A fermented portulaca oleracea preparation against diarrhea and a preparation method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BEIJING UNIV OF AGRI
- Filing Date
- 2026-02-12
- Publication Date
- 2026-06-09
Smart Images
Figure CN122163667A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to an antidiarrheal fermented purslane preparation, its preparation method, and its application. Background Technology
[0002] In clinical treatment, diarrhea interventions primarily rely on oral rehydration salts (ORS) to prevent dehydration, supplemented by antibiotics for infection control and antidiarrheal medications to relieve symptoms. However, these traditional approaches have significant limitations: the overuse and misuse of antibiotics have led to severe gut microbiota dysbiosis, resulting in the proliferation of drug-resistant strains, which not only reduces treatment effectiveness but also exacerbates the global antimicrobial resistance crisis. Simple antidiarrheal medications often relieve symptoms by inhibiting intestinal motility, but may delay the elimination of pathogens and toxins from the gut, failing to address the core issues of intestinal mucosal damage and microbiota imbalance—treating the symptoms but not the root cause. Therefore, developing a novel antidiarrheal agent that can target and regulate the gut microbiota, inhibit pathogen proliferation, repair the intestinal mucosa, and has minimal side effects has become a crucial issue urgently needing to be addressed in clinical medicine and public health.
[0003] Purslane (Portulaca oleracea L.), a traditional plant used for both food and medicine, has a long history of consumption and medicinal use in many parts of the world. In traditional medicine, it is often used to treat gastrointestinal diseases such as diarrhea, dysentery, and enteritis, with its efficacy verified through long-term practice. Modern pharmacological studies have shown that the medicinal value of purslane stems from its rich content of various bioactive components, including flavonoids, alkaloids, organic acids, and polysaccharides. Among these, purslane polysaccharides have been proven to have significant immunomodulatory functions and prebiotic effects, promoting the growth of beneficial intestinal bacteria and inhibiting the colonization of harmful bacteria, providing a solid material basis for its anti-diarrheal applications. However, the natural form of purslane and its existing applications have significant drawbacks: its active ingredients are mostly in large molecular form, resulting in low bioavailability and limited absorption efficiency by the human body; traditional extraction processes are complex and costly, and the content of active ingredients in the extracted products is unstable, leading to large fluctuations in its anti-diarrheal effects and making it difficult to achieve standardized and large-scale application, severely limiting its in-depth development and promotion in the field of anti-diarrheal medicine.
[0004] Lactic acid bacteria fermentation technology, as a mature bioprocessing method, has been widely applied in the food, health product, and pharmaceutical fields. In recent years, the use of probiotics to ferment and modify traditional Chinese medicinal materials or plants that are both food and medicine has become a research hotspot. This technology has been proven to enhance the biological value of raw materials from multiple dimensions: on the one hand, the enzyme system such as β-glucosidase produced by probiotics during fermentation can convert macromolecular active substances (such as flavonoid glycosides) in plants into small molecule aglycones, significantly improving their biological activity and human absorption rate, thus achieving "synergistic effect"; on the other hand, the fermentation process can decompose potentially harmful components such as oxalic acid in the raw materials, reducing their physiological toxicity, thus achieving "detoxification"; in addition, probiotic metabolism also produces novel beneficial substances such as lactic acid, bacteriocins, and short-chain fatty acids. These components can directly inhibit the growth of intestinal pathogens, regulate intestinal pH, and nourish the intestinal mucosa, further enhancing the physiological functions of the raw materials.
[0005] Against this backdrop, the limitations of existing diarrhea treatments, the natural advantages and application shortcomings of purslane, and the modification potential of probiotic fermentation technology present a significant technological gap. Combining compound probiotic fermentation technology with the anti-diarrheal potential of purslane, and optimizing the fermentation process to achieve efficient conversion and enhancement of purslane's active ingredients, a novel anti-diarrheal fermented preparation can be developed. This not only compensates for the shortcomings of existing treatments but also fully leverages the synergistic effects of this medicinal and edible plant with probiotics, providing a new pathway for the development of anti-diarrheal products and possessing significant clinical value and market application prospects. Therefore, this invention aims to address the problems existing in the prior art by providing a fermented purslane preparation with significant anti-diarrheal effects, high safety, and good bioavailability, along with its preparation method and applications. Summary of the Invention
[0006] The purpose of this invention is to provide a fermented purslane preparation with significant anti-diarrheal effect, high safety and good bioavailability, as well as its preparation method and application.
[0007] The objective of this invention is achieved through the following technical solution: This invention provides a method for preparing an anti-diarrheal fermented purslane preparation, comprising the following steps: (1) Pretreatment of purslane: Mix purslane with water, soak, boil and then cool to obtain purslane decoction; (2) Activation of strains: Lactobacillus plantarum and Bacillus subtilis were cultured and activated in their respective culture media for later use; (3) Fermentation: The activated Lactobacillus plantarum and Bacillus subtilis were inoculated into the purslane decoction in proportion and fermented statically at a suitable temperature; (4) Post-processing: After fermentation, centrifuge and take the supernatant to obtain the fermented purslane preparation.
[0008] Furthermore, in step (1), the ratio of purslane to water is 100g:1L, the soaking time is 1h, and the boiling time is 90min.
[0009] Furthermore, in step (2), the *Lactobacillus plantarum* subsp. *plantarum* Zhang-LL was deposited on December 4, 2012, at the China General Microbiological Culture Collection Center (CGMCC); the address of the collection unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; the accession number is CGMCC No. 6936. The *Bacillus subtilis* was *Bacillus subtilis* SH21, which was deposited on September 23, 2019, at the China General Microbiological Culture Collection Center (CGMCC); the address of the collection unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; the accession number is CGMCC No. 18612.
[0010] Furthermore, in step (3), the inoculation ratio of *Lactobacillus plantarum* to *Bacillus subtilis* is 1:1, and the total inoculation amount is 2 × 10⁻⁶. 8 The fermentation concentration was CFU / mL, the fermentation temperature was 37℃, and the fermentation time was 50h.
[0011] Furthermore, in step (4), the centrifugation speed is 8000 rpm / min and the centrifugation time is 5 min.
[0012] The present invention also provides an antidiarrheal fermented purslane preparation based on the preparation method described above.
[0013] Furthermore, the fermented purslane preparation contains 691 metabolites, of which 220 metabolites are significantly different from those in unfermented purslane. These differential metabolites include at least one of lipids and lipid molecules, phenylpropanoids and polyketides, benzene ring compounds, organic oxygen compounds, and heterocyclic compounds.
[0014] The present invention also provides the application of the aforementioned antidiarrheal fermented purslane preparation in the preparation of antidiarrheal products.
[0015] Furthermore, the antidiarrheal product is used to relieve diarrhea symptoms, regulate serum potassium and sodium ion levels in diarrhea patients, and repair intestinal mucosal damage in at least one of the following ways.
[0016] Furthermore, the dosage form of the antidiarrheal product is any one of oral liquid, powder, granules, or capsules.
[0017] Beneficial effects: The antidiarrheal fermented purslane preparation, its preparation method, and its application provided by this invention effectively overcome the technical deficiencies of existing diarrhea treatments and purslane applications, demonstrating significant technical advantages. In existing technologies, the bioavailability of purslane's active ingredients is low, and its effects are unstable. Furthermore, antibiotic abuse easily leads to intestinal flora imbalance and drug resistance, and simple antidiarrheal drugs are insufficient to eradicate the root cause. This invention utilizes a synergistic fermentation technology of *Lactobacillus plantarum* and *Bacillus subtilis*. This not only utilizes the enzyme system produced by the strain's metabolism to convert large-molecule active substances in purslane into small-molecule aglycones, significantly improving its bioactivity and human absorption rate, but also generates novel beneficial substances such as lactic acid, bacteriocins, and short-chain fatty acids during the fermentation process. These substances synergistically interact with the flavonoids and polysaccharides in purslane itself, achieving a "1+1>2" antidiarrheal effect. Simultaneously, the fermentation process decomposes potentially harmful components in purslane, reducing physiological toxicity. Moreover, the preparation is derived from medicinal and edible plants and probiotics, ensuring high safety and no obvious side effects, thus resolving the safety concerns of traditional drugs. Furthermore, this invention determined the optimal fermentation process through single-factor optimization and response surface methodology, ensuring the stable presence of 691 metabolites in the formulation. The synergistic effect of 220 differential metabolites (mainly lipids and lipid molecules, phenylpropanoids and polyketides) further guarantees the stability and reliability of the formulation's efficacy, providing a solid foundation for large-scale production.
[0018] The fermented purslane preparation of this invention demonstrates outstanding practical value in anti-diarrheal applications, exhibiting significantly superior anti-diarrheal effects compared to unfermented purslane and positive control drugs. Animal experiments have confirmed that this preparation effectively reduces the diarrhea index, rapidly regulates serum potassium and sodium ion imbalances caused by diarrhea, restores electrolyte balance, and significantly reverses inflammatory damage such as intestinal mucosal structural disorders, villous atrophy, and lymphocyte infiltration, repairing the intestinal mucosal barrier function. Compared to existing treatment regimens, this invention's preparation not only alleviates diarrhea symptoms but also addresses the root causes by regulating the intestinal microecology, inhibiting pathogens, and repairing intestinal damage, providing a comprehensive solution. Furthermore, the preparation process is simple and cost-controllable, and it can be formulated into various dosage forms such as oral liquids and powders, suitable for high-risk groups such as infants, the elderly, and those with weakened immune systems, providing a new pathway for the development of anti-diarrheal products. It not only possesses solid theoretical support and clear experimental data verification but also boasts broad clinical application prospects and market value, playing a significant role in reducing the burden of diarrheal diseases and improving public health security. Attached Figure Description
[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1 The graph shows the results of total antioxidant activity (T-AOC) detection of purslane fermented by different strains in this invention; Figure 2 This is a diagram showing the single-factor optimization results of the strain ratio of Lactobacillus plantarum subsp. plantarum Zhang-LL and Bacillus subtilis SH21 in the co-fermentation of purslane in this invention. Figure 3 This is a diagram showing the single-factor optimization results of the inoculum quantity of Lactobacillus plantarum subsp. plantarum Zhang-LL and Bacillus subtilis SH21 in the co-fermentation of purslane in this invention. Figure 4 This figure shows the single-factor optimization results of the co-fermentation time of purslane by Lactobacillus plantarum subsp. plantarum Zhang-LL and Bacillus subtilis SH21 in this invention. Figure 5 This is a response surface methodology diagram illustrating the effect of fermentation conditions on the total antioxidant activity of purslane in this invention. Figure 5 A is the response surface plot of the interaction between the proportion of bacterial species (A) and the inoculation amount (B). Figure 5 B is the response surface plot of the interaction between the proportion of microbial strains (A) and fermentation time (C). Figure 5 C is the response surface plot of the interaction between inoculum quantity (B) and fermentation time (C); Figure 6 This is a principal component analysis (PCA) diagram of bioactive substances from fermented and unfermented purslane in this invention; Figure 7 This is a metabolomics volcano diagram of bioactive substances from fermented and unfermented purslane in this invention. Figure 8 This is a statistical chart showing the classification of differentially bioactive substances in fermented purslane in this invention; Figure 9 The graph shows the evaluation results of the effects of fermented and unfermented purslane on relieving diarrhea in mice in this invention. The horizontal axis represents the experimental groups (control group, model group, positive drug group, low / medium / high dose group of unfermented purslane, and low / medium / high dose group of fermented purslane), and the vertical axis represents the diarrhea index, which is used to compare the relief effects of different groups on diarrhea symptoms in mice. Figure 10The figure shows the results of detecting the effect of fermented and unfermented purslane on serum potassium levels in mice with diarrhea in this invention. Figure 11 The figure shows the results of detecting the effect of fermented and unfermented purslane on serum sodium ion levels in mice with diarrhea in this invention. Figure 12 The figure shows the effect of fermented and unfermented purslane on the serum inflammatory factor IL-β level in mice with diarrhea in this invention. Figure 13 The figure shows the effect of fermented and unfermented purslane on the serum inflammatory factor IL-6 level in mice with diarrhea in this invention. Figure 14 The figure shows the effect of fermented and unfermented purslane on the serum TNF-α level in diarrheal mice in this invention. Figure 15 This is a histopathological HE staining image of mouse small intestine tissue in this invention (magnification 20×). Detailed Implementation
[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0026] Unless otherwise specified, the test methods used in the following implementations are all commonly used test methods in this field; Unless otherwise specified, the test materials used in the following implementations are all commonly used test materials in this field.
[0027] Example 1: Preparation of an antidiarrheal fermented purslane preparation 1.1 Experimental Materials and Instruments 1.1.1 Strains The strains used and the culture conditions are shown in Table 1. Among them, *Lactobacillus plantarum* subsp. *plantarum* Zhang-LL was deposited on December 4, 2012, at the China General Microbiological Culture Collection Center (CGMCC); address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 6936; and *Bacillus subtilis* SH21 was deposited on September 23, 2019, at the China General Microbiological Culture Collection Center (CGMCC); address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 18612.
[0028] Table 1. Main bacterial strains and culture conditions
[0029] 1.1.2 Raw Materials and Reagents Purslane; ultrapure water; MRS medium, LB medium; Total antioxidant activity assay kit (Catalog No. A015-3-1, Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.); Potassium ion assay kit (Catalog No. C001-2-1, Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.); Sodium ion assay kit (Catalog No. C002-2-1, Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.); IL-1β assay kit (Catalog No. H002-1-2, Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.); IL-6 assay kit (Catalog No. H007-1-1, Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.); TNF-α assay kit (Catalog No. H052-1-2, Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.); Acetylcholine assay kit (Catalog No. A105-2-1, Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.); Senna leaf; Roperamide; physiological saline; 4% formaldehyde solution; Hematoxylin-eosin (H&E) staining reagent.
[0030] 1.1.3 Instruments and Equipment Ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry (UHPLC-Qtrap 6500+, AB SCIEX); liquid chromatography column (Waters HSS T3, 2.1×100 mm, 1.8 μm); high-speed centrifuge; constant temperature incubator; electronic balance; UV-Vis spectrophotometer; biosafety cabinet; pathological sectioning machine; microscope; Prism 9.0 statistical analysis software; Progenesis QI metabolomics processing software.
[0031] 1.2 Experimental Procedure 1.2.1 Activation and preparation of strains Each strain in Table 1 was inoculated into its corresponding liquid culture medium and cultured for 24 hours under suitable conditions. Then, it was passaged twice at an inoculation rate of 2%. The viable cell count (CFU / mL) of each strain was determined by plate counting method and kept for later use.
[0032] 1.2.2 Preparation of Purslane Decoction Take 100g of purslane, add 1L of ultrapure water, soak for 1 hour, then boil for 90 minutes. After cooling, obtain the purslane decoction for later use.
[0033] 1.2.3 Screening of fermentation strains The six activated bacterial strains were respectively subjected to 1.0 × 10⁶... 7CFU / mL viable bacteria were inoculated into purslane decoction and fermented statically for 48 h at the optimal temperature for each strain. After fermentation, the mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was collected. The total antioxidant activity (T-AOC) of each fermentation supernatant was detected using a total antioxidant activity assay kit (catalog number A015-3-1). Unfermented purslane decoction was used as the control group (CK) to screen for strains with the best antioxidant activity enhancement.
[0034] The final result is as follows Figure 1 As shown, the T-AOC values of the fermentation broths of the six strains after fermenting purslane were significantly higher than those of the unfermented group (CK). Among them, the T-AOC values of the fermentation broths of Bacillus subtilis SH21 and Lactobacillus plantarum subsp. plantarum Zhang-LL were 9.16 mM and 9.74 mM, respectively, which were significantly higher than those of the other four lactic acid bacteria (p<0.01), and there was no significant difference between the two (p>0.05). Therefore, these two strains were selected for co-fermentation.
[0035] 1.2.4 Single-factor optimization of synergistic fermentation by compound probiotics The selected Lactobacillus plantarum subsp. plantarum Zhang-LL and Bacillus subtilis SH21 were selected for co-fermentation. The ratio of strains, inoculum amount and fermentation time were optimized by single factors, and the total antioxidant activity (T-AOC) of the fermentation supernatant was used as the evaluation index.
[0036] (1) Single-factor optimization of bacterial strain ratio: Lactobacillus plantarum subsp. plantarum Zhang-LL and Bacillus subtilis SH21 were inoculated into purslane decoction at ratios of 3:1, 2:1, 1:1, 1:2 and 1:3, respectively, with a total inoculum of 2.0 × 10⁻⁶. 7 The concentration of CFU / mL was used for static fermentation at 37℃ for 48 hours. After fermentation, the mixture was centrifuged at 8000 rpm for 5 minutes, and the supernatant was used to detect T-AOC.
[0037] The final result is as follows Figure 2As shown, when the inoculation ratio of Lactobacillus plantarum subsp. plantarum Zhang-LL and Bacillus subtilis SH21 was 1:1, the T-AOC value of fermented purslane was 16.81 mM, which was significantly higher than that of the other four inoculation ratios (p<0.01).
[0038] (2) Single-factor optimization of inoculum size: Lactobacillus plantarum subsp. plantarum Zhang-LL and Bacillus subtilis SH21 were inoculated into purslane decoction at a ratio of 1:1. The total inoculum size was set to 5.3 lg CFU / mL, 6.3 lg CFU / mL, 7.3 lg CFU / mL, 8.3 lg CFU / mL and 9.3 lg CFU / mL respectively. The mixture was statically fermented at 37℃ for 48 h. After fermentation, the mixture was centrifuged at 8000 rpm for 5 min and the supernatant was used to detect T-AOC.
[0039] The final result is as follows Figure 3 As shown, when the total inoculum was 8.3 lgCFU / mL, the T-AOC value of fermented purslane was 18.86 mM, which was significantly higher than the other four inoculum values (p<0.01).
[0040] (3) Single-factor optimization of fermentation time: Lactobacillus plantarum subsp. plantarum Zhang-LL and Bacillus subtilis SH21 were inoculated into purslane decoction at a ratio of 1:1, and the total inoculum was set at 2×10⁻⁶. 7 CFU / mL, fermentation time was set at 37℃ for 24h, 36h, 48h, 60h and 72h respectively. After fermentation, centrifugation was performed at 8000rpm for 5min, and the supernatant was taken to detect T-AOC.
[0041] The final result is as follows Figure 4 As shown, the T-AOC values for fermentation times of 48h and 72h were 16.24mM and 16.85mM, respectively, which were significantly higher than those for the other three fermentation times (p<0.01).
[0042] 1.2.5 Response Surface Optimization of Fermentation Process Based on the results of single-factor optimization, the inoculum ratio (A), inoculum quantity (B, 1g CFU / mL), and fermentation time (C, h) were selected as influencing factors. The total antioxidant activity of the fermentation broth was used as the response value. A three-factor, three-level response surface experiment was conducted using a Box-Behnken design. The factors and levels are shown in Table 2.
[0043] Table 2 Box-Behnken Design Factors and Levels
[0044] Fermentation experiments were conducted according to the experimental scheme shown in Table 3. The total antioxidant activity of each experimental group was measured. The influence of each factor on the response value was analyzed by regression model to determine the optimal fermentation process conditions.
[0045] Table 3. Response Surface Experimental Design and Results
[0046] Regression analysis and analysis of variance were performed on the response surface experimental data. The results of the analysis of variance are shown in Table 4. The effectiveness of the model was verified, and the optimal fermentation process conditions were finally determined and verified by experiments.
[0047] Table 4. Analysis of Variance of the Regression Model
[0048] The final results are shown in the response surface curves of different fermentation conditions on the total antioxidant activity of purslane, as follows: Figure 5 As shown, by Figure 5 The regression model was highly significant (p<0.01), showing a good fit to the response values. The model's lack-of-fit term had a p-value of 0.6542, indicating it was not significant. The influence of the three factors on the T-AOC of fermented purslane was B (inoculum quantity) > A (inoculum ratio) > C (fermentation time). The optimized process conditions obtained through model analysis were: inoculum ratio 1:0.89, inoculum quantity 2×10^7.85 CFU / mL, and fermentation time 50.6 h. For ease of implementation, the actual optimal process combination was determined to be an inoculum ratio of 1:1, inoculum quantity 2×10^8 CFU / mL, and fermentation time 50 h. Under this process, the total antioxidant activity of fermented purslane reached 19.4 mM.
[0049] 1.2.6 Preparation of Fermented Purslane Preparation The optimal process conditions obtained through response surface methodology are a 1:1 inoculum ratio and an inoculum quantity of 2 × 10⁻⁶. 8 The purslane decoction was subjected to static fermentation at 37℃ for 50 hours with a concentration of CFU / mL. After fermentation, the mixture was centrifuged at 8000 rpm for 5 minutes, and the supernatant was collected. This supernatant is the antidiarrheal fermented purslane preparation.
[0050] 1.3 Metabolomics analysis of fermented purslane preparations Ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry (UHPLC-Qtrap) was used to analyze the supernatant samples of the control group (unfermented purslane decoction, CK) and the fermented purslane preparation (Hun).
[0051] 1.3.1 Chromatographic and Mass Spectrometric Conditions Chromatographic conditions: ExionLC AD system, Waters HSS T3 (2.1×100 mm, 1.8 μm) liquid chromatography column, column temperature 35℃, injection volume 1 μL; mobile phase A was 0.1% formic acid water, and mobile phase B was 0.1% formic acid acetonitrile.
[0052] Mass spectrometry conditions: AB SCIEX QTRAP 6500+, positive / negative mode detection, Curtain Gas (CUR) 35, Collision Gas (CAD) Medium, IonSpray Voltage (IS) +5500 / -4500, Temperature (TEM) 550, Ion Source Gas1 (GS1) 55, Ion Source Gas2 (GS2) 55.
[0053] 1.3.2 Data Processing and Analysis Raw LC-MS data were imported into the metabolomics processing software Progenesis QI for baseline filtering, peak identification, integration, retention time correction, and peak alignment to obtain a data matrix of retention time, mass-to-charge ratio, and peak intensity. MS and MS / MS mass spectrometry information were matched with the HMDB database (http: / / www.hmdb.ca) and the Metlin database (https: / / metlin.scripp-s.edu) to obtain metabolite information. Differential metabolites were annotated using the KEGG database (https: / / www.kegg.jp / kegg / pathway.html), and pathway enrichment analysis was performed using the Python package scipy.stats. Fisher's exact test was used to identify the biological pathways most relevant to fermentation treatment.
[0054] A total of 691 metabolites were identified from both unfermented and fermented purslane. Principal component analysis (PCA) showed significant differences in the content of active ingredients between the unfermented and fermented groups (e.g., ...). Figure 6 As shown); after probiotic fermentation, the content of 220 metabolites in purslane changed significantly (p<0.05), of which 129 metabolites were upregulated, 91 metabolites were downregulated, and 471 metabolites remained unchanged (e.g. Figure 7 As shown). Figure 8 As shown, the identified differentially expressed compounds can be mainly divided into 10 categories. Lipids and lipid-like molecules were the most numerous, totaling 35 types, accounting for 21.60%, indicating their dominant role in metabolic composition. Phenylpropane and polyketides followed, totaling 31 types (19.14%), while benzene ring compounds totaled 27 types (16.67%). Together, these two categories accounted for more than one-third of the total, suggesting that phenylpropane metabolism and polyketide biosynthesis pathways are relatively active. Organic oxygen compounds (11.73%) and heterocyclic compounds (12.35%) accounted for 19 and 20 types respectively, also representing a significant proportion. Organic acids and their derivatives totaled 12 types (7.41%), and nucleosides and their analogues totaled 10 types (6.17%). Other categories, such as alkaloids and their complexes (3.03%), organic nitrogen compounds (1.29%), and hydrocarbons (0.63%), were fewer in number, each accounting for less than 5%.
[0055] Example 2: Evaluation of the antidiarrheal effect of fermented purslane preparation 2.1 Laboratory Animals and Grouping Ninety male SPF-grade Kunming mice, weighing 20±2g, were randomly divided into 9 groups of 10 mice each. All mice were acclimatized for 7 days under pathogen-free conditions, with free access to food and water. Before the experiment, the mice were fasted for 8–10 hours.
[0056] The grouping is as follows: Group 1: Control group (CK), no senna leaf modeling was given, and no drug treatment was given; Group 2: Model group, senna leaves were administered to establish the model, and 0.2 mL of physiological saline was administered by gavage; Group 3: Positive drug group (Smecta), senna leaf was administered to establish the model, and 0.2 mL of ropivacaine (0.5 mg / mL) was administered by gavage. Group 4: Low-dose unfermented purslane group (UFP-L), senna leaves were administered to establish the model, and 0.2 mL of unfermented purslane decoction (0.5 mg / mL) was administered by gavage. Group 5: Unfermented purslane medium-dose group (UFP-M), senna leaves were administered to establish the model, and 0.2 mL of unfermented purslane decoction (1 mg / mL) was administered by gavage. Group 6: High-dose unfermented purslane group (UFP-H), senna leaves were administered to establish the model, and 0.2 mL of unfermented purslane decoction (2 mg / mL) was administered by gavage. Group 7: Low-dose fermented purslane group (FP-L), senna leaves were administered to establish the model, and 0.2 mL of the fermented purslane preparation (0.5 mg / mL) prepared in Example 1 was administered by gavage. Group 8: Fermented purslane medium-dose group (FP-M), senna leaves were administered to establish the model, and 0.2 mL of the fermented purslane preparation prepared in Example 1 (1 mg / mL) was administered by gavage. Group 9: High-dose fermented purslane group (FP-H), senna leaves were administered to establish the model, and 0.2 mL of the fermented purslane preparation (2 mg / mL) prepared in Example 1 was administered by gavage.
[0057] 2.2 Modeling and Drug Administration Except for the control group, all other groups of mice were administered 0.2 ml of senna leaf concentrate (1 g / mL) by gavage to establish the model. Two hours after gavage, the mice were treated with the corresponding drugs according to the above grouping.
[0058] 2.3 Indicator Detection and Observation 2.3.1 Diarrhea Index Measurement Animals were observed for defecation 4 hours after administration. Filter paper was placed under each cage to record the characteristics of diarrheal stool and calculate the diarrhea index.
[0059] Depend on Figure 9 It can be seen that gavage with low-dose unfermented purslane (UFP-L) can significantly reduce the diarrhea index from 2.3 in the model group to 1.4 (p<0.01), while the diarrhea index of the low-dose fermented purslane (FP-L) gavage group can significantly reduce to 1.0 (p<0.01), and the diarrhea index of the FP-L group is significantly lower than that of the UFP-L group (p<0.05), and its antidiarrheal effect is better than that of unfermented purslane and positive control drug (Smecta).
[0060] 2.3.2 Sample Collection Four hours later, blood was collected from all mice under the eyes, and the serum was separated by centrifugation at 3000 rpm for 15 minutes for later use. The mice were then euthanized by cervical dislocation, and the jejunum, ileum, and colon tissues were collected for later use.
[0061] 2.3.3 Serum electrolyte level detection The potassium and sodium ion levels in serum were determined using a potassium ion detection kit (catalog number C001-2-1) and a sodium ion detection kit (catalog number C002-2-1). For specific operating procedures, please refer to the kit instructions.
[0062] Depend on Figure 10-11 It can be seen that the serum potassium ions (K+) in the model group mice are high. + ) and sodium ions (Na +The concentration of potassium ions in the UFP-L group was significantly reduced (p<0.05); the serum potassium ion content in the UFP-L group increased from 3.85 mmol / L in the model group to 4.30 mmol / L, and the serum potassium ion content in the FP-L group increased to 4.85 mmol / L, both significantly higher than that in the model group (p<0.05); the serum sodium ion level in the UFP-L group increased from 78.23 mmol / L in the model group to 94.42 mmol / L, and the serum sodium ion level in the FP-L group increased to 130.50 mmol / L, both significantly higher than that in the model group (p<0.05), and the sodium ion level in the FP-L group was significantly higher than that in the UFP-L group (p<0.05).
[0063] 2.3.4 Detection of serum inflammatory factor levels The levels of IL-1β, IL-6 and TNF-α in mouse serum were detected using the IL-1β assay kit (catalog number H002-1-2), the IL-6 assay kit (catalog number H007-1-1), and the TNF-α assay kit (catalog number H052-1-2). For specific operation, please refer to the kit instructions.
[0064] Depend on Figure 12-13 It was found that, compared with the control group (CK), the serum concentrations of IL-1β and IL-6 in the model group mice were significantly increased (p<0.05); the serum IL-1β level in the UFP-H group was significantly decreased from 21.5 ng / L in the model group (p<0.05) to 8.95 ng / L, and the IL-6 concentration was significantly decreased from 17.6 ng / mL in the model group (p<0.05) to 10.7 ng / mL. Further analysis... Figure 14 The serum TNF-α concentration in the model group mice was 170 pg / mL, while the serum TNF-α concentration in the UFP-H group mice was significantly reduced (p<0.05) to 142 pg / mL. This indicates that fermented purslane can significantly reduce inflammatory factors in diarrheal mice.
[0065] 2.3.5 Pathological observation of intestinal tissue The collected jejunum, ileum, and colon tissues were fixed with 4% formaldehyde solution, then stained with hematoxylin and eosin (H&E) to prepare pathological sections. The pathological changes such as intestinal mucosal structure, villus morphology, lamina propria lymphocyte infiltration, and goblet cell count were observed under a microscope.
[0066] Depend on Figure 15The results showed that, compared with the control group, the colon, jejunum, and ileum mucosal structures of the model group mice were disordered, with widespread atrophy, shortening, and even fusion of villi. A large number of lymphocytes diffusely infiltrated the lamina propria, and the number of goblet cells was significantly reduced, indicating inflammatory damage. The unfermented purslane group (UFP) and the fermented purslane group (FP) significantly reversed the inflammatory pathology of the small intestine, and no signs of toxicity such as death, excessive urination, or respiratory changes were observed. These results indicate that fermented purslane can effectively alleviate intestinal damage caused by diarrhea.
[0067] 2.4 Statistical Analysis All test data were statistically analyzed and plotted using Prism 9.0 software.
[0068] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. A method for preparing a fermented purslane preparation for treating diarrhea, characterized in that, Includes the following steps: (1) Pretreatment of purslane: Mix purslane with water, soak, boil and then cool to obtain purslane decoction; (2) Activation of strains: Lactobacillus plantarum and Bacillus subtilis were cultured and activated in their respective culture media for later use; (3) Fermentation: The activated Lactobacillus plantarum and Bacillus subtilis were inoculated into the purslane decoction in proportion and fermented statically at a suitable temperature; (4) Post-processing: After fermentation, centrifuge and take the supernatant to obtain the fermented purslane preparation.
2. The preparation method according to claim 1, characterized in that, In step (1), the ratio of purslane to water is 100g:1L, the soaking time is 1h, and the boiling time is 90min.
3. The preparation method according to claim 1, characterized in that, In step (2), the *Lactobacillus plantarum* subsp. *plantarum* Zhang-LL was deposited on December 4, 2012, at the China General Microbiological Culture Collection Center (CGMCC); the address of the collection unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; the accession number is CGMCC No. 6936. The *Bacillus subtilis* was *Bacillus subtilis* SH21, which was deposited on September 23, 2019, at the China General Microbiological Culture Collection Center (CGMCC); the address of the collection unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. Accession number: CGMCC No.18612.
4. The preparation method according to claim 1, characterized in that, In step (3), the inoculation ratio of *Lactobacillus plantarum* to *Bacillus subtilis* is 1:1, and the total inoculation amount is 2 × 10⁻⁶. 8 The fermentation concentration was CFU / mL, the fermentation temperature was 37℃, and the fermentation time was 50h.
5. The preparation method according to claim 1, characterized in that, In step (4), the centrifugation speed is 8000 rpm / min and the centrifugation time is 5 min.
6. A fermented purslane preparation for treating diarrhea, prepared according to any one of claims 1-5.
7. The fermented purslane preparation for treating diarrhea according to claim 6, characterized in that, The fermented purslane preparation contains 691 metabolites, of which 220 metabolites are significantly different from those in unfermented purslane. These differential metabolites include at least one of lipids and lipid molecules, phenylpropanoids and polyketides, benzene ring compounds, organic oxygen compounds, and heterocyclic compounds.
8. The use of a fermented purslane preparation for treating diarrhea as described in any one of claims 6-7 in the preparation of an antidiarrheal product.
9. The application according to claim 8, characterized in that, The antidiarrheal product is used to relieve diarrhea symptoms, regulate serum potassium and sodium ion levels in patients with diarrhea, and repair intestinal mucosal damage at least one of the following:
10. The application according to any one of claims 8-9, characterized in that, The dosage form of the antidiarrheal product is any one of oral liquid, powder, granule, or capsule.