CN115089641A - Enema for treating ulcerative colitis and preparation method thereof - Google Patents

Enema for treating ulcerative colitis and preparation method thereof Download PDF

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CN115089641A
CN115089641A CN202210576519.9A CN202210576519A CN115089641A CN 115089641 A CN115089641 A CN 115089641A CN 202210576519 A CN202210576519 A CN 202210576519A CN 115089641 A CN115089641 A CN 115089641A
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enema
polysaccharide
extract
radix rehmanniae
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CN115089641B (en
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刘昌�
刘彤
廖侠
贾庆安
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First Affiliated Hospital of Medical College of Xian Jiaotong University
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Abstract

The invention discloses an enema for treating ulcerative colitis and a preparation method thereof, wherein the enema comprises the following components in percentage by mass: 0.03% -0.16% of seclenic acid D; 20.03 to 0.09 percent of the bitter herb of rockfoil M; 2.5 to 3.8 percent of radix rehmanniae extract; 0.13 to 0.5 percent of butyrin; nicotine 0.008% -0.032 ‰; 0.48 to 1.29 percent of houttuynia polysaccharide; 0.62 to 1.8 percent of tetrandrine; hydrocortisone 0.03% -0.3 ‰; 10% -14% of thiolated chitosan; 8% -12% of N-isopropyl acrylamide; 16% -20% of glycerol; 0.8 to 1 percent of potassium metabisulfite and the balance of water, wherein the total mass is 100 percent and the pH value is 6 to 7. The invention makes up the vacancy that no convenient and effective enema treatment for the existing intractable ulcerative colitis exists.

Description

Enema for treating ulcerative colitis and preparation method thereof
Technical Field
The invention belongs to the technical field of medicine research and development, and particularly relates to an enema for treating ulcerative colitis difficultly and a preparation method thereof.
Background
Refractory Ulcerative Colitis (RUC) is defined as ulcerative colitis patients (UC patients) who are not effective in hormone treatment or hormone-dependent and cannot tolerate hormone, has the characteristics of being still ineffective after standardized and systematic internal treatment, being incapable of relieving the disease for a long time, being easy to repeatedly attack and the like, and has the occurrence rate accounting for about 30 percent of patients with severe UC. Chronic inflammation, which is difficult to control for a long time, provides a suitable microenvironment for tumorigenesis, and canceration is a significant cause of death of patients with UC. Epidemiological research shows that UC is a high-risk factor for colorectal cancer, the longer the course of disease is, the greater the risk of canceration is, the course of disease is 8-15 years, and the canceration rate is about 22%.
Patients with UC have no response to the conventional treatment of aminosalicylic acid drugs and glucocorticoids due to the influence of drug resistance, physical factors and the like. The main mechanisms are as follows: combining Cytomegalovirus (CMV) infection results in a reduction in the effective number of GRs. CMV causes T cell dysfunction, allowing CD4+ T cells to produce large amounts of Th 1-type cytokines, such as IFN- γ and TNF- α, which exacerbates the inflammatory response. In addition, CMV also activates the NF- κ B signaling pathway, increasing expression of proinflammatory cytokines, leading to worsening of the inflammatory response. Inflammatory factors IL-2, IL-4 and the like can activate and induce the expression increase of AP-1 and NF-kappa B, so that the expression increase of AP-1 and NF-kappa B and GR form a complex, thereby reducing the effective number and the effect of GR and generating hormone resistance; ② Glucocorticoid Receptor (GR) dysfunction results in impaired glucocorticoid signaling pathway. The pharmacological action of glucocorticoid is dependent on GR, and GR has two types of GR alpha and GR beta, wherein GR alpha can bind to glucocorticoid and has physiological pharmacological activity, and GR beta has no biological function but has the function of antagonizing GR alpha. The relation between the expression of GR alpha and GR beta in intestinal mucosa cells of patients with ulcerative colitis and the response and severity of glucocorticoid treatment is analyzed by Zhang tiger and the like, and the GR alpha/GR beta of the patients with hormone resistance is obviously reduced. It is suggested that in addition to the overall GR expression level in vivo affecting the hormonal response of the patient, dysregulation of the subtype is also a significant cause of glucocorticoid resistance.
In addition, as an inflammatory disease, the intestinal mucosa is also severely damaged by an abnormal inflammatory reaction, and the condition cannot be relieved. Moreover, most studies currently believe that the development of ulcerative colitis-associated carcinogenesis (UCAC) is subject to a pattern of "inflammation-atypical hyperplasia-cancer", the risk of carcinogenesis being directly proportional to the duration and severity of the inflammatory response.
Therefore, the intractable ulcerative colitis has the following problems: first, various mechanisms result in a decrease in the effective number of GR and maladjustment of subtype ratio, eventually leading to no conventional medication available; secondly, the inflammatory reaction is severe, the integrity of the intestinal mucosa is seriously damaged, the speed of relieving the disease condition is delayed, the disease course is prolonged, and the carcinogenic risk is increased.
The existing treatment mainly comprises surgical treatment and oral drug treatment, but the existing treatment has defects. Although the preventive colectomy can prevent canceration, the life quality of a patient is greatly influenced; the drug therapy is divided into two types of western medicines and traditional Chinese medicines, the immunosuppressant has many adverse reactions, such as myelosuppression and nephrotoxicity, and particularly when the immunosuppressant is used in combination with aminosalicylic acid, the adverse reactions are increased; novel pharmacotherapies such as biologies: anti-TNF- α monoclonal antibody: infliximab (IFX), adalimumab (ADA), and the like; alpha is alpha 4 β 7 Integrin monoclonal antibody: vedolizumab (vedolizumab); non-selective JAK inhibitors: tofacitinib (Tofacitinib) is costly to treat patients and is also a treatment for patientsAdverse reactions such as IFX can cause opportunistic infections, delayed allergic reactions, drug-induced lupus, and elevated cholesterol levels in tofacitinib. Most of the traditional Chinese medicine treatment schemes require the clinical typing of traditional Chinese medicine for the disease and then treatment. However, a unified typing standard is not formed so far, and the prescription is mostly added or subtracted from a self-prepared prescription or a traditional prescription, mainly an empirical prescription, and a recognized standard is not formed so far, so that a compound medicine with an optimal medicine ratio is lacking clinically at present, and the compound medicine cannot be popularized and applied.
At present, the local enema therapy of traditional Chinese medicines or western medicines is also available clinically, and good effects are achieved. As an enema, the enema treatment method itself has advantages. Most of the pathological changes of ulcerative colitis are located in rectum and lower segment of colon, the enema treatment effect is direct, the curative effect is obvious, and the advantages are obvious and: on one hand, the enema administration can lead the medicine to directly reach the focus and lead the medicine to fully contact the focus, thereby rapidly improving the blood concentration of the lesion part, leading the medicine to be rapidly absorbed and fully exerting the local treatment effect of the medicine; on the other hand, after the drug is absorbed by the colon and rectum, most of the drug can directly enter the systemic circulation without passing through the liver, thereby playing a therapeutic role on the whole body and reducing the influence of the detoxification process of the liver on the drug effect. Rectal administration can also prevent damage of gastrointestinal acid-base and digestive enzymes to the medicine, can realize medicine decrement, avoids serious systemic adverse reaction caused by large-dose medicine treatment, and is especially of great significance to the elderly and the weak, or patients suffering from digestive system diseases and unsuitable for oral administration of the medicine. However, the intestinal adhesion of the enema is insufficient, so that the frequency of enema needs to be increased or the patients need to cooperate with the prolonging of the intestinal retention time of the enema to achieve the drug effect, and the enema is often used as an adjuvant therapy of intractable ulcer and nodule in combination with oral drugs. The enema has the defects of short intestinal retention time, complex operation, frequent use and the like, and influences the medical experience and treatment compliance of patients, so that the treatment effect of the enema is further influenced, and the advantage of the enema is difficult to fully exert. More importantly, the components of the current enema can not play an effective treatment effect on the intractable ulcerative colitis.
Therefore, there is a need to provide a new enema for ulcerative colitis which is difficult to treat.
Disclosure of Invention
In view of the above, the present invention provides an enema for ulcerative colitis which is difficult to treat and a preparation method thereof.
In order to solve the technical problems, the invention discloses a refractory ulcerative colitis enema which comprises the following components in percentage by mass: 0.03-0.16% of seclenic acid D; 20.03 to 0.09 percent of the bitter herb of rockfoil M; 2.5 to 3.8 percent of radix rehmanniae extract; 0.13% -0.5% of butyrin; 0.008% -0.032% of nicotine; 0.48 to 1.29 percent of houttuynia polysaccharide; 0.62 to 1.8 percent of tetrandrine; hydrocortisone 0.03% -0.3 ‰; 10% -14% of thiolated chitosan; 8% -12% of N-isopropyl acrylamide; 16% -20% of glycerol; 0.8 to 1 percent of potassium metabisulfite, and the balance of distilled water, wherein the total mass is 100 percent, and the pH value is 6 to 7.
Optionally, the paint comprises the following components in percentage by mass: 0.09% -0.1% of seclenic acid D; 20.06% -0.07% of echeveria glauca extract M; 3% -3.2% of radix rehmanniae extract; 0.2 to 0.4 percent of butyrin; 0.01 to 0.03 per mill of nicotine; 0.7 to 1.1 percent of houttuynia polysaccharide; 0.62 to 1.8 percent of tetrandrine; hydrocortisone 0.2 ‰; 12% of thiolated chitosan; 11% of N-isopropylacrylamide; 18% of glycerol; 0.9 percent of potassium metabisulfite and the balance of water, wherein the total mass is 100 percent, and the pH value is 6-7.
The invention also discloses a preparation method of the intractable ulcerative colitis enema, which comprises the following steps:
step 1, preparing a radix rehmanniae extracting solution:
step 2, preparing houttuynia cordata polysaccharide:
step 3, weighing the following components in percentage by mass: 0.03% -0.16% of seclenic acid D; 20.03 to 0.09 percent of bitter pholidota ananas extract M; 2.5 to 3.8 percent of radix rehmanniae extract; 0.13 to 0.5 percent of butyrin; nicotine 0.008% -0.032 ‰; 0.48 to 1.29 percent of houttuynia polysaccharide; tetrandrine 0.62-1.8%; hydrocortisone 0.03% -0.3 ‰; 2% -4% of thiolated chitosan; 8% -12% of N-isopropyl acrylamide; 16% -20% of glycerol; 0.8 to 1 percent of potassium metabisulfite and the balance of distilled water;
step 4, preparing enzyme temperature double-sensitive type hydrogel components: adding weighed N-isopropyl acrylamide, glycerol and thiolated chitosan into 35% of distilled water, and magnetically stirring until the mixture is uniformly swelled, so that the mixture is fully swelled and reacts; adding weighed potassium metabisulfite, secalinic acid D, bitter herb extract M2, radix rehmanniae extract, butyrin, nicotine, houttuynia polysaccharide, tetrandrine and hydrocortisone, stirring at room temperature, adding water to full volume, adding pH regulator, regulating pH to 6-7, and stirring to obtain enzyme temperature double-sensitive hydrogel component;
step 5, homogenizing: homogenizing by a pipeline type homogenizer, and uniformly stirring;
step 6, filling: filling into an administration device at sterile room temperature, and externally packaging.
Optionally, the preparing of the radix rehmanniae extracting solution in the step 1 specifically comprises: baking rehmanniae radix at 40-50 deg.C until eighty percent dry, pulverizing, sieving with 40 mesh sieve, adding 70% ethanol at a liquid-material ratio of 15: 1-25: 1(mL/g), ultrasonic treating at 25-35 deg.C for 35-45min, extracting for 2 times, filtering, separating, concentrating to obtain extractive solution, and vacuum drying to constant weight to obtain radix rehmanniae extract.
Optionally, the preparing of the houttuynia cordata polysaccharide in the step 2 specifically comprises: sun drying herba Houttuyniae, pulverizing, sieving with 100 mesh sieve to obtain herba Houttuyniae dry powder, placing herba Houttuyniae dry powder in a conical flask, adding 70% ethanol at ultrasonic power of 72W and ultrasonic temperature of 60-70 deg.C for 20-30min at liquid-material ratio of 35: 1-45: 1(mL/g), filtering, and separating to obtain extractive solution; putting the extracting solution into a centrifuge tube, centrifuging for 15-25min at 3500r/min of 2500-; adding 8mL of absolute ethanol into the concentrated solution, standing at 4 ℃ for 12h for alcohol precipitation, centrifuging at 3500r/min of 2500-.
Optionally, the houttuynia cordata dry powder is prepared by soaking the houttuynia cordata dry powder in petroleum ether through a Soxhlet extractor, heating and refluxing for 4 times under a voltage of 70-80V by a heating sleeve, degreasing, air-drying for 12 hours, heating and refluxing for 5 times by using methanol, decoloring and finally naturally air-drying.
Optionally, the magnetic stirring time in the step 4 is 50-60 ℃, and the rotation speed is 350-450 r/min.
Optionally, the swelling reaction time in the step 4 is 10-15 h.
Compared with the prior art, the invention can obtain the following technical effects:
aiming at the etiological mechanism of intractable ulcerative colitis, the invention applies effective pharmaceutical ingredients to relieve the problem of glucocorticoid resistance by up-regulating the expression of colon mucosal cells GR alpha, activating downstream reaction of the colon mucosal cells GR alpha and relieving the influence of inflammation on the effective number of GR alpha. Meanwhile, the enzyme-temperature double-sensitive hydrogel is prepared to be used as an enema, so that the intestinal mucosa is adhered to the enema, the medicine is slowly and durably released, the intestinal irritation and treatment discomfort of a patient caused by short retention time, troublesome operation and more use times of the enema are overcome, the enema can be used as a main treatment means in ulcer and nodule treatment, the treatment advantage of combining traditional Chinese medicines and western medicines is exerted, and the defect that the conventional intractable ulcerative colitis has no convenient and effective enema treatment at present is overcome.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention to the right. In the drawings:
FIG. 1 is the cumulative release of tetrandrine;
FIG. 2 shows the body weight change of mice in each group according to the present invention.
Detailed Description
The following embodiments are provided to explain the present invention in detail, so as to fully understand and implement the technical solution of the present invention and achieve the technical effects.
The invention discloses an enema for treating intractable ulcerative colitis, which comprises the following components in percentage by mass: 0.03-0.16% of seclenic acid D; extract M of herba Ixeritis Sonchifoliae 2 0.03% -0.09%; 2.5 to 3.8 percent of radix rehmanniae extract; 0.13% -0.5% of butyrin; nicotine 0.008% -0.032 ‰; 0.48 to 1.29 percent of houttuynia polysaccharide; 0.62 to 1.8 percent of tetrandrine; hydrocortisone 0.03% -0.3 ‰; 12% -14% of thiolated chitosan; 8% -12% of N-isopropyl acrylamide; 16% -20% of glycerol; 0.8 to 1 percent of potassium metabisulfite and the balance of water, wherein the total mass of the potassium metabisulfite and the water is 100 percent; extract M of herba Ixeritis Sonchifoliae 2 According to the research on the action and mechanism of natural products M2 and V11 for treating ulcerative colitis (on steel, the research on the action and mechanism of natural products M2 and V11 for treating ulcerative colitis [ D)]Jiangsu, Nanjing university, 2016).
Wherein, 1) the seclenic acid D, the bitter herb extract M2 and the dried rehmannia root are used for enhancing the expression and the action of the GR alpha of the colon cells. Seclenone acid D is a natural product of microbial origin, has antifungal and antitumor activities, has agonistic activity on GR alpha transcription regulation and control action, and can obviously up-regulate mRNA expression quantity; the bitter stonecrop herb extract M2 can target GR, promote GR to enter the nucleus and enhance the GR alpha function.
Radix rehmanniae preparata can adjust the binding force of GR alpha upwards, realize the use of low-concentration glucocorticoid to play a therapeutic role, and greatly lighten the body adverse reaction caused by the glucocorticoid, and the traditional Chinese medicine is mostly applied to the treatment of other autoimmune diseases except ulceration. However, in the meantime, the contents of active ingredients in the prepared rehmannia root processed by various methods are reduced due to the influence of high temperature and processing time, and a large amount of 5-hydroxymethyl-2-furfural (5-HMF) is produced, and the pharmacological effect thereof is changed from beneficial to harmful according to the increase of dosage, especially, the pharmacological effect of the prepared rehmannia root which has dosage dependence on colon, such as the stimulation of abnormal growth of colon bursa, and the increase of carcinogenic risk. Compared with the traditional prepared rehmannia root processed by the traditional method, the method for preparing the traditional prepared rehmannia root by the warm extraction of the raw rehmannia root avoids the damage to the effective components in the early processing process, greatly reduces the content of the 5-HMF, avoids the toxic action from being exerted, and properly keeps the improvement capability of the traditional prepared rehmannia root on hemorheology.
2) The traditional Chinese medicine monomer components such as butyric acid, nicotine, houttuynia cordata polysaccharide, tetrandrine and the like are used for repairing intestinal mucosa, inhibiting inflammation, reducing canceration risk and also relieving the influence of inflammation activation on the effective number of GR alpha.
Butyrate is a metabolic substrate of the colon mucosa and is a necessary condition for synthesizing the tight junction protein by the epithelial cells of the intestinal mucosa, and finally, the proliferation and differentiation of the epithelial cells of the intestinal mucosa are promoted to improve the integrity of the intestinal mucosa; can stimulate colon mucous membrane cells to absorb sodium water. The colonic epithelium of ulcerative colitis may be in some energy deficient state and butyrate may help in the treatment of RUC by correcting this colonic epithelium energy deficiency, promoting cellular repair and inflammation relief. Butyric acid also has an anti-inflammatory effect and the intrinsic mechanism is: the continuous abnormal activation of T lymphocytes in intestinal wall tissues in UC patients causes the injury of the intestinal wall, butyric acid can activate transcription factors STAT3 and mTOR pathways by enhancing the acetylation of histone H3 in a Foxp3 promoter and combining GPR43 of intestinal epithelial cells, promotes the differentiation of CD4+ T cells for producing IL-10 and Th1 and Th17 cells, and inhibits the immune response of intestinal mucosa to play an anti-inflammatory role; butyric acid can also reduce the expression of proinflammatory factors TNF-alpha and IL-1 beta by inhibiting NF-kB pathway, and is also helpful to relieve glucocorticoid resistance.
The nicotine can stimulate vagus nerve to release acetylcholine, and act on nicotinic acetylcholine receptor to reduce excitability of intestinal sensory neuron. In addition, nicotine also has effects in improving microcirculation, inhibiting the production of arachidonic acid metabolite, regulating intestinal motility, and promoting the synthesis of intestinal mucin. CO also has anti-inflammatory effects, and its mechanism may be inhibition of neutrophil aggregation, reduction of expression of colon TNF, IL-4, and promotion of the production of anti-inflammatory cytokine IL-10 through heme oxygenase-1 (HO-1) pathway.
The Houttuynia Cordata Polysaccharide (HCP) contains rich monosaccharide components such as glucose, rhamnose, arabinose, galactose, galacturonic acid and the like, and is proved to have a plurality of active functions such as inflammation diminishing, bacteria inhibiting, virus resisting, immunoregulation, intestinal protection, oxidation resisting and the like. And can relax intestinal smooth muscle, relieve intestinal spasm, and relieve clinical symptoms.
Tetrandrine can increase superoxide dismutase (SOD) activity, reduce Malondialdehyde (MDA) content and Myeloperoxidase (MPO) activity, improve organism oxidation resistance, and relieve intestinal mucosa injury, thereby treating UC.
The invention also discloses a preparation method of the intractable ulcerative colitis enema, which comprises the following steps:
step 1, preparing a radix rehmanniae extracting solution: baking rehmanniae radix at 40-50 deg.C to eighty percent dry, pulverizing, sieving with 40 mesh sieve, adding 70% ethanol at a liquid-material ratio of 15: 1-25: 1(mL/g), ultrasonic treating at 25-35 deg.C for 35-45min, extracting for 2 times, filtering, separating, concentrating to obtain extractive solution, vacuum drying to constant weight, and making into radix rehmanniae extract;
step 2, preparing houttuynia cordata polysaccharide: sun drying, pulverizing, sieving with 100 mesh sieve, weighing herba Houttuyniae dry powder, placing in a conical flask, adding 70% ethanol at liquid-material ratio of 35: 1-45: 1(mL/g), ultrasonic power of 72W, ultrasonic temperature of 60-70 deg.C, and ultrasonic time of 20-30min, filtering to separate extractive solution; putting the extracting solution into a centrifuge tube, centrifuging for 15-25min at 2500-3500r/min, collecting supernatant, pouring into a round-bottom flask, condensing, refluxing and concentrating to 2mL, transferring into a test tube, and cooling; adding 8mL of absolute ethyl alcohol into the concentrated solution, standing at 4 ℃ for 12h for alcohol precipitation, centrifuging at 3500r/min of 2500-;
wherein, the houttuynia cordata dry powder is soaked in petroleum ether by a Soxhlet extractor, is heated and refluxed for 4 times by an electric heating sleeve (70-80V) to complete degreasing, is heated and refluxed for 5 times by methanol after being dried for 12 hours for decolorization, and is finally naturally dried to prepare the houttuynia cordata.
Step 3, weighing the following components in percentage by mass: 0.03% -0.16% of seclenic acid D; 20.03 to 0.09 percent of the bitter herb of rockfoil M; 2.5 to 3.8 percent of radix rehmanniae extract; 0.13% -0.5% of butyrin; nicotine 0.008% -0.032 ‰; 0.48 to 1.29 percent of houttuynia polysaccharide; 0.62 to 1.8 percent of tetrandrine; hydrocortisone 0.03% -0.3 ‰; 12% -14% of thiolated chitosan; 8% -12% of N-isopropyl acrylamide; 16% -20% of glycerol; 0.8 to 1 percent of potassium metabisulfite; the balance of water, the total mass of the above is 100%;
step 4, preparing enzyme temperature double-sensitive type hydrogel components: adding the weighed N-isopropylacrylamide, glycerol and thiolated chitosan into 35 percent of distilled water, and magnetically stirring at the temperature of between 50 and 60 ℃ and 450r/min until the materials are uniformly swelled, so that the materials are fully swelled and react for 10 to 15 hours; adding weighed potassium metabisulfite, secalinic acid D, bitter herb extract M2, radix rehmanniae extract, butyrin, nicotine, houttuynia polysaccharide, tetrandrine and hydrocortisone, stirring at room temperature, adding the balance of distilled water to full volume, adding a pH value regulator, regulating the pH value to 6-7, and stirring to obtain the enzyme temperature double-sensitive hydrogel component;
step 5, homogenizing: homogenizing by a pipeline type homogenizer, and uniformly stirring;
step 6, filling: filling into an administration device at sterile room temperature, and packaging.
In the aspect of preparation of the dosage form, the invention particularly prepares the enzyme-temperature double-sensitive hydrogel as an enema, and has the advantages that: the enzyme-temperature dual-sensitive enema gel has the gelling temperature of about 37 ℃, is close to body temperature, can ensure that the enzyme-temperature dual-sensitive enema gel is quickly gelled and attached to the surface of an intestinal tract at the body temperature, and stays on the mucosa of the intestinal tract for a longer time compared with the traditional enema, thereby prolonging the action time of a medicament; in addition, the chitosan can be degraded by specific glycosidase at the colon, has strong targeting property, is used as natural polysaccharide, has higher biocompatibility, stability and no toxicity, and controls the release of the drug by regulating the degradation rate; the product obtained after sulfhydrylation is carried out on the side chain of the derivative can generate specific combination similar to receptor-ligand with rich cysteine in mucosa, the acting force is strong, the biological adhesion can be obviously improved, the cilium clearing effect is effectively slowed down, and the permeability of the medicament to cell membranes can be improved. The novel thiolated chitosan-N-isopropyl acrylamide gel has high biocompatibility, biodegradability with enzyme degradation specificity and better mucosa adhesiveness, so that the medicine is slowly and durably released, and intestinal irritation and treatment discomfort of patients caused by short retention time, troublesome operation and more use times of the enema are overcome.
The formulations of examples 1-10 and comparative examples 1-6 are shown in Table 1.
TABLE 1 formulation tables for examples 1-10 and comparative examples 1-6
Figure BDA0003662392360000091
Example 1
The ingredients of example 1 are shown in the data of example 1 in table 1, and the method for preparing the enema for the refractory ulcerative colitis in example 1 comprises the following steps:
step 1, preparing a radix rehmanniae extracting solution: baking rehmanniae radix at 45 deg.C to about eighty percent dry, pulverizing, sieving with 40 mesh sieve, and extracting radix rehmanniae component with ultrasonic wave assistance: performing ultrasonic treatment with 70% ethanol at a liquid-material ratio of 20: 1(mL/g) at 30 deg.C for 40min, extracting for 2 times, filtering, separating, concentrating to obtain extractive solution, and vacuum drying to constant weight to obtain radix rehmanniae extract;
step 2, preparing houttuynia cordata polysaccharide: drying in the sun, pulverizing, sieving with 100 mesh sieve, placing herba Houttuyniae dry powder in a conical flask, adding 70% ethanol at ultrasonic power of 72W and ultrasonic temperature of 65 deg.C for 25min at a liquid-material ratio of 40: 1(mL/g), filtering, and separating to obtain extractive solution; putting the extracting solution into a centrifuge tube, centrifuging for 20min at 3000r/min, collecting supernatant, pouring into a round-bottom flask, condensing, refluxing and concentrating to 2mL, transferring into a test tube, and cooling; adding 8mL of absolute ethyl alcohol into the concentrated solution, standing at 4 ℃ for 12h for alcohol precipitation, then centrifuging at 3000r/min for 20min, removing the supernatant, and collecting the precipitate to obtain houttuynia polysaccharide;
wherein, the houttuynia cordata dry powder is soaked in petroleum ether by a Soxhlet extractor, degreased by heating and refluxing for 4 times by a heating jacket under the voltage of 70-80V, decolored by heating and refluxing for 5 times by methanol after air drying for 12h, and finally air dried to prepare the houttuynia cordata oil.
Step 3, weighing the components according to the content of the components in the example 1 in the table 1;
step 4, preparing enzyme temperature double-sensitive type hydrogel components: adding weighed N-isopropyl acrylamide, glycerol and thiolated chitosan into 35% distilled water, magnetically stirring at 55 ℃ for 400r/min until the mixture is uniformly swelled, and fully swelling and reacting for 12 hours. Adding weighed potassium metabisulfite, secalinic acid D, bitter herb extract M2, radix rehmanniae extract, butyrin, nicotine, houttuynia polysaccharide, tetrandrine and hydrocortisone, stirring at room temperature, adding water to full volume, adding pH regulator, adjusting pH to 6-7, and stirring;
step 5, homogenizing: homogenizing by adopting a pipeline type homogenizer, and uniformly stirring;
step 6, filling: filling into an administration device at sterile room temperature, and packaging.
Example 2
The components of example 2 are shown in the data of example 2 in table 1, and the preparation method of the intractable ulcerative colitis enema in example 2 comprises the following steps:
step 1, preparing a radix rehmanniae extracting solution: baking rehmanniae radix at 40 deg.C until eighty percent dry, pulverizing, sieving with 40 mesh sieve, adding 70% ethanol at a liquid-material ratio of 25: 1(mL/g), ultrasonic treating at 25 deg.C for 45min, extracting for 2 times, filtering, separating, concentrating to obtain extractive solution, and vacuum drying to constant weight to obtain radix rehmanniae extract;
step 2, preparing houttuynia cordata polysaccharide: drying in the sun, pulverizing, sieving with 100 mesh sieve, placing herba Houttuyniae dry powder in a conical flask, filtering to separate extractive solution according to liquid-material ratio of 35:1(mL/g) 70% ethanol, ultrasonic power of 72W, ultrasonic temperature of 70 deg.C, and ultrasonic time of 20 min; putting the extracting solution into a centrifuge tube, centrifuging for 15min at 3500r/min, collecting supernatant, pouring into a round-bottom flask, condensing, refluxing and concentrating to 2mL, transferring into a test tube, and cooling; adding 8mL of absolute ethyl alcohol into the concentrated solution, standing at 4 ℃ for 12h for alcohol precipitation, then centrifuging at 2500r/min for 25min, removing the supernatant, and taking the precipitate to prepare the houttuynia polysaccharide; (ii) a
Wherein, the houttuynia cordata dry powder is soaked in petroleum ether by a Soxhlet extractor, degreased by heating and refluxing for 4 times by a heating jacket under the voltage of 70-80V, decolored by heating and refluxing for 5 times by methanol after air drying for 12h, and finally air dried to prepare the houttuynia cordata oil.
Step 3, weighing the components according to the content of the components in the example 2 in the table 1;
step 4, preparing enzyme temperature double-sensitive type hydrogel components: adding weighed N-isopropyl acrylamide, glycerol and thiolated chitosan into 35% distilled water, magnetically stirring at 50 ℃ for 450r/min until the mixture is uniformly swelled, and fully swelling and reacting for 10 h; adding weighed potassium metabisulfite, seclenic acid D, bitter lotus extract M2, radix rehmanniae extract, butyrin, nicotine, houttuynia polysaccharide, tetrandrine and hydrocortisone, stirring at room temperature, adding water to full volume, adding pH regulator, regulating pH to 6-7, and stirring;
step 5, homogenizing: homogenizing by adopting a pipeline type homogenizer, and uniformly stirring;
step 6, filling: filling into an administration device at sterile room temperature, and externally packaging.
Example 3
The components of example 3 are shown in the data of example 1 in table 3, and the preparation method of the intractable ulcerative colitis enema of example 3 comprises the following steps:
step 1, preparing a radix rehmanniae extracting solution: baking rehmanniae radix at 50 deg.C until eighty percent dry, pulverizing, sieving with 40 mesh sieve, adding 70% ethanol according to liquid-material ratio of 15:1(mL/g), ultrasonic treating at 35 deg.C for 35min, extracting for 2 times, filtering, separating, concentrating to obtain extractive solution, and vacuum drying to constant weight to obtain radix rehmanniae extract;
step 2, preparing houttuynia cordata polysaccharide: sun drying herba Houttuyniae, pulverizing, sieving with 100 mesh sieve to obtain herba Houttuyniae dry powder, placing herba Houttuyniae dry powder in a conical flask, adding 70% ethanol at liquid-material ratio of 45: 1(mL/g), ultrasonic power of 72W, ultrasonic temperature of 60 deg.C, and ultrasonic time of 30min, filtering to separate extractive solution; putting the extracting solution into a centrifuge tube, centrifuging for 25min at 2500r/min, collecting supernatant, pouring into a round-bottom flask, condensing, refluxing and concentrating to 2mL, transferring into a test tube, and cooling; adding 8mL of absolute ethyl alcohol into the concentrated solution, standing at 4 ℃ for 12h for alcohol precipitation, then centrifuging at 3500r/min for 15min, discarding the supernatant, and collecting the precipitate to obtain houttuynia polysaccharide;
step 3, weighing the components according to the content of the components in the example 3 in the table 1;
step 4, preparing enzyme temperature double-sensitive type hydrogel components: adding the weighed N-isopropyl acrylamide, glycerol and thiolated chitosan into 35% distilled water, magnetically stirring at 60 ℃ for 350r/min until the mixture is uniformly swelled, and fully swelling and reacting for 15 h; adding weighed potassium metabisulfite, secalinic acid D, bitter herb extract M2, radix rehmanniae extract, butyrin, nicotine, houttuynia polysaccharide, tetrandrine and hydrocortisone, stirring at room temperature, adding water to full volume, adding pH regulator, adjusting pH to 6-7, and stirring;
step 5, homogenizing: homogenizing by a pipeline type homogenizer, and uniformly stirring;
step 6, filling: filling into an administration device at sterile room temperature, and packaging.
The preparation methods of examples 4 to 10 and comparative examples 1 to 6 are the same as example 1.
The technical effects of the invention are illustrated below with reference to specific experimental data:
testing the performance of the hydrogel: examples 1, 8, 9, 10 and comparative examples 5, 6
1. Measurement of gel temperature and adhesion:
the gel temperature was determined by the inverted tube method: placing 2mL of prepared gel solution in a test tube, placing the test tube in a constant-temperature water bath, slowly raising the temperature at a rate of about 0.2 ℃/min, rapidly inverting the test tube when the temperature rises by 0.2 ℃, observing the flowing condition of the solution, defining the temperature when the solution in the test tube does not flow any more as the gel temperature, measuring each sample for 3 times, and taking an average value, wherein the result is shown in the following table 2:
table 2 gel temperature measurement results (n ═ 3) for each group
Figure BDA0003662392360000121
Figure BDA0003662392360000131
From table 2 above, it can be seen that: the gelling temperature is as follows from low to high: example 10 < example 9 < comparative 6 < example 1 < comparative 5 < example 8, example 1 being closest to the intestinal temperature.
As can be seen from the results of comparing the gelation temperatures of example 1 and examples 8-10, the mass percent of thiolated chitosan in the enema of the present invention is optimally 12%, and the mass percent of N-isopropylacrylamide in the enema of the present invention is optimally 11%, and the gelation temperature is closer to the intestinal tract core temperature, namely 37 ℃, so that the form conversion in the intestinal tract can be successfully completed. And with the great increase of the content of the N-isopropyl acrylamide, the gelling temperature of the N-isopropyl acrylamide is continuously increased, which has a certain relation with the reaction of the thiolated chitosan, and the proportion of the thiolated chitosan changes the rising amplitude of the gelling temperature to a certain extent. Compared with the example 1, the comparative example 6 only replaces enzyme sensitive materials, specifically, sulfhydrylated chitosan is replaced by hydroxypropyl methylcellulose, the difference between the two groups of gel temperatures is minimum (about 2 ℃), but the two groups of gel temperatures are still lower than the intestinal tract temperature, and the successful modification and adhesion during enema cannot be met; the hydrogel enema prepared by directly using common temperature-sensitive materials has larger temperature difference.
2. Simulated release time of the drug in vitro: taking the release amount of tetrandrine as an index, and detecting the tetrandrine by an HPLC method: 3g of gel is taken from each group and is placed in pH7.4 buffer solution, in the presence of glycosidase, in-vitro degradation test is carried out at constant temperature of 37 ℃, 5mL samples are taken from 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0 and 12.0h respectively, equal amount of pH7.4 buffer solution at 37 ℃ is added immediately, the content of tetrandrine is detected by an HPLC method, the cumulative release degree is calculated, each sample is measured for 3 times, and the average value is taken; the results are shown in FIG. 1 and Table 3 below.
Table 3 cumulative release of tetrandrine (%) (n ═ 3)
Figure BDA0003662392360000132
Figure BDA0003662392360000141
From the results of fig. 1 and table 3, it can be seen that: the release rates of the groups from fast to slow are as follows: example 10 > example 1 > example 9 > example 8 > comparative example 6 (different from the enzyme sensitive material of the invention but in the same amount) > comparative example 5 (a common temperature sensitive hydrogel).
As can be seen from the in vitro simulated release conditions of comparative example 1 and examples 8-10, the release rate of example 1 is more suitable for the drug to act on the intestinal mucosa for a long time, a higher release rate can be achieved after 6h, and the drug is more beneficial to maintaining the therapeutic blood concentration; example 10 achieved a very high release rate at 4 hours, which greatly shortened the duration of drug action and was not conducive to maintaining therapeutic blood levels. The cumulative release of examples 8 and 9 appeared to be too long in effect. Comparative example 6 compared with example 1, only the enzyme sensitive material was replaced, but the drug release rate was greatly reduced, and only 80% of the drug release rate could be reached until 12 hours, so that the effective concentration of the drug could not be reached well, and intestinal excretion could not occur in such a long time, so that the enema could be discharged in a large amount, and the disease condition of the patient per se could not be matched. The hydrogel enema prepared by directly using the common temperature-sensitive material has the same problems as the hydrogel enema prepared by the comparative example 6, and even has worse effect.
(II) chronic experimental colitis mouse model to investigate the effect of the drug of the invention:
preparation and administration method of mouse model
Healthy male BALB/c mice were selected 48, 6-8 weeks old, and weight (21 + -2) g, and all mice were acclimatized for 1 week prior to the experiment.
The molding method in the prior literature is specifically as follows: adopting 25mg/kg TNBS-50% ethanol single enema and 3% DSS self-drinking water for 6d to establish a refractory UC model; the successful establishment of the model is marked by the appearance of at least two symptoms of semi-dilute stool, diarrhea, fecal occult blood positive and macroscopic bloody stool in the mice. Next, the samples were randomly divided into 12 groups, i.e., a control group, examples 1 to 7, and comparative examples 1 to 4. The control group dose is normal saline with the same amount as the medicated gel, and is continued for 7 days; corresponding medicines are administrated to each group of enema, and the administration dosage is converted into equivalent mouse dosage by daily enema dosage for 7 days continuously.
(1) Reduction of mouse body weight: the change in body weight of the mice was observed every 2 days from the start of the test to before sacrifice, and the results are shown in fig. 2 and table 4.
Table 4 weight change of mice in each group (n ═ 4)
Figure BDA0003662392360000151
As can be seen from the data in fig. 2 and table 4, there was no statistical difference between the initial body weights of the mice in each group, and there was a significant decrease in body weight during the molding time (first 6 days), but the difference in body weight between the groups was not significant after the molding was completed. During the subsequent treatment, the inventor obviously discovers that: as a control group, the body weight of the mice still showed a downward trend due to the non-drug treatment, and the final slight fluctuation appeared as the days extended, but there was still a significant gap from the initial body weight. In the treatment groups (examples 1-7) containing the drugs of the present invention but different drug dose combinations, the fluctuation of body weight of the rats finally showed a certain rise compared to the last day after the molding, but the rise rate of each group was different. Among them, the weight regain of example 1, which is the optimal formulation, is most remarkable and shows a tendency of weight gain of mice beyond the initial weight, indicating that the recovery of the disease of this group of mice is very good, the recovery of the intestinal mucosa even promotes the good absorption of nutrients by the mice and counteracts the excessive consumption of the capacity by the inflammatory state, so that the supplementary diet can achieve the positive nitrogen balance of the mice. When the dosage of the drug was changed, the body weight recovery of the mice was significantly weaker than that of example 1. Further analysis: examples 4, 5, 6 are the three groups with the next effect to example 1 and their drug doses are closer to example 1. In examples 1 and 4, the dosage of the drug is reduced to a certain extent, so that the overall treatment effect is greatly reduced in the aspect of weight recovery; the difference between the drug ingredients in the anti-inflammatory effects of examples 4 and 5, i.e., the dose of example 4 is larger than that of example 5, is also reduced by the adjustment; examples 5-7 compared with the prior art, although the drug has enhanced dose dependence on the activation of glucocorticoid receptor, the drug component dose which plays an anti-inflammatory role is reduced, and the drug are balanced in drug cooperation, so that the effect is weakened; other group changes will also produce the same effect. Comparative example 1, which retained the anti-inflammatory drug components and the conventional glucocorticoid therapeutic amounts, found that the body weight of the mice did not rise back and still declined in the early period of treatment, indicating that it responded slowly to the treatment, although the body weight also showed a gradual rise back in the last two days, it was sufficient to show that it could not achieve the recovery effect within the expected treatment period of the drug, demonstrating that the treatment effect of the disease was not good. This may also reflect that if only inflammation is reduced to restore upregulation of the mouse's own glucocorticoid receptor and thereby reduce glucocorticoid resistance, the phenomenon is very slow and does not achieve good results; in comparative example 2, the effect is greatly reduced after the extraction method of the radix rehmanniae recen is replaced by the conventional radix rehmanniae preparata, the effect is still inferior to that of example 1 after the anti-inflammatory drug is replaced by the treatment drug mentioned in the literature in comparative example 3, and the effect is worse after the two major drugs are completely replaced in comparative example 4; and the difference between the comparative examples can also be seen, whether the extraction mode of the medicine is changed or the components are replaced, the final effect is influenced. In summary, the two main groups of drugs have certain drug dose dependence on the disease weight recovery, which is consistent with the early background investigation, and the experiments of the present invention further show that the effects of the two main groups of drugs must exist simultaneously and the doses are matched with each other to achieve the best therapeutic effect.
(2) Case of colon specimen:
after the mice are administered for the last time, fasting for 1d, anesthesia and sacrifice are carried out, dissection is carried out, the whole intestinal section from the anus end to the tail end of the cecum is taken out, tissue samples at the ulcer part are fixed by 4 percent paraformaldehyde, conventional dehydration and paraffin embedding are carried out, after 5um slicing, conventional HE staining is carried out,
the colon histopathology was examined under light microscopy and scored according to the criteria of table 5:
TABLE 5 intestinal histological damage score
Scoring device Degree of inflammation Depth of lesion Destruction of crypts Extent of disease (%)
0 Is free of Is free of Is free of Is free of
1 Mild degree of Submucosa layer The recess of the substrate 1/3 is destroyed 1-25
2 Of moderate degree Muscular layer The recess of the substrate 2/3 is destroyed 26-50
3 Severe degree Serous layer Only intact superficial epithelium 51-75
4 / / All crypts and epithelium are destroyed 76-100
Statistical analysis is carried out on the results by adopting SPSS 26.0 software, the measurement data are all expressed by mean soil SD, after the homogeneity of variance test, the difference between each group is compared by adopting One-way ANOVA (One-way AN0VA), the difference with P <0.05 has statistical significance, and the results are shown in Table 6.
Table 6 groups of mice colon histological damage scores (n ═ 4)
Figure BDA0003662392360000171
Figure BDA0003662392360000181
Note: p <0.05 compared to control group and P <0.05 compared to treatment group 1.
The invention further analyzes the repair of colonic mucosal tissue of each group of mice by drug treatment. The general appearance of the colon was visually observed in each group of mice when the entire intestinal segment from the anal end to the cecal end was removed, and the difference in the appearance of the intestinal mucosa was clearly seen: the control group of mice had rough and granular colon surface, thickened intestinal wall, distorted deformation, congestion and edema of intestinal mucosa, and slight to severe erosion and ulcer; the colon intestinal wall of the mice of the groups 1, 4 and 5 is thickened, the intestinal mucosa is slightly congested and edematous, and 2 mice can be slightly eroded but have no ulcer focus; the colon intestinal wall of mice of other examples and comparative examples is thickened, the intestinal mucosa is congested, edematous and slightly erosive, and small ulcer foci with different degrees and numbers can be seen, but the overall situation is between the control group and the examples 1, 4 and 5. From the scores in table 5, it can be seen that the colonic mucosa recovery is, in order from good to bad: example 1 > example 4 > example 6 > example 5 > example 3 > example 2 > example 7 > comparative example 2-comparative example 3 > comparative example 4 > comparative example 1. And the recovery of the colon mucosa of each group is obviously stronger than that of the control group, and the medicament treatment has certain help on the recovery of the mucosa injury. The specific analysis shows that: examples 1, 4, 5 and 6 belong to a group of experiments with a low colon histological damage score, i.e. a good disease recovery effect, wherein example 1 is the most preferable, the effect is the best, the effect of other groups is weakened, but the difference has no statistical significance in the result. The dosage of the medicine in the range is more appropriate, and better effect can be achieved in the aspect of pathology; when the difference between the drugs increases, the effect of compatibility is greatly reduced, and the recovery speed and recovery condition of mucosa are also hindered. In the comparative example, the same effect was greatly impaired by changing the composition of the drug or changing the extraction method, which was the same tendency as the previous change in body weight.
(3) Serum TNF- α and IL-6 concentrations at the end of mouse treatment: after treatment, the rats in each group are sacrificed after fasting for 1d, the pericardium puncture before the treatment is sacrificed, 3ml of blood is collected, the rats are kept stand in a refrigerator at 4 ℃ for 3h, and then 3000 r.min is carried out -1 Centrifuging for 15min, sucking out serum, and detecting the content of TNF-alpha: measuring an absorbance value at 410nm by using a sigma 960E type full-automatic enzyme standard instrument according to the kit specification by adopting an ELISA method, and obtaining the content of TNF-alpha by contrasting a standard curve; detection of IL-6 content: adopts a double-antibody sandwich ELISA method, uses a sigma 960E type full-automatic enzyme labeling instrument at 450n according to the kit instructionAnd measuring the light absorption value at m, and contrasting with a standard curve to obtain the IL-6 content.
Statistical analysis was performed on the results using SPSS 26.0 software, the measurement data were all expressed in mean soil SD, after the homogeneity of variance test, One-way ANOVA (One-way AN0VA) was used to compare the differences between groups, P <0.05 was used as the difference with statistical significance, and the results are shown in Table 7.
Table 7 change in mouse serum TNF- α and IL-6 concentrations (n ═ 4)
Figure BDA0003662392360000191
Note: p <0.05 compared to control group and P <0.05 compared to treatment group 1.
Next, the present invention analyzes the changes of drug treatment to the serum TNF-alpha and IL-6 concentrations of each group of mice. From the scores in table 6, it can be seen that the overall level of inflammation in the mice is, in order from low to high: example 1 < example 4 < example 5 < example 6 < example 3 < example 2 < example 7 < comparative example 2 ≈ comparative example 3 < comparative example 4 < comparative example 1. And the inflammation level of each group is obviously higher than that of the control group, and the medicine treatment has obvious help on the recovery of inflammation. Further analysis and comparison show that: examples 1, 4, 5 and 6 still belong to a group of experimental groups with a low level of inflammatory response, which is characterized by a significant reduction in the overall disease inflammatory response of mice, wherein example 1 is the most preferable, the results are the best, the effects of the other groups are all reduced, but the differences are not statistically significant in the results. The dosage of the medicine in the range is more appropriate, and the effect of reducing inflammation is better; when the difference between the drugs increases, the effect of reducing inflammation is greatly reduced by the compatibility of the drugs. In the comparative example, the same effect was lost by changing the composition of the drug or changing the extraction method, which is consistent with the above change.
The foregoing description shows and describes several preferred embodiments of the invention, but as aforementioned, it is to be understood that the invention is not limited to the forms disclosed herein, and is not to be construed as excluding other embodiments, and that the invention is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (8)

1. The enema for the intractable ulcerative colitis is characterized by comprising the following components in percentage by mass: 0.03% -0.16% of seclenic acid D; 20.03 to 0.09 percent of the bitter herb of rockfoil M; 2.5 to 3.8 percent of radix rehmanniae extract; 0.13% -0.5% of butyrin; nicotine 0.008% -0.032 ‰; houttuynia cordata polysaccharide 0.48% -1.29%; 0.62 to 1.8 percent of tetrandrine; hydrocortisone 0.03% -0.3 ‰; 10% -14% of thiolated chitosan; 8% -12% of N-isopropyl acrylamide; 16% -20% of glycerol; 0.8 to 1 percent of potassium metabisulfite, and the balance of distilled water, wherein the total mass is 100 percent, and the pH value is 6 to 7.
2. The enema for intractable ulcerative colitis according to claim 1, wherein the enema comprises the following components in percentage by mass: 0.09% -0.1% of seclenic acid D; 20.06 to 0.07 percent of bitter stonecrop extract M; 3% -3.2% of radix rehmanniae extract; 0.2% -0.4% of butyrin; 0.01 to 0.03 per mill of nicotine; 0.7 to 1.1 percent of heartleaf houttuynia herb polysaccharide; tetrandrine 0.62-1.8%; hydrocortisone 0.2 ‰; 12% of thiolated chitosan; 11% of N-isopropylacrylamide; 18% of glycerol; 0.9 percent of potassium metabisulfite and the balance of water, wherein the total mass is 100 percent, and the pH value is 6-7.
3. The preparation method of the enema for treating ulcerative colitis is characterized by comprising the following steps:
step 1, preparing a radix rehmanniae extracting solution:
step 2, preparing houttuynia cordata polysaccharide:
step 3, weighing the following components in percentage by mass: 0.03% -0.16% of seclenic acid D; 20.03 to 0.09 percent of the bitter herb of rockfoil M; 2.5 to 3.8 percent of radix rehmanniae extract; 0.13 to 0.5 percent of butyrin; 0.008% -0.032% of nicotine; 0.48 to 1.29 percent of houttuynia polysaccharide; 0.62 to 1.8 percent of tetrandrine; hydrocortisone 0.03% -0.3 ‰; 2% -4% of thiolated chitosan; 8% -12% of N-isopropyl acrylamide; 16% -20% of glycerol; 0.8 to 1 percent of potassium metabisulfite, the balance of distilled water, the total mass of the potassium metabisulfite and the distilled water is 100 percent, and the pH value is 6 to 7;
step 4, preparing enzyme temperature double-sensitive type hydrogel components: adding weighed N-isopropyl acrylamide, glycerol and thiolated chitosan into 35% of distilled water, and magnetically stirring until the mixture is uniformly swelled, so that the mixture is fully swelled and reacts; then adding weighed potassium metabisulfite, secalinic acid D, bitter herb extract M2, radix rehmanniae extract, butyrin, nicotine, houttuynia polysaccharide, tetrandrine and hydrocortisone, stirring uniformly at room temperature, adding the remaining 65% of distilled water to full volume, adding a pH value regulator, regulating the pH value to be within 6-7, and stirring uniformly to prepare the enzyme temperature double-sensitive hydrogel component;
step 5, homogenizing: homogenizing by a pipeline type homogenizer, and uniformly stirring;
step 6, filling: filling into an administration device at sterile room temperature, and packaging.
4. The method according to claim 3, wherein the raw rehmannia root extract prepared in the step 1 is specifically: baking rehmanniae radix at 40-50 deg.C until eighty percent dry, pulverizing, sieving with 40 mesh sieve, adding 70% ethanol at a liquid-material ratio of 15: 1-25: 1(mL/g), ultrasonic treating at 25-35 deg.C for 35-45min, extracting for 2 times, filtering, separating, concentrating to obtain extractive solution, and vacuum drying to constant weight to obtain radix rehmanniae extract.
5. The preparation method according to claim 3, wherein the preparation of the houttuynia cordata polysaccharide in the step 2 is specifically: sun drying herba Houttuyniae, pulverizing, sieving with 100 mesh sieve to obtain herba Houttuyniae dry powder, placing herba Houttuyniae dry powder in a conical flask, adding 70% ethanol at ultrasonic power of 72W and ultrasonic temperature of 60-70 deg.C for 20-30min at liquid-material ratio of 35: 1-45: 1(mL/g), filtering, and separating to obtain extractive solution; putting the extracting solution into a centrifuge tube, centrifuging for 15-25min at 3500r/min of 2500-; adding 8mL of absolute ethanol into the concentrated solution, standing at 4 ℃ for 12h for alcohol precipitation, centrifuging at 3500r/min of 2500-.
6. The preparation method of claim 5, wherein the houttuynia cordata dry powder is prepared by soaking in petroleum ether in a Soxhlet extractor, defatting is completed by heating and refluxing 4 times with a heating jacket under a voltage of 70-80V for 12h, decoloring is performed by heating and refluxing 5 times with methanol, and finally air-drying naturally.
7. The method as claimed in claim 3, wherein the magnetic stirring time in step 4 is 50-60 ℃ and the rotation speed is 350-450 r/min.
8. The method according to claim 3, wherein the swelling reaction time in step 4 is 10 to 15 hours.
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