CN113332489B

CN113332489B - Bletilla striata-coptis chinensis compound sponge dressing and preparation method and application thereof

Info

Publication number: CN113332489B
Application number: CN202110702861.4A
Authority: CN
Inventors: 王萌; 任晓亮; 刘亚男; 张德芹; 陈美玲; 孙立丽; 刘虹; 刘艺; 张鹏; 李娜
Original assignee: Tianjin University of Traditional Chinese Medicine
Current assignee: Tianjin University of Traditional Chinese Medicine
Priority date: 2021-06-24
Filing date: 2021-06-24
Publication date: 2022-06-03
Anticipated expiration: 2041-06-24
Also published as: CN113332489A

Abstract

The embodiment of the invention provides a bletilla striata-coptis chinensis compound sponge dressing and a preparation method thereof, wherein the bletilla striata-coptis chinensis compound sponge dressing comprises bletilla striata polysaccharide and coptis chinensis total alkaloids, and the bletilla striata polysaccharide and the coptis chinensis total alkaloids form a porous lamellar sponge structure. The rhizoma bletillae-coptis chinensis compound sponge dressing can be prepared into the sponge dressing with high porosity, good water absorption performance, good moisture retention, excellent elasticity and drug slow release effect through a one-step freeze drying method, the preparation process is simple, the production cost is low, and furthermore, additives such as a cross-linking agent and a modifying agent are not needed in the synthesis process of the rhizoma bletillae-coptis chinensis compound sponge dressing, so that the rhizoma bletillae-coptis chinensis compound sponge dressing has better biological safety.

Description

Bletilla striata-coptis chinensis compound sponge dressing and preparation method and application thereof

Technical Field

The invention relates to the technical field of dressings, in particular to a bletilla striata-coptis compound sponge dressing and a preparation method and application thereof.

Background

The wound surface refers to a series of pathophysiological changes such as wound surface blood seepage, pain, loss of protein and electrolyte, immunologic function reduction, external microorganism invasion and the like caused by the fact that the integrity of skin at a damaged focus part is damaged due to the fact that various injury factors act on the body surface skin, and then body tissues are directly exposed in the external environment. The number of acute and chronic wounds of all countries around the world is huge and is on the rise due to various mechanical factors, physicochemical factors and self-disease factors. In addition, improper wound care, diabetes, cardiovascular diseases, neurological diseases and other factors may cause slow wound healing, susceptibility to infection and long course of disease. At present, in the treatment and repair of acute and chronic wounds, a wound dressing is the most common and convenient treatment means at present. The dressing covers on the wound surface, plays the wound exposed surface and shelters from, avoids effects such as secondary damage and external microorganism invasion and so on. However, literature reports have so far developed over 3000 dressings, but only a few of the dressings that are widely used clinically.

The existing dressing has a plurality of types, wherein the solid sponge has good absorption performance and is suitable for the wound surface with more exudation, and meanwhile, the solid sponge generally has excellent air permeability and absorbability and proper moisture retention and has unique advantages in wound treatment. However, the performance of the solid sponge is greatly influenced by materials and synthesis processes, so that the research field of the wound dressing still focuses on finding materials which have simple preparation process, good biocompatibility, high safety and good physical properties and can meet the requirements of wound care.

Disclosure of Invention

The invention aims to provide a bletilla striata-coptis compound sponge dressing as well as a preparation method and application thereof.

The application provides a bletilla striata-coptis compound sponge dressing which comprises bletilla striata polysaccharide and coptis total alkaloids, wherein the bletilla striata polysaccharide and the coptis total alkaloids form a porous lamellar sponge structure.

The second aspect of the present application provides a preparation method of the bletilla striata-coptis compound sponge dressing of the first aspect of the present application, which comprises the following steps:

(1) dispersing bletilla striata polysaccharide with the purity of more than 95% in the coptis total alkaloid water solution at 800-1200rpm, and stirring for 8-20 min;

(2) pre-freezing at-60 to-100 ℃ for 10-15 h;

(3) freeze-drying to obtain the bletilla striata-coptis compound sponge dressing.

In a third aspect, the application provides a use of the bletilla striata-coptis compound sponge dressing in the first aspect of the application in preparing a wound treatment drug.

The bletilla striata-coptis chinensis compound sponge dressing provided by the invention has excellent performances of high porosity, high water absorption, high air permeability, low blood coagulation index, good degradability, biological safety and the like; furthermore, the bletilla striata polysaccharide has the characteristic of 'drug-adjuvant-in-one', and has the synergistic effect with the coptis total alkaloid, so that the bletilla striata polysaccharide plays roles of resisting inflammation, promoting healing, stopping bleeding and the like, has excellent performance in both physical performance and hemostatic performance, and is an ideal dressing for wound treatment.

Furthermore, the rhizoma bletillae-coptis chinensis compound sponge dressing can be used for preparing the sponge dressing with high porosity, strong absorption performance, good moisture retention, excellent elasticity and drug slow release effect by a freeze-drying method, and has the advantages of simple preparation process and low production cost.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, it is obvious that the drawings in the following description are only one embodiment of the present invention, and other embodiments can be obtained by those skilled in the art according to the drawings.

Fig. 1 is an appearance diagram of bletilla striata-coptis chinensis sponge and gelatin sponge of the present application.

Fig. 2A is an SEM image of the surface of the bletilla striata polysaccharide sponge of preparation 2.

Fig. 2B is an SEM image of the surface of bletilla striata-coptidis sponge of preparation example 1.

FIG. 3 is an SEM image of a lamellar cross-section of bletilla striata-Coptis chinensis sponge of preparation example 1.

Fig. 4 is a picture of a sample for air permeability test.

FIG. 5 shows the results of water vapor transmission rate measurement.

Fig. 6A shows the results of the elastic coefficient test of gelfoam and bletilla striata-coptis sponge.

FIG. 6B shows the results of Young's modulus tests on gelatin sponge and bletilla striata-Coptis chinensis sponge.

Fig. 6C is a force test result of fixed deformation points of gelfoam and bletilla striata-coptidis sponge.

FIG. 7 is a morphology chart of bletilla striata-berberine hydrochloride sponge with different drug loading rates.

Fig. 8 is a result of testing cumulative release rate of four alkaloids in bletilla striata-coptis chinensis sponge.

FIG. 9 shows the results of the surface blood-sucking performance test of bletilla striata-Coptis chinensis sponge and gelfoam.

Fig. 10A and 10B show the results of the blood sucking performance test of the coverage of rhizoma bletillae-coptis sponge and gelatin sponge.

FIG. 11 is the result of the sponge BCI assay, in which panel A is a full wavelength scanning UV map of the whole blood solution; and B, the BCI test results of the gelatin sponge and the bletilla striata-coptis chinensis sponge are shown in a picture B.

FIG. 12 shows the results of a rat liver hemostasis test.

FIG. 13 shows the measurement results of rat liver bleeding amount.

FIG. 14 is a line graph showing the in vitro degradation of bletilla striata-Coptis chinensis sponge.

FIG. 15 shows the results of in vitro cytotoxicity assay of bletilla striata-Coptis chinensis sponge.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and examples. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in this application are within the scope of protection of this application.

The Bletilla Striata Polysaccharide (BSP) in the application is also called Bletilla striata gum and Bletilla striata mannan, is a natural soluble glucomannan extracted from Bletilla striata (Thunb.) Reichb.f. tuber of Orchidaceae Bletilla striata, and is prepared from alpha-glucose and beta-mannose in a weight ratio of 1:4 proportion is polymerized. The source of the bletilla striata polysaccharide is not limited in the application, and the bletilla striata polysaccharide can be obtained by extracting through any existing extraction method, such as a water extraction and alcohol precipitation method, or can be directly purchased from commercial sources.

The total alkaloid refers to a chemical composition with relatively clear chemical compositions extracted, separated and enriched from Chinese medicinal materials, natural medicaments and compounds. The 'coptis total alkaloid' referred in the application refers to a solid extract which is extracted from coptis and contains alkaloid components higher than 45%, and the components mainly comprise berberine hydrochloride, coptisine hydrochloride, palmatine hydrochloride, epiberberine and the like. The preparation method of the coptis total alkaloids is not limited in the application, and the specific extraction method can be selected by a person skilled in the art according to actual needs as long as the purpose of the invention is achieved, and the coptis total alkaloids can be obtained by methods such as ethanol heating reflux extraction, macroporous resin purification, pH gradient extraction and the like.

The inventor finds that the bletilla striata-coptis chinensis compound sponge dressing can be used as a hemostatic sponge and a wound dressing, wherein the bletilla striata polysaccharide and the coptis chinensis total alkaloids have synergistic effect to play the roles of resisting inflammation, promoting wound healing and the like, and have a treatment effect on common wounds and wounds which are difficult to heal.

In some embodiments of an aspect of the present application, the porosity of the bletilla striata-coptis compound sponge dressing is 97% -99%. The inventor finds that the bletilla striata-coptis chinensis compound sponge dressing has high porosity and is an important precondition for the dressing to exert excellent air permeability and liquid absorption performance so as to have good hemostatic effect.

In some embodiments of an aspect of the present application, the rhizoma bletillae-rhizoma coptidis compound sponge dressing has a water absorption rate of 19 to 22. The inventor finds that the bletilla striata-coptis chinensis compound sponge dressing has high water absorption rate, can efficiently absorb wound blood and exudate when being used for wound surface, on one hand, absorbs water in the blood, increases the blood viscosity, promotes platelet aggregation, enables the blood to be coagulated and plays a role in physical hemostasis; on the other hand, the wound exudate can be effectively prevented from being gathered on the surface of the wound, so that the bacterial infection is caused.

In some embodiments of one aspect of the present application, the bletilla striata-coptis compound sponge dressing has a water vapor transmission rate of 1800-^-2·d^-1. The inventor finds that the bletilla striata-coptis chinensis compound sponge dressing has a proper water vapor transmission rate, and the bletilla striata-coptis chinensis compound sponge dressing is good in moisture retention and cannot cause wound drying.

In some embodiments of an aspect of the present application, the rhizoma bletillae-coptis compound sponge dressing has a whole blood coagulation index of 14-15%, which indicates that the rhizoma bletillae-coptis compound sponge dressing has excellent ability of blood coagulation.

(1) dispersing bletilla striata polysaccharide with the purity higher than 95% in the coptis total alkaloid water solution at 800-1200rpm, and stirring for 8-20 min;

(2) pre-freezing at-60 to-100 ℃ for 10-15 h;

The inventor finds in research that when high-purity bletilla striata polysaccharide is adopted, for example, the purity is higher than 95%, the spatial structure is uniform and compact, and the bletilla striata-coptis chinensis compound sponge dressing prepared by adopting the matrix has a more stable structure and excellent performance.

The inventor also finds that the bletilla striata-coptis chinensis compound sponge dressing with a porous lamellar sponge structure can be obtained in one step by dispersing the bletilla striata polysaccharide in the water solution of the coptis chinensis total alkaloids and then performing freeze drying. In particular, additives such as cross-linking agents and the like are not needed in the preparation process, the preparation process is simple, and the sponge has higher biological safety. The inventor also finds that in the bletilla striata-coptis compound sponge dressing obtained by dispersing bletilla striata polysaccharide in the aqueous solution of the coptis total alkaloids and completing drug loading through mutual dissolution of the matrixes, the coptis total alkaloids exist on the surface and inside of the sponge, so that the drug loading rate of the sponge dressing is increased.

The inventor finds that all the rhizoma coptidis total alkaloids in the preparation system of the rhizoma bletillae-rhizoma coptidis compound sponge dressing obtained by the method are transferred to the rhizoma bletillae-rhizoma coptidis sponge finished product; in addition, the inventor also finds that the bletilla striata-polysaccharide sponge can still maintain good mechanical properties when the drug loading rate reaches 60%.

In some embodiments of the second aspect of the present application, the concentration of the bletilla striata polysaccharide in the coptis total alkaloid aqueous solution is 1.5-2.5% (w/v).

In some embodiments of the second aspect of the present application, the concentration of the coptis total alkaloid aqueous solution is 0.25-1% (w/v).

In some embodiments of the second aspect of the present application, the purity of the coptis total alkaloid is greater than 45%.

Since the standards for solid sponges are not well defined, the gelatin sponge currently most widely used was selected as the control group. Gelatin, a protein partially hydrolyzed from collagen in the connective or epidermal tissue of animals, has many excellent physical and chemical properties, such as gel formation, and excellent binding properties, as a safe and easily prepared solid sponge material. The gelatin sponge has long hemostatic action time, soft texture after absorbing water, no mechanical stimulation of tissues, no obvious tissue reaction caused by the absorption of degradation products by tissues, unique hemostatic advantages and wide clinical application. Therefore, the gelatin sponge (manufactured by kuaikang medical instruments limited, Guangzhou city, batch number: 20200310) is used as a control group to examine various performance indexes of the bletilla striata-coptis chinensis compound sponge dressing (hereinafter referred to as bletilla striata-coptis chinensis sponge).

Preparation example 1 preparation of bletilla striata-coptis compound sponge dressing

1.1 extraction and purification of Total alkaloid of Coptis chinensis Franch

Crushing the coptis chinensis medicinal material, sieving the crushed coptis chinensis by a No. 2 sieve to obtain coptis chinensis coarse powder, weighing 50g of coptis chinensis coarse powder, adding 10 times (500mL) of 50% ethanol, heating, refluxing and extracting for 2 hours, filtering to obtain filtrate, and concentrating the filtrate on a rotary evaporator under reduced pressure to obtain a loading solution. Separating and enriching by using a D-101 macroporous resin column, wherein the resin dosage is as follows: 150mL, amount of sample loading: 25mL, the concentration of the sample loading solution is 1 g/mL^-1(medicinal material amount/water solution amount) the flow rate of sample loading is 2BV/h, and the resin diameter-height ratio is 1: 4. After the sample loading is finished, washing with water for 1 time of column volume; eluting with 50% ethanol solution 3 times of column volume; concentrating the ethanol eluate under reduced pressure to obtain extract, pre-freezing in a refrigerator at-80 deg.C for 12 hr, and freeze drying to obtain Coptidis rhizoma total alkaloids. UPLC-MS/MS is used for measuring the content of berberine hydrochloride, epiberberine, coptisine hydrochloride and palmatine hydrochloride in the coptis total alkaloid, and the purity of the coptis total alkaloid is calculated to be 47.32%.

1.2 preparation of bletilla striata-coptis compound sponge dressing

Dissolving the coptis total alkaloids obtained in the step 1.1 in deionized water to obtain a coptis total alkaloids water solution with the concentration of 0.75% (w/v), and then dispersing bletilla striata polysaccharides (Shanxi Panier Biotechnology Co., Ltd.) with the purity of 98% in the coptis total alkaloids water solution with the concentration of 2% (w/v); stirring with an electric stirrer at 1000rpm for 10min to obtain a gel solution. Pre-freezing in a refrigerator at-80 deg.C for 12h, and freeze-drying in a freeze-drying machine to obtain rhizoma Bletillae-Coptidis rhizoma compound sponge dressing.

Preparation example 2 preparation of bletilla polysaccharide sponge

Dispersing bletilla striata polysaccharide with the purity of 98% in deionized water to enable the concentration of the bletilla striata polysaccharide to be 2% (w/v); the mixture was stirred by an electric stirrer at 1000rpm for ten minutes to obtain a gel solution with uniform mixing. Pre-freezing at-80 deg.C for 12 hr, and freeze drying in a freeze dryer to obtain rhizoma Bletillae polysaccharide sponge.

Example 1 Observation of morphology of Compound sponge dressing of bletilla striata and Coptis chinensis

The appearance of the bletilla striata-coptis compound sponge dressing and the gelatin sponge prepared in the preparation example 1 is shown in figure 1, wherein the figure A is the bletilla striata-coptis sponge prepared in the preparation example 1, and the figure B is the gelatin sponge.

Scanning Electron Microscopy (SEM) is adopted to observe the surface structures of the bletilla striata-coptis compound sponge dressing prepared in the preparation example 1 and the bletilla striata polysaccharide sponge prepared in the preparation example 2. Before detection, the sample is firstly scattered and adhered on a conductive adhesive tape and sprayed with gold, the operation accelerating voltage of SEM is 3.0kV, and the result is shown in figure 2A and figure 2B, wherein figure 2A is SEM photograph of bletilla polysaccharide sponge; fig. 2B is an SEM photograph of the bletilla striata-coptis compound sponge dressing.

SEM image of the section of the rhizoma bletillae-rhizoma coptidis compound sponge dressing of the preparation example 1 is shown in figure 3.

As can be seen from fig. 2A, 2B and 3, the blank sponge has a network-like structure with numerous pores therein (fig. 2A). After loading with the sponge, the compactness between the structures increased, becoming a layered structure (fig. 2B). As can be seen from the layered section of the bletilla striata-coptis compound sponge dressing, a porous structure is formed between layers (figure 3), which is closely related to the absorption capacity of exudates of the sponge and the slow release effect of the medicine.

Example 2 porosity determination

Respectively taking proper amount (M) of rhizoma bletillae-rhizoma coptidis sponge and gelatin sponge in preparation example 1_S) Dipped into a pycnometer filled with ethanol and weighed (M)₁) Ultrasonic treating for 30min to make ethanol fully fill in pores of rhizoma Bletillae sponge, adding ethanol again, and weighing (M)₂). The sponge was removed and the remaining ethanol was weighed against the mass of the pycnometer (M)₃). The porosity is calculated according to the following formula:

the numerator represents the mass of ethanol having the same volume as the pores in the sponge, and the denominator represents the mass of ethanol having the same volume as the imaginary volume of the sponge (the sum of the actual volume of the sponge and the pore volume). The measurement results of the porosity of the bletilla striata-coptis chinensis compound sponge dressing and the gelatin sponge in preparation example 1 of the present application are shown in table 1.

TABLE 1 porosity determination results

As can be seen from table 1, the porosity of the bletilla striata-coptis compound sponge dressing and the gelatin sponge of the present application is 97.95 ± 0.75% and 94.81 ± 1.12%, respectively, which indicates that the bletilla striata-coptis sponge of the present application also has a structure with high porosity. The rhizoma bletillae-coptis chinensis sponge has high porosity, and the possibility of high air permeability and high water absorption is provided.

Example 3 Water absorption Capacity measurement

The water absorption rate reflects the liquid absorption performance of the sponge and is the essential physical performance of the hemostatic material. Efficient absorption of wound exudate from the wound surface of the skin is critical to wound dressings because the accumulation of wound exudate on the wound surface may initiate bacterial infection, thereby slowing the wound healing process. In addition, when uncontrollable bleeding is caused by wounds, the blood sucking performance is strong, the flowing blood can be absorbed quickly, blood clots are formed on the surface of the wounds quickly, the wounds are effectively blocked, and the effect of stopping bleeding quickly is achieved. The higher water absorption is a necessary condition that the material has better hemostatic ability, when the material meets blood, the material can absorb water in the blood in time, the viscosity and concentration of the blood are increased instantly, the flow rate of the blood is slowed down, the platelet aggregation and blood coagulation are promoted, and the physical hemostatic effect is exerted. In this example, referring to GB/T461.3-2005 standard, the vacuum dried rhizoma bletillae-rhizoma coptidis compound sponge dressing and gelatin sponge of preparation example 1 were accurately weighed and recorded as M₀Immersing into simulated skin exudate (containing 142mmol NaCl, 2.5mmol CaCl)₂Aqueous solution of (1)) for 2h, holding a little sponge with tweezers, taking out, standing for 1min to make skin simulation exudate not drip, weighing sponge to obtain M (6 times of experiment average value) The water absorption capacity of the sponge was calculated by the following formula, and the results are shown in Table 2.

TABLE 2 Water absorption Capacity measurement results

The results in table 2 show that the water absorption capacity of the gelatin sponge is 11.86 ± 1.30, and the water absorption capacity of the bletilla striata-coptis chinensis sponge of the present application is 21.28 ± 1.35, which is 1.79 times of the gelatin sponge, which indicates that the bletilla striata-coptis chinensis sponge of the present application has more excellent water absorption performance, without being limited to any theory, the inventor thinks that this may be due to the fact that the skeleton structure of the bletilla striata-coptis chinensis sponge has a compact and uniform layered porous three-dimensional structure, and further, the coptis alkaloid increases the polarity and the hydrophilicity of the bletilla striata polysaccharide sponge, thereby the bletilla striata-coptis chinensis sponge of the present application has higher water absorption than the gelatin sponge while having high porosity.

Example 4 breathability test

The Wound, pressure Wound and burn Guidelines, second edition of general Wound Guidelines (Wound, pressure and burn Guidelines-1: Guidelines for wounds in general, second edition), published by the skin Association of Japan in 2020, show that the environment of Wound surface micro-wetting helps to promote tissue regeneration and remodeling, accelerating Wound healing. The high air permeability can lead the surface of the wound to be quickly dehydrated, leave scars and delay healing; lower breathability tends to leave exudate on the wound surface, leading to an increased risk of bacterial growth.

The research is carried out according to the test method 2 part of the contact wound dressing of Chinese medicine industry standard YY/T0471.2-2004: and (4) detecting the air permeability of the prepared sponge material by the water vapor transmission rate of the breathable film dressing.

The rhizoma bletillae-rhizoma coptidis sponge and the gelatin sponge in the preparation example 1 are respectively cut into circles and then covered on a penicillin bottle filled with deionized water, double-faced adhesive tape is coated on the mouth of the penicillin bottle, the sponge is pressed on the double-faced adhesive tape to seal a contact surface, and the area of the contact surface is marked as S. Nothing was covered on the blank control (see fig. 4); the vial and sponge were then weighed as W1 and placed in an incubator at a relative humidity of 20% and a temperature of 37 ℃ for 24 h. The vial and sponge were then removed, weighed again and counted as W2. Each sample was run in 6 replicates. The Water Vapor Transmission Rate (WVTR) of the sponge was calculated as follows, and the water vapor transmission rate measurement results are shown in table 3 and fig. 5.

Water vapor transmission rate (g.m)^-2·day^-1)＝(W2-W1)/S

TABLE 3 determination of Water vapor Transmission Rate

As can be seen from the results, the bletilla striata-Coptis chinensis sponge of the present application has a water vapor transmission rate of 1800-^-2·d^-1And the range of air permeability (2000 g.m) suitable for wound healing reported in the literature^-2·d^-1Left and right), suitable air permeability is favorable for ensuring the skin moisture degree of the local wound surface, maintaining the micro-moist environment required by wound healing, and simultaneously reducing the collection of wound exudate at the wound, thereby avoiding the infection caused by wound impregnation.

Example 5 elastic Property test

The test method comprises the following steps:

rhizoma Bletillae-Coptis sponge of preparation example 1 was taken and shaped as a cylinder with a height of 0.4cm and a radius of 1.5 cm. The gelatin sponge is a cube with height of 0.4cm and length and width of 2 cm. Using a material testing machine (PT-501B computer type single arm tensile testing machine, puxet testing equipment limited, tom), a sensor was placed on the upper platen, and a platform was placed on the lower platen, the compressibility and elasticity of the material were tested by pressing down the upper platen with a force of 5mm, and by pressing down the upper platen with a spring constant and young's spring constant of 5mm, 3 times for each sample, and the spring constant results are shown in table 4 and fig. 6A (where P is less than 0.0001, which is a significant difference compared to gelatin sponge); the results of Young's modulus are shown in Table 5 and FIG. 6B; the force measurement results of the compression set deformation points of bletilla striata-coptis chinensis sponge and gelatin sponge are shown in table 6 and fig. 6C.

TABLE 4 elasticity coefficients of rhizoma Bletillae-Coptis sponge and gelatin sponge

TABLE 5 Young's modulus of rhizoma Bletillae-Coptis sponge and gelatin sponge

TABLE 6 compression force of rhizoma Bletillae-Coptis sponge and gelatinum oxhide to determine deformation point

The modulus of elasticity, young's modulus, is a physical quantity describing the resistance of a solid material to deformation, which depends only on the physical properties of the solid material itself, regardless of the external environment. The larger the modulus of elasticity, Young's modulus, the less likely the solid material is to deform. From the above results, the elastic property of the rhizoma bletillae-coptis sponge is superior to that of gelatin sponge, so that the sponge is kept in the original shape in the using process, and is not deformed due to pressing in the using process, and the structure, the performance and the like of the sponge are further prevented from being influenced.

Example 6 drug load test

The application adopts bletilla striata-berberine hydrochloride sponge, tests the drug-loading rate of the bletilla striata polysaccharide sponge prepared by the method: weighing berberine hydrochloride 0.1g, berberine hydrochloride 0.2g, and berberine hydrochloride 0.3g, respectively, dissolving in 10mL distilled water, heating to dissolve, adding 0.2g bletilla striata polysaccharide with purity of 98%, respectively, stirring with electric stirrer at 1000rpm for 10min to obtain uniformly mixed gel solution. Pre-freezing at-80 deg.C for 12 hr, and lyophilizing in a lyophilizer to obtain rhizoma Bletillae polysaccharide-berberine hydrochloride sponge.

The drug loading was calculated according to the following formula:

medicine-carrying capacity (%) - (bletilla striata polysaccharide + total medicine mass)

The morphology of bletilla striata-berberine hydrochloride sponge with different drug-loading rates is shown in figure 7, the drug-loading rates from left to right are 33.3%, 50% and 60%, and it can be seen that when the drug-loading rate reaches 60%, the sponge still has a complete structure and good mechanical properties.

Example 7 in vitro Release behavior Studies of bletilla striata-Coptis chinensis Spongilla

Pure water is used as a release medium, and the in-vitro release conditions of four alkaloids, namely palmatine hydrochloride, epiberberine, coptisine hydrochloride and berberine hydrochloride in the rhizoma bletillae-rhizoma coptidis sponge are determined by adopting a dynamic in-vitro dialysis method. 50mg of bletilla striata-coptis chinensis sponge is precisely weighed and placed in a ready-to-use regenerated cellulose dialysis bag (molecular weight cut-off is 3500Da, Shanghai-sourced leaf Biotechnology Co., Ltd., M08J11Y117396) and the two ends are clamped by a dialysis bag clamp to prevent the solution from flowing out, and the dialysis bag is fixed on a stirring paddle. The sample was placed in a 1000mL beaker and 500mL of release medium was added and 2.0mL of sample was removed at 5, 10, 15, 20, 30, 45, 60, 90, 120, 240, 480, 720, 1440min at 37 ℃ at 200rpm for 5, 10, 15, 20, 30, 45, 60, 90, 120, 240, 480, 720, 1440min with a fresh volume of dissolution medium at the same temperature being replenished. Diluting the sample at a subsequent time point, recording the dilution multiple D, filtering by a membrane, injecting, respectively measuring the content of four alkaloids by using UPLC-MS/MS, calculating the cumulative release rate according to the following formula, and carrying out three experiments in parallel.

X_n(％)＝C_n×V×D/W×100％

X(％)＝X_n+(X₁+X₂+……X_n-1)×Vi/V

Xn is the release rate at a single sampling point, Cn is the concentration determined for a single sample, V is the release medium volume (500mL), D is the dilution factor, W is the initial dose (50mg), X is the cumulative release rate, and Vi is the volume per sample (2 mL). The cumulative release rate results for the four alkaloids are shown in table 7.

The obtained results were processed with GraphPad Prism 7.0, and plotted with time as abscissa and cumulative release rate as ordinate, the results are shown in fig. 8, in which graph a is the cumulative release rate of palmatine hydrochloride; b is the cumulative release rate of epiberberine; c is the accumulative release rate of coptisine hydrochloride; and D is the cumulative release rate of berberine hydrochloride.

TABLE 7 cumulative release rates of four alkaloids in Coptis chinensis Franch

As can be seen from the results in table 7 and fig. 8, the rhizoma bletillae-coptis chinensis sponge of the present application can release the drug slowly, so that the carried drug can be released continuously in a certain time, and a local sustained-release therapeutic effect can be achieved.

The release mechanism is an important content of the research of the sustained and controlled release preparation, and a kinetic equation is generally used as a method for researching the release mechanism. The kinetic equation is based on Fick's diffusion law, and the common release kinetic equation model provided on the basis of some boundary conditions and assumptions is shown in Table 8; the results of the kinetic equation fitting of palmatine hydrochloride, epiberberine, berberine hydrochloride and coptisine hydrochloride are shown in Table 9.

TABLE 8 kinetics equations

TABLE 9 kinetic equation fitting results for each alkaloid component

In the above table, Q is the cumulative percent drug released, t is the sampling time, a is a constant, and k is a constant. Fitting the release rate data of four alkaloids of rhizoma bletillae-rhizoma coptidis sponge, and finding that the release process of berberine hydrochloride in rhizoma coptidis is relatively closer to a first-level release equation, the release process of table berberine relatively most conforms to a Higuchi plane diffusion mode equation, the release process of palmatine hydrochloride relatively most conforms to a Hixcon-Crowlell corrosion equation, the release fitting equation of berberine hydrochloride conforms to the Hixcon-Crowell corrosion equation and the first-level release equation, and correlation coefficients of the release fitting equation and the first-level release equation are close.

In general, the release process of the coptis total alkaloids accords with the release characteristics of a sustained-release preparation, and the release mechanism is the comprehensive action of skeleton erosion and drug diffusion. The drug in the erosion-type matrix system is dissolved or dispersed in the polymer, and the release rate depends on the degradation rate of the polymer and the release mode of the system. And the drug release process is related to the diffusion behavior of the drug in the polymer, and the diffusion release of the drug through the porous polymer is the key for obtaining the sustained release. The entrapment of the drug within the polymeric matrix randomly creates pores that form a network system to the surface of the matrix, and the drug within the system is released into the matrix by virtue of its ability to associate with the release medium. The skeleton material of the bletilla striata-coptis chinensis sponge is bletilla striata polysaccharide which has good water solubility, a certain degradation process exists when the bletilla striata-coptis chinensis sponge is contacted with a release medium, and a plurality of pore structures exist in the bletilla striata-coptis chinensis sponge, so that the conditions of skeleton erosion and drug diffusion are met.

Example 8 evaluation of blood sucking behavior

Cutting rhizoma Bletillae-rhizoma Coptidis sponge and gelatin sponge into the same volume (1 × 1 × 0.2 cm)³) And put into a culture dish as a sample. 25 μ L of SD rat (beijing wilsoniwa laboratory animal technology ltd) blood containing 3.8% sodium citrate anticoagulant was dropped on the surface of each sample, and blood-sucking thereof was observed after 0, 1, 2, 3, 4, 5min and recorded by photographing to examine the surface blood-sucking behavior of the sponge, and the result is shown in fig. 9.

25 μ L of blood containing anticoagulant was added to each of the three petri dishes, and an equal volume of bletilla striata-coptis sponge and gelfoam was covered on the blood, while the control group was not covered with anything. After 5 minutes, the sponge was lifted and 10mL of distilled water was slowly added to the petri dish, and the blood coagulation was recorded by photographing to examine the covering blood-sucking behavior of the sponge, and the result is shown in fig. 10A and 10B, in which fig. 10B is an enlarged view of the sponge in fig. 10A.

As can be seen from fig. 9, 10A and 10B, the bletilla striata-coptis sponge exhibits better blood absorbability in terms of surface hemostasis and covering hemostasis behavior than gelatin sponge, and can sufficiently absorb blood and turn into gel when contacting with blood. As can be seen in fig. 9, blood absorbed in the bletilla striata-coptis sponge at 5min has coagulated, without being bound to any theory, which the inventors believe may depend on the good hydrophilicity of the bletilla striata polysaccharides. The blood on the surface of the gelatin sponge is not absorbed, and the blood in a flowing state stays on the surface of the sponge after 5 min. As can be clearly seen from the sample diagram after sponge blood suction in fig. 10B, the side of the rhizoma bletillae-coptidis sponge contacting with blood after blood suction is in the form of viscous gel, while the gelatin sponge has relatively poor affinity to blood, the surface of the gelatin sponge is still in flowing state, and blood coagulation is less.

The above results show that the rhizoma bletillae-coptis chinensis sponge has better blood sucking performance.

Example 9 Whole blood dynamic coagulation index assay

The in vitro coagulation performance of the hemostatic material was evaluated using the coagulation index BCI (blood Clotting index) with reference to the study method of Shih M F et al (journal of pharmacological sciences,2006,327(1): 117-. Fresh blood from SD rats was drawn using a blood collection tube containing 3.8% sodium citrate anticoagulant. Adding 25 μ L of blood containing anticoagulant into 10mL centrifuge tube, and taking 1 × 1 × 0.2cm³Placing rhizoma Bletillae-Coptidis rhizoma sponge and gelatin sponge on blood surface layer, standing to make sponge and blood fully contact, adding 10mL deionized water into centrifuge tube after 10min, vortexing for 2min, respectively adding 200 μ L solutions in different centrifuge tubes into 96-well plate, measuring light absorption A at 414nm with microplate reader_{Sample (I)}The BCI of the two sponges was calculated separately in the following formula, and the experiments were performed in triplicate, with the results shown in panel B of fig. 11. (414nm is the maximum absorption of rat whole blood solutionWavelength, Whole blood solution full wavelength scanning ultraviolet Picture A of FIG. 11

BCI＝A_Sample(s)/A_{Reference to}×100

A_{Reference to}The absorbance at 414nm was 25 μ L of blood dissolved in 10mL of deionized water.

The whole blood coagulation index reflects the coagulation effect of the material, and the lower the coagulation index, the better the coagulation effect of the corresponding material, and the better the blood clot retention, and conversely, the worse the coagulation effect. As can be seen from panel B of FIG. 11, the clotting index of gelatin sponge is 86.18% and the clotting index of bletilla striata-Coptis sponge is 14.28%. This indicates that bletilla striata-coptis sponge has better ability to absorb blood and promote blood coagulation (P <0.0001) compared to gelatin sponge.

Example 10 rat liver hemostasis test

Rats were randomized into 2 groups: gelatin sponge group and rhizoma Bletillae-rhizoma Coptidis sponge group. Rats were anesthetized, and the liver was exposed through a left abdominal wall transverse incision, with the surface lacerated to bleed. Gelatin sponge and bletilla striata-coptis sponge are respectively placed on bleeding livers, hemostasis is performed by pressing, the hemostasis effect of different materials is recorded by photographing (the result is shown in figure 12), and the bleeding amount (the quality of the sponge before hemostasis and after blood suction is poor) is measured (the result is shown in figure 13).

As can be seen from fig. 12, the blood on the surface of the gelfoam is in an uncoagulated form, and the blood around the bleeding point can be observed to be dispersed on the whole piece of the gelfoam, while the interaction between the blood absorbed by the surface of the bletilla striata-coptis sponge and the gelfoam is converted into a gel form, and the blood at the bleeding point is less dispersed. The rhizoma bletillae-coptis chinensis sponge shows better mucous membrane adsorption capacity and strong hemostatic capacity. As can be seen from fig. 13, the amount of bleeding of the liver in the gelfoam group is about 400mg, while the amount of bleeding of the bletilla striata-coptis chinensis sponge group is only about 100mg, which indicates that the bletilla striata-coptis chinensis sponge of the present application has a significantly better hemostatic effect than gelfoam. Without being bound by any theory, the inventor believes that this is closely related to the excellent blood absorption and mucosal adsorption of bletilla striata-coptis sponge.

Example 11 in vitro degradation study

With PBS (phosphate buffer)) The pH 7.4 lysozyme was prepared at a concentration of 4mg/mL (shanghai source leaf biotechnology limited, cat #: r21037) as a degradation solution, and storing at-20 ℃. The bletilla striata-coptis sponge obtained in the preparation example 1 is cut into 1X 0.2cm³Size, weight notation M₀Sequentially putting the materials into a 5mL centrifuge tube, adding 1mL of degradation liquid, incubating at 37 ℃, replacing the degradation liquid once every 2 days, only replacing the degradation liquid at the upper part of the centrifuge tube during liquid replacement, respectively taking out sponges in the centrifuge tube on days I, 2, 4, 6 and 8, freezing in a refrigerator at-80 ℃ for 12h, freeze-drying in a freeze dryer, weighing and recording as M₁. The degradation rate was calculated according to the following formula, and the result is shown in FIG. 14.

(ii) degradation rate (%) [ (M)₀-M₁)/M₀]×100％

As can be seen from fig. 14, the bletilla striata-coptis chinensis sponge can be degraded by 48% in 24 hours in lysozyme environment, and 87% after 8 days (192 hours), which indicates that the bletilla striata-coptis chinensis sponge can be sufficiently degraded when applied to the skin, and indicates that the bletilla striata-coptis chinensis sponge has higher biological safety and lower irritation.

Example 12 in vitro cytotoxicity Studies

Since macrophages act as key regulatory cells and provide growth factor action during the inflammatory phase, the wound healing process will be severely slowed if macrophage infiltration of the wound site is prevented. RAW264.7 macrophages (mouse macrophage leukemia cells; purchased from the cell bank of the typical culture collection committee of the chinese academy of sciences) were therefore selected as a model for evaluating the in vitro cytotoxicity of bletilla striata-coptis sponges.

Cell culture: collecting cells growing to logarithmic phase, discarding DMEM complete medium (DMEM high-sugar medium containing 5% fetal calf serum), washing with PBS twice, scraping cells, centrifuging at 1000rpm for 3min, discarding supernatant, suspending cells in 1ml of total medium, counting, inoculating into 96-well plate at 12000/well, and culturing in cell culture box (5% CO)₂37 ℃ for 24 h. Sterilizing 10.44mg rhizoma Bletillae-Coptidis rhizoma sponge with ultraviolet irradiation for 2 hr, soaking in 1mL DMEM high sugar culture medium, extracting at 37 deg.C for 24 hr, and taking out the material to obtain 7.6mL extractAnd (3) extracting the solution. Diluting the leaching solution with DMEM complete culture medium to obtain a series of culture media with different leaching solution concentrations (7.8 μ g-mL) respectively based on concentration of Coptidis rhizoma total alkaloids^-1、19.5μg·mL^-1、39μg·mL^-1、78μg·mL^-1、195μg·mL^-1And 390. mu.g.mL^-1) The RAW264.7 cells cultured in the 96-well plate are divided into 3 groups (experimental group-leaching solution group with different concentration, control group-DMEM complete medium group, blank group-cell-free group), and each group has 3 multiple wells. When the test group cells are changed, a culture medium containing bletilla striata-coptis chinensis leaching liquor is added, and an equivalent DMEM complete culture medium is added into a control group. After 24h incubation, the medium was aspirated off, 100. mu.L of DMEM medium containing 10% CCK-8 was added to each well, coated with tinfoil and incubated in the dark for 2h (5% CO)₂At 37 ℃, the absorbance is measured at 450nm of the microplate reader. Cell viability (Cell viability) was calculated using the following formula.

Cell viability ═ [ (As-Ab)/(Ac-Ab) ] 100%

As: absorbance of the assay well; ac: absorbance of control wells; ab: absorbance of blank well

As shown in fig. 15, the cell survival values of RAW264.7 cells cultured with the leaching solution of bletilla striata-coptis chinensis sponge for 24h were all above 100%, indicating that bletilla striata-coptis chinensis sponge has no obvious cytotoxic effect.

The results show that the rhizoma bletillae-coptis chinensis sponge has excellent performances such as better air permeability, water absorbability, hemostatic performance and higher drug-loading rate, and therefore, the rhizoma bletillae-coptis chinensis sponge can be used for preparing wound treatment medicines, and is particularly suitable for hemostasis of wounds.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. A rhizoma Bletillae-Coptidis rhizoma compound sponge dressing comprises rhizoma Bletillae polysaccharide and Coptidis rhizoma total alkaloids, wherein the rhizoma Bletillae polysaccharide and Coptidis rhizoma total alkaloids form porous lamellar sponge structure; the purity of the bletilla striata polysaccharide is more than 95 percent, and the purity of the coptis total alkaloid is more than 45 percent;

the preparation method of the bletilla striata-coptis chinensis compound sponge dressing comprises the following steps: the bletilla striata-coptis compound sponge dressing can be obtained by dispersing the bletilla striata polysaccharide in the coptis total alkaloid aqueous solution and then carrying out freeze drying.

2. The bletilla striata-coptis compound sponge dressing as claimed in claim 1, wherein the porosity of the bletilla striata-coptis compound sponge dressing is 97% -99%.

3. The bletilla striata-coptis compound sponge dressing as claimed in claim 1, wherein the water absorption rate is 19-22.

4. The bletilla striata-coptis compound sponge dressing as claimed in claim 1, wherein the water vapor transmission rate is 1800-1900 g-m^-2·d^-1。

5. The bletilla striata-coptis compound sponge dressing as claimed in claim 1, wherein the whole blood coagulation index is 14-15%.

6. A preparation method of a bletilla striata-coptis chinensis compound sponge dressing comprises the following steps:

(2) pre-freezing at-60 to-100 ℃ for 10-15 h;

(3) freeze-drying to obtain the rhizoma bletillae-rhizoma coptidis compound sponge dressing; the purity of the coptis total alkaloid is more than 45 percent.

7. The method according to claim 6, wherein the concentration of the bletilla striata polysaccharide in the coptis total alkaloid aqueous solution is 1.5-2.5% (w/v).

8. The method according to claim 6, wherein the concentration of the coptis total alkaloid aqueous solution is 0.25-1% (w/v).

9. Use of the bletilla striata-coptis compound sponge dressing of any one of claims 1-5 in preparation of a wound treatment dressing.