Controlled-release preparation of an extract composition of *Oudemansiella raphanipies* for treating ulcerative colitis

Abstract

The present invention provides a controlled-release preparation for treating ulcerative colitis based on an extract composition of Oudemansiella raphanipes, and the controlled-release preparation is nanoparticles comprising the extract composition of Oudemansiella raphanipes as an active ingredient; the preparation method of the nanoparticles comprises the steps of: ultrasonically dispersing the extract composition of Oudemansiella raphanipes uniformly in ethanol; adding an aqueous solution of ferric sulfate heptahydrate with konjac glucomannan oxidized and gelatin as solutes, stirring evenly, and then performing ultrasonic dispersion treatment in a mixed solution composed of span80 and liquid paraffin; centrifuging, discarding the supernatant, blowing the precipitate with anhydrous methanol, discarding the supernatant after centrifugation, and taking the precipitate to obtain the nanoparticles. The extract composition of Oudemansiella raphanipes provided by the present invention has good curative effects on ulcerative colitis and can reduce side effects, having certain advantages compared with traditional typical western medicines; the present invention lays a certain foundation for the related research of Oudemansiella raphanipes and improves the medicinal value of Oudemansiella raphanipes.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug controlled release preparations, relates to the treatment technology of ulcerative colitis, and particularly relates to a controlled release preparation for treating ulcerative colitis based on an extract composition of Termitomyces albuminosus. Background Art

[0002] Ulcerative colitis is a chronic non-specific inflammatory bowel disease, the cause of which has not been clearly studied. The lesions are mostly located in the sigmoid colon and rectum, and can extend to the descending colon and even the entire colon. At present, in addition to surgical resection for the treatment of ulcerative colitis, the treatment methods mainly include the use of aminosalicylate drugs, glucocorticoids, immunosuppressants and anti-infective drugs. However, these drugs usually have defects such as unstable drug efficacy, more side effects and being unsuitable for long-term use.

[0003] In recent years, for the treatment of ulcerative colitis, in order to overcome the defects of the above traditional western medicines, some useful traditional Chinese medicine formulas have been gradually developed. However, traditional Chinese medicine formulas are limited by the constitutions of different people and the symptoms of the disease, and it is difficult to be applied on a large scale; at the same time, there are still many deficiencies in the current research of traditional Chinese medicine prescriptions, and it has not been able to well promote the further development of research in this area [1] . In addition to the research of traditional Chinese medicine prescriptions, developing corresponding extracts based on the effects of natural drugs is also an effective way. For example, Guangzhou University of Chinese Medicine found the application of the extract of Croton crassifolius Geisel in the preparation of drugs for preventing and / or treating ulcerative colitis in ZL202110401877.1, and the therapeutic effect of the low-dose extract of Croton crassifolius Geisel is similar to that of mesalazine; again, Guizhou Key Laboratory of Natural Product Chemistry, Chinese Academy of Sciences found that the related extract of Rosa roxburghii Tratt pomace can have an anti-colitis effect in ZL202110021889.1; and Yunnan University of Chinese Medicine found that the total flavonoid extract of Desmodium triflorum (L.) DC. has the effects of preventing and treating ulcerative colitis in ZL201811193741.0.

[0004] However, as far as the inventor knows, there are very few reports on natural drug extracts that can treat ulcerative colitis, and the extraction reagents and methods are different. People have not yet mastered the research rules, resulting in most of the relevant achievements being accidental discoveries, which to a certain extent limits the development of this field.

[0005] In addition, in the existing research reports on treating ulcerative colitis with natural drug extracts, there are few reports on developing corresponding sustained-release preparations. The possible reason is that the components of natural drug extracts are usually relatively complex, and the release rates of related different components may be affected by the carrier, resulting in poor therapeutic effects of using sustained-release preparations. Therefore, there are still difficulties in the research and development of sustained-release carriers for multi-component active substances such as natural drug extracts.

[0006] It is worth noting that traditional therapeutic drugs, such as mesalazine, are prone to side effects such as leukopenia [6], and there are few reports on how to reduce the side effects when using natural drug extracts to treat ulcerative colitis.

[0007] Oudemansiella raphanipes is an edible and medicinal mushroom, with a fresh, tender, mellow and fragrant taste, and is deeply loved by people. In the research on the pharmacological effects of Oudemansiella raphanipes, it is currently mainly focused on Oudemansiella raphanipes polysaccharide. For example, Chen Zhuojuan et al. [2] , Miao Jingnan et al. [3] both studied the extraction process of Oudemansiella raphanipes polysaccharide and its antioxidant activity; Zhao Huajie et al. [4] studied the repair effect of Oudemansiella raphanipes mycelium polysaccharide on chronic liver injury. As far as the inventor knows, there are very few reports on using Oudemansiella raphanipes extract to treat ulcerative colitis. The inventor only found that Kong Deyin [5] studied the treatment effect of Oudemansiella raphanipes mycorrhizal polysaccharide on ulcerative colitis in mice in his paper, but the curative effect of the obtained extract is limited, and there is a significant gap compared with the curative effect of mesalazine.

[0008] Therefore, whether it is possible to obtain extracts with better effects and corresponding sustained-release preparations for the treatment of ulcerative colitis based on Oudemansiella raphanipes is a research topic worthy of study.

[0009] References:

[0010] [1] Liu Zhenju, Li Yingqian, Li Huashan. Evaluation of the reporting quality of randomized controlled trials of oral traditional Chinese medicine prescriptions for ulcerative colitis based on the CONSORT statement [J]. Modern Digestion & Intervention, 2022(006):027.

[0011] [2] Chen Zhuojuan, Zhou Qian, Yang Zhiqiang. Study on the extraction process and antioxidant activity of Oudemansiella raphanipes polysaccharide [J]. China Condiment, 2023, 48(3):199-203.

[0012] [3] Miao Jingnan, Dong Jiamu, Liu Shumin et al. Optimization of ultrasonic-assisted extraction conditions of Oudemansiella raphanipes polysaccharide and study on its antioxidant activity [J]. Journal of Food Safety and Quality, 2023, 14(12):109-116.

[0013] [4] Zhao Huajie. Structural characterization of Oudemansiella raphanipes mycelium polysaccharide and its study on the repair of chronic liver injury [D]. Shandong Agricultural University, 2020.

[0014] [5] Kong Deyin. Optimization of the extraction of Oudemansiella raphanipes mycorrhizal polysaccharide and its effect on ulcerative colitis in mice [D]. Shandong Agricultural University, 2021.

[0015] [6] Liu Wen. Observation on the curative effect of mesalazine in the treatment of ulcerative colitis [J]. Inner Mongolia Journal of Traditional Chinese Medicine, 2013, 32(32). Summary of the Invention

[0016] The object of the present invention is to provide a controlled-release preparation which has better effects than traditional western medicines for treating ulcerative colitis and can overcome their side effects in view of the deficiencies and research needs of the existing technology. A more specific research object is to develop a controlled-release preparation based on Termitomyces albuminosa, which has excellent therapeutic effects on ulcerative colitis and can overcome its side effects.

[0017] In view of the above object, the present invention provides the following technical solutions:

[0018] A controlled-release preparation based on an extract composition of Termitomyces albuminosa for treating ulcerative colitis, wherein the controlled-release preparation is nanoparticles comprising the extract composition of Termitomyces albuminosa as an active ingredient;

[0019] The preparation method of the nanoparticles comprises the steps of: (1) ultrasonically dispersing the extract composition of Termitomyces albuminosa uniformly in 50 v / v% ethanol; (2) adding an aqueous solution of ferric sulfate heptahydrate with konjac glucomannan oxidized and gelatin as a solute in a weight ratio of 2:1 to the product obtained in step (1), stirring evenly, and then adding it to a mixed solution composed of span80 and liquid paraffin in a volume-to-weight ratio of 2 g:40 mL for ultrasonic dispersion treatment; (3) centrifuging the product obtained in step (2), discarding the supernatant, blowing the precipitate with absolute methanol, discarding the supernatant after centrifugation, and taking the precipitate to obtain the product;

[0020] The extract composition of Termitomyces albuminosa is composed of a total flavonoid extract of Termitomyces albuminosa and an aqueous extract of Termitomyces albuminosa in a weight ratio of 5-7:1;

[0021] Among them, the total flavonoid extract of Termitomyces albuminosa is obtained by taking the dried powder of Termitomyces albuminosa fruiting body as an extraction substrate, 70 v / v% ethanol as an extraction reagent, at a liquid-to-solid weight ratio of 25:1, extracting at 70°C for 2 hours under ultrasonic assistance, and taking the dry powder after rotary evaporation of the extract;

[0022] The aqueous extract of Termitomyces albuminosa is obtained by taking the dried powder of Termitomyces albuminosa fruiting body as an extraction substrate, water as an extraction reagent, at a liquid-to-solid weight ratio of 10:1, extracting at 80°C for 3 hours, and taking the dry powder after rotary evaporation of the extract.

[0023] Preferably, the weight ratio of the total flavonoid extract of Termitomyces albuminosa to the aqueous extract of Termitomyces albuminosa is 6:1.

[0024] Preferably, when obtaining the dried powder of Termitomyces albuminosa fruiting body, first dry the Termitomyces albuminosa fruiting body, and then pulverize it and pass through a 40-mesh sieve.

[0025] Preferably, when preparing the nanoparticles, in step (1), the weight ratio of the black-skinned Convolvulus argenteus extract to 50 v / v% ethanol is 1:10.

[0026] Preferably, when preparing the nanoparticles, in step (2), the weight fraction of the solute composed of oxidized konjac glucomannan and gelatin in a weight ratio of 2:1 is 15%.

[0027] Preferably, when preparing the nanoparticles, the weight ratio of the product obtained in step (1) to the aqueous solution of ferric sulfate heptahydrate whose solute is composed of oxidized konjac glucomannan and gelatin in a weight ratio of 2:1 is 1:3; and / or, when preparing the nanoparticles, in step (3), the precipitate is blown with anhydrous methanol, the supernatant is discarded after centrifugation, and the precipitate is taken, and the treatment process is repeated at least three times.

[0028] Preferably, when preparing the nanoparticles, the concentration of ferric sulfate heptahydrate in the ferric sulfate heptahydrate aqueous solution is 50 mg / mL.

[0029] The treatment of ulcerative colitis includes alleviating colon damage, reducing the content of pro-inflammatory factors in serum, and alleviating blood in stool and diarrhea.

[0030] The present invention also provides the use of the controlled-release preparation for treating ulcerative colitis based on the black-skinned Coniscentrum argenteus extract composition in treating ulcerative colitis.

[0031] As shown in the experimental examples in the specific embodiments of the present invention, although the present invention initially discovered that the total flavonoid extract of Alpinia arborescens also has the effect of treating ulcerative colitis, similar to the "Optimization of Extraction of Mycorrhizal Polysaccharides from Alpinia arborescens and Its Effect on Ulcerative Colitis in Mice", the resulting extract does not have an advantage in therapeutic efficacy compared to traditional Western medicine. Through continuous research on extraction methods and combinations of extracts from Alpinia arborescens, the inventors ultimately discovered that the technical solution of the present invention can achieve significant therapeutic effects for ulcerative colitis, while as shown in Comparative Examples 1-4 of the present invention, the related therapeutic effects of other extraction methods and extract combinations are less than satisfactory.

[0032] It is worth noting that, compared with other natural medicinal extracts that are effective in treating ulcerative colitis, the present invention also overcomes the defect that these extracts are difficult to achieve or exceed the efficacy of traditional related Western medicines, and solves the shortcomings in the research field of natural medicinal extracts for treating ulcerative colitis.

[0033] In addition, in response to the problem that people have insufficient research on black-skinned chicken mushroom, the present invention provides a basis for the research on the relevant effects of black-skinned chicken mushroom in a certain sense.

[0034] Importantly, as shown in the related embodiments and experimental examples of the present invention, through exploratory research on sustained-release preparations, it is found that the use of the controlled-release preparation of the present invention can further improve the therapeutic effect of ulcerative colitis. When using other sustained-release preparations, the impact on the therapeutic effect is not significant, and even the therapeutic effect may be reduced.

[0035] It is worth pointing out that the present invention also finds that the extract composition of Termitomyces heimii can also produce a synergistic effect with 5-aminosalicylic acid in the treatment of ulcerative colitis, and the sustained-release preparation can also reduce the side effects during the treatment process.

[0036] Advantages of the present invention:

[0037] The controlled-release preparation for treating ulcerative colitis provided by the present invention based on the extract composition of Termitomyces heimii has a good therapeutic effect on ulcerative colitis and has certain advantages compared with traditional typical western medicines; the controlled-release preparation of the present invention can reduce the side effects during the treatment of ulcerative colitis; the present invention has laid a certain foundation for the related research of Termitomyces heimii and enhanced the medicinal value of Termitomyces heimii. Detailed implementation manners

[0038] Raw materials used in the following examples and comparative examples:

[0039] Fruiting bodies of Termitomyces heimii, commercially available; ethanol, water, owned by the laboratory; mesalazine, 5-aminosalicylic acid, commercially available.

[0040] Example 1

[0041] (1) Dry the fruiting bodies of Termitomyces heimii, then crush them and pass through a 40-mesh sieve;

[0042] (2) Using the dried powder of the fruiting bodies of Termitomyces heimii as the extraction substrate, 70 v / v% ethanol as the extraction reagent, at a liquid-solid weight ratio of 25:1, extract at 70 °C for 2 hours under ultrasonic assistance (60 w), perform rotary evaporation on the extract, and take the dry powder to obtain the total flavonoid extract of Termitomyces heimii;

[0043] (3) Using the dried powder of the fruiting bodies of Termitomyces heimii as the extraction substrate, water as the extraction reagent, at a liquid-solid weight ratio of 10:1, extract at 80 °C for 3 hours, perform rotary evaporation on the extract, and take the dry powder to obtain the water extract of Termitomyces heimii;

[0044] (4) Mix the total flavonoid extract of Termitomyces heimii and the water extract of Termitomyces heimii according to a weight ratio of 6:1 to obtain the extract composition.

[0045] Example 2

[0046] (1) Dry the fruiting bodies of Termitomyces heimii, then crush them and pass through a 40-mesh sieve;

[0047] (2) Using the dried powder of the fruiting bodies of *Oudemansiella raphanipies* as the extraction substrate, ethanol at 70 v / v% as the extraction reagent, at a liquid-solid weight ratio of 25:1, with ultrasonic assistance (60 w), extract at 70 °C for 2 hours. After rotary evaporation of the extract, take the dried powder to obtain the total flavonoid extract of *Oudemansiella raphanipies*;

[0048] (3) Using the dried powder of the fruiting bodies of *Oudemansiella raphanipies* as the extraction substrate, water as the extraction reagent, at a liquid-solid weight ratio of 10:1, extract at 80 °C for 3 hours. After rotary evaporation of the extract, take the dried powder to obtain the water extract of *Oudemansiella raphanipies*;

[0049] (4) Mix the total flavonoid extract of *Oudemansiella raphanipies* and the water extract of *Oudemansiella raphanipies* according to a weight ratio of 5:1 to obtain an extract composition.

[0050] Example 3

[0051] (1) Dry the fruiting bodies of *Oudemansiella raphanipies*, then pulverize and pass through a 40-mesh sieve;

[0052] (2) Using the dried powder of the fruiting bodies of *Oudemansiella raphanipies* as the extraction substrate, ethanol at 70 v / v% as the extraction reagent, at a liquid-solid weight ratio of 25:1, with ultrasonic assistance (60 w), extract at 70 °C for 2 hours. After rotary evaporation of the extract, take the dried powder to obtain the total flavonoid extract of *Oudemansiella raphanipies*;

[0053] (3) Using the dried powder of the fruiting bodies of *Oudemansiella raphanipies* as the extraction substrate, water as the extraction reagent, at a liquid-solid weight ratio of 10:1, extract at 80 °C for 3 hours. After rotary evaporation of the extract, take the dried powder to obtain the water extract of *Oudemansiella raphanipies*;

[0054] (4) Mix the total flavonoid extract of *Oudemansiella raphanipies* and the water extract of *Oudemansiella raphanipies* according to a weight ratio of 7:1 to obtain an extract composition.

[0055] Example 4

[0056] Prepare the *Oudemansiella raphanipies* extract composition according to Example 1, and prepare the sustained-release preparation according to the following steps:

[0057] (1) Add the *Oudemansiella raphanipies* extract composition to 50 v / v% ethanol according to a weight ratio of 1:10, disperse evenly by ultrasonic wave, and take 50 mL;

[0058] (2) Add 150 mL of an aqueous solution of ferric sulfate heptahydrate (the concentration of ferric sulfate heptahydrate is 50 mg / mL) with a solute composed of konjac glucomannan oxide and gelatin in a weight ratio of 2:1 to the product obtained in step (1). The solute weight fraction is 15%. After stirring evenly, add it to a mixture composed of span80 and liquid paraffin in a volume-weight ratio of 2 g:40 mL (the volume ratio of this mixture to the previously stirred and evenly mixed solution is 5:1) for ultrasonic dispersion treatment;

[0059] (3) Centrifuge the product obtained in step (2) (4000 rpm, 5 min), and discard the supernatant; pipette the precipitate with anhydrous methanol, centrifuge (4000 rpm, 3 min), discard the supernatant, take the precipitate, repeat the aforementioned pipetting - centrifugation treatment 3 times to obtain, and prepare it into an aqueous suspension.

[0060] Example 5

[0061] Prepare the extract composition of Oudemansiella raphanipies by following Example 2, and prepare the sustained - release preparation according to the following steps:

[0062] (1) Add the extract composition of Oudemansiella raphanipies to 50 v / v% ethanol according to a weight ratio of 1:8, disperse it evenly by ultrasonic wave, and take 50 mL.

[0063] (2) Add 150 mL of an aqueous solution of ferric sulfate heptahydrate (the concentration of ferric sulfate heptahydrate is 50 mg / mL) with a solute composed of oxidized konjac glucomannan and gelatin in a weight ratio of 2:1 to the product obtained in step (1). The weight fraction of the solute is 13%, stir evenly, and then add it to a mixture composed of span80 and liquid paraffin in a volume - weight ratio of 2 g:40 mL (the volume ratio of this mixture to the previously stirred - evenly solution is 5:1) for ultrasonic dispersion treatment.

[0064] (3) Centrifuge the product obtained in step (2) (4,000 rpm, 5 min), and discard the supernatant; pipette the precipitate with anhydrous methanol, centrifuge (4,000 rpm, 3 min), discard the supernatant, take the precipitate, repeat the aforementioned pipetting - centrifugation treatment 3 times to obtain, and prepare it into an aqueous suspension.

[0065] Example 6

[0066] Prepare the extract composition of Oudemansiella raphanipies by following Example 3, and prepare the sustained - release preparation according to the following steps:

[0067] (1) Add the extract composition of Oudemansiella raphanipies to 50 v / v% ethanol according to a weight ratio of 1:12, disperse it evenly by ultrasonic wave, and take 50 mL.

[0068] (2) Add 150 mL of an aqueous solution of ferric sulfate heptahydrate (the concentration of ferric sulfate heptahydrate is 50 mg / mL) with a solute composed of oxidized konjac glucomannan and gelatin in a weight ratio of 2:1 to the product obtained in step (1). The weight fraction of the solute is 16%, stir evenly, and then add it to a mixture composed of span80 and liquid paraffin in a volume - weight ratio of 2 g:40 mL (the volume ratio of this mixture to the previously stirred - evenly solution is 5:1) for ultrasonic dispersion treatment.

[0069] (3) Centrifuge the product obtained in step (2) (4000 rpm, 5 min), and discard the supernatant; pipette the precipitate with anhydrous methanol, centrifuge (4000 rpm, 3 min) and then discard the supernatant, take the precipitate, repeat the aforementioned pipetting - centrifugation treatment 3 times to obtain; prepare it into an aqueous suspension.

[0070] Example 7

[0071] Use the mixture of the Termitomyces albuminosus extract composition and 5 - aminosalicylic acid (weight ratio 1:1) in Example 1 as the active ingredient to replace the Termitomyces albuminosus extract composition in Example 5, and the rest is the same as in Example 5.

[0072] Comparative Example 1

[0073] (1) Dry the fruiting bodies of Termitomyces albuminosus, then pulverize and sieve through a 40 - mesh sieve.

[0074] (2) Use the dried powder of Termitomyces albuminosus fruiting bodies as the extraction substrate, use 70 v / v% ethanol as the extraction reagent, at a liquid - to - solid weight ratio of 25:1, extract at 70 °C for 2 hours with ultrasonic assistance (60 w), perform rotary evaporation on the extract and then take the dry powder to obtain the total flavonoid extract of Termitomyces albuminosus as the extract.

[0075] Comparative Example 2

[0076] On the basis of Example 1, change the ratio of the total flavonoid extract of Termitomyces albuminosus and the aqueous extract of Termitomyces albuminosus to 1:1, and the rest is the same as in Example 1.

[0077] Comparative Example 3

[0078] On the basis of Example 1, change the ratio of the total flavonoid extract of Termitomyces albuminosus and the aqueous extract of Termitomyces albuminosus to 5:2, and the rest is the same as in Example 1.

[0079] Comparative Example 4

[0080] Adjust the method for obtaining the aqueous extract of Termitomyces albuminosus as follows:

[0081] Dry the fruiting bodies of Termitomyces albuminosus, then crush them and sieve through a 40-mesh sieve; use the dried powder of Termitomyces albuminosus fruiting bodies as the extraction substrate, and add the dried powder of Termitomyces albuminosus fruiting bodies to water at a material-to-water ratio of 1 g:30 ml, and place it at 90 °C for 1 hour; then cool it to room temperature, add a composite enzyme with a weight fraction of 2% (the weight ratio of 10 U / mg cellulase, 500 U / mg pectinase and 800 U / mg papain is 4:3:3), and enzymolyze it for 2 hours at 50 °C and pH 5.5, then adjust the pH = 7, and then heat it to 90 °C to inactivate the enzyme for 10 minutes; then place the enzymolyzed solution in an ultrasonic instrument and ultrasonically treat it for 20 minutes at 60 W and 50 °C. Centrifuge the enzymolyzed solution to obtain the supernatant, add 4 times the volume of absolute ethanol to precipitate and let it stand overnight, centrifuge at 5000 rpm / min for 10 minutes, take the precipitate, wash the precipitate with absolute ethanol twice, and dry it to obtain the crude polysaccharide of Termitomyces albuminosus.

[0082] The rest is the same as in Example 1.

[0083] Comparative Example 5

[0084] Refer to Example 4 to prepare the sustained-release preparation, the difference is that gelatin is not added when preparing the sustained-release preparation. The specific scheme is as follows:

[0085] Prepare the Termitomyces albuminosus extract composition according to Example 1, and prepare the sustained-release preparation according to the following steps:

[0086] (1) According to a weight ratio of 1:10, add the Termitomyces albuminosus extract composition to 50 v / v% ethanol, ultrasonically disperse it evenly, and take 50 mL;

[0087] (2) Add 150 mL of an aqueous solution of ferric sulfate heptahydrate of oxidized konjac glucomannan (the concentration of ferric sulfate heptahydrate is 50 mg / mL) with a solute weight fraction of 15% to the product obtained in step (1), stir evenly, and then add it to a mixture composed of span80 and liquid paraffin at a volume-to-weight ratio of 2 g:40 mL (the volume ratio of this mixture to the previously stirred and evenly mixed solution is 5:1) for ultrasonic dispersion treatment;

[0088] (3) Centrifuge the product obtained in step (2) (4000 rpm, 5 min), discard the supernatant; blow the precipitate with absolute methanol, centrifuge (4000 rpm, 3 min) and then discard the supernatant, take the precipitate, repeat the aforementioned blowing-centrifugation treatment 3 times to obtain, and prepare it into a water suspension.

[0089] Experimental Example 1

[0090] 1. Animal model of ulcerative colitis

[0091] Sixty-six male C57BL / 6J mice of SPF grade weighing 18 - 20 g were adaptively fed for one week under 12-hour day-night cycle conditions. The mice were randomly divided into 11 groups, namely blank control group, model group, positive control group 1, positive control group 2, experimental groups (one group for each of Examples 1 - 3), and comparative experimental groups (one group for each of Comparative Examples 1 - 4), with 6 mice in each group. The experimental period was 16 days.

[0092] Blank control group: From day 1 to day 16, they had free access to water and food, and each mouse was gavaged with 100 μl of deionized water daily.

[0093] Model group: From day 1 to day 16, they had free access to water and food, and each mouse was gavaged with 100 μl of deionized water daily. Meanwhile, from day 7 to day 12, 3.5% DSS was used instead of drinking water for feeding to induce ulcerative colitis.

[0094] Positive control group 1: From day 1 to day 16, they had free access to water and food, and each mouse was gavaged with 100 μl of 5-aminosalicylic acid (the daily dose was converted according to the drug instruction manual and the body weight of the mice) daily. Meanwhile, from day 7 to day 12, 3.5% DSS was used instead of drinking water for feeding to induce ulcerative colitis.

[0095] Positive control group 2: From day 1 to day 16, they had free access to water and food, and each mouse was gavaged with 100 μl of mesalazine (the daily dose was converted according to the drug instruction manual and the body weight of the mice) daily. Meanwhile, from day 7 to day 12, 3.5% DSS was used instead of drinking water for feeding to induce ulcerative colitis.

[0096] Experimental groups: From day 1 to day 16, they had free access to water and food, and each mouse was gavaged with 100 μl of the corresponding extract composition of the example (50 mg / kg) daily. Meanwhile, from day 7 to day 12, 3.5% DSS was used instead of drinking water for feeding to induce ulcerative colitis.

[0097] Comparative experimental groups: From day 1 to day 16, they had free access to water and food, and each mouse was gavaged with 100 μl of the corresponding extract or extract composition of the comparative example (50 mg / kg) daily. Meanwhile, from day 7 to day 12, 3.5% DSS was used instead of drinking water for feeding to induce ulcerative colitis.

[0098] The body weight and feces of the mice in each group were recorded every day, and DAI (disease activity index) evaluation was carried out (the standard is shown in Table 1). After 16 days, at the end of the experimental period, the mice were sacrificed, and mouse blood was taken. After centrifuging at 4000 r / min for 10 min, the upper serum was aspirated, and ELISA kits were used to measure the levels of inflammatory factors MOP, IL-1β, IL-4, IL-6, IL-17, INF-γ, and TGF-β1. Meanwhile, the colon was taken to measure the colon length.

[0099] Table 1

[0100]

[0101]

[0102] As shown in Table 2, except for the blank control group, the DAI values of the other groups all increased to varying degrees, with the highest increase in the model group. At the same time, it can be seen that positive control group 1 treated with 5-aminosalicylic acid and positive control group 2 treated with mesalazine can better reduce the DAI value and reduce the shortening of the colon length. As shown by the results of Comparative Example 1, although the total flavonoid extract of Termitomyces albuminosa can also treat ulcerative colitis to a certain extent, the effect is not obvious compared with the positive control group. As shown by the experimental results of Experimental Groups 1-3, the extract composition obtained in the present invention can effectively treat ulcerative colitis, significantly reducing the DAI value and the shortening length of the colon.

[0103] Table 2

[0104]

[0105] As shown in Table 3, the anti-inflammatory factors TGF-β1 and IL4 in Examples 1-3 of the present invention were significantly increased, and the increase level was higher than that of positive control group 1 and positive control group 2; the pro-inflammatory factors IL-1β, IL6, IL-17, INF-γ and MOP were significantly decreased, and the decrease amplitude was higher than that of the positive control group. Similarly, the experimental results of the DAI score and colon length were similar. Comparing Examples 1-4, the effect on the increase of anti-inflammatory factors and the decrease of pro-inflammatory factors was limited.

[0106] Table 3 Unit: pg / mL

[0107]

[0108]

[0109] From the above experimental results, it can be seen that the Termitomyces albuminosa extract composition provided by the present invention has excellent effects in treating ulcerative colitis, which can provide a corresponding basis for the subsequent development of the medicinal value of Termitomyces albuminosa.

[0110] Experimental Example 2

[0111] The experiment was carried out by referring to Experimental Example 1, but with slight adjustments. The specific scheme is as follows:

[0112] 1. Animal model of ulcerative colitis

[0113] Fifty-four SPF male C57BL / 6J mice weighing 18 - 20 g were adaptively fed for one week under 12-hour day-night cycle conditions. The mice were randomly divided into 8 groups, namely blank control group, model group, positive control group 1, positive control group 2, experimental groups (one group for each of Examples 4 - 7), and comparative example 5, with 6 mice in each group. The experimental period was 16 days.

[0114] Blank control group: From day 1 to day 16, freely drank water and ate food, and each mouse was intragastrically administered 100 μl of deionized water per day.

[0115] Model group: From day 1 to day 16, freely drank water and ate food, and each mouse was intragastrically administered 100 μl of deionized water per day. Meanwhile, from day 7 to day 12, 3.5% DSS was used instead of drinking water for feeding to induce ulcerative colitis.

[0116] Positive control group 1: From day 1 to day 16, freely drank water and ate food, and each mouse was intragastrically administered 100 μl of 5-aminosalicylic acid (the daily dose was converted according to the drug instruction manual and the body weight of the mice) per day. Meanwhile, from day 7 to day 12, 3.5% DSS was used instead of drinking water for feeding to induce ulcerative colitis.

[0117] Positive control group 2: From day 1 to day 16, freely drank water and ate food, and each mouse was intragastrically administered 100 μl of the extract composition of Example 1 (50 mg / kg) per day. Meanwhile, from day 7 to day 12, 3.5% DSS was used instead of drinking water for feeding to induce ulcerative colitis.

[0118] Experimental groups: From day 1 to day 16, freely drank water and ate food, and each mouse was intragastrically administered 100 μl of the products obtained from Examples 4 - 7 (50 mg / kg) per day respectively. Meanwhile, from day 7 to day 12, 3.5% DSS was used instead of drinking water for feeding to induce ulcerative colitis.

[0119] Comparative experimental group: From day 1 to day 16, freely drank water and ate food, and each mouse was intragastrically administered 100 μl of the product obtained from the corresponding comparative example 5 (50 mg / kg) per day. Meanwhile, from day 7 to day 12, 3.5% DSS was used instead of drinking water for feeding to induce ulcerative colitis.

[0120] The body weight and feces of the mice in each group were recorded every day, and DAI (disease activity index) evaluation was carried out (the standard is shown in Table 1). After 16 days, at the end of the experimental period, the mice were sacrificed, blood of the mice was taken, centrifuged at 4000 r / min for 10 min, the upper serum was aspirated, and ELISA kits were used to measure the levels of inflammatory factors MOP, IL-1β, IL-4, IL-6, IL-17, INF-γ, TGF-β1; meanwhile, the colon was taken and the colon length was measured.

[0121] Table 4

[0122]

[0123]

[0124] Table 5

[0125]

[0126]

[0127] As can be seen from Table 4 and Table 5, when the sustained-release preparation of the present invention is adopted, the therapeutic effect on ulcerative colitis has been significantly improved. However, it can also be seen from the experimental results of Comparative Example 5 that when sustained-release preparations are prepared using different carrier materials, the obtained experimental effects are different. More importantly, the sustained-release preparation of Comparative Example 5 is only slightly better than Positive Control Group 1 (the drug is 5-aminosalicylic acid) in terms of the levels of IL-4, TGF-β1 and MOP, and is even inferior to Positive Control Group 1 in terms of the levels of IL-1β, IL-6, IL-7 and INF-γ. It can be seen therefrom that the sustained-release preparation of Comparative Example 5 limits the therapeutic effect of the active ingredient.

[0128] In addition, the inventors investigated the white blood cell count of each group in Table 4 and Table 5. Blood was taken on the 0th day and the 16th day to measure the white blood cell content. No case was found in which the white blood cell count did not change significantly (±1%) in the blank control group and the test mice in Examples 4-7. In Positive Control Group 1, 3 cases were found in which the white blood cell count decreased (more than 5%), and in Positive Control Group 2, 2 cases were found in which the white blood cell count decreased (more than 5%). It can be seen therefrom that the sustained-release preparation of the present invention can effectively reduce side effects.

Claims

1. A controlled-release preparation for treating ulcerative colitis based on a composition of a black-skinned Convolvulus aconitifolia extract, characterized in that: The controlled-release preparation is a nanoparticle comprising a composition of a black-skinned Aconitum aviculare extract as an active ingredient; The preparation method of the nanoparticles comprises the following steps: (1) uniformly dispersing the black-skinned dwarf fir extract composition in 50 v / v% ethanol by ultrasonication; (2) adding an aqueous solution of ferric sulfate heptahydrate containing oxidized konjac glucomannan and gelatin in a weight ratio of 2:1 to the result obtained in step (1), stirring the mixture, and then adding a mixed solution containing span80 and liquid paraffin in a volume weight ratio of 2g:40mL to perform ultrasonic dispersion treatment; (3) centrifuging the result obtained in step (2), discarding the supernatant, blowing the precipitate with anhydrous methanol, discarding the supernatant after centrifugation, and taking the precipitate to obtain the nanoparticles. The black-skinned Aconitum extract composition is composed of a black-skinned Aconitum total flavonoid extract and a black-skinned Aconitum water extract in a weight ratio of 5 to 7:1; The total flavonoids extract of Aconitum fusca is prepared by using dried powder of Aconitum fusca fruiting bodies as the extraction substrate and 70 v / v% ethanol as the extraction reagent. The extraction is carried out under ultrasonic assistance at a liquid-to-solid weight ratio of 25:1 at 70°C for 2 hours, and the extract is subjected to rotary evaporation to obtain a dry powder. The aqueous extract of Aconitum carmichaelii is prepared by using the dried powder of Aconitum carmichaelii fruiting bodies as an extraction substrate and water as an extraction reagent. The extraction is carried out at 80°C for 3 hours at a liquid-to-solid weight ratio of 10:1, and the extract is subjected to rotary evaporation to obtain the dry powder.

2. The controlled-release preparation for treating ulcerative colitis based on the extract composition of Aconitum carmichaelii according to claim 1, characterized in that: The weight ratio of the total flavonoids extract of Aconitum aviculare and the water extract of Aconitum aviculare is 6:

1.

3. The controlled-release preparation for treating ulcerative colitis based on the black-skinned Aconitum extract composition according to claim 1, wherein when obtaining the dried powder of the black-skinned Aconitum fruiting body, the black-skinned Aconitum fruiting body is first dried, then crushed and passed through a 40-mesh sieve.

4. The controlled-release preparation for treating ulcerative colitis based on the extract composition of Aconitum carmichaelii according to claim 1, characterized in that: When preparing the nanoparticles, in step (1), the weight ratio of the black-skinned Aconitum extract to 50v / v% ethanol is 1:

10.

5. The controlled-release preparation for treating ulcerative colitis based on the extract composition of Aconitum carmichaelii according to claim 1, characterized in that: When preparing the nanoparticles, in step (2), the weight fraction of the solute composed of oxidized konjac glucomannan and gelatin in a weight ratio of 2:1 is 15%.

6. The controlled-release preparation for treating ulcerative colitis based on the extract composition of Aconitum carmichaelii according to claim 5, characterized in that: When preparing nanoparticles, the weight ratio of the product obtained in step (1) to the aqueous solution of ferric sulfate heptahydrate whose solute is composed of oxidized konjac glucomannan and gelatin in a weight ratio of 2:1 is 1:3; and / or, when preparing nanoparticles, in step (3), the precipitate is blown with anhydrous methanol, the supernatant is discarded after centrifugation, and the precipitate is taken, and the treatment process is repeated at least 3 times.

7. The controlled-release preparation for treating ulcerative colitis based on the extract composition of Aconitum carmichaelii according to claim 6, characterized in that: When preparing the nanoparticles, the concentration of ferric sulfate heptahydrate in the ferric sulfate heptahydrate aqueous solution is 50 mg / mL.

8. The controlled-release preparation for treating ulcerative colitis based on the extract composition of Aconitum carmichaelii according to claim 1, characterized in that: The treatment of ulcerative colitis includes alleviating colon damage, reducing the content of pro-inflammatory factors in serum, and alleviating blood in stool and diarrhea.

9. The controlled-release preparation for treating ulcerative colitis based on the extract composition of Aconitum carmichaelii according to claim 1, characterized in that: The active ingredients are a black-skinned Convolvulus arvense extract composition and 5-aminosalicylic acid in a weight ratio of 1:

1.

10. Use of the controlled-release preparation for treating ulcerative colitis based on the extract composition of Aconitum carmichaelii according to claims 1 to 9 in the preparation of a drug for treating ulcerative colitis.

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