CN117106622B - Lactobacillus plantarum and application of composition thereof in preparation of medicines for treating internal hemorrhoids - Google Patents
Lactobacillus plantarum and application of composition thereof in preparation of medicines for treating internal hemorrhoids Download PDFInfo
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- CN117106622B CN117106622B CN202310745600.XA CN202310745600A CN117106622B CN 117106622 B CN117106622 B CN 117106622B CN 202310745600 A CN202310745600 A CN 202310745600A CN 117106622 B CN117106622 B CN 117106622B
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A61K35/66—Microorganisms or materials therefrom
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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Abstract
The invention belongs to the technical field of microorganism application, and in particular relates to application of a live bacteria preparation in preparation of a medicine for treating internal hemorrhoids. The viable bacteria preparation disclosed by the invention can effectively improve the intestinal flora diversity after operation, and the relative abundance of the thick-walled bacteria phylum (Firmicutes) is improved at the gate level; the relative abundance of bifidobacteria (bifidobacteria), megamonas (Megamonas) and Lactobacillus (Lactobacillus) is increased at the genus level; improves the improvement rate of the internal hemorrhoid endoscopic hardening treatment, and has higher clinical safety.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and in particular relates to application of a live bacteria preparation in preparation of a medicine for treating internal hemorrhoids.
Background
Internal hemorrhoids are the most common type of hemorrhoids, well developed in adults over 50 years of age, and are mainly characterized clinically by bleeding and prolapse. The occurrence of internal hemorrhoid symptoms is usually caused by drinking, spicy diet and abnormal intestinal habit (especially constipation). The costs associated with the onset of long-term symptoms and with the treatment of internal hemorrhoids, lead to serious problems in the daily life of the patient and may increase the risk of complications.
Endoscopic sclerotherapy has been an improvement over 100 years, and is now widely used in minimally invasive therapy for internal hemorrhoids, providing a simple and effective alternative to traditional hemorrhoidectomy. The principle of endoscopic sclerotherapy is to induce aseptic inflammation and fibrosis by endoscopically injecting sclerosant to reduce the volume of internal hemorrhoids. Although endoscopic sclerotherapy has good clinical efficacy, postoperative complications including pain, bleeding, infection, and recurrence are still common, and it is reported that about 40% of patients still have varying degrees of bleeding within one month after surgery. In addition, endoscopic surgery can cause post-operative intestinal dysfunction, and intestinal preparation prior to endoscopic surgery can also cause significant disturbance of the intestinal flora, with recovery times exceeding two weeks, possibly exacerbating symptoms of internal hemorrhoids. The occurrence of postoperative symptoms severely reduces the quality of life of the patient, and methods for reducing the occurrence rate of such symptoms and regulating the dysbacteriosis are sought to further improve the efficacy of endoscopic sclerotherapy.
Probiotics are defined as active microorganisms that benefit a host upon ingestion in sufficient amounts. Lactobacillus plantarum (original name Lactobacillus plantarum) is an important probiotic genus, has excellent acid resistance and intestinal mucosa adhesion, and can play roles by producing antibacterial metabolites, changing intestinal microecological environment, enhancing immune system and other mechanisms. Lactobacillus plantarum has been shown to significantly improve intestinal function, reduce intestinal transit time, increase the number of spontaneous bowel movements (SBM, spontaneous bowel movement), and improve stool consistency. In addition, both in vivo and in vitro studies have shown that lactobacillus plantarum can effectively reduce intestinal inflammation and improve the diversity and richness of intestinal flora. Our previous studies have also demonstrated that oral probiotics are effective in alleviating gastrointestinal symptoms following intestinal surgery and promoting recovery of intestinal flora. Based on this, the use of lactobacillus plantarum after endoscopic sclerotherapy may have an adjuvant therapeutic effect, but lacks clinical relevant research reports.
The auxiliary materials are basic materials and important components of the pharmaceutical preparation, and play a vital role in the preparation technology, the pharmaceutical dosage form and the production of medicines. Therefore, the preparation not only endows the medicine with a certain dosage form, but also has great influence on the administration route and the product quality, including the acting speed, the absorption speed, the bioavailability, the toxic and side effects and the like after the medicine is taken. In pharmaceutical dosage forms, the pharmaceutical activity and physical and chemical stability are the main part, determining the overall efficacy of the formulation. The auxiliary materials are used for realizing the research and development purposes of the preparation technology and ensuring that the medicine is released at a certain position in vivo at a certain speed and time. Therefore, the preparation consisting of the proper auxiliary materials plays a vital role in the practical application and the curative effect of the medicine.
Based on the analysis, the lactobacillus plantarum combined adjuvant is hopeful to be developed for auxiliary treatment after an endoscopic sclerosis treatment operation, including the influence on postoperative internal hemorrhoid symptoms and intestinal functions, and the potential treatment mechanism is analyzed through high-throughput sequencing of postoperative intestinal flora. The study aims at filling the blank of the prior knowledge and provides valuable insight for using the probiotic preparation as an auxiliary treatment for internal hemorrhoid treatment.
Disclosure of Invention
The terms in the present invention:
The lactobacillus plantarum composition is a living bacteria preparation.
"Lactobacillus plantarum" is equivalent to "Lactobacillus plantarum"
The "Shannon index" evaluates the abundance and uniformity of species composition in a sample. A larger value indicates a richer species of the environment and a more uniform distribution of each species.
The "Simpson index" randomly takes two OTUs from one sample data, which are probabilities of belonging to different species. The greater this probability means the higher the species diversity of the sample, and vice versa. The index allows assessment of the status and role of dominant species in the community.
"Chao1 index" estimates an index of the number of OTUs contained in a sample, with a larger number representing more species contained in the sample.
"OTU", operational taxonomic units, is one of the most common terms in microbiological studies, and in order to facilitate the study of species composition diversity information of samples, the effective sequences of the samples need to be clustered, CLEAN TAGS is clustered into OTUs under default given similarity (default 97%), the sequence of each cluster is obtained, and a number (OTU ID) is randomly assigned to each sequence. One OTU can generally be considered to correspond to one species.
The "PCoA analysis" (PRINCIPAL COORDINATE ANALYSIS ) is to find principal coordinates based on a distance matrix, and extract and visualize the most dominant elements and structures from complex data by dimension reduction of multi-dimensional data.
The "Goligher classification" method, goligher, proposed in 1980, to divide internal hemorrhoids into stages I-IV according to the degree of prolapse of the hemorrhoids, suggesting outpatient treatment for stage I and II patients, and hemorrhoidectomy for stages III and IV.
The "n-HDSS score" is a modified version of the study based on HDSS score, including five symptoms (bleeding, pain, itching, staining and prolapse), with a score of 0-3 per item (0=less than once per month, 1=less than once per week, 2=1-6 days per week, 3=daily or always).
The "SHS score" consists of four questions (severity, impact on life, attention, and well-being), each with a score of 1-7,1 = asymptomatic, 7 = severe symptoms.
"SBM" (spontaneous bowel movement ) is defined as bowel movement without the use of laxatives. "SBMs" is defined as the number of spontaneous bowel movements.
"HLS" (hard or lumpy stools) is defined as the spontaneous bowel movement type with type 0-1 bowel movement (Bristol bowel movement scale).
The results of an "ITT analysis" (Intention-to-TREAT ANALYSIS, intent analysis), a clinical trial, contained follow-up data for all randomized, grouped patients.
In order to solve the problems, the invention discloses a viable bacteria preparation which can effectively improve the intestinal flora diversity after operation, and the relative abundance of the thick-walled bacteria phylum (Firmicutes) is improved at the gate level; the relative abundance of bifidobacteria (bifidobacteria), megamonas (Megamonas) and Lactobacillus (Lactobacillus) is increased at the genus level; improving the improvement rate of the internal hemorrhoid endoscopic hardening treatment.
In one aspect, the invention provides a live bacteria preparation, which comprises lactobacillus plantarum MH-301 and auxiliary materials;
the preservation number of the lactobacillus plantarum MH-301 is CGMCC NO.18618;
The auxiliary materials include, but are not limited to: resistant dextrins, fructooligosaccharides, stachyose, xylitol and inulin.
Specifically, the viable count of the lactobacillus plantarum MH-301 is more than or equal to 1 multiplied by 10 9 cfu/g.
Specifically, the mass ratio of the resistant dextrin, fructo-oligosaccharide, stachyose, xylitol and inulin is 8-14:5-12:8-10:4-6:4-8.
Preferably, the mass ratio of the resistant dextrin, fructo-oligosaccharide, stachyose, xylitol and inulin is 14:5:10:4:8.
Preferably, the mass ratio of the resistant dextrin, fructo-oligosaccharide, stachyose, xylitol and inulin is 4:6:4:3:2.
Specifically, the mass ratio of the lactobacillus plantarum MH-301 to the auxiliary materials is 5-8:38-41.
Preferably, the mass ratio of the lactobacillus plantarum MH-301 to the auxiliary materials is 5:41.
On the other hand, the invention also provides application of the live bacteria preparation in preparing medicines for treating internal hemorrhoids.
In particular, the viable bacteria preparation is used for improving intestinal microbiota.
Further specifically, the viable bacteria preparation is used for improving intestinal flora diversity.
In yet another aspect, the invention provides the use of the aforementioned live bacterial formulation in food or health care products.
In particular, the health care product is used for improving intestinal microbiota.
Further specifically, the health care product is used for improving intestinal flora diversity.
The invention has the technical effects that:
(1) Improving the rate of the hardening treatment of the internal hemorrhoid endoscope;
(2) Shortens the first spontaneous defecation (SBM) time of patients and reduces the number proportion of patients with HLS of more than or equal to 25% SBMs;
(3) The use rate and complications of laxatives in the course of disease are reduced;
(4) At the gate level, the relative abundance of the firmicutes gate (Firmicutes) is increased;
(5) The relative abundance of bifidobacteria (bifidobacteria), megamonas (Megamonas) and Lactobacillus (Lactobacillus) is increased at the genus level;
(6) Has higher clinical safety.
Drawings
FIG. 1 is a bar graph of improvement rate of endoscopic sclerosis treatment of internal hemorrhoids.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Lactobacillus plantarum MH-301, accession number: CGMCC No.18618.
Maltodextrin, rui Chengkang medicine technology (Shanxi Co., ltd.), product number: pharmaceutical grade maltodextrin.
Resistant dextrin, shenzhen Lefu Biotechnology Co., ltd., product number: ZCJ0704.
Fructooligosaccharides, marKamick technologies Co., ltd., product number: 308066-66-2.
Stachyose, shanghai Kogyo commercial Co., ltd., product number: FK3198.
Xylitol, xian jin Xiang pharmaceutical excipients Co., ltd., product number: 180-9270-5451.
Inulin, chongqing Rui Yao Biotechnology Co., ltd., product number: 9005-80-5.
Preparation of basic experiment lactobacillus plantarum MH-301 bacterial powder
The lactobacillus plantarum MH-301 powder is prepared by a conventional method, and the method comprises the following steps:
And (3) taking a seed freezing tube of lactobacillus plantarum MH-301, activating and continuously activating the seed freezing tube in an MRS culture medium for three generations at 37 ℃, inoculating the activated lactobacillus plantarum MH-301 into a fermentation tank containing the MRS culture medium for fermentation, centrifuging by a centrifuge after the fermentation is finished, collecting thalli, freeze-drying in a freeze dryer, and crushing to obtain bacterial powder, wherein the number of viable bacteria of the prepared bacterial powder is more than or equal to 1 multiplied by 10 10 cfu/g.
Example 1
The present example provides a viable bacteria preparation, the formulation is shown in Table 1:
TABLE 1
Component (g) | Lactobacillus plantarum MH-301 powder | Resistant dextrins | Fructooligosaccharides | Stachyose | Xylitol | Inulin |
Dosage of | 0.5 | 1.4 | 0.5 | 1 | 0.4 | 0.8 |
Example 2
This example provides a viable bacteria formulation, the formulation is shown in table 2:
TABLE 2
Component (g) | Lactobacillus plantarum MH-301 powder | Resistant dextrins | Fructooligosaccharides | Stachyose | Xylitol | Inulin |
Dosage of | 0.8 | 0.8 | 1.2 | 0.8 | 0.6 | 0.4 |
Comparative example
The formulation of the viable bacteria preparation of the comparative example is shown in Table 3
TABLE 3 Table 3
Component (g) | Lactobacillus plantarum MH-301 powder | Resistant dextrins | Fructooligosaccharides | Stachyose | Xylitol | Inulin |
Comparative example 1 | 0.5 | 1.4 | 0.5 | 1.4 | 0 | 0.8 |
Comparative example 2 | 0.5 | 1.4 | 0.5 | 0 | 1.4 | 0.8 |
Comparative example 3 | 0.5 | 1.4 | 0 | 1 | 0.4 | 1.3 |
Comparative example 4 | 0.5 | 1.4 | 1.3 | 1 | 0.4 | 0 |
Example 2 clinical trial
2.1 Patient data information and grouping
2.1.1 Subject enrollment criteria
Age >18 years old; patients who met Golighter's classification as class I-III parallel endoscopic sclerotherapy. The invention is approved by the ethical committee of hospitals, all patients agree informed, and good life work and rest time and eating habits can be maintained during the trial period.
A total of 360 patients assessed as eligible for this study were enrolled from 2021-05-01 to 2022-10-31, with 338 (16 disqualified, 6 rejected participation) eventually randomized and enrolled 42 per ITT analysis experimental group, control group (C) 44.
The 8 groups of patients had no significant differences in age, sex, BMI, golight's classification. There was no statistical difference between the pre-operative n-HDSS score and the SHS score of the patient. The baseline data for the patients in the group are shown in tables 4 and 5.
TABLE 4 Table 4
TABLE 5
2.1.2 Groupings and experimental designs were as follows:
The placebo for the control group was maltodextrin. The positive medicine is mailing tablet, each tablet contains 150mg of horse chestnut extract, and the manufacturer: the German Gift Dada pharmaceutical factory; approval document: the national medicine standard character size ZJ20140002. The specific usage amounts are shown in Table 6.
TABLE 6
Group of | Numbering device | Drug name | Dosage of usage |
Control group (n=44) | A | Maltodextrin | 1 G/time, 3 times daily |
Positive drug group (n=42) | B | Mailing tablet | 2 Tablets/time, 2 times daily |
Example 1 (n=42) | C | Example 1 | 1 G/time, 3 times daily |
Example 2 (n=42) | D | Example 2 | 1 G/time, 3 times daily |
Comparative example 1 (n=42) | E | Comparative example 1 | 1 G/time, 3 times daily |
Comparative example 2 (n=42) | F | Comparative example 2 | 1 G/time, 3 times daily |
Comparative example 3 (n=42) | G | Comparative example 3 | 1 G/time, 3 times daily |
Comparative example 4 (n=42) | H | Comparative example 4 | 1 G/time, 3 times daily |
Each group needs to complete the taking follow-up for 4 weeks, and provides a fecal sample (S) for high-throughput sequencing and metabonomics analysis (after taking for 1 week), the collected fecal sample is placed in a sterile plastic container and stored to a laboratory at 4 ℃; the samples were kept for less than 8 hours before reaching the laboratory and the faeces were stored in a-80 refrigerator immediately after reaching until DNA was extracted.
2.1.3 Measurement index
(1) Follow-up index: ① 4 weeks post-operative good turnover, n-HDSS score, SHS score; ② Intestinal function 4 weeks after surgery: the first time of SBM, SBMs, HLS is more than or equal to 25 percent SBMs, and the use condition of cathartic; ③ Postoperative complications and safety indexes.
(2) High throughput and metabonomic analysis of faeces: and (5) analyzing the microbial diversity, the abundance, the species composition classification and the like of the feces.
2.2 Analysis of results
2.2.1 Evaluation of postoperative symptom improvement
The experimental results are shown in Table 7, and the improvement rate of the endoscopic sclerosis treatment of internal hemorrhoids is higher in the examples 1-2 compared with the control group, and the use rate and the complications of the cathartic are lower than those of the control group; examples 1-2 showed slightly lower improvement than the positive group, but the laxative use rate and complications were much lower than the positive group; examples 1-2 have a shorter time to first SBM (spontaneous bowel movement) and a lower proportion of patients with HLS (hard or lumpy stools) ≡25% SBMs than the control and positive drug groups. The results show that the examples 1-2 can effectively improve the bad symptoms of internal hemorrhoids after operation, the improvement rate reaches more than 88 percent, and the number proportion of patients with HLS more than or equal to 25 percent SBMs is low because of short time of first SBM (styrene butadiene rubber) although the improvement rate is slightly lower than that of positive drugs; and the usage rate and complications of the cathartic are lower than half of those of the positive medicine, so that the safety is higher.
As can be seen from the comparison of examples 1-2 and examples 1-4, the improvement rates of examples 1-2, the time of the first SBM, and the proportion of the number of patients with HLS of 25% SBMs are all better than those of comparative examples 1-4; but the laxative use rate and complications were not significantly different. As is clear from comparative examples 1-2 and comparative examples 3-4, stachyose and xylitol in the auxiliary materials have a larger influence on the effect of the live bacteria preparation; fructooligosaccharides and inulin also have a certain influence, but relatively small influence. The results show that the addition of auxiliary materials and the addition amount of the auxiliary materials can influence the drug effect to a certain extent, the bioavailability of the drug, the acting time or the acting time.
TABLE 7
2.2.2 Postoperative intestinal flora diversity analysis
Examples 1-2 were high in Shannon and Simpson indices of α -diversity compared to the control group, while there was no significant difference in Chao1 and good coverage indices. In terms of β -diversity, principal coordinate analysis (PCoA) showed that examples 1-2 had significant microbiota composition clusters, indicating that examples 1-2 had significant impact on intestinal microbiota. At the genus level, LEfSe (LDA effect size) analysis showed that the relative abundance of bifidobacteria (Bifidobacterium), megamonas (Megamonas) and Lactobacillus (Lactobacillus) of examples 1-2 was higher. The relative abundance of Bifidobacterium and Lactobacillus in the positive drug group was decreased compared to the control group. Examples 1-2 were not significantly different from comparative examples 1-4.
Analysis of species composition at the gate level showed that examples 1-2 had higher relative abundance of firmicutes gate (Firmicutes) compared to the control, while bacteroidetes gate (Bacteroidetes) did not differ significantly. At the genus level, the groups of examples 1-2 also had higher relative abundance of Bifidobacterium, megamonas and Lactobacillus, but there was no significant difference between the two groups among Prevotella, dorea and ruminococcus (Ruminococcus). At the portal level, firmicutes and Bacteroidetes were both reduced in the positive group compared to the control group; at the genus level, the relative abundance of Bifidobacterium and Lactobacillus was also reduced compared to the control. Examples 1-2 were not significantly different from comparative examples 1-4.
The results show that the viable bacteria preparations of the examples and the comparative examples improve the diversity of intestinal bacteria at a certain level; the positive medicine group reduces the diversity of intestinal bacteria to a certain extent, which can be the reason of side effects of medicines such as stomachache, gastrectasia, diarrhea, vomiting, dyspepsia and the like.
2.2.3 Security analysis
There were no significant differences in the incidence of adverse reactions during follow-up in the 8 groups of patients, and 2 cases of gastroesophageal reflux disease in the positive drug group; example 1 has 1 insomnia and 1 vertigo; comparative example 2 had 1 pneumonia and 1 insomnia; the control group had 2 cases of pneumonia, 1 case of gastroesophageal reflux disease and 1 case of vertigo, all symptoms were grade 1 (mild). It was initially thought that these symptoms might not be associated with the use of live bacterial preparations, and that no serious complications or deaths occurred during follow-up.
Claims (6)
1. The live bacteria preparation for treating internal hemorrhoids is characterized by comprising lactobacillus plantarum MH-301 and auxiliary materials; the preservation number of the lactobacillus plantarum MH-301 is CGMCC NO. 18618; the auxiliary materials comprise resistant dextrin, fructo-oligosaccharide, stachyose, xylitol and inulin; the mass ratio of the resistant dextrin to the fructo-oligosaccharide to the stachyose to the xylitol to the inulin is 8-14:5-12:8-10:4-6:4-8; the mass ratio of the lactobacillus plantarum MH-301 to the auxiliary materials is 5-8:38-41.
2. The viable bacteria preparation of claim 1, wherein the viable bacteria number of lactobacillus plantarum MH-301 is more than or equal to 1X 10 9 cfu/g.
3. The viable bacteria preparation according to claim 1, wherein the mass ratio of the resistant dextrin, fructo-oligosaccharide, stachyose, xylitol and inulin is 14:5:10:4:8.
4. The viable bacteria preparation according to claim 1, wherein the mass ratio of the resistant dextrin, fructo-oligosaccharide, stachyose, xylitol and inulin is 4:6:4:3:2.
5. The viable bacteria preparation of claim 1, wherein the mass ratio of lactobacillus plantarum MH-301 to auxiliary materials is 5:41.
6. Use of a live bacterial formulation according to any one of claims 1 to 5 for the preparation of a medicament for the treatment of internal hemorrhoids.
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