Use of a shenquo food digesting oral composition in the preparation of a medicine for regulating intestinal flora

Abstract

The application discloses application of a Shenquo Shixi oral composition in preparation of a medicine for regulating intestinal flora, compared with a control group, the Butyricimonas, Candidatus Soleaferrea, NK4A214 group, Rikenella and Ruminococcus abundances of an experimental group of the Shenquo Shixi oral composition significantly decrease after intragastric administration, and the Corynebacterium, GCA900066575, Peptococcus, Psychrobacter and Turicibacter abundances significantly increase, wherein the abundances of the Corynebacterium, Psychrobacter, Rikenella and other species recover to be equivalent to those of a blank group, and the composition disclosed by the application is helpful to adjustment and recovery of intestinal flora of FD rats.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine compound preparations, specifically relating to the application of a Shenqu (medicated leaven) digestive oral composition in the preparation of drugs for regulating intestinal flora. Background Technology

[0002] Functional dyspepsia (FD) typically presents with upper abdominal pain and discomfort after meals, postprandial fullness, and early satiety, without obvious organic lesions, and is mostly caused by gastrointestinal motility disorders. Functional dyspepsia can be divided into three categories based on pathophysiology and etiology:

[0003] (1) Postprandial discomfort syndrome (PDS) is clinically manifested as abdominal distension after eating a normal amount of food, or a significant reduction in food intake due to early satiety. It is usually caused by gastric motility disorders and gastrointestinal motility coordination disorders.

[0004] (2) Upper abdominal pain syndrome (EPS) is characterized by symptoms such as upper abdominal pain, nausea, and vomiting. The etiology is not fully understood, but it may be related to a variety of factors, such as improper diet, food allergies, and abnormal gastrointestinal motility.

[0005] (3) Types that combine the characteristics of PDS and EPS.

[0006] With changes in dietary structure and lifestyle, as well as increasing social pressure, the incidence of functional dyspepsia is gradually rising, with a global prevalence exceeding 10%. Although functional dyspepsia is a non-fatal disease, many patients also experience anxiety, insomnia, lethargy, and other overlapping symptoms, which affect their quality of life, frequency of medical visits, and mental well-being.

[0007] The human and animal gut contains a complex microbial community composed of bacteria, fungi, viruses, and archaea. These microbial members are commonly referred to as the gut microbiota, or intestinal flora. During childbirth, some gut microbiota (including Bacteroides) are passed from mother to child, thus becoming part of the individual's gut microbiota in the early stages of life. The microbiota structure subsequently changes according to the host's life events, such as diet, medications, lifestyle habits, and age. The gut microbiota can be considered a "dynamic organ," participating in the regulation of various physiological processes in the host, such as nutrient absorption, immune responses, and disease development. However, disruption of this balance can also have adverse effects on the host, leading to functional gastrointestinal disorders.

[0008] Functional dyspepsia is one of the most common functional gastrointestinal disorders. Previous studies have shown that the occurrence of functional dyspepsia is related to multiple pathophysiological mechanisms, including gastric and duodenal motility disorders, mild duodenal inflammation, visceral hypersensitivity, brain-gut interactions, and genetic susceptibility. However, new research has found differences in the gut microbiota between patients with functional dyspepsia and healthy controls, suggesting that gut ecological imbalance may be related to gastrointestinal dysfunction.

[0009] Chinese patent CN106620485B discloses a method for preparing an oral composition of Shenqu (a type of fermented medicinal herb) for digestion. The composition prepared by this method promotes small intestinal propulsion in normal mice and mice with spleen deficiency, and also significantly promotes gastric emptying in mice with spleen deficiency. However, it does not disclose the application of the oral composition of Shenqu for digestion in the preparation of drugs for regulating intestinal flora. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of the Shenqu Xiaoshi oral composition in the preparation of drugs for regulating intestinal flora.

[0011] The technical solution of this invention is summarized as follows:

[0012] Application of a digestive oral composition for treating intestinal flora in the preparation of a drug.

[0013] The aforementioned Shenqu digestive oral composition is prepared according to the preparation method of the Shenqu digestive oral composition disclosed in Chinese Patent CN106620485 B.

[0014] Advantages of the present invention

[0015] At the genus level, compared with the control group, the abundance of Butyricimonas, Candidatus Soleaferrea, NK4A214 group, Rikenella, and Ruminococcus was significantly reduced in the experimental group treated with Shenqu Xiaoshi oral liquid, while the abundance of Corynebacterium, GCA 900066575, Peptococcus, Psychrobacter, and Turicibacter was significantly increased. Among them, the abundance of species such as Corynebacterium, Psychrobacter, and Rikenella recovered to a level comparable to that of the blank group, indicating that the Shenqu Xiaoshi oral liquid composition helps to adjust and restore the intestinal flora of rats with functional dyspepsia. Attached Figure Description

[0016] Figure 1The changes in immune factors in the gastric tissue and blood of rats under the intervention of Shenqu Xiaoshi oral liquid are shown. Among them, A is the concentration of IL-10 in the gastric tissue of rats, B is the concentration of IL-10 in the serum of rats, C is the concentration of IL-6 in the gastric tissue of rats, D is the concentration of IL-6 in the serum of rats, and E is the concentration of sIgA in the serum of rats.

[0017] Figure 2 The results are from the Lefse analysis of the gut microbiota of rats. Detailed Implementation

[0018] The Shenqu digestive oral composition of the present invention (also known as Shenqu digestive oral liquid) is disclosed in Chinese Patent CN106620485B, which was applied for and authorized by Jiangsu Longfengtang Pharmaceutical Co., Ltd. of Yangtze River Pharmaceutical Group. The invention is entitled: Preparation method of Shenqu digestive oral composition.

[0019] A method for preparing a digestive oral composition, wherein the composition is prepared from the following traditional Chinese medicine raw materials in the indicated weight ratios: (the following weight parts are specifically in grams).

[0020] 15 parts by weight of roasted hawthorn, 15 parts by weight of roasted Shenqu (medicated leaven), 15 parts by weight of roasted malt, 25 parts by weight of white peony root, 15 parts by weight of codonopsis root, 15 parts by weight of poria cocos, 8.5 parts by weight of roasted atractylodes macrocephala, 8 parts by weight of costus root, 6 parts by weight of amomum villosum, 12 parts by weight of vinegar-processed corydalis rhizome, and 10 parts by weight of roasted licorice root.

[0021] The preparation method steps are as follows:

[0022] Step 1: Add 20 times the amount of water to Amomum villosum, Aucklandia lappa, and Atractylodes macrocephala (or any value between 8 and 40 times, such as 8, 10, 15, 25, 32, 35, or 40 times), extract and collect the volatile oil;

[0023] Step 2: Collect the dregs and liquid after the volatile oil is removed, and decoct them together with the remaining medicinal materials twice (or three times). Each time, add 8 times the amount of water (or any value between 6 and 10 times, such as 6 times or 10 times) and decoct for 2 hours (or any value between 1 and 3 hours, such as 1 hour or 3 hours). Combine the decoctions, filter, and concentrate.

[0024] Step 3: Add ethanol to make the alcohol content reach 65% (or any value between 55% and 75%, such as 55% or 75%), refrigerate for 24 hours (or any time between 10 and 48 hours, such as 10 hours or 48 hours), precipitate out, filter, and recover the ethanol from the filtrate under reduced pressure until there is no alcohol odor.

[0025] Step 4: Add volatile oil and appropriate amount of surfactant, then add 1 part by weight of taurine (or 2 parts by weight), appropriate amount of asparagine methyl ester (or appropriate amount of sucralose), appropriate amount of preservative, adjust with distilled water to a total of 1000 parts by weight, refrigerate, filter, fill the filtrate, sterilize to obtain oral liquid.

[0026] Example 1: Shenqu Xiaoshi Oral Liquid Relieves Inflammation in Rats with Functional Dyspepsia

[0027] 1. Dilute hydrochloric acid-induced functional dyspepsia (FD) model

[0028] SPF-grade male SD rats were acclimatized for one week under the following conditions: temperature 20℃~26℃, humidity 40%~70%, with 12-hour light-dark alternation. The SPF-grade male SD rats used were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., and were 6-8 weeks old, weighing 180g~200g. All rats were randomly divided into a blank group, a control group, and an experimental group, with 12 rats in each group. All rats were provided with 30g of feed per day according to their required intake.

[0029] The control group rats had free access to food and water every day;

[0030] Rats in both the control and experimental groups were fed on one day and fasted on two days, with free access to 0.1M hydrochloric acid solution.

[0031] Observe the rats' fur color, mental state and activity level every day, as well as their weight, food intake and water intake. Record the data every three days and continue feeding for four weeks.

[0032] 2. Animal model grouping and drug administration

[0033] After successful model establishment, the drug administration phase began. The experimental group animals were divided into subgroups, and each group of rats was treated as follows: During the drug administration period, all rats ate and drank normally. The color of their fur, mental state, and activity level were observed daily, and their weight, food intake, and water intake were recorded. Drug administration continued for 4 weeks.

[0034] Control group: Healthy rats were given 2 ml of physiological saline by gavage at the same time every morning, once a day.

[0035] Control group: FD model rats were administered 2 ml of physiological saline by gavage at the same time every morning, once a day.

[0036] Experimental group: FD model rats were administered 2ml of Shenqu Xiaoshi oral liquid by gavage at the same time every morning, once a day.

[0037] 3. Detection of rat immune factors

[0038] Six rats were randomly selected from each of the blank group, control group, and experimental group. After the last gavage, the rats were fasted and deprived of water for 24 hours. The rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg / kg). After the rats became pliable, they were fixed on the operating table. The abdominal skin was cut open with surgical scissors, the abdominal cavity was opened, and blood was collected from the abdominal aorta using a blood collection needle and vacuum blood collection tube. After blood collection, the rats were euthanized by cervical dislocation, and the gastric antrum tissue was removed.

[0039] Rat gastric antrum tissue was collected, rinsed in physiological saline to remove blood, blotted dry with absorbent paper, and weighed. The gastric antrum tissue was minced, and pre-cooled physiological saline was added at a ratio of 1 ml of physiological saline per 50 mg of tissue sample. The mixture was homogenized using a tissue homogenizer, centrifuged at 5000 rpm for 10 min, and the supernatant was retained as the gastric tissue homogenate supernatant. Rat aortic blood samples were centrifuged at 5000 rpm for 10 min, and the supernatant was the serum. The levels of immunoglobulin sIgA, interleukin 10 (IL-10), and interleukin 6 (IL-6) in the tissue homogenate supernatant and serum were detected using rat secretory immunoglobulin A (sIgA), rat interleukin 10 (IL-10), and rat interleukin 6 (IL-6) ELISA kits, respectively. Specific procedures were performed according to the kit instructions. All kits were purchased from Beijing Wolais Biotechnology Co., Ltd.

[0040] Results of ELISA detection of immune factors as follows Figure 1 The levels of IL-10 in the gastric tissue and serum of rats in the control group were significantly lower than those in the blank group, while the levels of IL-10 in the gastric tissue and serum of rats in the experimental group were significantly higher than those in the control group (see...). Figure 1 A and Figure 1 B). IL-6 levels showed results opposite to those of IL-10 (see...). Figure 1 C and Figure 1 D) The levels of IL-6 in the gastric tissue and serum of rats in the control group were significantly higher than those in the blank group, while the levels of IL-6 in the gastric tissue and serum of rats in the experimental group were significantly lower than those in the control group. The sIgA detection results in tissue samples from rats in the blank group, control group, and experimental group were all negative, while the sIgA level in the serum of rats in the blank group was the highest (see...). Figure 1 E) There was no significant difference between the control group and the experimental group, but the results were not statistically significant. IL-10 is a pleiotropic cytokine that can inhibit inflammatory factors (such as IL-6) by activating macrophages. Shenqu Xiaoshi oral liquid increased the IL-10 level in the gastric tissue and serum of rats with functional dyspepsia, while decreasing the IL-6 level. There was no significant difference in the level of immunoglobulin sIgA in the blood, indicating that Shenqu oral liquid could not promote the secretion of immunoglobulin sIgA.

[0041] Example 2: Regulating the Intestinal Flora of Rats with Shenqu Digestive Oral Liquid

[0042] Following steps 1 and 2 of Example 1, the remaining 6 rats were selected from the blank group, control group, and experimental group. After the last administration, the rats were placed in metabolic cages, and fresh feces were collected from each rat in the blank group, model group, and experimental group separately using the metabolic cages. The feces were transferred to sterile centrifuge tubes with forceps, frozen in liquid nitrogen, and stored at -80°C. Beijing Biomarker Biotechnology Co., Ltd. was then commissioned to perform 16S amplicon sequencing and sequencing data processing on the microbial community in the fecal samples.

[0043] Experimental results:

[0044] (1) Species annotation and taxonomic analysis

[0045] Taxonomic annotation of feature sequences was performed using Silva.138 as a reference database, yielding species classification information for each feature. Subsequently, the species composition of the bacterial communities in each sample was statistically analyzed at the phylum, class, order, family, genus, and species levels. At the phylum level, Firmicutes, Bacteroidetes, Actinobacteriota, and Proteobacteria comprised over 90% of the bacteria in the rat gut microbiota, similar to the human gut microbiota. Compared to the control group, oral administration of Shenqu Xiaoshi oral liquid did not significantly change the abundance of these four phyla (Table 1).

[0046] At the genus level, compared with the control group, the abundance of Butyricimonas, Candidatus Soleaferrea, NK4A214 group, Rikenella, and Ruminococcus was significantly reduced in the experimental group treated with Shenqu Xiaoshi oral liquid by gavage, while the abundance of Corynebacterium, GCA 900066575, Peptococcus, Psychrobacter, and Turicibacter was significantly increased (see Table 2). Among them, the abundance of species such as Corynebacterium, Psychrobacter, and Rikenella recovered to a level comparable to that of the blank group, indicating that Shenqu Xiaoshi oral liquid helps to adjust and restore the intestinal flora of rats with functional dyspepsia.

[0047] Table 1. Species composition of the gut microbiota at the phylum level in rats

[0048]

[0049] Table 2 Abundance of some species in the horizontal gut microbiota of rats

[0050]

[0051] (2) Intergroup differences in rat gut microbiota

[0052] Between-group difference analysis, also known as between-group significance test, is a commonly used statistical method in microbial data analysis to compare differences between two or more groups.

[0053] This invention uses Lefse analysis to identify differences in gut microbiota among the blank group, control group, and experimental group of rats.

[0054] LEFSE analysis is an analytical technique that combines the Kruskal-Wallis test and Wilcoxon rank-sum test with linear discriminant analysis (LDA) effect size. It can identify species (i.e., biomarkers) with significant differences in abundance between different groups. The LDA score represents the magnitude of each species' abundance effect on the differential effect; generally, an LDA > 2 is considered a biomarker species with significant differences between groups. Figure 2 The bar chart shows the abundance of marker species among groups under the condition of LDA>3. At the genus level, the abundance of *Roseburia*, *Prevotella 9*, uncultured *Barnesiella sp.*, *Lachnospira*, *Parasutterella*, and uncultured *rumen bacterium* in the gut microbiota of rats in the control group was significantly higher than that in the experimental and control groups. The abundance of unclassified *Lachnospiraceae*, *Quinella*, *Treponema*, and *Akkermansia* in the gut microbiota of rats in the experimental group was significantly higher than that in the control group. The abundance of *Ruminococcus*, unclassified *Eubacterium coprostanoligenes group*, and unclassified *DTU014* in the gut microbiota of rats in the control group was significantly higher than that in the control and experimental groups.

[0055] Akkermansia colonizes the intestinal mucosa, participates in host metabolic and immune regulation, and maintains the integrity of intestinal epithelial cells and the thickness of the mucus layer.

[0056] Ruminococcus is a common species in the gut microbiota of mammals and is involved in the digestion and breakdown of dietary starch. However, studies have found that its abundance is positively correlated with diseases such as Crohn's disease, ulcerative colitis, and irritable bowel syndrome.

Claims

1. The application of a digestive oral composition for treating intestinal flora in the preparation of a drug for regulating intestinal flora.

2. The application according to claim 1, characterized in that: The aforementioned Shenqu digestive oral composition is prepared according to the preparation method of the Shenqu digestive oral composition disclosed in Chinese Patent CN106620485B.

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