Gegen Qinlian decoction nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to a Gegen Qinlian decoction nanoparticle as well as a preparation method and application thereof. The Gegen Qinlian decoction nanoparticle is a natural nanoparticle formed by self-assembling a Gegen Qinlian decoction water extract, the particle size is 50-200nm, and the zeta potential is-(10-40) mV. The preparation method comprises the following steps: S1, dissolving the water extract of the Gegen Qinlian decoction, centrifuging, and carrying out microporous filtration on supernate to obtain filtrate; s2, the filtrate is placed in a dialysis bag for dialysis, and retentate in the dialysis bag is obtained; and S3, carrying out freeze-drying on the retentate, so as to obtain the Gegen Qinlian decoction nanoparticles. The Gegen Qinlian decoction nanoparticles have good biocompatibility and biodegradability, can significantly improve intestinal absorption and tissue distribution of active components of the Gegen Qinlian decoction, and have a significant anti-UC effect.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine preparations, and in particular to Gegenqinlian decoction nanoparticles and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) that originates in the rectum and extends to the colon, with clinical symptoms such as abdominal pain, diarrhea, bloody stools with mucus, pus, and malabsorption. UC has a slow onset, with varying degrees of severity and prone to relapse. Up to 50% of IBD patients have at least one extraintestinal manifestation (EIM), such as hepatobiliary disease.

[0003] At present, the means of treating UC are mainly related to regulating autoimmunity, reducing the expression of inflammatory cytokines, regulating T cells, anti-oxidative stress, regulating intestinal flora, etc. There is no clinical treatment method that can completely cure UC. Drugs such as aminosalicylic acid preparations, immune or biological inhibitors, and adrenal glucocorticoids are commonly used for treatment. However, the above therapies require long-term maintenance treatment and are prone to relapse. Long-term use of drugs has adverse effects on hematopoiesis, liver, kidney and other functions, and there are large individual differences in drug treatment, and the effect is unstable.

[0004] The Chinese Guidelines for the Diagnosis and Treatment of Ulcerative Colitis (2023 Edition) recommends the use of traditional Chinese medicine for patients with mild to moderate active left-sided UC, because traditional Chinese medicine has the characteristics of multiple components, multiple targets, and few adverse reactions. Gegen-Qinlian decoction (GQD) is a classic Chinese medicine prescription prepared with Pueraria root, Scutellaria baicalensis, Coptis chinensis, and Licorice in a mass ratio of 8:3:3:2 or 5:3:3:2. Clinically, GQD alone can relieve UC symptoms and prevent recurrence, and can also synergistically enhance the efficacy of modern drugs. In UC model mice, oral administration of GQD can significantly improve UC by reducing inflammation and oxidative stress, restoring colonic mucosal homeostasis, and regulating intestinal microbiota. In addition, GQD has a variety of other pharmacological effects, including liver protection and anti-lung injury, which means that GQD may alleviate the extraintestinal manifestations of UC.

[0005] After oral administration of the water extract of Gegenqinlian decoction, puerarin, berberine, baicalin, baicalein (the main metabolite of baicalin), glycyrrhizic acid and glycyrrhetinic acid (the main metabolite of glycyrrhizic acid) were exposed at high levels in the colon and liver of mice, and all of these components have potential anti-UC effects. In addition, the small molecule mixture of Gegenqinlian decoction, which is mainly composed of these components, can alleviate UC through anti-inflammatory and antioxidant mechanisms (Xu, BL, GJZhang, YBJi. Active components alignment of Gegenqinlian decoction protects ulcerative colitis by attenuating inflammatory and oxidative stress. Journal of Ethnopharmacology, 2015, 162: 253-260). These results indicate that the above components may be the pharmacological material basis for the anti-UC effect of Gegenqinlian decoction. However, the permeability and metabolic stability of puerarin, baicalin, berberine and glycyrrhizic acid are poor, which affects the efficacy of Gegenqinlian decoction.

[0006] In addition, the extracts of Gegenqinlian decoction and its modified prescriptions prepared by traditional decoction process cannot effectively deliver active ingredients to the distal colon after oral administration, and it is difficult to exert the maximum anti-UC efficacy. When administered by enema, patient compliance is poor and it is not conducive to the treatment of diffuse ulcerative colitis. The composition of ingredients prepared based on the main small molecule active ingredients of Gegenqinlian decoction is not conducive to its intestinal absorption, and its colon targeting and anti-UC efficacy are not clear, and it cannot exert the role of macromolecular components such as proteins and polysaccharides in Gegenqinlian decoction. In addition, the oral drug delivery system of the effective ingredients of Gegenqinlian decoction reported so far usually requires the addition of organic reagents, which poses a safety hazard. Therefore, it is of great significance to develop a new traditional Chinese medicine preparation of Gegenqinlian decoction to improve its efficacy for the treatment of UC. Summary of the invention

[0007] The purpose of the present invention is to provide Gegenqinlian decoction nanoparticles and their preparation method and application in view of the deficiencies in the prior art, so as to solve the problems of poor drug absorption, poor activity and poor safety of Gegenqinlian decoction in the related art. The present invention discloses the characteristics and potential advantages of natural nanoparticles in Gegenqinlian decoction for treating UC, and provides an innovative Gegenqinlian decoction component composition that can be taken orally for anti-UC and has the characteristics of drug-carrier integration.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is: to provide a Gegen Qinlian Decoction nanoparticle, which is a natural nanoparticle self-assembled from the Gegen Qinlian Decoction water extract; its particle size is 50-200nm, and the zeta potential is -(10-40)mV.

[0009] According to some embodiments of the present invention, the particle size of the Gegenqinliantang nanoparticles is 80-200 nm, and the zeta potential is -(14-30) mV.

[0010] According to some embodiments of the present invention, the Gegen Qinlian Decoction water extract is obtained by mixing Pueraria root, Astragalus, Coptis chinensis and Licorice in a weight ratio of (5-8):3:3:2 and then extracting with water.

[0011] According to some embodiments of the present invention, the Gegen Qinlian Decoction nanoparticles contain 5-20 mg / g of puerarin, 40-80 mg / g of baicalin, 10-40 mg / g of berberine, and 10-30 mg / g of glycyrrhizic acid. Preferably, the Gegen Qinlian Decoction nanoparticles contain 6.5-7.5 mg / g of puerarin, 60-70 mg / g of baicalin, 16-20 mg / g of berberine, and 10-15 mg / g of glycyrrhizic acid. More preferably, the Gegen Qinlian Decoction nanoparticles contain 6.94 mg / g of puerarin, 65.4 mg / g of baicalin, 18.3 mg / g of berberine, and 12.7 mg / g of glycyrrhizic acid.

[0012] In a second aspect of the present invention, a method for preparing Gegenqinlian decoction nanoparticles is provided, comprising the following steps:

[0013] S1, dissolving the Gegenqinlian decoction water extract and centrifuging it, taking the supernatant and performing microfiltration to obtain a filtrate;

[0014] S2, placing the filtrate in a dialysis bag for dialysis to obtain a retentate in the dialysis bag;

[0015] S3. After freeze-drying the retentate, the Gegenqinliantang nanoparticles are obtained.

[0016] According to some embodiments of the present invention, in step S2, placing the filtrate in a dialysis bag for dialysis specifically comprises the following steps: placing the filtrate in a dialysis bag with a molecular weight cutoff of 3000-10000Da, and the dialysis time is 24-72h.

[0017] According to some preferred embodiments of the present invention, in step S2, the filtrate is placed in a dialysis bag for dialysis, which specifically includes the following steps: placing the filtrate in a dialysis bag with a molecular weight cutoff of no more than 10,000 Da, placing the dialysis bag in a beaker containing 8-12 times the volume of pure water, placing a magnetic stirrer in the beaker and stirring at 200-400 rpm, changing the water every 8-16 hours, and the continuous dialysis time is 24-72 hours.

[0018] According to some preferred embodiments of the present invention, in step S2, the molecular weight cutoff of the dialysis bag is 10000Da, and the dialysis time is 24h; or

[0019] According to some preferred embodiments of the present invention, in step S2, the molecular weight cutoff of the dialysis bag is 3500 Da, and the dialysis time is 72 h.

[0020] According to some preferred embodiments of the present invention, in step S2, the stirring speed is 200 rpm.

[0021] According to some embodiments of the present invention, in step S1, the initial concentration of the Gegenqinlian decoction water extract is 0.1-0.2 g / mL.

[0022] According to some preferred embodiments of the present invention, the initial concentration of the Gegenqinlian decoction water extract is 0.15-0.2 g / mL.

[0023] According to some embodiments of the present invention, in step S1, the dissolution is performed by ultrasonic assisted dissolution for 1-2 hours.

[0024] According to some embodiments of the present invention, in step S1, the centrifugal rotation speed is 3000-5000 rpm, and the time is 5-15 min.

[0025] According to some embodiments of the present invention, in step S1, the microporous filtration is filtration using a 0.22 μm microporous filter membrane.

[0026] According to some embodiments of the present invention, the Gegen Qinlian Decoction water extract is obtained by mixing Pueraria root, Astragalus, Coptis chinensis and Licorice in a weight ratio of (5-8):3:3:2 and then decocting in water.

[0027] According to some preferred embodiments of the present invention, the method for preparing the Gegenqinlian decoction water extract specifically comprises:

[0028] S01, take the pueraria root slices and boil them separately with 8-12 times the volume of boiling water for 10-30 minutes, then mix them with scutellaria, coptis and liquorice in a weight ratio of (5-8):3:3:2, and boil them with 8-12 times the volume of boiling water for 0.5-2h, and filter the obtained extract through gauze;

[0029] S02, re-extract the residue with 8-12 volumes of water for 0.5-2h;

[0030] S03. Combine the decoctions from the two extractions, and dry them in a vacuum at 55° C.-65° C. to obtain a powder of the Gegenqinlian decoction water extract.

[0031] According to some more preferred embodiments of the present invention, the method for preparing the Gegenqinlian decoction water extract specifically comprises:

[0032] S01, take the pueraria root slices and boil them alone with 10 times the volume of boiling water for 20 minutes, then mix them with scutellaria, coptis and liquorice in a weight ratio of 8:3:3:2 or 5:3:3:2, boil them with 10 times the volume of boiling water for 1 hour, and filter the obtained extract through gauze;

[0033] S02, re-extract the residue with 8-12 volumes of water for 1 hour;

[0034] S03. Combine the decoctions from the two extractions, and dry them under vacuum at 60° C. to obtain the powder of the Gegenqinlian decoction water extract.

[0035] The Gegenqinlian decoction nanoparticles of the present invention are prepared by the above method.

[0036] In the third aspect of the present invention, a medicine or a pharmaceutical composition is provided, comprising the above-mentioned Gegenqinliantang nanoparticles.

[0037] In the fourth aspect of the present invention, a use of Gegenqinliantang nanoparticles in the preparation of a drug for treating ulcerative colitis is proposed.

[0038] According to one technical solution of the method technical solution of the present invention, at least the following beneficial effects are achieved:

[0039] (1) The Gegenqinlian decoction nanoparticles prepared by the present invention do not contain any organic reagent;

[0040] (2) The Gegenqinlian decoction natural nanoparticles prepared by the present invention have good biocompatibility and biodegradability;

[0041] (3) The preparation method of the natural nanoparticles of Gegenqinlian decoction provided by the present invention is simple and the quality is controllable;

[0042] (4) The natural nanoparticles of Gegenqinlian decoction prepared by the present invention can be used for oral administration and significantly improve the intestinal absorption and tissue distribution of the active ingredients of Gegenqinlian decoction;

[0043] (5) The natural nanoparticles of Gegenqinlian decoction prepared by the present invention have significant anti-UC effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0045] Figure 1 This is the scanning electron microscope observation result (20,000×) of the GQD-Nnps-N powder of the present invention.

[0046] Figure 2 This is the scanning electron microscope observation result (20,000×) of the GQD-Nnps-L powder of the present invention.

[0047] Figure 3 The graph is the concentration-time curve of the six active ingredients in the systemic circulation of mice after oral administration of the extract of Gegenqinlian decoction, the monomer mixture or GQD-Nnps-N of the present invention (mean±SD, n=5); AF are puerarin, berberine, baicalin, baicalein, glycyrrhizic acid and glycyrrhetinic acid, respectively.

[0048] Figure 4 This is the concentration-time curve of three components of Gegenqinlian decoction in the liver of mice after oral administration of the Gegenqinlian decoction extract, monomer mixture or GQD-Nnps-N of the present invention (mean ± standard deviation, n = 5); AC is puerarin, berberine and baicalin, respectively.

[0049] Figure 5 The present invention is the effect on the time-dependent changes in body weight of mice with ulcerative colitis (Mean±SD, n=8-10); ##, p<0.01 vs. normal; **, p<0.01 vs. Model.

[0050] Figure 6 : The effect of the present invention on the colon length of ulcerative colitis mice (Mean±SD, n=8-10); ##, p<0.01 vs. normal; *, p<0.05, **, p<0.01 vs. GQD-Nnps-L.

[0051] Figure 7 The figure shows the effect of the present invention on the DAI score of ulcerative colitis mice (Mean±SD, n=8-10); ##, p<0.01 vs. normal; **, p<0.01 vs. Model.

[0052] Figure 8 The present invention is a representative pathological section of the colon of ulcerative colitis mice (20×); A, normal group; B, model group; C, 5-ASA group; D, GQD extract group; E, GQD-Nnps-L group; F, monomer mixture group.

[0053] Fig. 9 The figure shows the effect of the present invention on the colon pathological section scores of ulcerative colitis mice; ##, p<0.01 vs. normal; **, p<0.01 vs. Model.

[0054] Fig.10This is the effect of the present invention on the spleen organ index of ulcerative colitis mice; ##, p<0.01 vs. normal; *, p<0.05 vs. Model. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solutions and advantages of the present application clearer, the present application is described and illustrated below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. Based on the embodiments provided in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present application.

[0056] Obviously, the drawings described below are only some examples or embodiments of the present application. For ordinary technicians in this field, the present application can also be applied to other similar scenarios based on these drawings without creative work. In addition, it can also be understood that although the efforts made in this development process may be complicated and lengthy, for ordinary technicians in this field related to the content disclosed in this application, some changes in design, manufacturing or production based on the technical content disclosed in this application are just conventional technical means, and should not be understood as insufficient content disclosed in this application.

[0057] Reference to "embodiments" in this application means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those of ordinary skill in the art that the embodiments described in this application may be combined with other embodiments without conflict.

[0058] Unless otherwise defined, the technical terms or scientific terms involved in this application should be understood by people with ordinary skills in the technical field to which this application belongs. The words "one", "a", "a", "the" and the like involved in this application do not indicate a quantitative limitation, and may represent the singular or plural. The terms "include", "comprise", "have" and any of their variations involved in this application are intended to cover non-exclusive inclusions; for example, a process or method that includes a series of steps or units (units) is not limited to the listed steps or units, but may also include steps or units that are not listed, or may also include other steps or units inherent to these processes, methods, and products. The "multiple" / "several" involved in this application refers to two or more. "And / or" describes the association relationship of associated objects, indicating that there may be three relationships, for example, "A and / or B" can represent: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the objects associated with each other are in an "or" relationship. The terms "first", "second", "third", etc. involved in this application are only used to distinguish similar objects, and do not represent a specific ordering of objects.

[0059] Unless otherwise specified, the reagents and consumables described in the present invention are all commercially available.

[0060] Example 1

[0061] In this embodiment, a kind of Gegenqinlian decoction natural nanoparticles was prepared, which were separated from the Gegenqinlian decoction water extract.

[0062] A method for preparing Gegenqinlian decoction natural nanoparticles, the specific preparation method is as follows:

[0063] ① Soak the slices of Pueraria lobata (Willd.) Ohwi (variety Pueraria lobata (Willd.) Ohwi, origin Anhui, batch number 230718), Scutellaria baicalensis Georgi (variety Scutellaria baicalensis Georgi, origin Shanxi, batch number 230807), Coptis chinensis Franch (variety Coptis chinensis Franch, origin Chongqing, batch number 230517), and Glycyrrhiza uralensis Fisch (variety Glycyrrhiza uralensis Fisch, origin Xinjiang, batch number 230614) purchased from Shanghai Kangqiao Chinese Medicine Piece Co., Ltd. for 0.5 hours. First, take the Pueraria lobata slices and decoct them separately with 10 volumes of boiling water for 20 minutes, then mix them with Scutellaria baicalensis, Coptis chinensis, and Glycyrrhiza uralensis in a weight ratio of 8:3:3:2, decoct them with 10 volumes of boiling water for 1 hour, and filter the obtained extract through 4 layers of gauze. Then re-extract the residue with 10 volumes of water for 1 hour. The decoctions of the two extractions were combined and dried in vacuum at 60°C to obtain the Gegenqinlian decoction water extract (named as GQD-extract) powder.

[0064] ② Dissolve GQD-extract in water at a certain mass concentration (such as 0.10, 0.15, 0.2 g / mL).

[0065] ③Ultrasonic dissolution for 1.5 h, centrifuge at 4,000 rpm for 10 min at room temperature, and collect the supernatant.

[0066] ④ Filter the supernatant with a 0.22 μm microporous filter membrane.

[0067] ⑤ Add the filtrate into a dialysis bag with a certain molecular weight cutoff (such as 3,500, 10,000Da), place the dialysis bag in a beaker containing 10 times the volume of pure water, put a magnetic stirrer in the beaker and stir at a certain speed (such as 200, 300, 400 rpm), change the water every 12 hours, and continue dialysis for a certain period of time (such as 24, 48, 72 hours).

[0068] ⑥ Freeze-dry the retained material in the dialysis bag at -45°C to obtain the Gegenqinlian decoction natural nanoparticle powder.

[0069] The above steps were used to optimize the conditions and prepare the natural nanoparticles of Gegenqinlian Decoction. The natural nanoparticles prepared under condition 1 (mass concentration 0.2 g / mL, dialysis bag molecular weight cutoff 10,000 Da, stirrer speed 200 rpm, dialysis for 24 hours) were named GQD-Nnps-N, and the natural nanoparticles prepared under condition 2 (mass concentration 0.15 g / mL, dialysis bag molecular weight cutoff 3,500 Da, stirrer speed 200 rpm, dialysis for 72 hours) were named GQD-Nnps-L.

[0070] Example 2

[0071] A method for preparing natural nanoparticles of Gegenqinlian decoction, the optimization process is as follows:

[0072] Three factors that have a greater impact on the preparation of Gegenqinlian decoction natural nanoparticles (Gegenqinlian decoction water extract concentration, dialysis time and stirring speed) were selected for orthogonal experiments, and three levels were designed for each factor, as shown in Table 1. The experiment was repeated three times using the orthogonal table, and the results were averaged.

[0073] Table 1. Orthogonal design factors and levels

[0074]

[0075]

[0076] The preparation steps are as follows:

[0077] (1) Dissolve the water extract of Gegenqinlian decoction in water at a certain concentration (e.g., 0.10, 0.15, 0.2 g / mL).

[0078] (2) Ultrasonic dissolution for 1.5 h.

[0079] (3) Centrifuge at 4,000 rpm for 10 min at room temperature and collect the supernatant.

[0080] (4) Filter the supernatant using a 0.22 μm microporous filter membrane.

[0081] (5) Add the filtrate to a dialysis bag with a molecular weight cutoff of 10,000 Da. Place the dialysis bag in a beaker containing 10 times the volume of pure water. Place a stirrer in the beaker and stir at a certain speed (such as 200, 300, 400 rpm). Change the water every 12 hours and continue dialysis for a certain period of time (such as 24, 48, 72 hours).

[0082] (6) The retentate in the dialysis bag was freeze-dried at -45°C.

[0083] An appropriate amount of the above-mentioned lyophilized powder was accurately weighed, and a 1 mg / mL nanoparticle solution was prepared with pure water, and the particle size and zeta potential of the nanoparticles were detected by dynamic light scattering technology. In addition, an appropriate amount of the above-mentioned lyophilized powder was accurately weighed, and a 1 mg / mL solution was prepared with 80% methanol solution, and the content of several major active ingredients therein was detected by LC-MS / MS.

[0084] The results are as follows.

[0085] (1) Table 2 shows that the concentration of Gegenqinlian decoction extract, dialysis time and stirring speed have a weak effect on the size and zeta potential of GQD-Nnps-N, but have a significant effect on the content and encapsulation efficiency of the four representative active ingredients.

[0086] Based on the above results, the optimal preparation process of GQD-Nnps-N was determined as follows: the initial concentration of the Gegenqinlian decoction extract was 200 mg / mL, the dialysis time was 24 hours, and the stirring speed was 200 rpm. From the experimental results, the stirring speed had little effect on the preparation of nanoparticles, while the initial concentration of the Gegenqinlian decoction extract and the dialysis time had a greater impact on the representative active ingredients in the nanoparticles and the encapsulation rate.

[0087] In Table 2, A, dialysis time (h); B, stirring speed (rpm / min); C, concentration of Gegenqinliantang extract (g / ml). Ber, berberine; Bai, baicalin; Pue, puerarin; Gly, glycyrrhizic acid.

[0088] Table 2. Orthogonal experiment results (mean ± SD, n = 3)

[0089]

[0090]

[0091] (2) After the GQD-Nnps-N nanoparticle powder was sprayed with gold and vacuum dried, the results were observed by scanning electron microscopy ( Figure 1 ) showed that GQD-Nnps-N was composed of spherical particles.

[0092] Example 3

[0093] A method for preparing Gegenqinlian decoction natural nanoparticles, the specific preparation steps are as follows:

[0094] (1) Weigh 144 g of Gegenqinliantang extract powder and dissolve it in 960 mL of pure water (i.e., 150 mg / mL), and stir thoroughly;

[0095] (2) Ultrasonic dissolution for 1.5 h;

[0096] (3) Centrifugation at 4,000 rpm for 10 min at room temperature;

[0097] (4) Filter the supernatant with a 0.22 μm microporous membrane;

[0098] (5) The filtrate was dialyzed continuously for 3 days using membrane dialysis (molecular weight cut-off 3,500 Da);

[0099] (6) The retentate in the dialysis bag was freeze-dried at -45°C to obtain a powder of Gegenqinlian decoction natural nanoparticles, which was named GQD-Nnps-L.

[0100] The initial concentration of the Gegenqinlian decoction extract, the molecular weight cutoff of the dialysis membrane, and the dialysis time in the preparation method are adjusted to obtain Gegenqinlian decoction natural nanoparticles.

[0101] 2. Evaluation indicators and results

[0102] An appropriate amount of the above-mentioned GQD-Nnps-L lyophilized powder was accurately weighed, and a 1 mg / mL nanoparticle solution was prepared with pure water, and the particle size and zeta potential in the solution were detected by dynamic light scattering experiment. In addition, an appropriate amount of the above-mentioned lyophilized powder was accurately weighed, and a 1 mg / mL nanoparticle solution was prepared with 80% methanol solution, and the content of several active ingredients therein was detected by LC-MS / MS.

[0103] The results are as follows:

[0104] (1) 144 g of Gegenqinlian decoction extract powder can produce 5.3 g of GQD-Nnps-L freeze-dried powder, with a yield of about 3.7%.

[0105] (2) The content of puerarin in this test was 6.94 mg / g, baicalin 65.4 mg / g, berberine 18.3 mg / g, and glycyrrhizic acid 12.7 mg / g. mg / g represents the content of the component per gram of GQD-Nnps-L freeze-dried powder. Due to batch differences, the content range of the components is: puerarin 5-20 mg / g, baicalin 40-80 mg / g, berberine 10-40 mg / g, and glycyrrhizic acid 10-30 mg / g in Gegenqinliantang nanoparticles.

[0106] (3) The particle size and zeta potential of the nanoparticles in the GQD-Nnps-L solution are shown in Table 3. In addition, the nanoparticles have good stability, and their particle size and zeta potential do not change significantly after being placed at 4°C and room temperature (22°C) for different time periods.

[0107] Table 3. Size and Zeta potential of GQD-Nnps-L under storage conditions

[0108]

[0109]

[0110] Table 3 shows the particle size and Zeta potential (mean ± SD, n = 3) of GQD-Nnps-L stored at 4 ° C or 22 ° C for different time periods after preparation. Control refers to the data of the newly prepared nanoparticles. PDI (Polydispersity Index) refers to the polydispersity index. For a nanoparticle solution, the PDI value of GQD-Nnps-L is low, indicating that the particle size is relatively uniform and the particle size distribution is narrow.

[0111] (4) After the GQD-Nnps-L nanoparticle powder was sprayed with gold and vacuum dried, the results were observed by scanning electron microscopy ( Figure 2 ) showed that GQD-Nnps-L was composed of spherical particles.

[0112] Example 4

[0113] This example verifies the effect of Gegenqinliantang nanoparticles and active ingredients on the pharmacokinetics of mice blood. The animal experiments were approved by the Ethics Committee of Shanghai University of Traditional Chinese Medicine / Shanghai Institute of Traditional Chinese Medicine, with the approval number ZSHUTCM211018015. The animal experiments were conducted at the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine in accordance with the guidelines of the Ethics Committee.

[0114] DSS was used to induce UC in mice. Ten male C57BL / 6J mice were used as the control group and given normal drinking water. In addition, 135 male C57BL / 6J mice were given a 3% DSS aqueous solution, which was changed every 2 days for 7 consecutive days. The success of UC induction was evaluated by monitoring the body weight of the mice.

[0115] On day 8, UC mice were randomly divided into 3 large groups according to body weight, and each group was further divided into 9 small groups (n = 5 in each group). The 3 large groups were given 6.1 g / kg (clinically effective dose equivalent dose) of Gegenqinlian decoction extract (first batch), a mixture of 4 small molecule components of Gegenqinlian decoction (equivalent to the dose of each component in Gegenqinlian decoction extract, namely 0.247 g / kg of puerarin, 0.108 g / kg of berberine, 0.277 g / kg of baicalin and 0.028 g / kg of glycyrrhizic acid) and 4.197 g / kg of GQD-Nnps-N (the dose of each component was 0.016 g / kg of puerarin, 0.130 g / kg of berberine, 0.235 g / kg of baicalin and 0.029 g / kg of glycyrrhizic acid) by gavage. At predetermined time points after administration (0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24 h), mice were anesthetized with Shutai, and blood was collected to prepare plasma with heparin anticoagulation; the liver was removed, rinsed with distilled water, and quickly frozen in liquid nitrogen. Plasma and liver samples were stored at -80°C. Liver homogenates were prepared with 5-10 times the volume of water. The concentrations of four components and two metabolites (i.e., baicalin and glycyrrhetinic acid) in biological samples were determined using LC-MS / MS methods.

[0116] like Figure 3 As shown in Table 4, each component showed a second peak at 8 or 12 h after administration, indicating that it was absorbed in the colon. It is worth noting that the second peak was significantly higher than the first peak, indicating that the colon plays a dominant role in the intestinal absorption of the components of Gegenqinlian decoction during UC. In addition, compared with the Gegenqinlian decoction extract group, GQD-Nnps-N( Figure 3 and Figure 4 The time delay to reach the second absorption peak in the GQD-Nnps) group and the monomer mixture group is expressed in . In terms of exposure level, the concentration of all components in the Gegenqinlian decoction extract group was the lowest. The exposure levels of puerarin and glycyrrhizic acid were the highest in the monomer mixture group, while the exposure levels of berberine, baicalin, baicalein and glycyrrhetinic acid were the highest in the GQD-Nnps-N group. After correction by the dosage, the exposure level of puerarin in the GQD-Nnps-N group was also the highest.

[0117] Depend on Figure 4It can be seen that only puerarin, berberine and baicalein can produce complete CT curves in the liver. Table 4 shows the detailed pharmacokinetic parameters, which are the pharmacokinetic parameters (Mean, n=5) of the six components of Gegenqinlian decoction in the systemic circulation and liver after oral administration of Gegenqinlian decoction extract, monomer mixture or GQD-Nnps-N to mice. Similar to the systemic circulation, the CT curves of these components are bimodal, and the second peak (i.e., the highest peak) appears 8 hours after administration. The exposure level of puerarin in the Gegenqinlian decoction extract group is the highest, while the exposure level of baicalein in the GQD-Nnps-N group is the highest. If calibrated by the dosage, the exposure level of puerarin in the GQD-Nnps-N group is also the highest.

[0118] Table 4. Pharmacokinetic parameters in systemic circulation and liver

[0119]

[0120]

[0121] In Table 4, ND means not detected. The data in brackets were obtained by normalizing the experimental data to the doses of the corresponding components in the GQD extract. Puerarin: Puerarin; Berberine: Berberine; Baicalin: Baicalin; Baicalein: Baicalein; Glycyrrhizic acid: Glycyrrhizic acid; Glycyrrhetinic acid: Glycyrrhetinic acid.

[0122] The pharmacokinetic experimental results show that GQD-Nnps-N can promote the absorption of active ingredients from the colon of UC mice and significantly increase the exposure levels of several active ingredients in the blood and liver of mice.

[0123] Although this example only uses GQD-Nnps-N nanoparticles to conduct pharmacokinetic experiments in mouse blood, since GQD-Nnps-L nanoparticles have similar structures and properties, it can be seen that GQD-Nnps-L nanoparticles should have similar pharmacokinetic properties.

[0124] Example 5

[0125] This example verifies the anti-UC effect of Gegenqinliantang nanoparticles in mice. The animal experiment was approved by the Ethics Committee of Shanghai University of Traditional Chinese Medicine / Shanghai Institute of Traditional Chinese Medicine, with the approval number PZSHUTCM2502180001. The animal experiment was conducted at the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine in accordance with the guidelines of the Ethics Committee.

[0126] The mice were divided into 6 groups (n=10 per group), namely normal group, model group, positive control (5-ASA, 5-aminosalicylic acid) group, Gegenqinlian decoction extract group and GQD-Nnps-L group. The normal group was given ordinary drinking water, and the other groups were given 2.5% DSS aqueous solution and drank freely to prepare UC model. Drug administration was started at the same time as modeling: the normal group and the model group were gavaged with 0.2 mL / 10 g body weight of pure water every day; the 5-ASA group was gavaged with 200 mg / kg of 5-ASA every day; the Gegenqinlian decoction extract group was gavaged with 5.6 g / kg (clinically effective dose equivalent dose) of Gegenqinlian decoction extract every day (the second batch; the total content of puerarin, baicalin, berberine, and glycyrrhizic acid was 116.6 μg / mg extract, and the total dose was 653 mg / kg); the GQD-Nnps-L group was gavaged with 2.6 g / kg every day (the total content of puerarin, baicalin, berberine, and glycyrrhizic acid was 103.2 μg / mg extract, and the total dose was 268.3 mg / kg).

[0127] The disease activity index (DAI) of mice was scored every day according to the weight change of mice, the consistency of feces and the color of the test results of fecal occult blood kit until the end of the experiment. After 7 days of modeling, the 2.5% DSS aqueous solution in each group was replaced with ordinary drinking water and the drug administration continued. 4 hours after the last drug administration on the ninth day, all mice were killed and blood and tissue samples were collected.

[0128] The test results are as follows:

[0129] (1) Figure 5 As shown, on the eighth day of modeling, the body weight of mice in the model group was significantly decreased compared with that of mice in the normal group (p<0.01), while the body weight of mice in the GQD extract (p<0.01), monomer mixture (p<0.01) and GQD-Nnps-L (p<0.01) administration groups were significantly higher than that of the model group.

[0130] (2) Figure 6 As shown, compared with the normal group, the colon length of the model group mice was significantly shortened (p<0.01); there was no significant difference in the colon length between the GQD extract (p>0.05) and monomer mixture (p>0.05) administration groups and the model group; while the colon of the GQD-Nnps-L administration group was significantly longer than that of the model group (p<0.01), 5-ASA group (p<0.01) and Gegenqinlian decoction extract group (p<0.05).

[0131] (3) Figure 7As shown, on the eighth day of modeling, the DAI scores of mice in the model group were significantly higher than those in the normal group (p<0.01), while the DAI scores of mice in the GQD extract (p<0.01), monomer mixture (p<0.01) and GQD-Nnps-L (p<0.01) administration groups were significantly lower than those in the model group.

[0132] (4) HE staining results of mouse colon sections ( Figure 8 ) showed that compared with the normal group, the colon tissue of the model group mice was severely damaged, accompanied by tissue necrosis and shedding, and obvious inflammatory cell infiltration. The crypt structure of the colon of the mice in the GQD extract, GQD-Nnps-L and monomer mixture administration groups was intact, the inflammatory cell infiltration was significantly reduced, and the pathological score ( Fig. 9 ) were significantly lower than those in the model group (all p<0.01).

[0133] (5) Fig.10 As shown, the spleen index of mice in the model group was significantly greater than that in the normal group (p<0.01), while the spleen index of mice in the GQD-Nnps-L and monomer mixture administration groups was significantly lower than that in the model group (all p<0.05), indicating that the activation of the systemic immune system of mice in the two administration groups was alleviated.

[0134] The experimental results show that GQD-Nnps-L has a very significant anti-UC effect; moreover, in terms of the effect of protecting colon length, GQD-Nnps-L is stronger than the extract of Gegenqinlian decoction and the mixture of four small molecule components.

[0135] In the present invention, a natural nanoparticle (named GQD-Nnps-N) was first isolated from the water extract of Gegenqinlian Decoction, and its pharmacokinetic characteristics in UC model mice induced by dextran sodium sulfate (DSS) after oral administration were studied. By optimizing the preparation method, another natural nanoparticle (named GQD-Nnps-L) was isolated from the GQD extract, and its anti-UC effect on DSS-induced model mice was fully identified and evaluated.

[0136] The embodiment of the present invention separates natural nanoparticles from the water extract of Gegenqinlian decoction and optimizes its preparation process to obtain natural nanoparticles derived from Gegenqinlian decoction. The experimental results of the embodiment verify that the Gegenqinlian decoction nanoparticles can promote the absorption of active ingredients from the colon of UC mice and can significantly increase the exposure levels of several active ingredients in the blood and liver of mice; the Gegenqinlian decoction nanoparticles have a very significant anti-UC effect, especially in terms of the effect of protecting the length of the colon, the Gegenqinlian decoction nanoparticles are stronger than the Gegenqinlian decoction extract and the mixture of four small molecule components.

[0137] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A Gegenqinlian decoction nanoparticle, characterized in that: The Gegenqinlian decoction nanoparticles are natural nanoparticles formed by self-assembly of the Gegenqinlian decoction water extract; the particle size is 50-200nm, and the zeta potential is -(10-40)mV.

2. The Gegenqinliantang nanoparticles according to claim 1, characterized in that: The water extract of Gegenqinlian decoction is obtained by mixing Pueraria root, Astragalus, Coptis chinensis and Licorice in a weight ratio of (5-8):3:3:2 and then extracting with water.

3. A method for preparing Gegenqinliantang nanoparticles, characterized in that: The following steps are involved: S1, dissolving the water extract of Gegenqinlian decoction and centrifuging it, taking the supernatant and performing microfiltration to obtain a filtrate; S2, placing the filtrate in a dialysis bag for dialysis to obtain a retentate in the dialysis bag; S3. After freeze-drying the retentate, the Gegenqinliantang nanoparticles are obtained.

4. The preparation method according to claim 3, characterized in that: In step S2, the filtrate is placed in a dialysis bag for dialysis, which specifically includes the following steps: the filtrate is placed in a dialysis bag with a molecular weight cutoff of 3000-10000Da, and the dialysis time is 24-72h.

5. The preparation method according to claim 4, characterized in that: The molecular weight cut-off of the dialysis bag is 10000Da, and the dialysis time is 24h; or The molecular weight cut-off of the dialysis bag is 3500Da, and the dialysis time is 72h.

6. The preparation method according to claim 3, characterized in that: In step S1, the initial concentration of the Gegen Qinlian Decoction water extract is 0.1-0.2 g / mL; preferably, the initial concentration of the Gegen Qinlian Decoction water extract is 0.15-0.2 g / mL.

7. The preparation method according to claim 3, characterized in that: In step S1, the dissolution is performed by ultrasonic dissolution for 1-2 hours; and / or The centrifugal speed is 3000-5000 rpm and the time is 5-15 min; and / or The microporous filtration is carried out by using a 0.22 μm microporous filter membrane.

8. The preparation method according to claim 3, characterized in that: The Gegenqinlian decoction water extract is obtained by mixing Pueraria root, Astragalus, Coptis chinensis and Licorice in a weight ratio of (5-8):3:3:2 and then decocting in water; and / or The preparation method of the Gegenqinlian decoction water extract specifically comprises: S01, take the pueraria root slices and boil them separately with 8-12 times the volume of boiling water for 10-30 minutes, then mix them with scutellaria, coptis and liquorice in a weight ratio of (5-8):3:3:2, and boil them with 8-12 times the volume of boiling water for 0.5-2h, and filter the obtained extract through gauze; S02, re-extract the residue with 8-12 volumes of water for 0.5-2h; S03. Combine the decoctions from the two extractions, and dry them in a vacuum at 55° C.-65° C. to obtain a powder of the Gegenqinlian decoction water extract.

9. A drug or a pharmaceutical composition, characterized in that The medicine or pharmaceutical composition comprises the Gegenqinlian decoction nanoparticles as described in claim 1 or 2 and / or the Gegenqinlian decoction nanoparticles prepared by the preparation method according to any one of claims 3-8.

10. Use of Gegenqinliantang nanoparticles in the preparation of a drug for treating ulcerative colitis, characterized in that: The Gegenqinlian decoction nanoparticles are the Gegenqinlian decoction nanoparticles as described in claim 1 or 2 and / or the Gegenqinlian decoction nanoparticles prepared by the preparation method according to any one of claims 3 to 8.

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