuging for 4 times, and washing for 3 times. At this time, the procyanidine-doped calcium carbonate microspheres with good dispersion and uniform size can be observed under a microscope.

Adding 3mL of acetic acid solution with the concentration of 1mol/L into a proper amount of the procyanidine-doped calcium carbonate microsphere, swirling while adding acid liquor for 20s, sequentially performing centrifugal washing at 3500rpm for 4min, performing centrifugation for 4 times, and washing for 4 times. Freeze drying at-45deg.C for 300min to obtain procyanidin microsphere.

Example 9

Preparation of procyanidine (OPC) microspheres:

mixing 1mL of pure water, 1mL of procyanidine solution with the concentration of 90mg/mL and 1mL of ammonium carbonate solution with the concentration of 1mol/L at 25 ℃, placing on a magnetic stirrer, regulating the stirring rotation speed to 1200rpm, adding 2mL of calcium nitrate solution with the concentration of 0.5mol/L in 3s, continuously stirring for 50s, standing for 30min, centrifuging for 4min at 1500rmp, washing with ultrapure water, centrifuging for 4 times, and washing for 3 times. At this time, the procyanidine-doped calcium carbonate microspheres with good dispersion and uniform size can be observed under a microscope.

15mL of nitric acid solution with the concentration of 1mol/L is added into a proper amount of the procyanidine-doped calcium carbonate microsphere, vortex is carried out while acid liquor is added for 3s, centrifugal washing is sequentially carried out, the rotating speed is 3500rpm, the time is 4min, the centrifugal washing is carried out for 4 times, and the washing is carried out for 4 times. Freeze drying at-45deg.C for 300min to obtain procyanidin microsphere.

Test example 1

And (3) selecting a total antioxidant capacity determination kit (T-AOCAssaykit), and determining the total antioxidant capacity of the procyanidine (OPC) microsphere by using an FRAP method. The experiments were performed according to the instructions provided by the manufacturer. Procyanidine (OPC) microsphere diluent (250 mug/mL) and working solution are uniformly mixed, incubated for 3min at 37 ℃, and absorbance at 593nm is detected by an enzyme-labeled instrument. The higher the number, the greater the total antioxidant capacity of the material. The test results are shown in fig. 7, wherein "×" indicates that there was a significant difference between the two groups (P < 0.0001).

As can be seen from fig. 7, the short-term total antioxidant capacity of procyanidin (OPC) microspheres is 3.14 times that of procyanidins (OPC).

Test example 2

Determination of procyanidine (OPC) microsphere clearance H by Hydrogen peroxide decomposition FOX method ₂ O ₂ Is provided). First, H with different concentrations is prepared ₂ O ₂ The concentrations were 0. Mu. Mol/L, 10. Mu. Mol/L, 20. Mu. Mol/L, 30. Mu. Mol/L, 40. Mu. Mol/L, 50. Mu. Mol/L, 60. Mu. Mol/L and 70. Mu. Mol/L, respectively. The 50 mu LH is then applied to ₂ O ₂ Rapidly mixed with 950 mu LFOX reagent and incubated for 30min at room temperature. The absorbance at 560nm was recorded and H was plotted ₂ O ₂ Is a standard curve of (2). 20. Mu.L of procyanidin (OPC) microsphere dilution (250. Mu.g/mL) was combined with H ₂ O ₂ Mixing the solution (60. Mu.M), shaking at 60rpm for 10H, and mixing 100. Mu.L procyanidine (OPC) microsphere dilution with H ₂ O ₂ Is centrifuged at 3000rpm for 5min, 50. Mu.L of the supernatant of the mixture is taken and rapidly mixed with 950. Mu.LFOX reagent and incubated at room temperature for 30min. The absorbance of the mixture at 560nm was measured by UV-visible spectroscopy. According to H ₂ O ₂ Obtaining H of procyanidine (OPC) microsphere ₂ O ₂ Scavenging activity. The test results are shown in FIG. 8, wherein "/indicates a significant difference between the two groups (P < 0.0001)

As can be seen from FIG. 8, short-term clearance H of procyanidin (OPC) microspheres ₂ O ₂ Is 2.77 times higher than procyanidins (OPC).

Test example 3

The capability of procyanidine (OPC) microspheres to remove superoxide anion free radicals is determined by using a total SOD activity detection kit (WST-8 method). The experiments were performed according to the instructions provided by the manufacturer. Procyanidine (OPC) microspheres (100. Mu.g/mL) were mixed with working fluid and incubated at 37℃for 30min. The absorbance value of the sample was measured at 450 nm. The calculation formula of the sample superoxide anion radical clearance is as follows:

Wherein Abs blank 1 is absorbance value of WST working solution and enzyme working solution, abs blank 2 is absorbance value of solvent ultrapure water and buffer solution, abs sample is absorbance value of sample mixed with WST working solution and enzyme working solution, abs blank 3 is absorbance value of sample mixed with solvent: absorbance values after mixing ultrapure water and buffer.

The test results are shown in fig. 9, where "×" indicates that there was a significant difference between the two groups (P < 0.0001).

As can be seen from fig. 9, the short-term superoxide anion radical scavenging capacity of procyanidin (OPC) microspheres is 5.34 times that of procyanidins (OPC).

Test example 4

The ability of procyanidin (OPC) microspheres to scavenge DPPH radicals was determined using a DPPH radical scavenging ability assay kit. The experiments were performed according to the instructions provided by the manufacturer. Procyanidine (OPC) microsphere diluent (100 mug/mL) and working solution are uniformly mixed, kept away from light at room temperature, and kept stand for 30min, and absorbance at 515nm is measured by an enzyme-labeled instrument. Absorbance values for blank tube, control tube and sample tube were recorded as Abs blank, abs control and Abs sample, respectively. The calculation formula of the DPPH free radical clearance of the sample is as follows:

the Abs blank is the absorbance value of the working fluid, the Abs sample is the absorbance value of the sample after the sample is mixed with the working fluid, and the Abs control is the absorbance value of the sample. The test results are shown in fig. 10, where "×" indicates that there was a significant difference between the two groups (P < 0.0001).

As can be seen from fig. 10, the short term DPPH scavenging capacity of procyanidin (OPC) microspheres is 3.99 times that of procyanidins (OPC).

Test example 5

The ability of procyanidin (OPC) microspheres to scavenge ABTS free radicals was determined using an ABTS free radical scavenging ability assay kit. The experiments were performed according to the instructions provided by the manufacturer. Procyanidine (OPC) microspheres (100 ug/mL) are mixed with a working solution, and after being fully and uniformly mixed, the mixture is kept away from light for 6min at room temperature, and the absorbance value of a sample at 405nm is measured. Absorbance values for blank tube, control tube and sample tube were recorded as Abs blank, abs control and Abs sample, respectively. The formula for calculating the radical scavenging rate of the sample ABTS is as follows:

the Abs blank is the absorbance value of the working fluid, the Abs sample is the absorbance value of the sample after the sample is mixed with the working fluid, and the Abs control is the absorbance value of the sample. The test results are shown in fig. 11, where "×" indicates that there was a significant difference between the two groups (P < 0.0001).

As can be seen from fig. 11, the short-term ABTS radical scavenging ability of procyanidin (OPC) microspheres is 2.22 times that of procyanidins (OPC).

Test example 6

To evaluate the biocompatibility of procyanidin (OPC) microspheres in mice, the toxicity of procyanidin (OPC) and procyanidin (OPC) microspheres to both mouse mononuclear macrophages (RAW 264.7) and mouse dendritic cells (DC 2.4) in common cells of inflammatory experiments was tested using a Cell Counting Kit-8 (CCK-8) kit. When cells grew to about 80%, the medium was discarded and the walls of the flask were washed twice with 2mL of room temperature PBS. Then adding 1mL of 0.25% pancreatin, and digesting for 0-1 min to discard pancreatin. 3mL of complete medium was added, the walls were gently blown using a pipette, and the cells on the walls were collected and mixed well. 10. Mu.L of the cell suspension was placed in a new centrifuge tube, 90. Mu.L of complete medium was added to dilute the cells and mixed well, 10. Mu.L of diluted cell suspension was removed and dropped onto a cell counting plate, and the cell number was counted by a microscope. Diluting the cell stock to 5X 10 ⁴ Each cell per milliliter. A96-well plate is selected, 100 mu L of diluted cell suspension is added into each well, and the mixture is placed in an incubator for culturing for 24 hours. Procyanidine (OPC) solutions and procyanidine (OPC) microsphere dilutions (0 mg/mL, 0.03mg/mL, 0.06mg/mL, 0.12mg/mL, 0.25mg/mL, 0.5 mg/mL and 1 mg/mL) with different concentrations prepared by using fresh culture media are used, the plates are taken out after 24 hours of culture, the procyanidine (OPC) solutions and procyanidine (OPC) microsphere dilutions prepared above are added, and the plates are continuously placed in an incubator for culture. Three control wells were set for each concentration. After incubation for 24h or 48h, old medium was aspirated using a pipette, 100. Mu.L of basal medium containing 10% CCK-8 was added to each well, the well plate was placed in a cell incubator for incubation for 2h, absorbance at 450nm was detected by a microplate reader, and the viability of cells at different concentrations was calculated by comparison with the control group. The test results are shown in FIG. 12, which(a) is a survival rate test chart of RAW264.7 cells at 24 h; (b) is a viability test pattern of RAW264.7 cells at 48 h; (c) viability test pattern of DC2.4 cells at 24 h; (c) viability test pattern of DC2.4 cells at 48 h. The experimental data all adopt repeated experimental mean values to indicate that the mean values are calculated by mean + -SD, and n=3.

As can be seen from fig. 12, both mouse mononuclear macrophages (RAW 264.7) and mouse dendritic cells (DC 2.4) showed good survival rates at 24h and 48h with procyanidin (OPC) microsphere dilutions, whereas the survival rate decreased with increasing concentration with procyanidin (OPC) solution. From this, it is known that procyanidine (OPC) microspheres exhibit lower biotoxicity and more excellent biocompatibility than procyanidins (OPC).

Test example 7

The CLSM method detects the degree of cellular internalization: RAW264.7, DC2.4 and NIH3T3 cells (5X 10) ⁴ Individual cells/well) were cultured in 24-well plates for 24 hours, and then a procyanidin (OPC) microsphere solution was added, the ratio of procyanidin (OPC) microsphere to cell number was 12:1. after further culturing for 24 hours, 300. Mu.L of 4% paraformaldehyde was added to each well to fix the cells for 10min. The cells were then washed three times with PBS, followed by cell staining. The specific dyeing process is as follows: (1) mu.L of 0.5% Triton X-100 was added to each well, and the cells were washed three times with PBS after 10min. (2) After adding 300uL of 5. Mu.g/mL rhodamine staining for 60min per well, cells were washed three times with PBS. (3) 300uL of 5. Mu.g/mLDAPI was added to each well for 10 minutes. The samples were then observed with a confocal laser microscope. The results of the test are shown in FIG. 13, wherein (a), (b) and (c) are endocytic behavior of RAW264.7, DC2.4 and NIH3T3 cells On Procyanidin (OPC) microspheres, respectively. Wherein the scale is 10 μm.

As can be seen from fig. 13, after 24h of culture of the cells with procyanidin microsphere, the morphology of RAW264.7, DC2.4 and NIH3T3 cells was not changed, and more green circles appeared inside the red cell membrane and outside the blue cell nucleus, thus it was found that more procyanidin (OPC) microsphere was successfully endocytosed by the cell within 24 h.

Test example 8

Procyanidine (OPC) microsphere pair H ₂ O ₂ Preservation of treated cellsThe protection effect is as follows: procyanidin (OPC) microsphere pair H was evaluated using the CellCounting Kit-8 (CCK-8) Kit ₂ O ₂ Protective effect of the treated cells. After RAW264.7 cells and DC2.4 cells were inoculated on 96-well plates and cultured for 24 hours (5X 103 cells/well), different concentrations of procyanidin (OPC) microspheres were added to the cells (procyanidin (OPC) microspheres to cell count ratios of 3:1, 6:1, 12:1, 25:1 and 50:1) at a volume of 100. Mu.L per well. RAW264.7 cells were cultured for 12 hours, while DC2.4 cells were cultured for 24 hours, after which the medium was removed and 0.5. 0.5mMH was added to each well ₂ O ₂ Incubate for 30min. Cells were then rinsed with PBS and replaced with fresh medium. After the plates were incubated at 37℃for 24h, cell viability was detected with CCK8 reagent. Only by 0.5. 0.5mMH ₂ O ₂ Treated cells served as controls. The results of the test are shown in FIG. 14, which is a graph showing the change in cell viability after hydrogen peroxide treatment of RAW264.7 cells and DC2.4 cells, respectively.

As can be seen from FIG. 14, H is added ₂ O ₂ The survival rate of RAW264.7 and DC2.4 cells after treatment was reduced to 52% and 75%, while the survival rate of RAW264.7 cells after addition of procyanidin (OPC) microspheres was increased to 79%, the survival rate of DC2.4 cells was increased to 100%, and the effect was dose-dependent. From this, it was found that procyanidine (OPC) microsphere pair H ₂ O ₂ The treated cells have good protective effect.

Test example 9

2',7' -dichlorofluorescein diacetate (DCF-DA) determination of intracellular ROS levels: RAW264.7 cells and DC2.4 cells were inoculated on 24-well plates, respectively, and cultured for 24 hours (5X 10) ⁴ Individual cells/well), with H ₂ O ₂ (500. Mu.g/mL) stimulated cells for 30min. The cells were then washed with PBS and a solution of procyanidin (OPC) microspheres (diluted with medium) was added with a procyanidin (OPC) microsphere to cell number ratio of 12:1. the volume added per well was 100 μl. After 24 hours incubation, DCF-DA (10M) was added and incubated for 30 minutes. Finally, the cells were washed with PBS and stained with 300. Mu.L of 5. Mu.g/mLDAPI per well for 10 min. Confocal laser microscopy was used to observe fluorescent signals within cells. The test results are shown in FIG. 15, and (a) and (b) are DCFH-DA fluorescent probes, respectivelyCLSM map to detect active oxygen levels in RAW264.7 cells and CLSM map to detect active oxygen levels in DC2.4 cells with DCFH-DA fluorescent probe.

As can be seen from FIG. 15, the green fluorescence in RAW264.7 and DC2.4 cells after addition of procyanidin (OPC) microspheres was not increased relative to the control, whereas H was added ₂ O ₂ Green fluorescence in RAW264.7 and DC2.4 cells after treatment was increased 6-fold and 7-fold, respectively, compared to control, and procyanidine (OPC) microspheres and H were added ₂ O ₂ The green fluorescence level of the post group is obviously reduced, and the ROS level and pure H in RAW264.7 and DC2.4 cells are obviously reduced ₂ O ₂ The group was reduced by 78.5% and 41.67%, respectively. From this, procyanidine (OPC) microspheres are effective in reducing intracellular ROS levels.

Test example 10

Cell migration: evaluation of H by scratch wound experiments ₂ O ₂ Migration ability of DC2.4 cells after treatment. DC2.4 cells were seeded on 12-well plates (2X 10) ⁵ Individual cells/well) for 24h, the DC2.4 cells were plated in the wells of the culture plate to form a monolayer of cells. By H ₂ O ₂ After 30min of stimulation of the cells (500. Mu.g/mL), the monolayer of cells was streaked in a straight line with a 200. Mu.L pipette tip, simulating an incision. The floating cells and debris were then rinsed with PBS. The cells were then cultured for an additional 24h with fresh DMEM or DMEM containing procyanidin (OPC) microspheres. The difference in wound surface area between 0h and 24h was observed under an optical microscope to quantify the degree of closure of the scratches. A white light image taken by fluorescence microscopy of DC2.4 cell migration is shown in FIG. 16.

As can be seen from fig. 16, the cell mobility of the control group increased to 43% compared to 0h for the cultured cells 24h, and the migration rate of the procyanidin (OPC) microsphere group was comparable to that of the control group, about 41%, so that no significant change in the cell migration rate was caused after the addition of the microspheres. To add H ₂ O ₂ The migration rate of the treated cell group was 16%, and procyanidin (OPC) microspheres and H were added ₂ O ₂ Cell migration rate of 27.76% compared to H) ₂ O ₂ The group increased 11.76%. From the results, procyanidine (OPC) microspheres can significantly improve H ₂ O ₂ State of cells after addition.

Test example 11

ELISA kit for detecting intracellular inflammatory factor level: the mouse IL-6ELISA kit and the mouse TNF-alpha ELISA kit are used for evaluating the inflammatory factor level of RAW264.7 cells after lipopolysaccharide induction. The specific flow is as follows:

(1) RAW264.7 cells were inoculated into 24-well plates and cultured for 24 hours (5X 10) ⁴ Individual cells/wells) and subsequently pre-treating the cells with different concentrations of procyanidin (OPC) microspheres (the ratio of procyanidin (OPC) microspheres to number of cells is: 6: 1. 12: 1. 25:1 and 50: 1) The supernatant was then removed and the cells were further cultured by adding 2. Mu.g/mL of lipopolysaccharide solution to a volume of 500. Mu.L per well for 24 hours.

(2) After 24h incubation, the supernatant from each well was aspirated into 1.5mL centrifuge tubes, and the levels of cytokines (IL-6 and TNF-. Alpha.) in the supernatant were measured by ELISA and cells treated with LPS alone were used as positive controls. The results of the test are shown in FIG. 17, (a) and (b) are inflammatory factors IL-6 and TNF- α levels, respectively, in supernatants of different groups of RAW264.7 cells after LPS induction. Where "+" indicates a large difference between the two groups (P < 0.05), "+" indicates a significant difference between the two groups (P < 0.001), both expressed as mean±sd, and n=3.

As can be seen from FIG. 17, the expression level of inflammatory factors IL-6 and TNF- α in RAW264.7 cells after LPS stimulation was increased, and the IL-6 expression level after addition of procyanidin (OPC) microspheres was increased from 530 pg.mL ^-1 Reduced to 280 pg.mL ^-1 TNF-alpha expression level was determined to be 870 pg.mL ^-1 Reduced to 540 pg.mL ^-1 . From this, procyanidine (OPC) microspheres show a better anti-inflammatory effect on cells.

Test example 12

To evaluate the anti-inflammatory activity of procyanidin (OPC) microspheres, a surgical osteoarthritis model was established and procyanidin (OPC) microspheres were used for treatment.

C57 male mice were purchased at 6 weeks of age. Grouping mice: the mice were divided into 4 groups of 10 mice each. The normal group, the control group and the experimental group with two concentrations are respectively named as a normal group, a control group, an L-microsphere group and an H-microsphere group. Procyanidin (OPC) microspheres required in the experiment were washed three times with physiological saline in advance. The experiment comprises the following steps:

Mice were anesthetized with isoflurane. After fixing the mice, the joints of the hind legs of the mice were cut, the meniscus on one side of the mice was pulled out, and then the wound was sutured. The normal group only needs to cut the skin and then suture. The control group was injected with 10. Mu.L of physiological saline, and the experimental group was respectively injected with 10. Mu.L of procyanidine (OPC) microsphere dilutions at concentrations of 0.2mg/kg and 2 mg/kg. The administration mode is joint cavity injection administration.

After normal feeding of mice for four weeks, the mice were anesthetized with isoflurane and sacrificed using cervical dislocation. The leg joints of the mice are taken out, and the adhered tissues on the joints are cleared as much as possible. After decalcification treatment of the joints for four weeks, paraffin embedding, tissue sections, H & E staining and qPCR testing were performed. The test results are shown in FIG. 18, wherein (a) is a staining chart of osteoarthritis model H & E; (b) Is a graph for detecting the expression level of IL-6, IL-1 beta, TNF-alpha and MMP-13 in an osteoarthritis model.

As can be seen from FIG. 18 (a), H & E staining showed that the synovial membrane of the control group was inflamed and severely damaged. And after procyanidine (OPC) microspheres are added, the synovial degeneration induced by operation is obviously improved, and the damage of articular cartilage cells is obviously reduced.

As can be seen from fig. 18 (b), qPCR test results show that the expression of IL-6, IL-1β, TNF- α and MMP-13 in the L-microsphere set and the H-microsphere set is reduced in concentration dependency, which indicates that procyanidine (OPC) microspheres can effectively reduce the levels of pro-inflammatory factors in joints, and achieve the effect of alleviating inflammation. The above test results all show that procyanidin (OPC) microspheres exhibit excellent anti-osteoarthritis effects.

Test example 13

To evaluate the anti-inflammatory activity of procyanidin (OPC) microspheres, an acute pancreatitis C57BL/6 mouse model was established and treated with procyanidin (OPC) microspheres.

6-week-old male mice were purchased, divided into an experimental group and a control group, after 1 week of adaptive feeding, the abdominal cavity was fed with 1. Mu.g of a rana grahami peptide (product number: IC4660, available from Solibao) at a concentration of 50. Mu.g/kg 7 times per hour, 2. Mu.g of a lipopolysaccharide (product number: L8880, available from Solibao) at an interval of 1 hour, and 5mg of a procyanidin (OPC) microsphere diluent at a concentration of 20mg/kg at an interval of 1 hour after the lipopolysaccharide injection, and the control group was fed with an equivalent amount of physiological saline. C57BL/6 mice were anesthetized after 12 hours, fixed, and pancreatic tissue was removed after opening the abdomen for paraffin embedding, tissue sections, and H & E staining. The experimental results are shown in FIG. 19, wherein the scale is 50. Mu.m.

As can be seen from fig. 19, the control group pancreatic tissue was severely damaged, while the experimental group pancreatic acinar cells were significantly increased in number, and the morphology of the secretion of acinar cell aggregates was more complete. Indicating that procyanidine (OPC) microspheres exhibit excellent anti-pancreatitis effects.

Test example 14

To evaluate the anti-inflammatory activity of procyanidin (OPC) microspheres, a chronic obstructive pulmonary disease C57BL/6 mouse model was established and treated with procyanidin (OPC) microspheres.

A model of chronic pneumonia C57BL/6 mice was established using the Kobayashi method: 6-week-old male mice were purchased, divided into an experimental group and a control group, after 1 week of adaptive feeding, a lipopolysaccharide (product number: L8880, available from Soy pal) of 10mg/kg in 20. Mu.l physiological saline was fasted for 12 hours, nasal feeding drops were injected into the lungs of the mice, and after 1 hour interval, the experimental group was injected with a 5mg procyanidin (OPC) microsphere dilution of 20mg/kg concentration, and the control group was injected with an equivalent physiological saline. After 72 hours, C57BL/6 mice were anesthetized, fixed, and lung tissue was removed after chest opening for paraffin embedding, tissue sections, and H & E staining. The experimental results are shown in FIG. 20.

As can be seen from fig. 20, the experimental group showed a reduction in abnormally enlarged alveoli, a reduction in the areas of lung thickening and bleeding injury, and a more regular and uniform pulmonary alveoli shape, compared to the control group. Indicating that procyanidine (OPC) microspheres exhibit excellent anti-pneumonia effects.

Test example 15

To evaluate the anti-inflammatory activity of procyanidin (OPC) microspheres, an acute wound C57BL/6 mouse model was established and treated with procyanidin (OPC) microspheres.

C57 male mice were purchased at 6 weeks of age. Grouping mice: the mice were divided into three groups of 10 mice each. The control group and the two concentration material groups (L-microsphere group and H-microsphere group) were respectively used. Procyanidin (OPC) microspheres required in the experiment were washed three times with physiological saline in advance. The experiment comprises the following steps: after mice were anesthetized with isoflurane, the mice were fixed, wounds of 8mm were made on the backs of the mice using a punch, and each mouse was coated with a drug at the wound site, and 30 μl of material was coated on the backs of each mouse. The control group was smeared with physiological saline. Mice were kept normally and mice were continuously monitored for skin wound healing on days 0, 3, 7 and 14 of the manufacturing wound. On day 14 of molding, after isoflurane anesthetizing the mice, the mice were sacrificed using cervical dislocation. Taking a wound of about 8mm near the wound, embedding paraffin, and staining the tissue section by H & E. In vivo evaluation of the therapeutic effect of skin wounds on the back of normal mice: wound contraction and inflammation were observed and evaluated. All observations and evaluations were performed by the same observer under blind conditions. The experimental results are shown in fig. 21, which are respectively a skin wound white light chart and a wound H & E staining chart of an acute wound model control group, an L-microsphere group and an H-microsphere group.

As can be seen from fig. 21, according to the white light pattern and the H & E staining pattern of the skin wound surface, the addition of procyanidine (OPC) microspheres significantly accelerates the healing of skin wound from 3 days to 14 days after operation, and the lengths of the sloughed scab areas are respectively: control group > L-microsphere group > H-microsphere group. The procyanidin (OPC) microspheres were shown to exhibit excellent effect in treating acute skin wounds.

Test example 16

To evaluate the anti-inflammatory activity of procyanidin (OPC) microspheres, a chronic wound C57BL/6 mouse model was established and treated with procyanidin (OPC) microspheres.

C57 male mice were purchased at 6 weeks of age. Grouping mice: a total of 3 groups of 10 mice each. The control group and the two concentration material groups (L-microsphere group and H-microsphere group) were respectively used. Procyanidin (OPC) microspheres required in the experiment were washed three times with physiological saline in advance. The experiment comprises the following steps: (1) preparing a citric acid-sodium citrate buffer solution: the concentrations of citric acid and sodium citrate are 10mM, the sodium citrate and the citric acid are dissolved by sterile water, and then the sodium citrate is prepared according to the volume ratio: citric acid = 28:22 such that the final ph=4.2 to 4.5, then filtered sterilized with a needle filter, and stored at 4 ℃. (2) then dissolving streptozotocin: streptozotocin is said to be protected from light and stored in a refrigerator at-20 ℃. Just prior to injection, streptozotocin was dissolved in ice-cold buffer, vortexed until clear, the final solution concentration was 5mg/mL, and the solution was placed in an ice-water mixture, with streptozotocin being prepared as quickly as possible (completed within 30min as possible) from the time of preparation to injection. (3) Mice were given intraperitoneal injections in an amount of 10. Mu.L/g (body weight), and after 5 days of continuous injection, were fed normally for one week, and die-making was considered successful when fasting blood glucose was measured, with blood glucose above 250 mg/dL. Starvation was required for 12h (no water forbidden) before injection, and feeding was continued for 2h after injection and 2h after injection.

The skin wound molding process comprises the following steps: after mice were anesthetized with isoflurane, the mice were fixed, wounds of 8mm were made on the backs of the mice using a punch, and each mouse was coated with a drug at the wound site, and 30 μl of material was coated on the backs of each mouse. The control group was smeared with physiological saline. Mice were kept normally and mice were continuously monitored for skin wound healing on days 0, 3, 7 and 14 of the manufacturing wound. On day 14 of molding, after isoflurane anesthetizing the mice, the mice were sacrificed using cervical dislocation. Taking a wound of about 8mm near the wound, embedding paraffin, and staining the tissue section by H & E. In vivo evaluation of the therapeutic effect of skin wounds on the back of normal mice: wound contraction and inflammation were observed and evaluated. All observations and evaluations were performed by the same observer under blind conditions. The experimental results are shown in fig. 22, which are respectively a skin wound white light chart and a wound H & E staining chart of a control group, an L-microsphere group and an H-microsphere group after 14 days of operation.

As can be seen from fig. 22, the addition of procyanidin (OPC) microspheres significantly accelerated skin wound healing from 3 days to 14 days post-surgery compared to the control group, and the skin healing rate was faster for the H-microsphere group than for the L-microsphere group. H & E staining of the damaged skin area at 14 days post-surgery showed that the scab shedding lengths were respectively: control group > L-microsphere group > H-microsphere group, indicating that procyanidin (OPC) microspheres exhibit excellent effect of treating chronic diabetic skin wounds.

From the above embodimentsAnd test examples show that the plant polyphenol microsphere provided by the invention has the diameter of 1-500 mu m, and maintains good porous microsphere morphology by the structure of the plant polyphenol microsphere. According to experimental data, the plant polyphenol microsphere provided by the application has low biotoxicity, good stability, good biocompatibility and high bioavailability, and correspondingly, the plant polyphenol microsphere has excellent total antioxidant capacity and H removal capacity ₂ O ₂ The ability to clear DPPH, and the ability to combat osteoarthritis, pancreatitis, pneumonia. That is, the plant polyphenol microsphere provided by the application has excellent antioxidant and anti-inflammatory effects, and has good research and application prospects in the field of biomedical materials.

Although the foregoing embodiments have been described in some, but not all embodiments of the invention, other embodiments may be obtained according to the present embodiments without departing from the scope of the invention.

Claims

1. The plant polyphenol microsphere is characterized by comprising plant polyphenol and calcium ions distributed in the plant polyphenol; the plant polyphenol is procyanidine;

The plant polyphenol microsphere has a porous structure;

the preparation method of the plant polyphenol microsphere comprises the following steps:

2. The plant polyphenol microsphere according to claim 1, wherein the plant polyphenol microsphere has a diameter of 1 to 500 μm.

3. The method for preparing the plant polyphenol microsphere according to claim 1 or 2, which is characterized by comprising the following steps:

firstly mixing a plant polyphenol solution, a carbonate solution and a calcium salt solution, and standing to obtain plant polyphenol doped calcium carbonate microspheres; the plant polyphenol solution is procyanidine solution;

4. The method according to claim 3, wherein the carbonate solution has a molar concentration of 0.1 to 5mol/L;

the molar concentration of the calcium salt solution is 0.1-5 mol/L;

The concentration of the plant polyphenol solution is 0.1-300 mg/mL.

5. A method of preparing according to claim 3, wherein the first mixing comprises: mixing plant polyphenol solution and carbonate solution, and adding calcium salt solution;

the stirring speed is 200-5000 rpm;

the frequency of the ultrasonic wave is 10-90 kHz.

6. The method of claim 3, wherein the acid solution comprises one or more of a hydrochloric acid solution, a nitric acid solution, and an acetic acid solution;

the molar concentration of the acid liquor is 0.1-5 mol/L.

7. Use of the plant polyphenol microsphere according to claim 1 or 2 or the plant polyphenol microsphere obtained by the preparation method according to any one of claims 3 to 6 in the preparation of antioxidant preparations and anti-inflammatory preparations.

8. The use of claim 7, wherein the antioxidant formulation comprises a hydrogen peroxide scavenging formulation, a superoxide anion radical scavenging formulation, a DPPH radical scavenging formulation, or an ABTS radical scavenging formulation.

9. The use of claim 7, wherein the anti-inflammatory agent comprises an anti-osteoarthritis agent, an anti-acute pancreatitis agent, an anti-chronic obstructive pulmonary agent, an anti-acute wound inflammation agent, or an anti-chronic wound inflammation agent.