CN118141852A - Mulberry extract temperature-sensitive gel and preparation method and application thereof - Google Patents

Abstract

The invention discloses a mulberry extract temperature-sensitive gel and a preparation method and application thereof. The mulberry extract temperature-sensitive gel is prepared from the following raw materials in percentage by mass: 16-18% of temperature sensitive material, 2.5-4% of temperature regulator, 0.3-0.7% of biological adhesive, 1-5% of mulberry extract and the balance of water. The thermosensitive gel provided by the invention can be rapidly gelled in a physiological state, has a certain slow release performance, has no irritation to intestinal mucosa, can improve intestinal inflammation or UC symptoms, has a repairing effect on the intestinal mucosa, and has a great application prospect.

Description

Mulberry extract temperature-sensitive gel and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a mulberry extract temperature-sensitive gel, and a preparation method and application thereof.

Background

Intestinal inflammation is a defensive reaction of the intestinal tract to stimulus, and intestinal chronic inflammation can influence the number of endocrine cells in the intestinal tract and secretion of intestinal hormone by changing the permeability of the intestinal barrier, influence the nerve regulation signal path of the brain-intestinal axis, or participate in the occurrence and development of insulin resistance and type 2 diabetes mellitus and the like. At present, medicines for treating intestinal inflammation mainly comprise 5-aminosalicylic acid, glucocorticoid, anti-tumor necrosis factor-alpha medicines, anti-adhesion preparations, small molecule medicines such as pro-inflammatory factor inhibitors, JAK kinase inhibitors and the like, and auxiliary treatment methods such as supplements, probiotics, enteral nutrition and the like, but the methods have poor treatment effects on a large part of patients or have serious side effects, so that patients cannot tolerate the medicines. Ulcerative Colitis (UC) is a chronic recurrent disease (Liu,Y.,et al.,Potential activity of Traditional Chinese Medicine against Ulcerative colitis:A review.J Ethnopharmacol,2022.289:p.115084), mainly characterized by superficial mucosal ulcers of the rectum and colon, which are mainly clinical manifestations of abdominal pain, increased water content of stool and concentrated blood of mucus, and are accompanied by different degrees of damage to joints, skin, eyes, liver and gall and other parts, and even with the development of disease conditions, the possibility of canceration is even present. A meta-analysis shows that the incidence rate of colon cancer of UC patients increases year by year along with the extension of the disease course, and the probability of colon cancer of patients after 10 years, 20 years and 30 years is 1.6 percent, 8.3 percent and 18.4 percent in sequence. The disease is well developed in the 45-59 year old people, and has no difference in sex. In recent years, with the change of resident eating and living habits, the incidence of UC in China is remarkably increased. The pathogenesis of the disease is related to immune inflammation, oxidative stress, cell apoptosis, and disruption of epithelial barrier function. At present, traditional 5-aminosalicylic acid, sulfasalazine, glucocorticoid, antibiotics, immunosuppressant and the like are still main medicines for preventing and treating UC, but have the limitations of long treatment course, large adverse reaction and high recurrence rate. Based on the above, research and development of a traditional Chinese medicine with small side effects and capable of improving the prognosis effect and the immune function of patients are important. The traditional Chinese medicine is widely paid attention to clinically as an auxiliary means for treating intestinal inflammation.

Earlier studies found that mulberry extract has a certain effect in oral treatment of intestinal inflammation and UC, can significantly alleviate intestinal tissue inflammation, reduce proinflammatory factor level, alleviate colonic atrophy and the like (see patent CN113143996B, CN114601865 a), the mulberry extract is partially degraded by enzymes in the gastrointestinal tract through the gastrointestinal tract, and is absorbed when passing through the small intestine, and the amount of mulberry extract reaching the colon is greatly reduced, so that the mulberry extract has a certain limit in treatment of intestinal inflammation and UC. Rectal administration is a method of administration that delivers drugs through the anus into the intestinal canal for release to exert a therapeutic effect in order to treat systemic or local diseases. Related applications are mainly focused on analgesia, anesthesia, fever abatement and the like; or the medicine is directly contacted with colon mucous membrane, thereby playing the roles of anti-inflammatory, hemostasis and the like. Compared with oral administration, rectal administration avoids symptoms such as nausea, vomiting and gastrectasia caused by direct stimulation of the stomach by the medicine, the pain of the patient in oral administration of the traditional Chinese medicine can be relieved by rectal administration, and most of the medicine can be absorbed by intestinal mucosa and directly enter the blood circulation without passing through the liver, so that the hepatotoxicity and side effects of the medicine are reduced. Compared with injection administration, the rectal administration has higher safety and smaller side effect.

At present, the dosage forms for domestic and foreign rectal administration are mainly enemas, suppositories and gels. The enema needs medical staff to operate, and has long administration time, easy leakage of medicine and poor compliance; after the suppository is melted, the diffusion distribution range is limited, so that the effect of the medicine is insufficient; the foam agent has wider distribution range in intestinal tracts and is not easy to leak, but the problems of inconvenient use, complex administration device and quicker elimination exist; the gel has high viscosity, which makes administration difficult. The in-situ temperature-sensitive gel is a new dosage form with solution-gel transition property, is characterized in that the gel is liquid at room temperature, reduces the administration difficulty, can be widely distributed on the surface of a colorectal section mucous membrane, can realize rapid gelation and close adhesion to an administration part under the action of body temperature, has long retention time, can realize positioning targeting and continuous administration, and becomes a hot spot direction for researching new dosage forms for rectal administration.

Disclosure of Invention

The invention aims to provide a mulberry extract temperature-sensitive gel.

The mulberry extract thermosensitive gel provided by the invention is prepared from the following raw materials in percentage by mass: 16-18% of temperature sensitive material, 2.5-4% of temperature regulator, 0.3-0.7% of biological adhesive, 1-5% of mulberry extract and the balance of water.

Further, the temperature-sensitive material is poloxamer 407 and/or chitosan; the temperature regulator is poloxamer 188 and/or polyethylene glycol; the bioadhesive is one or more selected from hydroxypropyl methylcellulose, sodium alginate and carbomer.

Further, the mulberry extract temperature-sensitive gel is prepared from the following raw materials in percentage by mass: 16% -18% of poloxamer 407 (P407), 2.5% -4% of poloxamer 188 (P188), 0.3% -0.7% of hydroxypropyl methylcellulose (HPMC), 1% -5% of mulberry extract and the balance of water.

Furthermore, the mulberry extract temperature-sensitive gel is prepared from the following raw materials in percentage by mass: 17% -18% of poloxamer 407 (P407), 2.5% -2.8% of poloxamer 188 (P188), 0.65% -0.7% of hydroxypropyl methylcellulose (HPMC), 1% -5% of mulberry extract and the balance of water.

Optimally, the mulberry extract temperature-sensitive gel is prepared from the following raw materials in percentage by mass: 17.80% of poloxamer 407 (P407), 2.8% of poloxamer 188 (P188), 0.7% of hydroxypropyl methylcellulose (HPMC), 2% of mulberry extract and the balance of water.

Optionally, the mulberry extract temperature-sensitive gel also contains electrolyte, wherein the electrolyte is sodium salt; the concentration of the sodium salt is preferably 0.0125-0.2mol/L; the sodium salt is preferably NaCl and/or Na ₂CO₃.

In the raw materials for preparing the mulberry extract thermosensitive gel, P407 is a thermosensitive material, P188 is a thermosensitive regulator, and HPMC is a bioadhesive material.

The mulberry extract temperature-sensitive gel is liquid at room temperature, and is converted into a semi-solid state when reaching the gelation temperature, which is beneficial to rectal administration.

The gelation temperature of the mulberry extract thermosensitive gel is 32+/-0.5 ℃.

The second object of the present invention is to provide a method for preparing the above mulberry extract temperature-sensitive gel.

The preparation method of the mulberry extract temperature-sensitive gel provided by the invention adopts a cold dissolution method, and comprises the following steps: dissolving HPMC in water to disperse uniformly, sequentially adding prescribed amounts of P188, P407 and mulberry extract in ice water bath (0-4deg.C), stirring uniformly, and refrigerating in a refrigerator at 2-6deg.C for 12-24 hr to swell thoroughly to obtain clear and uniformly dispersed medicinal gel solution.

Further, the mulberry extract gel also contains an electrolyte, wherein the electrolyte is sodium salt; the concentration of the sodium salt is preferably 0.0125-0.2mol/L; the sodium salt is preferably NaCl and/or Na ₂CO₃.

In the above method, the electrolyte is added after the mulberry extract.

The third object of the present invention is to provide the use of the above mulberry extract temperature-sensitive gel.

The application of the mulberry extract thermosensitive gel provided by the invention is as described in any one of the following (a 1) to (a 4):

(a1) Application of mulberry extract thermosensitive gel in preparing products for treating intestinal inflammation;

(a2) Application of mulberry extract thermosensitive gel in preparing products for preventing intestinal inflammation;

(a3) Use of mulberry extract temperature-sensitive gel in treating intestinal inflammation;

(a4) An application of mulberry extract temperature-sensitive gel in preventing intestinal inflammation is provided.

In one embodiment of the present invention, the application of the mulberry extract temperature-sensitive gel provided by the present invention is as described in any one of the following (b 1) to (b 4):

(b1) Application of mulberry extract thermosensitive gel in preparing ulcerative colitis treatment products;

(b2) Application of mulberry extract thermosensitive gel in preparing ulcerative colitis prevention products;

(b3) The application of mulberry extract temperature-sensitive gel in treating ulcerative colitis;

(b4) An application of mulberry extract temperature-sensitive gel in preventing ulcerative colitis is provided.

The prevention and/or treatment of ulcerative colitis is embodied in at least one of the following:

1) Inhibiting colonic atrophy in patients with ulcerative colitis;

2) Inhibiting colonic edema in a patient suffering from ulcerative colitis;

3) Inhibit weight loss of patients with ulcerative colitis.

Preferably, the product is a medicament or pharmaceutical formulation.

In the invention, the mulberry extract is mulberry twig extract, mulberry bark extract and/or mulberry leaf extract.

In one embodiment of the present invention, the mulberry extract mainly comprises alkaloids, and also comprises polysaccharides, flavones and amino acids.

Preferably, the alkaloid comprises at least one of 1-deoxynojirimycin (1-deoxynojirimycin or DNJ), N-methyl-1-deoxynojirimycin (N-methly-1-deoxynojirimycin), fagomine or FAG, 3-epi-Fagomine (3-epi-Fagomine), 1,4-dideoxy-1, 4-imino-D-ribitol (1, 4-dideoxy-1,4-imino-D-arabinitol or DAB), calpain B2 (calysteginB 2), calystegine C1 (CALYSTEGINC 1), 2-oxo- (alpha-D-galactopyranosyl) -1-deoxynojirimycin (2-O- (. Alpha. -D-galactopyranosyl) -1-deoxynojirimycin), 6-oxo- (beta. -D-glucopyranose) -1-deoxynojirimycin (6-O- (. Beta. -D-glucoranosyl) -1-deoxynojirimycin), 1,4-dideoxy-1, 4-oxo-2- (beta. -D-glucopyranosyl) -1-oxamine (2-O- (. Beta. -D-34-glucopyranosyl) -1, 4-oxo-2- (beta. -D-glucopyranosyl) -1-O- (2-34-2-O) -. Beta. -glucopyranosyl) -1-O- (2-oxamine).

Wherein, preferably, the weight percentage of DNJ is not less than 50% (preferably 60-99%) of total alkaloids.

Preferably, the heavy metal content of the mulberry extract is not more than 10ppm.

Preferably, in the present invention, the mulberry extract contains an alkaloid in an amount of 3% by weight or more (optionally contains an alkaloid in an amount of 3 to 99% by weight, further optionally contains an alkaloid in an amount of 15 to 99% by weight, further preferably contains an alkaloid in an amount of 30 to 99% by weight, further optionally contains an alkaloid in an amount of 40 to 99% by weight, further optionally contains an alkaloid in an amount of 50 to 99% by weight, further optionally contains an alkaloid in an amount of 60 to 99% by weight, further optionally contains an alkaloid in an amount of 35 to 70% by weight, further optionally contains an alkaloid in an amount of 60 to 75% by weight) based on 100% by weight of the sum of the components of the mulberry extract;

And/or comprises not more than 70% by weight of polysaccharide (optionally comprises 0.2-70% by weight of polysaccharide, further optionally comprises 0.2-50% by weight of polysaccharide, further optionally comprises 0.2-35% by weight of polysaccharide, further optionally comprises 0.2-25% by weight of polysaccharide, further optionally comprises 0.2-23% by weight of polysaccharide, further optionally comprises 20-25% by weight of polysaccharide);

and/or, containing flavone in an amount of not more than 10% by weight (optionally containing flavone in an amount of 0-10% by weight, further optionally containing flavone in an amount of 0.05-5% by weight, further optionally containing flavone in an amount of 0-2% by weight, further optionally containing flavone in an amount of 0.05-2% by weight, further optionally containing flavone in an amount of 0.5-1.5% by weight, further optionally containing flavone in an amount of 0-1% by weight, further optionally containing flavone in an amount of 0.05-1% by weight);

And/or comprises not more than 50% by weight of amino acids (optionally comprises 0-30% by weight of amino acids, further optionally comprises 0-25% by weight of amino acids, further optionally comprises 0-20% by weight of amino acids, further optionally comprises 0-5% by weight of amino acids, further optionally comprises 3-25% by weight of amino acids or further optionally comprises 5-20% by weight of amino acids);

and/or the other components (preferably 0-25% by weight, further optional 0-20% by weight, further optional 0-15% by weight, further optional 0-11% by weight, further optional 2-20% by weight, further optional 4-8% by weight).

Wherein each component refers to all components in the plant extract including alkaloid, polysaccharide, flavone and amino acid, namely the plant extract contains other components besides alkaloid, polysaccharide, flavone and amino acid.

In an alternative embodiment of the present invention, the weight percentage of the sum of the components of the mulberry extract is 100%, and the weight percentage of the components of the mulberry extract is:

preferably, the weight percentage of each component in the mulberry extract is calculated as 100 percent of the sum of each component in the mulberry extract:

preferably, the weight percentage of each component in the mulberry extract is calculated as 100 percent of the sum of each component in the mulberry extract:

preferably, the weight percentage of each component in the mulberry extract is calculated as 100 percent of the sum of each component in the mulberry extract:

preferably, the weight percentage of each component in the mulberry extract is calculated as 100 percent of the sum of each component in the mulberry extract:

preferably, the weight percentage of each component in the mulberry extract is calculated as 100 percent of the sum of each component in the mulberry extract:

preferably, the weight percentage of each component in the mulberry extract is calculated as 100 percent of the sum of each component in the mulberry extract:

Further optionally, the weight percentage of each component in the mulberry extract is 100%, based on the total weight percentage of each component in the mulberry extract:

Further optionally, the weight percentage of each component in the mulberry extract is 100%, based on the total weight percentage of each component in the mulberry extract:

The weight content of each component can be any combination of the above values.

Preferably, the mulberry extract can be prepared by the method described in CN 110393738 a. In one embodiment, the preparation of the mulberry extract comprises the steps of: preparing Moraceae plant crude extract; optionally, separation by cationic and/or anionic resins; optionally, subjecting the resin effluent to an alcohol precipitation treatment and collecting the supernatant; and optionally concentrating and/or drying the supernatant.

Preferably, in the present invention, the preparation of the mulberry extract comprises the steps of: 1) Preparing Moraceae plant crude extract; 2) Separating the crude extract by cationic resin and/or optional anionic resin to obtain a resin effluent, optional step 3): carrying out alcohol precipitation treatment on the resin effluent liquid in the step 2), and collecting supernatant; 4) Concentrating and/or drying the supernatant. Optionally, the resin effluent of step 2) is concentrated and/or dried before being subjected to an alcohol precipitation treatment.

Preferably, the Moraceae plant is one or more of Morus alba (Morus multicaulis Perrott.), morus alba (Morus alba L.), morus guangdong (Morus atropurpurea Roxb), morus reevesii (Morusmizuho Hotta), morus longifolia (Morus wittiorum Hand Mazz.), morus longifolia (Morus LAEVIGATA WALL), morus nigra (Morus nigra Linn.), hua Sang (Morus cathayana hemsi.), morus gracilis (Morus serrata Roxb.), meng Sang (Morus mongolica Schneid.), morus alba (Morus bombycis Koidz.), morus Sichuan (Morus notabilis Schneid.), tang Guisang (Morus nigriformis Koidz.), morus yunnanensis (Morusyunnanensis Koidz.), morus australis Poir), morus alba (Morus mongolica (Burr.) Schneid var. Digra), morus She Sang, morus mori (Morus alba Variana) Dippel), morus alba, and a hybrid species selected from the above;

preferably, the Moraceae plant is one or more selected from Morus guangdong, morus alba, morus tenuifolia, morus alba or Morus alba, and the Morus alba is preferably Morus cantonensis 11, gui Sangyou, or Sang Teyou. The mulberry branch, mulberry leaf or mulberry bark may be used in the leaves, roots, branches, bark, buds, stems, fruits, etc.

In the present invention, the mulberry extract is preferably a mulberry branch extract, a mulberry bark extract, a mulberry leaf extract or a mixed extract thereof.

1) Preparing crude extract of plant

The plant may be subjected to crude extraction with a solvent such as alcohol water, an alkaline aqueous solution or an acidic aqueous solution, and preferably, the solvent used for the crude extraction is water.

During extraction, the plant is preferably crushed and then added into water for heat extraction, and the extraction time is preferably 0.5-3h each time, and the extraction times are 1-3 times.

In a preferred embodiment, the crushed plants may be fed into an extraction tank for extraction.

The more the solvent is added during extraction, the higher the extraction rate of the plant is, but the excessive solvent addition can increase the difficulty of subsequent separation and purification, preferably the solvent addition is 3-20 times, more preferably 4-15 times, the weight of the plant raw material, the plant extract can be obtained to the greatest extent, and the difficulty of subsequent treatment is increased without excessively increasing the volume of the solution.

The extraction can be performed by decoction, ultrasonic extraction or reflux extraction, preferably by decoction or reflux extraction; more preferably, extraction is carried out using a more sophisticated decoction method in industrial equipment.

Optionally, the extraction may be repeated and the extracts combined.

Preferably, the extract is filtered to remove insoluble matter to obtain a crude plant extract.

Optionally, the concentrated crude extract may also be subjected to an alcohol precipitation treatment prior to the resin separation treatment of step 2). And during the alcohol precipitation treatment, adding ethanol into the crude extract, stirring and uniformly mixing, stopping stirring and standing for a certain time to precipitate insoluble substances in the crude extract. Preferably, the volume to mass ratio of ethanol added to the plant raw material is 0.2-20 times, optionally 0.4-10 times, in terms of L/kg. Further preferably, the alcohol precipitation treatment is performed using an alcohol precipitation tank. Preferably, the stirring speed in the alcohol precipitation treatment is 10 to 600rpm, preferably 40 to 500rpm, and more preferably 80 to 400rpm or 300rpm.

2) Separation by cationic and optionally anionic resins

After the cation resin column is packed, the activation is carried out according to the sequence of acid solution washing, alkaline solution washing and acid solution washing. Preferably, the alkaline solution is washed to a pH of the eluate of 8.0-9.5, preferably 8.5-9.5; preferably, the alkaline solution is selected from an aqueous ammonia solution, a sodium hydroxide solution, a potassium hydroxide solution or a sodium carbonate solution; preferably, the concentration of the alkaline solution is 0.5 to 4mol/L, preferably 1 to 2mol/L. Preferably, the acidic solution is washed until the pH of the eluate is 3.0-7.0, preferably 3.5-6.5 or 4.5-6.5. Preferably, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, disodium hydrogen phosphate-citric acid buffer, preferably, the concentration of the acidic solution is 0.5-4mol/L, preferably 1.5-2mol/L. Optionally, the cationic resin may also be rinsed with 3-5 column volumes of deionized water after the last acidic solution wash.

Preferably, the cationic resin is selected from one or more of a strong acid cationic exchange resin, a weak acid cationic exchange resin and a strong base quaternary ammonium type cationic resin;

Preferably, the cation resin is one or a combination of 732 type strongly acidic styrene cation exchange resin, 002SC type strongly acidic styrene cation exchange resin, 734 type strongly acidic styrene cation exchange resin, D001 type macroporous strongly acidic styrene cation exchange resin or D113 type macroporous weak acid cation exchange resin and D254 type macroporous strong basic quaternary ammonium type cation exchange resin.

Preferably, the cation resins are 732 type strongly acidic styrenic cation exchange resins, 734 type strongly acidic styrenic cation exchange resins, and D001 type macroporous strongly acidic styrenic cation exchange resins.

Preferably, the cationic resin is used in an amount to plant raw material feed weight ratio of 1:1-30 (alternatively, 1:1-25, 1:2-20,1:2-15,1:2-10,1:2-7, 1:2-3).

And (3) loading the crude plant extract to the cationic resin, and eluting the loaded cationic resin by using an eluent. Preferably, the eluent is a salt solution or an alkaline solution containing cations, and one or more of sodium chloride, ammonia sulfate, ammonia nitrate, ammonia water, potassium chloride and sodium hydroxide can be selected.

Preferably, the concentration of the eluent is 0.04 to 5mol/L (preferably 0.5 to 2.5mol/L,0.2 to 3mol/L, more preferably 0.5 to 2.5 mol/L).

Preferably, the eluent flow rate is 1-15BV/h (preferably 5-10BV/h, more preferably 5-6 BV/h).

Preferably, the weight of the eluent used for separating the cationic resin is 0.1-30 times of the weight of the plant raw material, and preferably, the eluent is 0.5-10 times of the weight of the plant raw material.

The collection starting point may be determined according to the pH of the cation resin effluent, for example, when an alkaline solution such as ammonia water is selected for elution, the eluent is collected when the pH of the cation column effluent is detected to be >7 or the collection starting point of the effluent is determined according to a color development or precipitation reaction; preferably, the collection is stopped when the volume of the collected liquid reaches 0.1 to 10 times (further preferably, 0.2 to 5 times) the charged weight of the plant raw material, and the collected liquid is optionally purified by passing through an anion column.

In the case of purification by passing through an anion column, it is preferable that the anion resin is packed and then activated in the order of alkaline solution washing, acidic solution washing and alkaline solution washing.

Preferably, the alkaline solution is washed to a pH of the eluate of 8.0-9.5, preferably 8.5-9.5;

Preferably, the alkaline solution is selected from an aqueous ammonia solution, a sodium hydroxide solution, a potassium hydroxide solution or a sodium carbonate solution; preferably, the concentration of the alkaline solution is 0.5 to 4mol/L, preferably 1 to 2mol/L.

Preferably, the acidic solution is washed until the pH of the eluate is 3.0-7.0, optionally 3.5-6.5 or 4.5-6.5. Preferably, the acidic solution is selected from the group consisting of hydrochloric acid solution, phosphoric acid solution, disodium hydrogen phosphate-citric acid buffer, preferably, the concentration of the acidic solution is 0.5-4mol/L, preferably 1-2mol/L.

Preferably, the anion resin is one or a combination of a plurality of strong basic anion exchange resins, weak basic anion exchange resins or weak acidic anion exchange resins;

Preferably, the anion resin is one or a combination of 711 type strong basic styrene anion resin, 717 type strong basic styrene anion resin, D201 type macroporous strong basic styrene anion resin or D218 type macroporous strong basic acrylic anion resin, D301-G type macroporous weak acid type styrene anion resin and D301 type macroporous weak basic styrene anion resin.

Preferably, the anionic resin is used in an amount to plant raw material feed weight ratio of 1:1 to 80 (alternatively, 1:1 to 64,1:1 to 32,1:1 to 24,1:5 to 16, 1:3).

Collection begins when liquid flows out of the anionic resin. Preferably, the collection is stopped when the volume of the collection liquid reaches 0.05-10 times (optionally, 0.1-5 times) the weight of the charged plant material.

3) Concentrating the collecting liquid obtained in the step 2)

Further, before the alcohol precipitation treatment, the method further comprises the steps of centrifugally removing impurities from the anion resin effluent or filtering impurities by a microfiltration membrane, and then concentrating by a reverse ion permeable membrane. The specific gravity of the concentrated liquid may be 1.0 to 1.3, preferably 1.1 to 1.25.

4) Alcohol precipitation treatment

Preferably, the ethanol used for the alcohol precipitation treatment is fed in a weight ratio of 1:4 to 600 (preferably 1:20 to 300, more preferably 1:20 to 50,1:40,1:80, 1:22) to the plant raw material. In the alcohol precipitation treatment, the stirring speed is 10 to 600rpm (preferably 40 to 500rpm,80 to 400 rpm). The time of the alcohol precipitation treatment is 12-24 hours.

5) Concentrating and drying

Optionally, the extraction method further comprises: step 5) concentrating and drying.

Filtering the ethanol precipitation solution to remove insoluble substances, concentrating under reduced pressure to obtain plant extract or drying to obtain dried extract.

In one embodiment, the mulberry extract is prepared according to the following steps: pulverizing Moraceae plant, reflux-extracting with water and/or alcohol solution or acid water under heating with solvent amount 3-20 times that of the raw materials, repeatedly extracting for 1-3 times, mixing extractive solutions, concentrating, loading onto cation exchange resin, eluting with 0.2-3N ammonia water, concentrating and/or drying to obtain extract.

In one embodiment, the mulberry extract is prepared according to the following steps: pulverizing Moraceae plant, reflux extracting with water and/or alcohol solution or acid water under heating with solvent amount 3-20 times of that of the raw materials, repeatedly extracting for 1-3 times, mixing extractive solutions, concentrating, loading onto cation exchange resin, eluting with 0.2-3N ammonia water, loading onto anion exchange resin, collecting non-adsorbed fraction, concentrating and/or drying to obtain extract.

In one embodiment, the mulberry extract is prepared according to the following steps: pulverizing Moraceae plant, reflux extracting with water and/or alcohol solution or acid water under heating with solvent amount 3-20 times of that of the raw materials, repeatedly extracting for 1-3 times, mixing extractive solutions, concentrating, loading onto cation exchange resin, eluting with 0.2-3N ammonia water, loading onto anion exchange resin, collecting non-adsorbed part, adding ethanol, precipitating to remove impurities, concentrating and/or drying to obtain extract.

In one embodiment, the mulberry extract is prepared according to the following steps: pulverizing Moraceae plant, extracting with water under reflux under heating with solvent 3-20 times (preferably 4-15 times, further optionally 4-12 times) of the raw materials, repeatedly extracting for 1-3 times (preferably 0.5-3 hr each time, further preferably 1-2 hr each time), mixing extractive solutions, concentrating, loading onto cation exchange resin, eluting with 0.2-3N ammonia water, loading onto anion exchange resin, collecting non-adsorbed fraction (i.e. anion resin effluent), adding ethanol, precipitating to remove impurities, concentrating and/or drying to obtain extract.

The invention has the following beneficial effects:

the mulberry extract temperature-sensitive gel is prepared, the gelation temperature is used as an index, and a star point response surface method is adopted to optimize the prescription; the mulberry twig extract (SZ-a) temperature sensitive gel was characterized using fourier transform infrared spectrometer (FT-IR), differential Scanning Calorimetry (DSC), rheological properties, gel strength, pH, and moisture retention; taking purified water as a release medium to examine the in-vitro drug release rule; analyzing the targeting result of the temperature-sensitive gel in vivo through fluorescence imaging of the in vitro tissues; evaluating the safety of the temperature-sensitive gel in vivo by continuous seven days of rectal administration; and the treatment effect of the SZ-A temperature-sensitive gel on Ulcerative Colitis (UC) model rats induced by a TNBS/ethanol composite model is researched; and the cytotoxicity of the SZ-A temperature sensitive gel was analyzed from the in vitro level. Results: the mulberry extract temperature-sensitive gel is suitable for rectal administration; the in-vitro release accords with the Higuchi equation, and a certain slow release performance is reflected; the retention time of the fluorescent dye in the body can be remarkably prolonged; no apparent hyperemia and pathological changes were observed with rectal tissue administration for seven consecutive days, indicating that the formulation was non-irritating to the rectal mucosa; in-vitro and in-vivo experiments show that the mulberry extract temperature-sensitive gel has a certain curative effect in rectal administration and has no cytotoxicity in a concentration range.

The results show that the temperature-sensitive gel prepared by the invention can be rapidly gelled in a physiological state, has certain slow release performance, has no irritation to intestinal mucosa, can improve intestinal inflammation or UC symptoms, has a repairing effect on the intestinal mucosa, and has great application prospect.

Drawings

FIG. 1 is a flow chart of the preparation of the SZ-A temperature-sensitive gel of the invention;

FIG. 2 shows the effect of different temperature sensitive materials on gelation temperature; (a) a P407 dose trial, (b) a P188 dose trial, (c) an HPMC dose trial;

FIG. 3 is a two-dimensional effect surface graph and a three-dimensional contour graph of the influence of the addition amounts of P407 and P188 and HPMC on the gelation temperature of the temperature-sensitive gel;

FIG. 4 change in appearance of temperature sensitive gel;

FIG. 5 is a quality assessment of different prescriptions; (a) measuring the pH value of the temperature-sensitive gel; (b) Temperature-sensitive gel moisture retention rate measurement (c) temperature-sensitive gel strength measurement;

FIG. 6 is a graph of cumulative erosion rates and dissolution rates for different prescription drugs; (a) Cumulative erosion rate of blank gel (b) cumulative erosion rate of different prescription drugs; (c) cumulative dissolution rate of different prescription drugs;

FIG. 7 is a linear viscoelastic region survey of different prescriptions; (a) linear viscoelastic region investigation of prescription one; (b) linear viscoelastic region investigation of prescription two; (c) linear viscoelastic region investigation of prescription three;

FIG. 8 is a frequency sweep for different prescriptions; (a) frequency sweep of prescription one; (b) frequency sweep of prescription two; (c) frequency sweep of recipe three;

FIG. 9 is a diagram of viscosity measurements for different formulations; (a) viscosity measurement after different prescriptions are glued; (b) viscosity measurement before gel formation of different prescriptions;

FIG. 10 shows the pH and gel temperature of different drug-loaded gels (a) pH of different drug-loaded gels (b) gel temperature of different drug-loaded gels;

FIG. 11 is a linear viscoelastic area scan of gels of different drug loads;

FIG. 12 is a frequency sweep of different drug loading gels;

FIG. 13 is a linear viscoelastic area scan, frequency and viscometry for a 2% drug loading gel; (a) linear viscoelastic zone scanning of 2% gel drug loading (b) frequency scanning of 2% sz-a gel (c) viscometry of 2% sz-a gel at 4 ℃ (d) viscometry of 2% sz-a gel at 37 ℃;

FIG. 14 is an effect of electrolyte on temperature sensitive gel viscosity;

FIG. 15 is a FT-IR spectrum of SZ-A, blank gel, SZ-A temperature sensitive gel;

FIG. 16 is a DSC spectrum of SZ-A, blank gel, SZ-A temperature sensitive gel;

FIG. 17 shows colon imaging results in rats;

FIG. 18 shows the fluorescence intensities of IR-820 (oral), IR-820 (rectal) and IR-820 (hydrogel group) at 0.5h, 1h, 3h and 5 h; ig is oral, CS is enema;

FIG. 19 shows HE staining results;

FIG. 20 shows the levels of IL-1. Beta. And IL-6 cytokines in blood;

FIG. 21 shows the levels of MPO, IL-6, IL-1. Beta. Cytokines in colon tissue;

FIG. 22 is a graph showing the change in body weight of rats in different groups;

FIG. 23 is a graph showing colon length for different groups of rats;

FIG. 24 shows the colon length trend for different groups of rats;

FIG. 25 shows the effect of (a) different concentrations of SZ-A gel extract on cell viability; (b) effect of different concentrations of SZ-a on cell viability;

FIG. 26 is a graph showing the effect of SZ-A on cell viability as observed by staining live dead cells.

Detailed Description

The present invention will be described in further detail by way of examples. The features and advantages of the present invention will become more apparent from the exemplary description. The present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The starting materials are available from published commercial sources unless otherwise specified.

The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

In addition, the technical features described below in the different embodiments of the present invention may be combined with each other as long as they do not collide with each other.

The materials and animals involved in the following examples are as follows:

Poloxamer 407 (P407) and poloxamer 188 (P188) were purchased from BASF, germany; hydroxypropyl methylcellulose is available from DOW in the united states; fluorenylmethyl 9-chloroformate was purchased from Sigma; TNBS was purchased from Shanghai Jizhui Biochemical technologies Co., ltd; CCK-8 kit was purchased from Beijing Sophora; new indocyanine green was purchased from microphone; rat IL-1 beta, IL-6, MPO were purchased from Huamei organism CUSABIO; the sulfasalazine enteric coated tablet is purchased from Shanghai Yi balance pharmaceutical Co., ltd; male SD rats weighing 180-220g were purchased from Beijing Wallkukangjie Biotech Co., ltd, and all animal experiments were approved by the animal laboratory ethical committee.

Analysis of data in the following examples: all data subjected to statistical analysis were obtained from at least three parallel experiments, data were processed and analyzed using GRAPHPAD PRISM 8.0.2 software, where quantitative data were expressed as averages, group comparisons were made using one-way anova, and at p <0.05, the average differences were considered statistically significant.

The mulberry extract used in the following examples was prepared as follows (preparation 1 of mulberry extract of example 1 in CN 113143997A):

1000kg of fresh mulberry twig (number Sang Yuesang of fine teeth) is taken, crushed, 4000L of water is added, the heating reflux method is used for extracting for 2 hours, the extracting solutions are combined, and insoluble matters are removed by filtration, so that crude extracting solution is obtained. The crude extract is thermally concentrated until the solid mass percentage content reaches 4%, and the crude extract is used as the sample loading liquid of the cation resin column after heat preservation at 50 ℃.

150Kg of D113 macroporous weak acid styrene cationic resin is used for filling a column, and 2mol/L hydrochloric acid solution is used for washing until the pH value of an eluate is 4.5; washing with 1mol/L sodium hydroxide solution until the pH of the eluate is 8.5; washing with 2mol/L hydrochloric acid solution until the pH of the eluate is 4.5; and then washing with deionized water with the volume of 5 times of the column volume to complete activation. Loading the concentrated extractive solution, eluting with 1000L of 2.5mol/L ammonia water at eluting speed of 6BV/h, collecting eluate when pH of the cation column effluent is detected to be >7, stopping collecting when the collected solution reaches 900L, and purifying the collected solution directly by anion column.

62.5Kg of D218 macroporous strong-base acrylic anionic resin is used for filling a column, and 1.5mol/L sodium hydroxide solution is used for washing until the pH value of an eluate is 9.0; washing with 1.5mol/L hydrochloric acid solution until the pH of the eluate is 3.5; washing with 1.5mol/L sodium hydroxide solution until the pH of the eluate is 9.0; the activation is completed. And loading the collected cation resin eluent to anion resin, and collecting effluent until the effluent reaches 870L.

And (3) centrifuging the collected liquid to remove impurities, concentrating the collected liquid by a reverse ion osmosis membrane, transferring the concentrated liquid into an alcohol precipitation tank with the specific gravity of 1.25, and adding 25L of absolute ethyl alcohol under the stirring paddle of 500 rpm. Stopping stirring after ethanol addition is completed, precipitating with ethanol for 24 hr, collecting supernatant, and concentrating under reduced pressure to obtain extract.

Concentrating the effluent liquid under reduced pressure to obtain extract (SZ-A) of ramulus Mori, wherein the alkaloid content is 52% (the content of DNJ in alkaloid is 69.5%, DAB content is 11.5%, FAG content is 15%), polysaccharide content is 22%, flavone content is 0.8%, and amino acid content is 20%.

Example 1 prescription screening and preparation of SZ-A temperature sensitive gel

Preparation of 1SZ-A temperature-sensitive gel

1.1 Preparation method

The SZ-A temperature-sensitive in-situ gel is prepared by adopting a cold dissolution method, as shown in figure 1, HPMC is dissolved in a small amount of water to be uniformly dispersed, P188 and P407 with different concentrations and the mulberry extract are sequentially added in ice water bath, and the mixture is stirred uniformly and then is placed in a refrigerator at 4 ℃ for refrigeration for 24 hours, so that the mixture is fully swelled, and a clear and uniformly dispersed medicine gel solution is obtained.

1.2 Method for determining the gel temperature (Tgel)

The measurement was performed by the tilt method.

1.3 Gel time determination

2ML of gel is measured and placed in a test tube, the gel is placed in a constant temperature water bath kettle with the temperature being controllable to about 37 ℃, and after the solution is gelled, the gelation time is recorded.

1.4 Prescription optimization

1.4.1 Single factor investigation

P407 (16%, 17%, 18%, 19%, 20%), P188 (1.0%, 2.5%, 3.5%, 4.0%, 5.0%) and HPMC (0.3%, 0.5%, 0.8%, 1.0%, 1.2%) were single-factor examined with Tgel and gel time as examination indexes, and the fixed drug loading amounts were all 2% based on the mulberry extract.

Results: p407 is taken as a temperature sensitive material, P188 is fixed to be 2% (w/w), HPMC is fixed to be 0.7% (w/w), temperature sensitive gels with different mass concentrations of P407 are respectively prepared, the gelation temperature is measured, and the influence of P407 on the gelation temperature is shown as a in figure 2. The results show that as the concentration of P407 increases, the gelation temperature decreases, and as the concentration of P407 increases to 19%, the gelation temperature decreases to 28 ℃, and the gel can be converted into gel at a lower temperature. Therefore, the dosage range of P407 is 16-18% to be optimized; to the prepared 18% P407 and 0.7% HPMC solutions, P188 was added at different mass concentrations to prepare gel solutions, and the effect of P188 on the gelation temperature was shown in FIG. 2 b. The results show that as the concentration of P188 increases, the gelation temperature increases, when the concentration of P188 is as low as 1%, the gelation temperature is as low as 28 ℃, the gel can be converted into gel at a lower temperature, when the concentration reaches 5%, the gelation temperature increases to 36 ℃, and the temperature is too high, so that the dosage range of P188 is selected to be 2.5% -4% for optimization. To the prepared 18% P407 and 2% P188 solutions, HPMC was added at different mass concentrations to prepare gel solutions, and the results are shown in FIG. 2 c. The results showed that the gelation temperature decreased with increasing HPMC concentration, with HPMC usage in the range of 0.3% -0.7% being selected. The factor levels are shown in table 1.

TABLE 1 factors and level design Table

1.4.2Box-Behnken response surface method optimization prescription

Through single factor investigation, in order to further determine the influence of the proportion of P407, P188 and HPMC on the gelation temperature of the temperature-sensitive gel, the gelation temperature is taken as an investigation index, the dosage of P407 (A), the dosage of P188 (B) and the dosage of HPMC (C) are taken as investigation factors, the optimization range of the three is determined, the temperature-sensitive gel prescription is optimized through a three-factor three-level star point design experiment, and finally the optimal proportion is determined.

1.4.2.1 Experimental design

According to the single factor experimental result, the dosage of P407, P188 and HPMC in the prescription is taken as investigation factors, the optimization ranges of the dosage, the P407, the P188 and the HPMC are respectively 16% -18%, 2.5% -4% and 0.3% -0.7%, the 3-factor 3 horizontal star point design experiment is used for optimizing the temperature-sensitive gel prescription, the gelation temperature (T) is taken as a response variable, and the experimental design and the gelation temperature measurement result are shown in Table 2.

Table 2 experimental design results and gel temperature determination

1.4.2.2 Model fitting

Model fitting was performed on experimental data using Design Expert 8.0.6 software, and on the gelation temperature and the respective variables as

T＝35.26-3.22×A+1.01×B-0.89×C+0.67×A×B-0.22×A×C+0.000×B×C-0.61×A²-0.88B²+0.17C²(R²＝0.9839,p<0.0001). As can be seen from the equation, the gelation temperature decreases significantly with increasing P407 concentration and increases with increasing P188, HPMC concentration within the range of the assay. The model P <0.0001 indicated a very significant difference. The mismatch term p=0.1938 >0.05, without significance, indicates that the equation fitting degree and the reliability are good. The three-dimensional effect surface graph and the two-dimensional contour graph of the experimental result are drawn by using software and are shown in fig. 3.

1.4.2.3 Analysis of variance and significance test

The analysis results of ANOVA are obtained by the significance test of each regression coefficient of the table 3, and P407, P188 and HPMC in the model equation have significant influence on the gelation temperature.

TABLE 3 response surface analysis of variance results

1.4.2.4 Optimization process verification

The accuracy of the optimization method was verified and evaluated by analyzing the results and substituting Tg at 32±0.5 ℃ into the simulation equation, selecting three representative prescriptions among the optimized prescriptions, and determining the actual gelation temperature, and the experimental results are shown in table 4. The result shows that the deviation between the measured value and the predicted value of the gelation temperature of each prescription is within 5%, which shows that the predictability of the fitting equation is good.

Table 4 recipe verification and optimization results

2. Gel preparation using HPMC as gel matrix

The prescription ratio is as follows: HPMC 1% (w/w), glycerol 10% (w/w), water in balance, and drug loading rate of 2%.

The preparation method comprises the following steps: 1% HPMC is measured, 10% glycerol is used for wetting, a proper amount of water is added for stirring to fully swell, 2% SZ-A is taken for dissolving with a proper amount of water, and then the solution is slowly added into the blank matrix, and the solution is stirred while being clear and transparent, and the total amount of the solution is regulated to be 10g.

Experimental results:

Appearance color: the gel has a flat surface and brown color

Uniformity: the gel has a small particle size, is less round and has poor transparency

Viscosity: the gel is relatively viscous and has moderate fluidity, which is unfavorable for rectal injectable administration.

3. Gel preparation using carbomer 980 as gel matrix

CP 980% (w/w), glycerol 1% (w/w), the remainder being water, the drug loading was 2%.

CP 980.9% (w/w), glycerol 1% (w/w), the remainder being water, the drug loading was 2%.

CP 980.8% (w/w), glycerol 1% (w/w), the remainder being water, the drug loading was 2%.

The preparation method comprises the following steps: and spreading carbomer-980 with different mass concentrations of 1%, 0.9% and 0.8% on the surface of 10% of glycerol respectively, fully wetting the glycerol, adding 2% of SZ-A, and stirring uniformly until the total amount reaches 10 g.

Experimental results: the gel prepared by the three proportions is sticky and poor in fluidity, and is unfavorable for rectal injectable administration.

EXAMPLE 2 quality evaluation of SZ-A temperature sensitive gel

1 Evaluation of appearance

And observing the fluidity of the temperature-sensitive gel after the room temperature and the gelation temperature are reached, and visually observing whether the temperature-sensitive gel is uniformly clarified.

At room temperature, the blank sol is a liquid, clear and transparent solution with good fluidity. The drug-loaded sol was a yellow-brown uniformly dispersed solution with good flowability reaching LCST (temperature >32 ℃), the three prescription drug-loaded gels in table 4 turned into yellow-brown solids with good morphology and no flow, and the experimental results are shown in fig. 4.

2PH measurement

The pH of the temperature-sensitive in-situ gel was directly measured in triplicate (see table 4) using a pH meter to investigate whether the pH met rectal injection requirements.

The results are shown in FIG. 5a, which shows that the pH of the SZ-A temperature-sensitive in-situ gel can reach the rectal injection requirement (4-9).

3 Measurement of moisture retention

Taking three prepared prescriptions, uniformly placing the prescriptions in an open centrifuge tube, placing the prescriptions in a constant-temperature airtight dryer at 37 ℃, weighing the mass (m ₁) of the prescriptions before drying, taking out the prescriptions after 24 hours, weighing the prescriptions to be dried to constant mass (m ₂), and evaluating the moisture retention of the three thermosensitive gels according to a formula m ₂/m₁.

As shown in fig. 5 b, the results of the experiments show that the moisturizing rates of the three prescriptions in table 4 can reach more than 90%, which proves that the gel has better moisturizing property.

4 Gel strength determination

15ML of gel was placed in a graduated cylinder and stabilized at 37℃for 30min, and then a weight was placed on the gel, the gel strength being determined by the time required for the weight (10 g) to sink a certain distance through the gel in the graduated cylinder.

The greater the gel strength, the longer the residence time at the site of administration; the smaller the gel strength, the faster the drug release rate. The results are shown in fig. 5 c, with moderate gel strength and no significant differences for the three formulas in table 4.

Example 3 evaluation of in vitro drug release Properties of SZ-A temperature sensitive gel

The content of 1-deoxynojirimycin in the test sample was measured according to the method in CN 111077247A.

TABLE 5 gradient elution procedure

2 Release degree measurement

By adopting a weight loss method, distilled water is used as an erosion release medium, 2.0g of each of the temperature-sensitive gels of the three prescriptions in table 4 are weighed into a glass bottle, and are sealed and placed into a constant temperature water bath oscillator at 37 ℃. After complete gelation, 10mL of distilled water at constant temperature of 37 ℃ is added respectively, and the mixture is oscillated reciprocally at a frequency of 100 r/min. And wiping the inner surface and the outer surface of the glass bottle at 0.5h, 1h, 1.5h, 2h, 3h, 3.5h, 4.5h, 6h and 6.5h, rapidly weighing, supplementing 10mL of distilled water for oscillation, and plotting the accumulated gel erosion rate against the sampling time to obtain a gel erosion curve over time. The content of the released release medium was measured by HPLC, and the cumulative drug release percentage was plotted against the sampling time to obtain a gel drug release profile.

3 Cumulative drug release results

The cumulative erosion rate of the blank gel is shown in fig. 6 a, the cumulative erosion rate of the drug-loaded gel is shown in fig. 6 b, and the cumulative drug release rate is shown in fig. 6 c, and the dissolution curves of the three prescriptions (see table 4) are fitted by using the origin pro software with zero order, first order, and Higuchi distribution models, respectively. The fitting results are shown in Table 6, and the results indicate that the formulations fit well with Higuchi, indicating that SZ-A can be released from the gel. T50 and Td respectively show the time required for dissolution of 50% and 63.2%, see Table 7, T50 is above 3h, and shows a certain slow release performance, and the result shows that the dissolution difference among the prescriptions is not large, and the cumulative dissolution rate of blank gel without medicine is slightly increased to a certain extent.

TABLE 6 model fitting results

Table 7 Higuchi equation fitting results

Example 4 rheological characterization of different prescriptions of SZ-A temperature sensitive gel

Unlike conventional gels, temperature sensitive gels have two rheological properties, viscous liquid, represented by the rheological parameter viscous modulus G ", and elastic solids, represented by the rheological parameter elastic modulus G'. The viscoelasticity interval of the sample was measured by amplitude sweep (strain 0.1% -100% and frequency 1 Hz) using Kinexus Lab ⁺ rotary rheometer, the frequency sweep range was 0.1Hz-10Hz and strain 0.5%, and the pre-gel (25 ℃) and post-gel (37 ℃) viscosities of the different formulations were measured simultaneously.

The maximum deformation which can be borne by the broken internal three-dimensional network structure of the gel can be obtained through the test result of the gel linear viscoelastic region (LVER), the amplitude scanning can be used for representing the stability of the gel structure, the gel is not broken when G' is not changed, and all the measurement needs to be carried out in the linear viscoelastic region. The measurement results of each prescription (see Table 4) are shown in FIG. 7, which shows that the linear viscoelastic regions of the three prescriptions are not significantly changed; frequency scanning is intended to describe the time-dependent behavior of a sample over a non-destructive deformation range, high frequency meaning rapid movement over a shorter time scale, and low frequency meaning slow movement or stationary state over a longer time scale, useful for characterizing the long-term stability of the gel internal structure. The experimental results are shown in fig. 8, and the internal structure of each prescription gel is relatively stable, while the prescription 1G' is higher, which indicates that the elastic modulus is higher; and simultaneously measuring the viscosity after the glue formation and the viscosity before the glue formation of different prescriptions to prepare and select the optimal prescription, wherein the experimental result is shown in figure 9. As shown by experimental results, the linear viscoelastic region among different prescriptions is not changed greatly, and the G' of prescription 1 is larger and the elasticity is better; and the viscosity after gel formation is larger, the viscosity before gel formation is moderate, and rectal administration is more facilitated.

Example 5 SZ-A temperature sensitive gel characterization

1 Drug loading investigation

1.1 Investigation of the influence of drug-carrying quantity on temperature-sensitive in-situ gel Property

Using formulation 1 in Table 4, the drug loading was examined and 1%, 2%, 3%, 4%, 5%, 7%, 9% of the drug loading was prepared as described above, and the appearance, centrifugal stability, pH and Tg were compared with the mulberry extract.

The pH and Tg of the temperature sensitive gel were compared for different drug loading amounts, and the results are shown in FIG. 10. From the graph, the pH of the temperature-sensitive in-situ gel tends to increase as the drug loading rate increases; when the drug loading is too large, the gelation temperature of the temperature-sensitive in-situ gel starts to drop, probably because the SZ-A has certain viscosity, so that the gelation temperature is reduced. And after the drug loading of the gel exceeds 7%, the temperature-sensitive gel has a small amount of sediment in appearance and is non-uniform and transparent.

1.2 Investigation of the influence of drug-loading on the gel viscoelastic region and frequency sweep Curve

By using Kinexus Lab ⁺ rotary rheometer, setting strain to 0.1% -100% by amplitude scanning, setting strain to 1% by frequency scanning, and setting frequency scanning range to 0.1-10 Hz.

The linear viscoelastic regions for different drug loads are shown in fig. 11, and the frequency sweep is shown in fig. 12. The graph shows that after medicine loading, the G' is relatively stable through the inspection of a linear viscoelastic region, and the gel structure is proved to be not destroyed; through frequency scanning, when the gel drug loading rate exceeds 5%, the gel structure is not stable, the fluctuation of G' modulus is large along with the frequency change, and the gel stability is poor; after the gel content exceeds 7%, the gel distribution is not uniform, and the drug loading is controlled below 5% by comprehensive consideration.

1.3 2% SZ-A temperature-sensitive gel linear viscoelastic region, frequency and viscosity investigation

According to the method, the gel drug loading rate is controlled to be 2%, and the linear viscoelastic region, the frequency scanning, the viscosity at 4 ℃ and the viscosity after gel formation are measured.

The results are shown in FIG. 13. From the experimental results, the SZ-A temperature-sensitive gel is a Newtonian fluid at 4 ℃, and is converted into a non-Newtonian fluid at 37 ℃ with the characteristic of shear thinning. The SZ-A temperature-sensitive gel has a wide linear viscoelastic region, stable internal structure of the gel and no obvious frequency dependence.

1.4 Effect of electrolyte addition on temperature sensitive gel viscosity

SZ-a temperature sensitive gels (2% drug loading based on mulberry extract) loaded with 0.0125, 0.025, 0.05, 0.1, 0.2mol/L NaCl and 0.0125, 0.025, 0.05, 0.1, 0.2mol/L Na ₂CO₃ were prepared, and the viscosity trend was determined as described above with respect to the electrolyte concentration.

The gel loaded with NaCl and Na ₂CO₃ with different concentrations is prepared, the electrolyte is added after the mulberry extract, the result is shown in figure 14, the viscosity of the temperature-sensitive gel gradually rises along with the increase of the concentration of the electrolyte, and the addition of the electrolyte can increase the viscosity of the temperature-sensitive gel to a certain extent, so that the scouring force of rectal peristalsis and diarrhea is better resisted.

2 FT-IR characterization

Mixing SZ-A, blank gel and SZ-A temperature sensitive gel (with drug loading of 2% based on ramulus Mori extract) with KBr, tabletting, measuring infrared spectrum of sample with Fourier transform infrared spectrometer, and setting wave number range to 4000-400cm ^-1.

The experimental results are shown in fig. 15, and no new absorption peak appears after drug loading, indicating that no new substance is generated.

3 DSC characterization

Weighing 3-5mg of SZ-A, SZ-A temperature-sensitive gel and blank gel which are dried to constant weight. Sealing the samples in aluminum discs respectively, taking a blank aluminum disc as a reference, setting the heating speed to be 10 ℃/min under the constant nitrogen flow rate (50 mL/min), and recording the heating curve of each sample when the heating range is 15-200 ℃.

The results of the experiment are shown in FIG. 16, which illustrates the interaction between the drug and the matrix.

Example 6 in vivo targeting study of SZ-A temperature sensitive gel

36 SPF-class male rats are taken, the weight of the SPF-class male rats is about 180g, the 36 rats are randomly divided into three groups, namely, an SZ-A temperature-sensitive gel rectal administration group added with new indocyanine green (IR-820), an SZ-A solution (purified water) common enema group added with IR-820 and an SZ-A solution (purified water) oral group added with IR-820, the preparation concentration of the IR-820 is 200 mug/g, and the dose of the IR-820 given to each rat is 0.25mg/kg. Four time periods of 0.5h, 1h, 3h and 5h are set for each group of rats, three rats are dissected in each time period on average, the rats are anesthetized by isoflurane gas before administration, and colon tissues of the rats are taken after dissection for in-vitro imaging observation.

The SZ-A temperature sensitive gel used in this example was prescription 1 in Table 4 and the drug loading was 2%.

The concentration of the mulberry extract (SZ-A) in the SZ-A solution (purified water) used in this example was 2%.

By dissecting the rat at different time points and observing in vitro imaging, the experimental results are shown in fig. 17, and the distribution and retention conditions of the oral administration group, the common enema group and the temperature-sensitive gel group in the colon of the rat can be clearly observed through fluorescence display, wherein the fluorescence signals of the oral administration group and the common enema group are weaker than those of the temperature-sensitive gel group; the experimental results show that the temperature-sensitive gel group has obvious differences compared with the oral administration group and the free SZ-A solution group in each time period, and as shown in figure 18, the temperature-sensitive gel can be proved to prolong the local residence time of the new indocyanine green.

EXAMPLE 7 evaluation of safety of SZ-A temperature sensitive gel

SD male rats weighing 200-250g are randomly divided into three groups of four, wherein SZ-A in-situ thermosensitive gel is given to group 1, physiological saline is given to group 2, and blank control is given to group 3. The SZ-A temperature-sensitive gel (same as the SZ-A temperature-sensitive gel used in example 6) and physiological saline were administered in a volume of 2mL/kg, rectally, 1 time a day, and continuously for 7 days. After 24 hours of administration on the seventh day, the rats were sacrificed, the rectum was dissected and removed, and first, whether the rectal tissue had red swelling, mucosal damage, bleeding and other pathological changes was visually observed. Taking out the rectum, washing the rectum tissue with normal saline, immersing in 4% paraformaldehyde fixing solution, embedding and fixing paraffin, slicing the tissue, staining HE, performing histopathological examination by an optical microscope, and evaluating the irritation of SZ-A temperature-sensitive gel to the rectum mucous membrane. Simultaneously, blood and colon were taken and relevant inflammatory cytokines were measured.

After 7 days of continuous stimulation of rectal administration, the rectum is dissected out, and no pathological changes such as red swelling, mucous membrane breakage and the like of rectal tissues are observed, and the pathological detection result is shown in fig. 19. The experimental results show that intestinal tissues among groups have complete colonic mucosa epithelium, clear structure and regular gland arrangement, and no obvious lesions are found. Meanwhile, the levels of the plasma and tissue related inflammatory factors are measured, and the results shown in figures 20 and 21 show that p is more than 0.05, no obvious difference exists among groups, and the SZ-A temperature-sensitive gel has no obvious mucous membrane irritation and higher safety in vivo.

EXAMPLE 8 in vivo pharmacodynamic evaluation of SZ-A temperature-sensitive gel

36 SPF-grade male SD rats weighing 200g-220g were randomly divided into 6 groups: control group (Control), model group (Model), positive SASP group (SASP), SZ-A oral administration group (SZ-A-Po), SZ-A temperature-sensitive gel rectal administration low dose group (SZ-A-L), SZ-A temperature-sensitive gel rectal administration high dose group (SZ-A-H) (SZ-A temperature-sensitive gel is the same as the prescription used in example 6), 6 rats in each group except for normal Control group all adopt TNBS/ethanol solution modeling method: after weighing, the isoflurane anesthetizing rats, lightly inserting a 12-gauge stomach filling needle into a position about 8cm away from the anus of the rats, uniformly mixing TNBS (5% TNBS and absolute ethyl alcohol according to the volume ratio of 1:1) according to the concentration of 25mg/kg TNBS, wherein the final concentration of TNBS is 2.5%, the administration volume of the rats is 0.1mL/100 g), continuously injecting air about 0.4mL after the filling, normally feeding the rats after the molding agent is fed, taking normal saline with the same volume as a contrast after the anesthesia, performing drug intervention after the molding 1d, continuously feeding 10d and 7d of the rest rats except the normal control group and the model group, again feeding TNBS/ethanol 0.1mL/200g, wherein during the administration period, the control group and the model group adopt physiological saline to perform stomach 3mL/200g, the administration dosage of the positive drug group is 350mg/kg, the SZ-A oral group (the administration concentration is 68mg/15mL, the administration dosage is 34mg/kg of alkaloid, and the rectal dose is 34 mg/34 mg of alkaloid, and the biological gel is high in the dosage and 17 kg of the biological agent is respectively; after 10d, rats were fasted, the abdominal aorta was sacrificed after blood was taken, colon tissue was taken and the length of the colon was measured, and the colon tissue was saved for later analysis.

1 Influence on the physiological State of rats

All groups of rats have no death condition, after the experimental group rats are modeled, the rats have symptoms of reduced activity, reduced appetite, listlessness, severe and even hematochezia, and after two days, the rats have black stool, and the stool is flat and oval, and some rats last until the day of material collection. The normal control group has normal appetite, no obvious change of stool and slightly more water to eat after modeling; the weight of each group of rats before molding is not significantly different through statistical analysis, as shown in fig. 22, the weight of the rats in the blank group is continuously increased after molding, and compared with the blank group, the weight of each other group is slowly increased, each administration group has the tendency of improving the weight of the rats, and the effect of improving the weight of the rats in the SZ-A temperature sensitive gel low-dose and high-dose rectal administration groups is equivalent to that of positive drugs.

Colon length change in rats of group 2

Compared with the control group, the model group has the changes of colon atrophy and edema, as shown in fig. 23, and the model group prompts that colonitis modeling is successful, and after the SZ-A temperature-sensitive gel rectal administration group is treated, colon atrophy caused by ulcerative colitis can be relieved, as shown in fig. 24, and the effect is equivalent to that of a positive control drug. The results show that the temperature-sensitive gel provided by the invention has certain advantages and certain curative effect in rectal administration.

Example 9, in vitro cytological experiments with SZ-A temperature sensitive gels

1 Cell culture

Macrophage Raw264.7 is cultured in a high-sugar DMEM medium containing 10% of fetal calf serum, 1% of streptomycin and penicillin in a cell culture box containing 5% of CO ₂ at 37 ℃ and is subjected to periodic liquid exchange and passage, and logarithmic phase cells are taken for experiment.

2 CCK-8 method for determining proliferation capacity of Raw264.7 cells

The 96-well cell culture plates are plated, six compound wells are arranged in each group, the cell density is adjusted to be 2 x 10 ⁵/mL, the SZ-A concentration is set to be 25, 50, 100 and 200 mug/mL, and the influence of the drugs on the proliferation capacity of macrophages is qualitatively analyzed through cell death staining Calcein-AM/PI.

The effect of different concentrations of SZ-a gel extract on cell proliferation was also determined. Preparation of SZ-A gel leaching solution: table 4 based on prescription 1 drug loading 1%, 2%, 3%, 4% temperature sensitive gel solution 1mL placed in 37 ℃ constant temperature shaking table until gel is completely formed, 10mL DMEM medium containing 10% FBS is added, after incubation for 24h at 37 ℃ 100% extract is obtained, after co-culture with cells for 24h, the effect of different concentrations of SZ-A extract on cell proliferation capacity is measured by CCK-8 kit.

The effect of SZ-a gel extract at different concentrations on cell viability is shown in fig. 25 a, the effect of SZ-a gel at different concentrations on cell viability is shown in fig. 25b, and the result of cell death staining is shown in fig. 26. From the experimental results, compared with the normal group, the medicines in each dose group have no obvious effect on the proliferation of Raw264.7 cells.

Claims (10)

1. The mulberry extract thermosensitive gel is prepared from the following raw materials in percentage by mass: 16-18% of temperature sensitive material, 2.5-4% of temperature regulator, 0.3-0.7% of biological adhesive, 1-5% of mulberry extract and the balance of water.

2. The mulberry extract temperature-sensitive gel of claim 1, wherein: the temperature-sensitive material is poloxamer 407 and/or chitosan; the temperature regulator is poloxamer 188 and/or polyethylene glycol; the biological adhesive is one or more selected from hydroxypropyl methylcellulose, sodium alginate and carbomer;

Or, the mulberry extract is selected from one or more of mulberry twig extract, mulberry bark extract and mulberry leaf extract.

3. The mulberry extract temperature-sensitive gel of claim 1 or 2, wherein:

The mulberry extract temperature-sensitive gel is prepared from the following raw materials in percentage by mass: 40716-18% of poloxamer, 2.5-4% of poloxamer 188, 0.3-0.7% of hydroxypropyl methyl cellulose, 1-5% of mulberry extract and the balance of water;

Preferably, the mulberry extract temperature-sensitive gel is prepared from the following raw materials in percentage by mass: 40717-18% of poloxamer, 2.5-2.8% of poloxamer 188, 0.65-0.7% of hydroxypropyl methyl cellulose, 1-5% of mulberry extract and the balance of water;

More preferably, the mulberry extract temperature-sensitive gel is prepared from the following raw materials in percentage by mass: poloxamer 407.80%, poloxamer 188.8%, hydroxypropyl methylcellulose 0.7%, mulberry extract 2% and the balance of water.

4. A mulberry extract temperature sensitive gel as claimed in any one of claims 1 to 3 wherein: the mulberry extract temperature-sensitive gel also contains electrolyte; the electrolyte is preferably a sodium salt; and/or, the concentration of the sodium salt is preferably 0.0125-0.2mol/L; and/or, the sodium salt is preferably NaCl and/or Na ₂CO₃.

5. The mulberry extract thermosensitive gel according to any one of claims 1 to 4, wherein: the mulberry extract temperature-sensitive gel is liquid at room temperature, and is converted into semi-solid when reaching the gelation temperature.

6. The mulberry extract thermosensitive gel according to any one of claims 1 to 5, wherein: the mulberry extract temperature-sensitive gel is used for rectal administration.

7. A method for preparing a mulberry extract temperature-sensitive gel as claimed in any one of claims 3 to 6, prepared by a cold dissolution method, comprising the steps of: dissolving hydroxypropyl methylcellulose in water to disperse uniformly, sequentially adding 2.5% -4% poloxamer 188, 16% -18% poloxamer 407 and 1% -5% mulberry extract at 0-4 ℃, stirring uniformly, and cooling at 2-6 ℃ for more than 24 hours to fully swell to obtain mulberry extract temperature-sensitive gel solution.

8. The method of manufacturing according to claim 7, wherein: the mulberry extract temperature-sensitive gel also contains electrolyte, and the electrolyte is preferably sodium salt; and/or, the concentration of the sodium salt is preferably 0.0125-0.2mol/L; and/or, the sodium salt is preferably NaCl and/or Na ₂CO₃;

and/or, the electrolyte is added after the mulberry extract in the method of claim 7.

9. The use of the mulberry extract thermosensitive gel as defined in any one of claims 1 to 6, as defined in any one of the following (a 1) to (a 4):

(a1) Application of mulberry extract thermosensitive gel in preparing products for treating intestinal inflammation;

(a2) Application of mulberry extract thermosensitive gel in preparing products for preventing intestinal inflammation;

(a3) Use of mulberry extract temperature-sensitive gel in treating intestinal inflammation;

(a4) An application of mulberry extract temperature-sensitive gel in preventing intestinal inflammation is provided.

10. Use of a mulberry extract temperature-sensitive gel according to any one of claims 1 to 6, as described in any one of the following (b 1) to (b 4):

(b1) Application of mulberry extract thermosensitive gel in preparing ulcerative colitis treatment products;

(b2) Application of mulberry extract thermosensitive gel in preparing ulcerative colitis prevention products;

(b3) The application of mulberry extract temperature-sensitive gel in treating ulcerative colitis;

(b4) Application of mulberry extract temperature-sensitive gel in preventing ulcerative colitis;

Preferably, the prevention and/or treatment of ulcerative colitis is embodied in at least one of the following:

1) Inhibiting colonic atrophy in patients with ulcerative colitis;

2) Inhibiting colonic edema in a patient suffering from ulcerative colitis;

3) Inhibiting weight loss in patients with ulcerative colitis;

Or, the product is a medicament or a pharmaceutical formulation.