Preparation of biological agent with lysozyme nanoparticles entrapped by honey vesicles and application of biological agent in intestinal diseases

Abstract

The invention provides a biological preparation with lysozyme nanoparticles entrapped by honey vesicles, the biological preparation comprises honey and lysozyme, the mass ratio of the honey to the lysozyme is 0.8: 1-1: 1, and the biological preparation is a suppository. According to the biological preparation with the lysozyme nanoparticles entrapped by the honey vesicles, the lysozyme and the honey are matched for use, so that the dynamic balance of intestinal flora can be effectively improved, the treatment cycle of various intestinal diseases can be shortened, and constipation can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of biological preparations, and in particular to the preparation of a biological preparation of honey vesicle-encapsulated lysozyme nanoparticles and its application in intestinal diseases. Background Art

[0002] There are a huge number of microorganisms in the human intestine. These microorganisms rely on the human intestine for life and help the human body complete a variety of physiological and biochemical functions. Intestinal microorganisms not only play an important bridging role between diet and host, but also regulate human health by themselves and their metabolites. Therefore, intestinal microorganisms are closely related to human health. Intestinal microorganisms achieve microecological balance through dynamic physiological interactions with the host, effectively preventing the translocation of bacteria and endotoxins in the intestine. When the normal microbial community is affected by the host and the external environment, the original balance is destroyed, the intestinal flora is imbalanced, and the types, quantity and proportion of normal intestinal flora change abnormally, turning into a pathological combination state, which in turn causes host disease. Current studies have shown that many diseases in the human body are closely related to intestinal microorganisms, such as intestinal diseases or constipation, depression and diabetes.

[0003] In addition to clinical symptoms such as difficulty in defecation, low defecation frequency, and hard and dry stool, constipation can also lead to intestinal flora imbalance. Constipation can change the composition of the intestinal microbiota, increase the permeability of the intestine to pathogens and endotoxins, and promote oxidation and inflammatory responses. Therefore, intestinal flora is closely related to constipation. At present, constipation in diseases such as stroke, elderly fractures, and functional constipation is more serious, and the general treatment period ranges from three weeks to half a year.

[0004] Intestinal mucositis (IM) is also defined as structural, functional and immunological changes caused by inflammation of the mucosa lining the digestive tract. IM is clinically manifested by diarrhea, abdominal pain, blood in the stool, nausea and vomiting, and various serious complications may also occur. Intestinal mucositis is one of the symptoms of radiation enteritis. Currently, antibiotics or mometasone furoate suppositories are mainly used for treatment. These two methods are used to treat radiation enteritis for a long period of time, generally 1-3 months, and both will damage the intestinal environment, resulting in side effects such as intestinal flora disorders and constipation.

[0005] The purpose of the present invention is to provide a biological preparation of honey vesicles encapsulating lysozyme nanoparticles, which can be used to treat constipation caused by various diseases and intestinal mucositis, and can shorten the treatment cycle. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a biological preparation of honey vesicles encapsulating lysozyme nanoparticles. The combination of lysozyme and honey can effectively improve the dynamic balance of intestinal flora and constipation, and can shorten the treatment period of various intestinal diseases.

[0007] The first aspect of the present invention is to provide a biological preparation of honey vesicles encapsulating lysozyme nanoparticles, which comprises honey and lysozyme, and the mass ratio of the two is 0.8:1-1:1, and the biological preparation is a suppository.

[0008] Specifically, the mass ratio of honey to lysozyme may be 0.8:1, 0.9:1 or 1:1, or other values ​​within this range.

[0009] Furthermore, the amount of each dose of the biological preparation of honey vesicles encapsulating lysozyme nanoparticles is 2-2.5 g.

[0010] Furthermore, the biological preparation of honey vesicles encapsulating lysozyme nanoparticles is prepared by the following method:

[0011] Step S1, dissolving lysozyme in purified water, stirring, and fully dissolving, the lysozyme concentration is 1000-3000 mg / L; specifically, the lysozyme concentration can be 1000 mg / L, 2000 mg / L or 3000 mg / L, or other values ​​within the range;

[0012] Step S2, placing honey in a container and heating it to 90-100° C. to liquefy the honey;

[0013] Step S3, rapidly cooling the honey to 55-65° C., adding the lysozyme aqueous solution of step S1 in proportion, and stirring evenly;

[0014] Step S4, adding sodium bicarbonate to adjust the pH of the mixed solution to 6.5-7.0;

[0015] Step S5, cooling and shaping to prepare the biological preparation.

[0016] Preferably, the mass ratio of honey to lysozyme is 1:1.

[0017] The second aspect of the present invention is to provide the use of the biological preparation of honey vesicles encapsulating lysozyme nanoparticles described in the first aspect in regulating intestinal flora disorders and alleviating constipation.

[0018] The third aspect of the present invention is to provide the use of the biological preparation of honey vesicles encapsulating lysozyme nanoparticles described in the first aspect in the treatment of intestinal mucositis.

[0019] Compared with the prior art, the biological preparation of honey vesicles encapsulating lysozyme nanoparticles provided by the present invention has the following beneficial effects:

[0020] 1. The biological preparation of honey vesicles encapsulating lysozyme nanoparticles provided by the present invention is used as a suppository by combining lysozyme nanoparticles and honey, wherein lysozyme can directly or indirectly regulate intestinal microorganisms, thereby maintaining the balance of intestinal flora. Direct regulation is to effectively disinfect harmful microorganisms in the intestine through its antibacterial effect, thereby promoting the growth and reproduction of probiotics. Indirect regulation is to reduce the stimulation of foreign antigens, reduce the occurrence of immune response and subclinical diseases, reduce the need for intestinal maintenance, and reduce the damage of harmful substances to the body's immune system; the main effect of honey is to reduce the occurrence of subclinical diseases, ... The function is to moisturize the intestines and relieve anorectal sphincter motility disorders. The bioactive vesicle nanoparticles it contains also have anti-inflammatory effects and can work synergistically with lysozyme nanoparticles. Honey is used as an excipient to carry the drug to the diseased area, making it convenient for drug administration. When used as a carrier, it can enhance the permeability of the drug, thereby increasing the bioavailability of the drug ingredients and enhancing the biological activity. At the same time, after the drug reaches the rectum, honey can promote intestinal peristalsis, effectively remove the metabolites of rectal inflammatory reactions and constipation, cooperate with lysozyme to inactivate harmful bacteria, and promote the discharge of products to clean the intestinal environment. Clinical trials have found that the synergistic effect of the two can effectively improve the dynamic balance of intestinal flora and constipation, have good anti-inflammatory effects, and can shorten the treatment cycle of various intestinal diseases.

[0021] 2. The biological preparation of honey vesicles encapsulating lysozyme nanoparticles provided by the present invention is non-toxic, residue-free, drug-resistant and has high biological safety. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention and to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are further described below.

[0023] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0024] Example 1

[0025] A biological preparation containing honey vesicles and lysozyme nanoparticles, comprising honey and lysozyme, wherein the mass ratio of the honey and lysozyme is 1:1. The biological preparation is a suppository and is prepared by the following method:

[0026] Step S1, dissolving lysozyme in purified water, stirring, and fully dissolving, the lysozyme concentration is 2000 mg / L;

[0027] Step S2, placing honey in a container and heating it to 100° C.;

[0028] Step S3, rapidly cooling the honey to 55-65° C., adding the lysozyme aqueous solution of step S1 in proportion, and stirring evenly;

[0029] Step S4, adding sodium bicarbonate to adjust the pH of the mixed solution to 6.5-7.0;

[0030] Step S5, cooling and shaping to prepare the biological preparation.

[0031] The amount of each dose of the biological preparation in this example is 2 g.

[0032] Animal Experimentation:

[0033] 1. Experimental purpose: To establish a rat model of chemotherapy-induced intestinal mucositis and intestinal dysbiosis by intraperitoneal injection of 5-Fu, observe the intestinal mucosal barrier indicators (DAO, ET and D-lactic acid) and intestinal flora changes of the model animals, and evaluate the therapeutic effect of this biological agent on chemotherapy-induced intestinal mucositis and intestinal dysbiosis.

[0034] 2. Model preparation and grouped drug administration

[0035] SPF Wistar rats, male. After 5 days of adaptive feeding, they were randomly divided into a blank control group, a model control group, and an experimental group, with 12 rats in each group. Each group was placed in the anus with a corresponding suppository (the blank control group used pure water (2 g per dose), the model control group used bisacodyl suppository (10 mg per dose), and the experimental group used the biological preparation of Example 1 (2 g per dose)), and the drug was administered continuously for 7 days, once a day.

[0036] 3. Detection method

[0037] (1) Intestinal mucosal barrier indicators

[0038] Rats were anesthetized by intraperitoneal injection, and blood was collected from the abdominal aorta. The blood was centrifuged at 3000 r / min*10 min, and the supernatant was collected and stored at 80°C for the detection of serum endotoxin (EX) and D-lactate acid (DLA) levels and diamine oxidase (DAO) activity.

[0039] (2) Intestinal flora

[0040] Rats were anesthetized by intraperitoneal injection and sacrificed by blood sampling from the abdominal aorta. About 0.51 g of feces were collected from the cecum and stored at 80°C for intestinal flora detection.

[0041] 4. Test results

[0042] (1) Intestinal mucosal barrier index, the experimental results are shown in Table 1:

[0043] Table 1: Effects of biological agents on intestinal mucosal barrier parameters in rats with chemotherapy-induced intestinal mucositis (X±S)

[0044]

[0045] As can be seen from Table 1, compared with the blank control group, the endotoxin, D-lactic acid, and diamine oxidase activity in the serum of rats in the model control group and the experimental group were significantly reduced, indicating that the preparations in the model group, the control group and the experimental group can protect the intestinal mucosal barrier of rats with chemotherapy-induced intestinal mucositis; compared with the model control group, the endotoxin, D-lactic acid, and diamine oxidase activity levels in the serum of rats using the biological preparation of honey vesicles encapsulating lysozyme nanoparticles of the present invention were significantly lower than those in the model control group, indicating that the biological preparation of the present invention has a more significant protective effect on the intestinal mucosal barrier of rats with chemotherapy-induced intestinal mucositis.

[0046] (2) Influence of intestinal flora

[0047] The Observed_species, Shannon and Simpson, Chao 1 and ACE of the rat intestinal flora were detected; Observed_species was the number of ASVs; Shannon and Simpson were the flora diversity indexes; Chao 1 and ACE were the flora richness indexes.

[0048] The test results are shown in Table 2:

[0049] Table 2: Effects of biological agents on the diversity and richness of intestinal flora in rats with chemotherapy-induced intestinal mucositis (X±S)

[0050] Group n Observed_species Shannon Simpson Chao1 ACE Blank control group 12 643.54±125.67 6.37±0.26 0.74±0.03 634.23±148.19 628.39±146.57 Model control group 12 526.03±132.49 5.85±0.57 0.59±0.05 516.45±134.68 520.38±132.74 Experimental Group 12 768.46±152.36 9.04±0.82 0.96±0.08 860.56±167.51 862.87±165.51

[0051] As can be seen from Table 2, compared with the blank control group, the Observed_species, Shannon and Simpson, Chao 1 and ACE of the intestinal flora of the rats in the model control group were significantly decreased, indicating that the drugs used in the model control group destroyed the intestinal flora of the rats; while the Observed_species, Shannon and Simpson, Chao 1 and ACE of the intestinal flora of the rats in the experimental group were significantly increased, indicating that the biological preparation of the present invention can effectively regulate the intestinal flora of rats.

[0052] Clinical trial I:

[0053] A total of 80 patients with functional constipation treated in a hospital in Hunan Province from 2021 to 2023 were selected as the research subjects, aged 30-75 years old, and the inclusion criteria were met.

[0054] Exclusion criteria: patients with organic intestinal obstructive lesions; patients with lack of clinical data; patients with allergic constitution or history of adverse reactions to trial drugs; pregnant and lactating women; patients with mental and intellectual disorders; patients with systemic diseases that affect the digestive system.

[0055] The subjects were randomly divided into a treatment group (n=40) and a control group (n=40), wherein the treatment group used the biological preparation of Example 1, twice a day (rectal delivery); the control group 1 used bisacodyl suppository (rectal delivery), once a day, each dose of 10 mg.

[0056] The treatment duration for both groups was 14 days.

[0057] Observation indicators:

[0058] (1) Efficacy criteria: ① Marked effect, the main symptoms and signs of constipation disappear or almost disappear, intestinal function is normal, bowel movement frequency is normal, and stool characteristics are normal; ② Effective, the main symptoms and signs of constipation are significantly improved, intestinal function is improved, bowel movement frequency is close to normal, and stool characteristics are close to normal; ③ The above criteria are not met or constipation symptoms worsen; Total effective rate = (number of markedly effective cases + number of effective cases) / total number of cases × = 100%.

[0059] (2) All patients were assessed for constipation using the Constipation Scale (CCS) before and after treatment based on multiple dimensions, including defecation frequency, defecation pain, defecation effort, defecation time, and completeness of the defecation process. The score range of the scale is 0-30 points, with higher scores representing more severe constipation.

[0060] (3) All patients underwent anorectal sphincter motility tests before and after treatment, including maximum anal sphincter pressure at rest (MRASP) and maximum squeeze pressure (MSP).

[0061] Statistical methods:

[0062] After the clinical data were collected, all the data were established in a database. The professional statistical software package SPSS25.0 was used for statistical analysis. The measurement data were described by mean and standard deviation (x±s), and P<0.05 was considered statistically significant.

[0063] result:

[0064] The comparison of the total effective rate is shown in Table 3:

[0065] Table 3: Comparison of total effective rate in different treatment cycles between the treatment group and the control group

[0066]

[0067] It can be seen from Table 3 that the use of the biological preparation of the present invention can not only improve the effect of treating functional constipation, but also shorten the treatment period.

[0068] Comparison of constipation scores is shown in Table 4:

[0069] Table 4: Comparison of constipation scores before and after treatment between the treatment group and the control group (x±s, points)

[0070] Group n Before treatment After treatment Treatment Group 40 22.46±2.65 3.94±0.47 Control group 40 21.86±3.12 12.59±4.78

[0071] Comparison of anorectal sphincter motility indices is shown in Table 5:

[0072] Table 5: Comparison of changes in anorectal sphincter motility indexes before and after treatment in the treatment group and the control group (x±s, mm Hg)

[0073]

[0074] The biological preparation of the present invention improves the symptoms of intestinal flora disorder and constipation of patients by regulating the dynamic balance of intestinal flora and promoting intestinal peristalsis. Honey can enhance the permeability of the preparation through rectal delivery, thereby improving the bioavailability of the drug and enhancing its biological activity, thereby shortening the treatment cycle. In addition to being used for functional constipation, it is also suitable for constipation after stroke and constipation due to fracture in the elderly.

[0075] Clinical Trial Ⅱ

[0076] A total of 60 patients with intestinal mucositis treated in a hospital in Hunan Province from 2021 to 2023 were selected as the research subjects, aged 40-70 years old, and the inclusion criteria were met.

[0077] Exclusion criteria: patients with allergic constitution or history of adverse reactions to trial drugs.

[0078] The subjects were randomly divided into a treatment group (n=30) and a control group (n=30), wherein the treatment group used the biological preparation of Example 1, with a dosage of 2 g twice a day (rectal delivery); the control group 1 used mometasone furoate suppository (rectal delivery), once a day, 2 g each time.

[0079] The treatment duration for both groups was 14 days.

[0080] Observation indicators:

[0081] (1) Efficacy criteria: ① markedly effective, the main symptoms of intestinal mucositis basically disappeared, and stool test was normal; ② effective, the main symptoms of intestinal mucositis were significantly improved, intestinal function improved, and stool was close to normal; ③ the above criteria were not met or symptoms worsened; total effective rate = (number of markedly effective cases + number of effective cases) / total number of cases × 100%.

[0082] (2) All patients underwent anorectal sphincter motility tests before and after treatment, including maximum anal sphincter pressure at rest (MRASP) and maximum squeeze pressure (MSP).

[0083] Statistical methods:

[0084] After the clinical data were collected, all the data were established in a database. The professional statistical software package SPSS25.0 was used for statistical analysis. The measurement data were described by mean and standard deviation (x±s), and P<0.05 was considered statistically significant.

[0085] result:

[0086] The comparison of the total effective rate is shown in Table 6:

[0087] Table 6: Comparison of total effective rate in different treatment cycles between the treatment group and the control group

[0088]

[0089] It can be seen from Table 6 that the biological preparation of the present invention can effectively treat intestinal mucositis and shorten the treatment period.

[0090] Comparison of anorectal sphincter motility indices is shown in Table 7:

[0091] Table 7: Comparison of changes in anorectal sphincter motility indexes before and after treatment in the treatment group and the control group (x±s, mm Hg)

[0092]

[0093]

[0094] The biological preparation of the present invention exerts its efficacy by regulating the dynamic balance of intestinal flora and promoting intestinal peristalsis. At the same time, both lysozyme and honey vesicles have anti-inflammatory effects, and honey has good permeability. It can directly reach the diseased area through rectal administration, improve the bioavailability of the preparation and enhance its biological activity, thereby improving the anti-inflammatory effect and shortening the treatment cycle. The biological preparation of the present invention can be applied to intestinal mucosal inflammation caused by radiation enteritis and the like.

[0095] The biological preparation of the present invention is non-toxic, has no residue, has no drug resistance, and has high biological safety.

[0096] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations made to these embodiments without departing from the principles and spirit of the present invention are still within the protection scope of the present invention.

Claims

1. A biological preparation of honey vesicles encapsulating lysozyme nanoparticles, characterized in that: The biological preparation comprises honey and lysozyme, and the mass ratio of the honey and lysozyme is 0.8:1-1:

1. The biological preparation is a suppository.

2. The biological preparation of honey vesicles encapsulating lysozyme nanoparticles according to claim 1, characterized in that: The amount of each dose of the biological preparation is 2-2.5 g.

3. The biological preparation of honey vesicles encapsulating lysozyme nanoparticles according to claim 1, characterized in that: The biological preparation is prepared by the following method: Step S1, dissolving lysozyme in purified water, stirring, and fully dissolving, the lysozyme concentration is 1000-3000 mg / L; Step S2, placing honey in a container and heating it to 90-100° C.; Step S3, rapidly cooling the honey to 55-65° C., adding the lysozyme aqueous solution of step S1 in proportion, and stirring evenly; Step S4, adding sodium bicarbonate to adjust the pH of the mixed solution to 6.5-7.0; Step S5, cooling and shaping to prepare the biological preparation.

4. The biological preparation of honey vesicles encapsulating lysozyme nanoparticles according to claim 1, characterized in that: The mass ratio of honey to lysozyme is 0.8:1-1:

1.

5. Use of the biological preparation of honey vesicles encapsulating lysozyme nanoparticles as claimed in claim 1 in regulating intestinal flora disorders and alleviating constipation.

6. Use of the biological preparation of honey vesicles encapsulating lysozyme nanoparticles as claimed in claim 1 in the treatment of intestinal mucositis.

Images