阅读说明：本技术 透明质酸修饰醇质体、含其的穴位给药系统及系统的应用 (Hyaluronic acid modified ethosome, acupoint drug delivery system containing same and application of system ) 是由 张永太 冯年平 张洪宇 王志 郭腾 侯晓琳 何泽慧 于 2021-07-23 设计创作，主要内容包括：本发明公开了一种透明质酸修饰醇质体、含其的穴位给药系统及系统的应用,透明质酸修饰醇质体为透明质酸以共价键接枝于二油酰磷脂酰乙醇胺分子上后形成的磷脂材料插入醇质体的磷脂膜中形成的。含其的穴位给药系统包括透明质酸修饰醇质体和包覆在透明质酸修饰醇质体内的一种以上温中散寒中药或其提取物。穴位给药系统可应用在治疗溃疡性结肠炎及因寒症引起的腹痛、腹泻、痛经等症状上。本发明以HA修饰醇质体,其能够减少所包载药物的渗漏,有效提升制剂稳定性,从而增加药物随载体向皮肤中的递送；选用HA-ES作为温中散寒中药或其提取物的经皮递送载体,其能够强化给药后的穴区特殊效应,进而用于治疗因寒症引起的腹痛、腹泻与痛经等症状。(The invention discloses a hyaluronic acid modified ethosome, an acupoint drug delivery system containing the same and application of the system. The acupoint drug delivery system containing the hyaluronic acid modified ethosome comprises a hyaluronic acid modified ethosome and more than one traditional Chinese medicine for warming middle-jiao and dispelling cold or extracts thereof coated in the hyaluronic acid modified ethosome. The acupoint drug delivery system can be applied to treating ulcerative colitis and symptoms such as abdominal pain, diarrhea, dysmenorrhea and the like caused by cold symptoms. According to the invention, the ethosome is modified by HA, so that the leakage of the encapsulated drug can be reduced, and the stability of the preparation is effectively improved, thereby increasing the delivery of the drug to the skin along with the carrier; HA-ES is selected as a transdermal delivery carrier of the traditional Chinese medicine for warming middle-jiao and dispelling cold or the extract thereof, which can strengthen the special effect of acupoint regions after administration, and further can be used for treating symptoms such as abdominal pain, diarrhea and dysmenorrheal caused by cold symptoms.)

1. The hyaluronic acid-modified ethosome is characterized in that a phospholipid material formed by grafting hyaluronic acid on a dioleoyl phosphatidylethanolamine molecule by a covalent bond is inserted into a phospholipid membrane of the ethosome.

2. The hyaluronic acid-modified ethosome according to claim 1, wherein the phospholipid material is obtained by activating carboxyl groups in hyaluronic acid with 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and then reacting the activated carboxyl groups with amino groups on dioleoylphosphatidylethanolamine to form amide bonds.

3. The method for preparing the hyaluronic acid-modified ethosome according to any one of claims 1 to 2, wherein the hyaluronic acid-modified ethosome is obtained by dissolving phospholipid and cholesterol in ethanol, stirring to form a uniform solution, slowly injecting the uniform solution into a phospholipid material solution, sealing, stirring, cooling and performing ultrasonic treatment.

4. The method of claim 3, wherein the phospholipid material is prepared by a method comprising the steps of:

a) adding hyaluronic acid into distilled water, and stirring overnight on a magnetic stirrer to fully swell and dissolve the hyaluronic acid to obtain a hyaluronic acid solution;

b) adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide into the swelled hyaluronic acid solution, adjusting the pH to 4.0, and stirring and activating for 1-5 hours at 700rpm in a water bath at 37 ℃;

c) adding an ethanol solution dissolved with dioleoyl phosphatidylethanolamine into the solution obtained in the step b), adjusting the pH to 8.6, and magnetically stirring the solution for 20-24 hours at 500rpm in a water bath at 37 ℃;

d) and after the reaction is finished, adding the reaction solution into a dialysis bag to remove redundant reactants and reaction byproducts, thus obtaining the phospholipid material.

5. The method according to claim 3, wherein the phospholipid and the cholesterol are dissolved in the ethanol and stirred in a water bath at 55-65 ℃ in a sealing manner;

the solvent of the phospholipid material solution is PBS buffer solution, and the solute is phospholipid material;

the cooling means cooling to room temperature.

6. An acupoint drug delivery system, which is characterized by comprising the hyaluronic acid modified ethosome according to any one of claims 1 to 2 and more than one traditional Chinese medicine for warming middle-jiao and dispelling cold or extracts thereof coated in the hyaluronic acid modified ethosome.

7. The acupoint drug delivery system according to claim 6, wherein the traditional Chinese medicine for warming spleen and stomach for dispelling cold is selected from at least one of dried ginger, cinnamon, evodia rutaecarpa, fennel, clove, galangal, pepper, zanthoxylum, piper longum, cubeb, aconite and mugwort leaf;

the acupoint drug delivery system is an umbilical drug delivery system.

8. The acupoint drug delivery system of claim 6, comprising a hyaluronic acid-modified ethosome and a volatile oil of Cinnamomum burmannii powder encapsulated in the hyaluronic acid-modified ethosome;

the particle size of the hyaluronic acid modified ethosome is 50-500 nm, and the drug-loading rates of the hyaluronic acid modified ethosome to cinnamaldehyde in cinnamon oil and eugenol in clove oil are 5-20 wt% and 10-46 wt%, respectively.

9. The method for preparing the acupoint drug delivery system according to claim 8, wherein the volatile oil of cinnamomum burmannii, phospholipid and cholesterol are dissolved in ethanol, sealed and stirred in a water bath at 55-65 ℃ to form a uniform solution, then the uniform solution is slowly injected into a PBS solution dissolved with phospholipid material, sealed and stirred continuously, cooled to room temperature, and subjected to uniform ultrasound to obtain the acupoint drug delivery system;

wherein the contents of the cinnamomum burmannii volatile oil, the phospholipid, the cholesterol, the ethanol and the HA-DOPE in the acupoint drug delivery system are respectively 0.2-4 w/v%, 0.2-5 w/v%, 0.05-2 w/v%, 20-45 v/v% and 0.1-1.5 w/v%.

10. The use of an acupoint drug delivery system as defined in any one of claims 6-8 in the treatment of ulcerative colitis and cold-induced abdominal pain, diarrhea, dysmenorrhea.

Technical Field

The invention belongs to the technical field of medicines, and relates to a hyaluronic acid modified ethosome, an acupoint drug delivery system comprising the same and application of the system.

Background

The transdermal administration through the channels and acupoints is an administration mode based on the theory of 'external treatment of traditional Chinese medicine', the international medical community has intensive research on the science, and the unique administration mode is called as a transdermal administration system (TTS), which is administered on the surface of the skin, and the medicine passes through all layers of the skin at a constant speed (or close to the constant speed) through pore expansion, enters the systemic circulation and generates the whole body or local treatment effect.

Umbilical region administration is one of the most distinctive Chinese medicine acupoint administration therapies, is guided by the meridian theory of traditional Chinese medicine, and is widely applied in clinic. The umbilicus part is close to the abdominal cavity, the pelvic cavity and a plurality of important nerve plexuses and ganglia of the human body, and the traditional Chinese medicine theory holds that the umbilical administration can adjust the functions of viscera and meridians through the comprehensive action of the medicines and acupuncture points, thereby achieving the purpose of preventing and treating diseases. The research in the literature finds that the umbilical administration is widely applied to the treatment of the diseases of the middle-jiao and the lower-jiao, and particularly the treatment of abdominal pain and diarrhea is the most common. The most commonly used navel-applying herbs are interior-warming herbs. In clinical practice of traditional Chinese medicine, clove and cinnamon are often applied to the navel through internal-warming medicine, so as to achieve the purpose of treating symptoms such as abdominal pain, diarrhea and dysmenorrhea caused by cold syndrome. Clove and cinnamon are prepared into clove and cinnamon powder, and are applied to the navel to treat infantile diarrhea caused by various enteritis, wherein clove and cinnamon are used as main medicines and are applied to the navel to treat infantile abdominal pain and diarrhea with definite curative effect. Ulcerative Colitis (UC) is one of common diseases causing abdominal pain and diarrhea, and interior-warming medicines represented by clove and cinnamon are used for treating yang deficiency or cold syndrome UC, so that a better clinical effect is achieved.

The deep layer of the acupoint region is a functional region which plays the effect of the acupoint, thereby promoting the delivery of the medicine to the deep layer of the acupoint region and being beneficial to improving the special treatment effect of the acupoint. Currently, the umbilical region administration preparation used in clinic has low level and poor absorption, and fails to embody the preparation characteristics of umbilical region administration. The main effective part of the clove cinnamon powder is volatile oil, the polarity of the volatile oil is small, the volatile oil is easy to be retained in a epidermal layer of a lipid environment after transdermal administration, and the volatile oil is slowly diffused to the deep layer of a acupoint area; meanwhile, the volatility is strong, so that the medicine loss is easily caused during administration.

Therefore, the development of an acupoint drug delivery system which can effectively encapsulate the effective part of the cinnamomum burmannii powder volatile oil and deliver the drug to the deep layer of the acupoint area so as to strengthen the special effect of the acupoint area after umbilical administration is of great practical significance.

Disclosure of Invention

The invention aims to overcome the defects that the existing acupoint drug delivery system is easy to stay on a skin layer in a lipid environment after transdermal drug delivery when coating the cinnamomum burmannii volatile oil, is slow in diffusion to a deep layer of a acupoint region and is easy to cause drug loss, and provides the acupoint drug delivery system which can effectively coat the effective part of the cinnamomum burmannii volatile oil and can deliver the drug to the deep layer of the acupoint region so as to strengthen the special effect of the acupoint region after umbilical drug delivery. Of course, the practical application of the acupoint drug delivery system is not limited to the entrapment of the cinnamomum zeylanicum volatile oil and the application of the volatile oil to umbilical administration, and a person skilled in the art can select proper drug components and application parts according to actual requirements.

In order to achieve the purpose, the invention provides the following technical scheme:

the hyaluronic acid modified ethosome (HA-ES) is formed by inserting a phospholipid material (HA-DOPE) formed after Hyaluronic Acid (HA) is grafted on a dioleoyl phosphatidylethanolamine (DOPE) molecule by a covalent bond into a phospholipid membrane of the Ethosome (ES).

The ethosome is a novel transdermal drug delivery system, and the liposome is dispersed in low-chain alcohol with medium and low concentration, so that the phospholipid vesicle has stronger deformability, the transdermal permeability of the phospholipid vesicle is improved, and various drugs can be carried to deep layers of the skin. Because a certain amount of low-chain alcohol exists in the system, the drug loading performance of the system on the fat-soluble drug is enhanced. The ethosome is used as a transdermal delivery carrier of the cinnamomum burmannii powder volatile oil, so that the feasibility is certain, but the volatile oil has stronger permeability and is easy to penetrate a phospholipid membrane, and the entrapment capacity of vesicles can be reduced. The present application thus employs hyaluronic acid for modification of ethosomes. Hyaluronic Acid (HA) is a major component of the extracellular matrix, is a linear acidic mucopolysaccharide formed by the alternate linkage of N-acetylglucosamine and β -D-glucuronic acid disaccharide units, and is widely distributed in vertebrate tissues. HA HAs good biocompatibility, biodegradability and no immunogenicity, and is widely applied to the fields of medical treatment, cosmetics and the like. The liposome in the HA-modified ethosome is used as a carrier for transdermal delivery of the drug, and an aqueous gel network formed on the surface of the liposome by the HA can be utilized to reduce leakage of the encapsulated drug and effectively improve the stability of the preparation, so that the delivery of the drug to the skin along with the carrier is increased; in addition, the moistening effect of hyaluronic acid can also enhance the hydration effect of the medicine-carrying vesicle and the skin stratum corneum, and promote the vesicle to pass through the stratum corneum, and the aqueous coat can also protect the vesicle from passing through the epidermis of lipid and entering into the aqueous dermis and loose subcutaneous tissues. The invention modifies HA molecules on the surface of an ethosome in a covalent bond mode to obtain a novel ethosome, namely the hyaluronic acid modified ethosome (HA-ES) of the invention, and compared with the prior art (HA is modified into liposome in a coating mode), the hyaluronic acid modified ethosome (HA-ES) of the invention HAs better preparation stability.

As a preferred technical scheme:

in the hyaluronic acid modified ethosome, the phospholipid material HA-DOPE in the HA-ES is obtained by activating carboxyl in a hyaluronic acid HA structure by 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and then reacting the carboxyl with amino on dioleoylphosphatidylethanolamine DOPE to generate an amide bond.

The invention also provides a method for preparing the hyaluronic acid modified ethosome, which comprises the steps of dissolving phospholipid and cholesterol in ethanol, stirring to form a uniform solution, slowly injecting the uniform solution into a phospholipid material solution, sealing, stirring, cooling and ultrasonically treating to obtain the hyaluronic acid modified ethosome.

As a preferred technical scheme:

the preparation method of the phospholipid material comprises the following steps:

a) adding Hyaluronic Acid (HA) into distilled water, and stirring overnight on a magnetic stirrer to fully swell and dissolve the Hyaluronic Acid (HA) to obtain a Hyaluronic Acid (HA) solution;

b) adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the swollen Hyaluronic Acid (HA) solution, adjusting the pH to 4.0 by using 0.1mol/L hydrochloric acid, and stirring and activating for 1-5 h at 700rpm in a water bath at 37 ℃;

c) adding an ethanol solution dissolved with dioleoyl phosphatidylethanolamine (DOPE) into the solution obtained in the step b), adjusting the pH to 8.6 by using 0.1mol/L sodium hydroxide solution, and magnetically stirring for 20-24 hours at 500rpm in a water bath at 37 ℃;

d) after the reaction is finished, the reaction solution is added into a dialysis bag (the molecular weight cutoff is 3500Da) to remove redundant reactants and reaction byproducts, and then the phospholipid material is obtained.

Specifically, in the step a), the concentration of HA is 1.0-4.0 mg/mL; in the step b), the concentration of EDC and the concentration of NHS are respectively 0.5-2.4 mg/mL and 0.2-1.5 mg/mL; in step d), the reaction solution obtained by dialysis is freeze-dried to obtain HA-DOPE, and the HA-DOPE is stored at the temperature of-20 ℃ for later use. The protection scope of the present application is not limited to the specific parameters in the above method, and those skilled in the art can select suitable parameters according to actual needs.

The method comprises the steps of dissolving the phospholipid and the cholesterol in the ethanol, and stirring in a water bath at 55-65 ℃ in a sealed manner;

the solvent of the phospholipid material solution is PBS buffer solution, and the solute is phospholipid material;

the cooling means cooling to room temperature.

The invention also provides an acupoint drug delivery system which comprises the hyaluronic acid modified ethosome and more than one traditional Chinese medicine for warming middle-jiao and dispelling cold or extracts thereof coated in the hyaluronic acid modified ethosome.

Specifically, the acupoint drug delivery system selects hyaluronic acid modified ethosome (HA-ES) as a transdermal delivery carrier of the traditional Chinese medicine for warming middle-jiao and dispelling cold or the extract thereof, can better encapsulate effective components of the traditional Chinese medicine for warming middle-jiao and dispelling cold or the extract thereof, and delivers the drug to the deep layer of the acupoint region, thereby strengthening the special effect of the acupoint region after drug delivery and having great application prospect.

As a preferred technical scheme:

the acupoint drug delivery system is characterized in that the traditional Chinese medicine for warming spleen and stomach and dispelling cold is selected from more than one of dried ginger, cinnamon, fructus evodiae, fennel, clove, galangal, pepper, fructus piperis longi, piper cubeba, monkshood, aconite and folium artemisiae argyi;

the acupoint drug delivery system is an umbilical drug delivery system. The protection scope of the present invention is not limited to this, and the above list is only a feasible technical scheme, and other traditional Chinese medicines can be applied to the present invention, and the acupoint drug delivery system of the present invention can be applied to other acupoints, not limited to umbilical region, and the specific effective components of the drugs for acupoints and loads can be selected by those skilled in the art according to actual needs.

The acupoint drug delivery system comprises a hyaluronic acid modified ethosome and cinnamomum burmannii volatile oil coated in the hyaluronic acid modified ethosome;

the particle size of the hyaluronic acid modified ethosome is 50-500 nm, the drug loading rates of the hyaluronic acid modified ethosome to cinnamaldehyde in cinnamon oil and eugenol in clove oil are 5-20 wt% (w/w) and 10-46 wt% (w/w), preferably the drug loading rates are 7-17 wt% and 36-46 wt%, and the encapsulation rates of the hyaluronic acid modified ethosome to cinnamaldehyde and eugenol are 74-84 wt% and 79-89 wt%, respectively.

In addition, the invention also provides a method for preparing the acupoint drug delivery system, which comprises the steps of dissolving the cinnamomum burmannii volatile oil, the phospholipid and the cholesterol in ethanol, carrying out sealed stirring in a water bath at 55-65 ℃ to form a uniform solution, slowly injecting the uniform solution into a PBS solution dissolved with a phospholipid material, continuing to carry out sealed stirring, cooling to room temperature, and carrying out ultrasonic homogenization to obtain the acupoint drug delivery system;

specifically, slowly injecting the mixed solution into a PBS solution dissolved with HA-DOPE, sealing and stirring for 0.5-1.5 h, cooling to room temperature, and performing ultrasonic treatment for 5-15 min by using a 200-300W probe to obtain the hyaluronic acid modified ethosome (acupoint drug delivery system) carrying the Tingguisan volatile oil;

wherein the contents of the cinnamomum burmannii volatile oil, the phospholipid, the cholesterol, the ethanol and the HA-DOPE in the acupoint drug delivery system are respectively 0.2-4 w/v%, 0.2-5 w/v%, 0.05-2 w/v%, 20-45 v/v% and 0.1-1.5 w/v%.

In addition, the invention also provides the application of the acupoint drug delivery system in treating Ulcerative Colitis (UC) and symptoms such as abdominal pain, diarrhea, dysmenorrheal and the like caused by cold syndrome.

Has the advantages that:

(1) according to the hyaluronic acid modified ethosome, the ethosome is modified by HA, so that the leakage of the encapsulated drug can be reduced, and the stability of a preparation is effectively improved, so that the delivery of the drug to the skin along with a carrier is increased;

(2) the acupoint drug delivery system comprising the hyaluronic acid modified ethosome selects the hyaluronic acid modified ethosome (HA-ES) as a transdermal delivery carrier of the traditional Chinese medicine for warming middle-jiao and dispelling cold or an extract thereof, can better encapsulate effective components of the traditional Chinese medicine for warming middle-jiao and dispelling cold or the extract thereof, and delivers the drug to the deep layer of an acupoint region, thereby strengthening the special effect of the acupoint region after drug delivery, being expected to be used as a carrier of the traditional Chinese medicine components/effective parts of the traditional Chinese medicine for warming middle-jiao and dispelling cold, further being used for treating symptoms such as abdominal pain, diarrhea and dysmenorrheal caused by cold syndrome, especially for treating yang deficiency or cold syndrome UC, and having great application prospect;

(3) the acupoint drug delivery system (including the volatile oil of the cinnamomum burmannii powder) containing the hyaluronic acid modified ethosome not only can deliver the volatile oil of the cinnamomum burmannii powder to the deep layer of the skin of a acupoint region, but also can enhance the absorption of the volatile oil of the cinnamomum burmannii powder in the acupoint region so as to improve the bioavailability of the volatile oil of the cinnamomum burmannii powder, and the HA-ES acupoint drug delivery system carrying the volatile oil of the cinnamomum burmannii powder can improve the gastrointestinal dynamics index of ulcerative colitis and reduce H in blood serum and colon tissues of UC rats₂O₂And NO level, increase acetylcholine (Ach) and ATP level in UC rat colon tissue, obviously reduce Vasoactive Intestinal Peptide (VIP) in model rat colon tissue and improve Stem Cell Factor (SCF) expression, thereby obviously improving the effect of treating rat ulcerative colitis and having medicinal prospect;

(4) the preparation method disclosed by the invention is simple in process, free of special equipment and harsh conditions, easy to realize large-scale production and high in practical value.

Drawings

FIG. 1 is an HA-ES TEM (A is ES, B is HA-ES) transmission electron micrograph of the volatile oil carrying Ting Gui san prepared in example 2 of the present invention;

figure 2 is a drug loaded ethosome obtained in example 3 of the invention: cumulative permeation of drugs in ES, HA-ES1, HA-ES2 and HA-ES3 through rat isolated skin over 8h versus time, wherein: a is eugenol, b is cinnamaldehyde, and c is cinnamic acid;

figure 3 is a drug loaded ethosome obtained in example 3 of the invention: a transdermal rate graph of the drugs of ES, HA-ES1, HA-ES2 and HA-ES3 through rat isolated skin within 8h, wherein: a is eugenol, b is cinnamaldehyde, and c is cinnamic acid;

figure 4 is a drug loaded ethosome obtained in example 3 of the invention: retention of drugs in ES, HA-ES1, HA-ES2 and HA-ES3 in rat exsomatize skin in 8h, wherein: a is eugenol, b is cinnamaldehyde, and c is cinnamic acid;

FIG. 5 is a graph showing cytotoxicity of free drug, physical mixture, HA-ES blank vehicle, ES and HA-ES at different administration concentrations, obtained in example 4 of the present invention, wherein: (A) CCC-ESF-1 cells, (B) HaCaT cells;

FIG. 6 is a graph showing the effect of C6-HA-ES, C6-ES and C6-Free on fluorescence intensity in HaCaT and CCC-ESF-1 cells obtained in example 4 of the present invention, in which: (A, C) HaCaT cells, (B, D) CCC-ESF-1 cells;

FIG. 7 is a CLSM map of C6-labeled HA-ES and ES obtained in example 4 of the present invention by HaCaT cell uptake and intracellular co-localization, wherein: a is HA-ES, B is ES, C is C620% ethanol solution.

FIG. 8 is a graph of the in vivo microdialysis of subcutaneous drug concentration versus time obtained in example 5 of the present invention, wherein: a is cinnamic acid, B is clove;

FIG. 9 is a graph of the plasma-time curve of rats obtained in example 5 of the present invention, wherein: a is CA and B is EUG;

FIG. 10 is a photograph of a HE stained histopathological section obtained in example 6 of the present invention, in which: (A) normal group, (B) normal group HA-ES umbilical administration, (C) model group, (D) model group HA-blank (empty vector) umbilical administration, (E) model group ES umbilical administration (F) model group HA-ES umbilical administration, (G) model group HA-ES paradoxical administration;

FIG. 11 shows an HA-ES umbilical region administration group, an ES umbilical region administration group, and an HA-ES collateral administration group obtained in example 6 of the present invention, in which H is observed in cold-syndrome ulcerative colitis in rats₂O₂(C, D in the figure) and NO (A, B in the figure) expression level;

FIG. 12 is a graph showing the results of the concentration of Ach, ATP, SCF, VIP in the UC rat serum and colon obtained in example 6 of the present invention, wherein: (A) concentration of Ach in the colon, (B) concentration of ATP in the colon, (C) concentration of SCF in serum, (D) concentration of SCF in colon tissue, (E) concentration of VIP in serum, (F) concentration of VIP in colon tissue;

FIG. 13 is a graph showing the positive rate of immunohistochemical detection of c-kit in rat colon tissue between different groups obtained in example 6 of the present invention;

FIG. 14 is a drawing of an expression section of colon tissue of c-kit protein obtained in example 6 of the present invention in rats of different groups, wherein: (A) normal group, (B) normal group HA-ES umbilical administration, (C) model group, (D) model group HA-blank (empty vector) umbilical administration, (E) model group ES umbilical administration, (F) model group HA-ES umbilical administration, (G) model group;

FIG. 15 is a Western-blot assay of c-kit expression in rat colon tissue obtained in example 6 of the present invention, in which: 1. a normal control group 2, a normal mouse HA-ES umbilical administration 3, a model control group 4, a model mouse blank HA-ES umbilical administration 5, a model mouse ES umbilical administration 6, a model mouse HA-ES umbilical administration 7 and a model mouse HA-ES side-by-side administration;

FIG. 16 is a graph showing the statistical results of the expression of c-kit in rat colon tissue by Western-blot assay obtained in example 6 of the present invention;

fig. 17 is a graph of the skin irritation response of guinea pigs obtained in this example, in which: integral skin normal saline group, injected damaged skin normal saline group, HA-ES. damaged skin HA-ES group, ES. damaged skin ES group.

Detailed Description

The invention is further illustrated by the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Example 1

Novel acupoint drug delivery system for hyaluronic acid modified ethosome (HA-ES) loaded with cinnamomum burmannii powder volatile oil

1.1 extraction of volatile oil of Dinggui powder

Referring to clove and cinnamon medicinal materials specified in Chinese pharmacopoeia of 2015 edition, 200.3g of clove and 200.6g of cinnamon medicinal materials are weighed and crushed into 20-mesh coarse powder, 8 times of water is added to soak the coarse powder for 1 hour, the coarse powder is extracted for 6 hours by a steam distillation method at 200 ℃, and volatile oil is collected. 3mL of cinnamon volatile oil and 15mL of clove volatile oil are obtained. Mixing them uniformly to obtain the invented volatile oil. HPLC detection shows that the contents of eugenol and cinnamaldehyde in the clove volatile oil and the cinnamon volatile oil are respectively 85.8 +/-0.43% and 93.1 +/-4.45%, and the quality standards of clove and cinnamon in 2015 version Chinese pharmacopoeia are met.

1.2 Synthesis of HA-DOPE

52mg of HA was weighed into 20mL of distilled water (pH 4.0), and stirred overnight on a magnetic stirrer to fully swell and dissolve. 24mg of EDC and 13.6mg of NHS were added, the pH was adjusted to 4.0 with 0.1mol/L hydrochloric acid, 37 ℃ C., 700rpm/min and activated for 2h in a water bath. 720 mul of ethanol solution dissolved with 1mg/mL DOPE is added into the swelled HA solution, the pH is adjusted to 8.6 by 0.1mol/L sodium hydroxide solution, the mixture is magnetically stirred in a water bath at 37 ℃ and 500rpm for 24 hours. After the reaction, the reaction solution was added to a dialysis bag (molecular weight cut-off was 3500Da) to remove excess reactants and reaction by-products. Freeze drying the reaction liquid to obtain HA-DOPE, and storing at-20 deg.C for use.

1.3 preparation of HA-ES

Prior to preparation, the prescribed amount of HA-DOPE was dissolved in PBS for 12 h. Accurately weighing appropriate amount of phospholipid and cholesterol in penicillin bottles, adding ethanol (containing volatile oil of Cinnamomum burmannii powder) at a prescription amount, sealing, magnetically stirring in 60 deg.C water bath at 300rpm for 15min to obtain homogeneous solution, slowly injecting PBS solution dissolved with HA-DOPE, sealing and stirring for 1h, cooling at room temperature, and performing ultrasonic treatment with 300W probe for 5min to obtain the final product. The obtained ethosome contains the volatile oil of the cinnamomum burmannii powder of 7.66mg/mL, the HA-DOPE content of 0.25mg/mL, the phospholipid content of 8mg/mL, the cholesterol content of 2.26mg/mL, the ethanol content of 21.55 v/v%, and the balance of PBS solution.

Example 2

Evaluation and characterization of hyaluronic acid modified ethosome (HA-ES) carrying cinnamomum burmannii volatile oil:

2.1 measurement of particle diameter and potential

And measuring the particle size distribution of each nanometer preparation by a Malvern particle size potential measuring instrument and a dynamic light scattering method. 3 samples were prepared in parallel and their particle size and Zeta potential were measured. The result shows that the HA-ES carrying the cinnamomum burmannii powder volatile oil HAs the average grain diameter of 299.5 +/-54.3 nm and the Zeta potential of-31.7 +/-0.47 mV.

2.2 Transmission Electron microscopy

Diluting each preparation with deionized water by a certain multiple, dripping on a copper net, after natural drying, negative dyeing with uranyl acetate for 2min, sucking away excessive negative dyeing liquid with filter paper, air drying, and observing the preparation form under a transmission electron microscope. The results are shown in FIG. 1.

FIG. 1 is a transmission electron microscope image of HA-ES of the volatile oil carrying Cinnamomum burmannii powder prepared in example 2 of the present invention, as can be seen from FIG. 1, the forms of HA-ES and ES lipid vesicles are similar to spherical or ellipsoidal, the lipid bilayer is obvious, and the vesicles are uniform and round in size, uniform in dispersion and free from adhesion. Compared with the ES vesicles, the HA-ES vesicles have darker color in the middle, which is probably caused by the fact that the surfaces of the HA-ES vesicles are covered with HA molecular gel networks and contain higher moisture.

2.3 encapsulation efficiency and drug Loading

The encapsulation efficiency and drug-loading capacity of the preparation are measured by an ultrafiltration centrifugation method. The results show that the drug-loading rates of HA-ES cinnamaldehyde and eugenol of the clove powder-loading volatile oil are respectively (10.9 +/-1.3)%, and (36.0 +/-5.2)%; the encapsulation efficiency is (74.9 +/-1.1)%, respectively, (79.3 +/-2.0)%.

Comparative example 1

Preparation of Ethosome (ES):

the preparation method of the embodiment 1 is the same, the HA-DOPE in the prescription is removed, and the dosage of other components in the prescription is fixed, thus obtaining the compound.

Comparative example 2

Preparation of drug-loaded HA-ES-2:

the preparation method of the embodiment 1 is the same, the HA-DOPE concentration in the prescription is set to be 0.50mg/mL, and the dosage of other components in the prescription is fixed, so that the traditional Chinese medicine composition is obtained.

Comparative example 3

Preparation of drug-loaded HA-ES-3:

the preparation method of the embodiment 1 is the same, the HA-DOPE concentration in the prescription is set to be 1.00mg/mL, and the dosage of other components in the prescription is fixed, so that the compound is obtained.

Example 3

In vitro transdermal behavior:

3.1 in vitro transdermal behavior

Taking SD rat, anesthetizing with 10% chloral hydrate, depilating with razor, scrubbing with normal saline, peeling abdominal skin with scalpel, removing subcutaneous tissue and fat, cleaning with normal saline, storing in-4 deg.C refrigerator, using within one week, and carefully checking the integrity of rat skin before use. The improved Franze diffusion pool is adopted, the in vitro rat skin is taken, the cuticle faces to the supply pool, and the subcutaneous tissue faces to the receiving pool. The receiving cell was filled with receiving solution (12.5mL of 10% PEG-200/PBS [ w/v ] solution), vacuolated, and equilibrated for 30 min. The supply tank was filled with 1mL of formulation, leaving the stratum corneum surface intact with the formulation. The supply cell was sealed with a sealing film, and the receiving solution was magnetically stirred at a temperature of 32 ℃ and 500 rpm. 1mL of receiving solution is quantitatively taken out from the receiving pool at 0.5 h, 1h, 2h, 4h, 6h and 8h, and fresh receiving solution with the same volume at 32 ℃ is supplemented into the receiving pool. HPLC measurement, drug concentration and cumulative permeation per unit area (Q) were calculated. The results are shown in FIGS. 2 and 3.

Figure 2 is the drug loaded ethosome obtained in this example: cumulative permeation of drugs in ES, HA-ES1, HA-ES2 and HA-ES3 through rat isolated skin over 8h versus time, wherein: a is eugenol, b is cinnamaldehyde, and c is cinnamic acid; figure 3 is a drug loaded ethosome obtained in this example: a transdermal rate graph of the drugs of ES, HA-ES1, HA-ES2 and HA-ES3 through rat isolated skin within 8h, wherein: a is eugenol, b is cinnamaldehyde, and c is cinnamic acid; as can be seen from FIG. 2, the in vitro transdermal amounts of eugenol and cinnamaldehyde in the volatile oil are both significantly improved after HA-DOPE modification. The transdermal amount of cinnamaldehyde is highest when the HA-DOPE concentration is 0.25mg/mL, and the transdermal amount is reduced along with the increase of the dosage. As shown in fig. 3(a is eugenol, b is cinnamaldehyde, and c is cinnamic acid), when HA-DOPE is 0.25mg/mL, the in vitro skin permeation rate of eugenol and cinnamaldehyde is significantly increased compared to other groups (p <0.01, p < 0.05).

3.2 skin Retention

After the in vitro transdermal experiment is finished, the skin of the effective transdermal area is carefully cut off, the residual liquid medicine is washed by 10mL PBS, then 1mL 10% DMSO is added for homogenization treatment, the centrifugation is carried out at 13000rpm for 10min, and the supernatant is absorbed and detected by HPLC. The results are shown in FIG. 4.

Figure 4 is a drug loaded ethosome obtained in this example: retention of drugs in ES, HA-ES1, HA-ES2 and HA-ES3 in rat exsomatize skin in 8h, wherein: a is eugenol, b is cinnamaldehyde, and c is cinnamic acid; as can be seen from fig. 4, the skin retention of each component in the ES group was higher than that of the HA-ES group, indicating that HA modification significantly promotes transdermal penetration of the drug. The skin retention of eugenol and cinnamaldehyde is reduced along with the increase of the dosage of HA-DOPE, and the influence of the dosage of HA-DOPE on the skin retention of cinnamic acid is not obvious. The result is identical with the research result of the transdermal cumulative penetration, namely the modification of HA-DOPE enhances the transdermal penetration capability of ethosome, thereby enhancing the transdermal penetration of the drug and reducing the retention in the epidermis.

Comparative example 4

Preparation of a 20% ethanol solution of free drug:

dissolving the volatile oil in 20% ethanol solution.

Comparative example 5

Preparation of a 20% ethanol solution of a physical mixture of lipid and drug:

weighing the components carrying the HA-ES prescription amount of the cinnamomum burmannii powder volatile oil, respectively adding the components into a 20% ethanol solution, and uniformly mixing to obtain the cinnamomum burmannii powder volatile oil.

Comparative example 6

Preparation of HA-ES blank formulation:

the preparation method is the same as the preparation method of the example 1, the volatile oil of the cinnamomum burmannii powder in the prescription is removed, and the dosage of other components in the prescription is fixed, so that the traditional Chinese medicine is obtained.

Example 4

The study on the interaction between the volatile oil HA-ES of the cinnamomum burmannii powder and skin cells:

4.1 cytotoxicity

MTT method is adopted to detect toxicity of free drug 20% ethanol solution, lipid and drug physical mixture 20% ethanol solution, HA-ES blank preparation, drug-loaded ES and HA-ES to CCC-ESF-1 and HaCaT cells. Taking a proper amount of preparation, adding DMEM, respectively diluting the preparation into solutions with the same concentration of 5-100 mu g/mL, and inspecting the toxicity of the preparation on skin cells.

By 1 × 10⁴Inoculating CCC-ESF-1 and HaCaT cells in logarithmic growth phase into 96-well plate, respectively, at 37 deg.C and 5% CO₂Culturing for 24h, discarding the culture solution, adding 5-100 μ g/mL free drug, physical mixture, blank vesicle, drug-loaded ES and HA-ES, each concentration operating in parallel for 3 parts, placing in an incubator at 37 deg.C and 5% CO₂Culturing for 24h under the condition. After the culture was completed, the supernatant was discarded, 200. mu.L of DMEM medium containing 500. mu.g/mL MTT was added to each well, incubation was performed at 37 ℃ for 4 hours, the supernatant in each well was aspirated, and 150. mu.L of DMSO was added to each wellThe solution was shaken in the dark for 3min to dissolve formazan in the cells sufficiently, and the absorbance value was measured at a wavelength of 570nm using an enzyme-labeling instrument. And (4) processing the blank cells without the added medicine in the same direction, measuring an absorbance value, and calculating the cell survival rate. The results are shown in FIG. 5.

FIG. 5 is a graph of cytotoxicity of free drug, physical mixture, HA-ES blank vehicle, ES and HA-ES obtained in this example at various dosing concentrations, wherein: (A) CCC-ESF-1 cells, (B) HaCaT cells; as can be seen from FIG. 5, the cytotoxicity increased with the increase of the concentration of the preparation, indicating that both ethanol and volatile oil in the preparation can exert certain toxic effect on cells. The cytotoxicity of the lipid vesicle group is obviously lower than that of the free drug group, particularly the cytotoxicity of the HA-ES group is the minimum, which indicates that the direct stimulation of the drug to cells is reduced by the encapsulation of the lipid vesicle, and the biocompatibility of the preparation can be better improved by the modification of HA.

4.2 preparation of coumarin-6 labeled preparation

Preparing 40 mug/mL Coumarin 6 ethanol solution (Coumarin-6, C6) with anhydrous ethanol, and replacing the volatile oil of BUTENG powder with 0.1mL solution, and preparing the fluorescence labeling preparation with the preparation method of ES and HA-ES.

4.3 cellular uptake

CCC-ESF-1 and HaCaT cells in logarithmic growth phase at 5X 10⁵And (3) respectively inoculating the density of each hole on a 6-hole plate, culturing for 24h, discarding the culture solution, washing with PBS for 2 times, respectively adding a certain amount of C6 labeled preparation, incubating with the cells for 2h in a dark place, discarding the culture solution, and washing with PBS for 3 times. 2mL of 0.25% pancreatin solution is added into each well, the mixture is transferred into a 5mL flow tube after digestion, the mixture is centrifuged at 1000rpm for 3min, cells are redispersed by 0.5mL of PBS and placed in the flow tube, the fluorescence uptake is detected by a flow cytometer, cells incubated by blank culture solution are used as a control, and 3 parts of each group are paralleled. The results are shown in FIG. 6.

FIG. 6 is a graph showing the effect of C6-HA-ES, C6-ES and C6-Free on fluorescence intensity in HaCaT and CCC-ESF-1 cells obtained in this example, in which: (A, C) HaCaT cells, (B, D) CCC-ESF-1 cells; as can be seen from fig. 6, compared to the free C6 group, the lipid vesicle can significantly promote cellular uptake of C6, which is probably due to the protection of C6 by the vesicle, and the C6 encapsulated by the vesicle is endocytosed by the cell. However, cellular uptake did not show significant differences between HA-ES and ES groups, possibly associated with shorter incubation times.

4.4 intracellular Co-localization

By 5X 10³Respectively inoculating CCC-ESF-1 and HaCaT cells in logarithmic growth phase into a laser confocal dish at 37 deg.C and 5% CO₂After 24h incubation, the culture medium was discarded, 100. mu. L C6 labeled preparation was added separately and incubated at 37 ℃ for 2 h. After incubation, the supernatant was discarded, the cell surface was washed with PBS 3 times, fixed with 1mL of 4% paraformaldehyde, incubated at 37 ℃ with 5% CO₂After culturing in dark for 20min, removing paraformaldehyde solution, washing the cell surface with PBS for 2 times, adding 1mL DAPI into each well, continuously dyeing in dark for 8min, and washing with PBS for 3 times. Finally, 0.5mL of PBS is added, laser confocal microscopy is carried out within 2h, the Ex/Em is 415nm/485nm and 498nm/568nm, and blue DAPI fluorescence labeling the cell nucleus and green C6 fluorescence labeling the cytoplasm are observed. The results are shown in FIG. 7.

FIG. 7 is a CLSM map of C6-labeled HA-ES and ES obtained in this example, taken up by HaCaT cells and co-localized intracellularly, in which: a is HA-ES, B is ES, C is C620% ethanol solution; as can be seen, C6 was mainly distributed in the cell membrane and cytoplasm after cellular uptake, but not in the nucleus. The intracellular co-localization of HA-ES and ES groups is not obviously different, and the lipid vesicles can promote cellular uptake, but do not change the distribution condition of C6 in cells.

Example 5

Study on the pharmacokinetics of umbilical administration of novel ethosome:

5.1 animal groups

Four groups are as follows: (1) HA-ES is administrated to the umbilical region of a normal group of rats, (2) HA-ES is administrated to the umbilical region of a model group of rats, (3) ES is administrated to the umbilical region of the model group of rats, and (4) HA-ES is administrated to the side of the model group of rats. Except for the normal group, the model groups are grouped after the model is successfully manufactured.

5.2 preparation of model of Ulcerative Colitis in rat (UC)

On day 1, rats in the model group were fasted without water prohibition, and 24 hours later were anesthetized by intraperitoneal injection of 3.5% chloral hydrate (1mL/100g), fixed on a rat dissecting table, a rubber infusion tube of 2.0mm in diameter and about 11cm in length was gently inserted from the anus to a depth of about 8cm, 5% 2, 4, 6-trinitrobenzene sulfonic acid (1mL/100g) and an equivalent 50% ethanol solution were slowly injected into the rat intestinal cavity for about 8cm, a little air was further injected, the rat tail was lifted up, and the model preparation was inverted for 30 seconds to sufficiently infiltrate the model preparation. Animals were raised normally after awakening. The abdomen of the rats in the model group was placed in an ice-water bath for 2min 1 time a day and 3 times a day from day 2. Daily body weight, food intake, water intake, hair gloss, stool characteristics and activity were observed and recorded.

5.3 administration and in vivo skin microdialysis

After 3 days of molding, rats with loose stools, weight loss, hair color withering and the like are selected as successfully molded rats, 3 rats are randomly selected, colon is dissected and checked, and whether molding is successful or not is judged. The picked model group rats were randomly divided into 3 groups. Rats in the normal group and the model group are respectively anesthetized with 20% urethane (0.6mL/100g), after the rats enter an anesthetic state, the rats are fixed on a rat board in a prone position, abdominal hairs are carefully shaved off by an electric shaver until the hairs are shorter than 2mm, the rats are scrubbed clean by physiological saline, a microdialysis probe is embedded under the skin of the umbilical region, and the rats are perfused by using 20% ethanol-water solution as perfusion fluid at the flow rate of 0.2 mL/h. The probe embedded in the abdomen of the rat is adhered with glue to the position with the area of about 1cm²The circular plastic cover of (2) is used as a dosing pool so as to fix the dosing area. After 30min of equilibration to a stable state, 1mL of ES and HA-ES preparations were administered to the umbilical region (centered on the navel and 0.5cm in radius) and the side-open area (0.5 cm area outside the umbilical region), respectively, and fixed with medical tape. Samples were collected every 30min for 10 h. The samples were directly analyzed by HPLC. Analysis of in vivo skin microdialysis pharmacokinetic data a non-compartmental model (noncromompartmental analysis) was used with the software Phenix winnonlin 2 pharmacokinetic software. Statistical analysis of data the differences between groups, p, were analyzed by ANOVA using the software SPSS 19.0 (IBM corporation, USA) using the LSD method<0.05 set as significant difference level, p<0.01 was set to a very significantly different level. The results are shown in figure 8, and the pharmacokinetic parameters of the cinnamic acid and the eugenol in the skin in vivo are shown in Table 1As shown.

TABLE 1 pharmacokinetic parameters of cinnamic acid and eugenol in vivo skin microdialysis

FIG. 8 is a graph of the in vivo microdialysis subcutaneous drug concentration versus time obtained in this example, wherein: a is cinnamic acid, B is eugenol; as shown in FIG. 8, the concentrations of cinnamic acid and eugenol were higher in the model group than in the other groups within 10 hours after umbilical administration of HA-ES, which is probably due to the decrease in body weight after molding of the rats in the model group, the thin umbilical skin, and the distribution of the effective substances in the tissues and blood of the rats in the normal group, which were the same administration area as that of the rats in the normal group, and therefore, the content of cinnamic acid and eugenol detected in the subcutaneous microdialysis receptor fluid was less than that in the model group. The significant increase in HA-ES over ES after umbilical administration of HA-ES and ES, respectively, in the model group may be due to the fact that HA protects the drug from leakage during its transdermal penetration and that HA is an aqueous macromolecule that better delivers the drug to the deep layers of the crypt area below the dermal layer.

5.4 pharmacokinetics in vivo

Animal grouping, modeling method and administration method are the same as those under the section "2.1.2" in this chapter. After administration, 200 μ L of blood is collected from 5min, 15min, 30min, 1h, 2h, 4h, 8h, 10h, 12h and 24h orbits respectively in a heparinized centrifuge tube of 1.5mL, 0.6mL of ethyl acetate and 10 μ L of internal standard substance (ferulic acid is dissolved in ethyl acetate, the concentration is 5 μ g/mL) are added into 200 μ L of drug-containing plasma, the mixture is swirled for 3min, 13000rpm is centrifuged for 5min, the supernatant is taken out in a centrifuge tube of 1.5mL, nitrogen is dried, 50 μ L of acetonitrile is used for redissolving, the mixture is swirled for 3min, 13000rpm is centrifuged for 5min, and the supernatant is taken out to be detected by HPLC. The in vivo pharmacokinetic data analysis method is the same as the in vivo skin microdialysis pharmacokinetic data analysis method. The results are shown in FIG. 9, and in tables 2 and 3.

Table 2 in vivo pharmacokinetic parameters of cinnamic acid (n ═ 6)

Parameter	Unit	1	2	3	4
						Tmax	h	1.25±0.6	1.6±1.5	1.2±0.8	2.4±2.8
Cmax	ng/mL	203.8±53.5	205.3±38.3	153.5±31.0	162.4±32.
						AUC0-t	h/ng/mL	1273.9±196.2	1485.3.7±197.3	1043.0±146.0	1140.1±246.7
MRT0-t	h	4.3±0.3	4.3±0.2	4.4±0.3	4.5±0.4

Table 3 eugenol in vivo pharmacokinetic parameters (n ═ 6)

Parameter	Unit	1	2	3	4
						Tmax	h	2.0±0	2.7±1.2	2.7±1.2	1.0±0.9
Cmax	ng/mL	259.8±33.2	254.8±51.9	178.2±7.0	163.3±41.9
						AUC0-t	h/ng/mL	1903.2±170.1	1617.9±136.1	1193.3±129.7	1232.2±270.4
MRT0-t	h	4.5±0.3	4.4±0.3	4.3±0.1	4.6±0.2

FIG. 9 is a graph of the plasma drug-time curve obtained in rats of the present example, in which: a is CA and B is EUG; as can be seen from the figure, after transdermal administration, the plasma concentration of the HA-ES umbilical region administration group is obviously higher than that of the side-by-side administration group, and the AUC is also obviously higher than that of the side-by-side administration group. In addition, the umbilical administration group data of the model group show that the blood concentration and AUC of the HA-ES group are obviously higher than those of the ES group, which indicates that the HA-ES can effectively promote the percutaneous absorption of the medicine. The results are all consistent with the results of in vitro transdermal experiments and in vivo microdialysis experiments. Umbilical administration aids in the transdermal absorption of the drug, and may be associated with its thin stratum corneum, where ethosomes vesicles readily penetrate the stratum corneum into the deep layers of the skin, thereby releasing the drug into systemic circulation. In addition, the modification of HA also protects the drug from leaking when penetrating the epidermal layer, so that the drug is more transported to the dermis and subcutaneous tissues with abundant microcirculation, and the absorption of the drug into the systemic circulation is promoted, thereby obviously improving the bioavailability of the drug.

Both in vivo microdialysis and in vivo pharmacokinetic experiments demonstrate that modification of HA protects the drug from leakage through the epidermal layerMore medicine is transported to dermis and subcutaneous tissue with abundant microcirculation, and the medicine is promoted to be absorbed and enter the systemic circulation, so that the bioavailability of the medicine is obviously improved. So that the model group HAs C after umbilical administration of HA-ES_maxAnd AUC significantly higher than those of the group administered umbilical region of ES and the group administered HA-ES side by side.

Example 6

Pharmacodynamic study of carrying the novel ethosome umbilical administration of the cinnamomum burmannii powder volatile oil on rat ulcerative colitis:

6.1 establishment and administration of model of cold syndrome ulcerative colitis in rat

70 SD male rats were randomly divided into 7 groups of 10 rats each, and the rats in each group were fasted for 24h before molding without water deprivation. The molding method was the same as in example 5. The administration was performed in groups of (1) normal group: no administration of the drug; (2) normal group: umbilical administration of HA-ES; (3) no drug was administered to the model group; (4) model group HA-blank administered umbilical; (5) model group umbilical administration of ES (6) model group umbilical administration of HA-ES; (7) HA-ES was administered by-pass for 7 consecutive days, during which time rat status was observed and body weight changes were recorded. On day 8, the rats were sacrificed and the colons, serum, and skin were stored at-80 ℃ for future use.

Appearance signs: the normal group showed normal, gradually increased body weight and good mental status. After the TNBS is injected into the model building group, the weight of the model building group is gradually reduced, and secretion appears in the anus, the mental state is not good, the movement is reduced, and the food intake and the water intake are reduced.

6.2 histopathological Observation

After the administration of the drug, the rat is sacrificed, the colon is taken out and cut into small segments, paraffin embedding is carried out after formalin fixation, hematoxylin/eosin staining is carried out on the segments, and the segments are sealed. Histological lesions of the colon area of each group of rats were observed under a microscope. The results are shown in FIG. 10.

Fig. 10 is a graph of HE stained histopathological sections obtained in the present example, in which: (A) normal group, (B) normal group HA-ES umbilical administration, (C) model group, (D) model group HA-blank (empty vector) umbilical administration, (E) model group ES umbilical administration (F) model group HA-ES umbilical administration, (G) model group HA-ES paradoxical administration; as can be seen from FIG. 10, no significant pathological changes were observed in the colon wall layers of the rats in the normal group (A) and the normal mouse HA-ES umbilical region administration group (B); the model group (C) rat colonic ulcer is formed, the ulcer surface is large, the mucosa layer is involved, the mucosa muscle layer is not reached, and the submucosa is congested, edematous and inflammatory cell infiltration; model mouse HA-blank umbilical administration group (D) colonic ulcer formation, involvement of the whole mucosal layer; the colon wall of the ES umbilical region administration group (E) of the model mouse is frequently ulcerated, the ulceration affects the whole mucous membrane layer, and local congestion and edema on the mucous membrane surface of the colon wall are improved compared with the model group; the HA-ES umbilical administration group (F) of the model mouse HAs necrosis, desquamation and erosion formation of local mucosal epithelium of colon wall, mild hyperemia and edema of erosion surface and inflammatory cell infiltration, and most of colon mucosal epithelium of the rat is frequently proliferated and repaired compared with the model group; the HA-ES side-opening administration group (G) of the model mouse is formed by congestion, edema, inflammatory cell infiltration, necrosis, desquamation and erosion of the mucosal epithelial part. The success of molding is proved by pathological sections.

6.3 detection of inflammatory factors and Colon kinetics

After the orbit blood is taken, the orbit blood is placed for 30min, is centrifuged for 3min at 3000rpm, and the supernatant is taken and is used for measuring the serum and the H in the colon by adopting an Elisa method₂O₂NO, Ach, ATP, SCF, VIP. The results are shown in FIGS. 11 and 12.

FIG. 11 shows the umbilical region administration set of ES, HA-ES, and side-by-side administration set of HA-ES obtained in this example for H in cold-syndrome ulcerative colitis in rats₂O₂And NO expression level; as can be seen, the umbilical administration of HA-ES significantly reduced H in serum and colon tissues of model mice₂O₂And NO level, showing that HA-ES HAs stronger effect of promoting the percutaneous absorption of the medicine.

FIG. 12 is a graph showing the results of the concentration of Ach, ATP, SCF, and VIP in the UC rat serum and colon obtained in the present example, wherein: (A) concentration of Ach in the colon, (B) concentration of ATP in the colon, (C) concentration of SCF in serum, (D) concentration of SCF in colon tissue, (E) concentration of VIP in serum, (F) concentration of VIP in colon tissue; as can be seen from FIG. 12, the content of Ach and ATP in colon tissue was significantly increased in the HA-ES umbilical region-administered group compared to the model group (p <0.05, p < 0.01); the umbilical and collateral administration groups of HA-ES showed a significant increase in serum SCF concentration (p <0.05 >); SCF expression was significantly increased in the ES group, umbilical administration group of HA-ES, and side-by-side group compared to the model group (p <0.01, p <0.0001, p < 0.0001). VIP expression was significantly reduced in ES, umbilical part of HA-ES and side-by-side dosed groups compared to the model group (p <0.001, p <0.001, p < 0.01).

6.4 immunohistochemistry

Taking a paraffin section, dewaxing, removing endogenous catalase, repairing antigen, sealing serum, respectively adding a primary antibody and a secondary antibody, then adding a DAB color developing agent, and sealing the section after hematoxylin counterstaining, dehydration and transparency. The positive rate of c-kit protein in rat colon tissue between different groups is shown in FIG. 13 and FIG. 14.

FIG. 13 is a graph showing the positive rate of immunohistochemical detection of c-kit in rat colon tissue between different groups obtained in this example; FIG. 14 is a sectional view showing the expression of the c-kit protein obtained in this example in colon tissues in rats of different groups, in which: (A) normal group, (B) normal group HA-ES umbilical administration, (C) model group, (D) model group HA-blank (empty vector) umbilical administration, (E) model group ES umbilical administration, (F) model group HA-ES umbilical administration, (G) model group; as can be seen, the positive rate of c-kit protein in the model control group was significantly decreased and was significantly increased after umbilical administration of HA-ES (p <0.05) compared to the normal group.

6.5Western-blot method for detecting contents of multiple indexes in colon tissue

Frozen colon tissue is precisely weighed, cut into small pieces, and added with precooled protein extraction reagent containing inhibitor. The tissue was homogenized by a homogenizer at a low speed for 30 seconds each time, with an ice bath interval of 1 minute for each homogenization until the tissue was completely lysed. The lysate was centrifuged at 14,000g for 15 minutes in a precooled centrifuge. The supernatant was immediately transferred to a new centrifuge tube and stored for further use. Determining the amount of protein concentration according to the BCA protein quantification kit instructions; adding a loading buffer solution to prepare a protein sample; after SDS-PAGE gel electrophoresis, the gel was transferred to an NC membrane, and 0.5ml of ponceau was added thereto and stained for 30 seconds to observe the effect of the transfer. The dye liquor was removed and the membrane was washed three times for 5 minutes each time with double steam. Sealing 5% skimmed milk powder at room temperature for 1 hr; the diluted primary antibody was added and left overnight at 4 ℃. The membranes were washed 3 times with TBST (5 minutes each). Diluted anti-antibody (1:5000) was added and incubated at room temperature for 1 hour with shaking. The membranes were washed 3 times with TBST (5 minutes each). After washing the membrane, the ECL luminescent reagent was taken out, and 1ml of each of solutions A and B was mixed. And (3) putting the film into an exposure instrument, dropwise adding a luminescent liquid, exposing for three times, each time for 5min, and selecting the overlapping value of the three exposures. The results of Western blot analysis of AKT, c-kit, Erk, P-AKT, P-Erk, P-STAT3 expression in colon tissues of each group are shown in FIGS. 15 and 16.

FIG. 15 is a graph showing the expression of c-kit in rat colon tissue measured by Western-blot, obtained in this example, wherein: 1. a normal control group 2, a normal mouse HA-ES umbilical administration 3, a model control group 4, a model mouse blank HA-ES umbilical administration 5, a model mouse ES umbilical administration 6, a model mouse HA-ES umbilical administration 7 and a model mouse HA-ES side-by-side administration; FIG. 16 is a graph showing the statistical results of the expression of c-kit in rat colon tissue by Western-blot assay obtained in this example; as can be seen from the graphs, the relative protein expression level of c-kit was significantly reduced in the model group (p <0.0001), compared with the normal group, in the ES umbilical region model group, in the HA-ES bypass model group, significantly increased in the non-administration model group (p <0.0001), and in the model group, the c-kit protein expression level of the HA-ES umbilical region administration group was significantly increased in the model group (p < 0.0001). The expression of the c-kit protein of the HA-ES umbilical administration group in the model group is obviously higher than that of other groups, which shows that HA-ES can relieve and repair ICC injury after umbilical administration, and simultaneously recover the quantity of HA-ES, thereby playing an important role in regulating gastrointestinal motility.

The above results indicate that umbilical administration has a certain specific effect on gastrointestinal motility, which may be related to better drug absorption in the umbilical region. Compared with the administration of the HA-ES by umbilical administration and the side-opening administration, the HA-ES HAs stronger regulating effect on gastrointestinal motility, mainly due to the excellent transdermal permeability of the HA-ES, effectively improves the transdermal absorption of the medicine, thereby enhancing the medicine effect.

6.6 skin irritation study

Guinea pigs were taken and, 12 hours before administration, hairs of abdominal skin were carefully removed with a razor and divided into (1) intact skin saline group, (2) damaged skin saline group, (3) damaged skin HA-ES group, and (4) damaged skin ES group. The damaged skin group is cut into a shape like a Chinese character 'jing' by a blade, preferably oozing blood, 1mL of the preparation is respectively smeared on the skin of each administration group, the administration area of each group is 2cm multiplied by 2cm, the administration groups are fixed for 4 hours by gauze and a medical bandage, the medicine is removed, and the change of the skin is observed for 24 hours, 48 hours and 72 hours after continuous administration for 3 days. The results of the experiment are shown in FIG. 17.

Fig. 17 is a graph of the skin irritation response of guinea pigs obtained in this example, in which: A. a complete skin normal saline group, a damaged skin HA-ES group and a damaged skin ES group; as can be seen from the figure, neither HA-ES nor ES is significantly irritating to the skin of rabbits and guinea pigs. After multiple 3d administration, no irritation such as obvious redness and edema appeared on the skin of the guinea pig.

Therefore, the novel acupoint drug delivery system provided by the invention is applied to acupoint delivery of traditional Chinese medicines for warming middle-jiao and dispelling cold, can effectively promote absorption of the medicines in acupoint regions, and strengthens special effects of the acupoint regions after acupoint drug delivery. The HA-ES acupoint transdermal drug delivery system HAs better performance of coating drugs deeply entering into acupoint regions, can protect the drugs from leaking when penetrating through epidermal layers, transports the drugs to more dermis and subcutaneous tissues with rich microcirculation, promotes the absorption of the drugs into systemic circulation, and thus obviously improves the bioavailability of the drugs. HA-ES umbilical administration carrying cinnamon volatile oil can remarkably reduce H in UC model rat serum and colon tissues₂O₂And NO levels, up-regulate Ach and ATP levels in UC murine colon tissue, such that VIP expression in colon tissue is significantly reduced, and SCF expression in colon tissue is significantly increased. The expression of colon c-kit protein of the HA-ES umbilical region administration group is obviously higher than that of other groups, which shows that HA-ES can relieve and repair ICC injury after umbilical region administration, and simultaneously recover the quantity of HA-ES, thereby playing an important role in regulating gastrointestinal motility. The novel acupoint drug delivery system is expected to be used as a carrier of traditional Chinese medicine components/effective parts for warming middle-jiao to dispel cold, is further used for treating symptoms such as abdominal pain, diarrhea and dysmenorrheal caused by cold syndrome, is especially used for treating yang deficiency or cold syndrome UC, and has obvious medicinal prospect.

Although specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these embodiments are merely illustrative and various changes or modifications may be made without departing from the principles and spirit of the invention.