Microcos paniculata methanol phase extract with antibacterial activity and application thereof
A methanol phase extract from Buza leaves, produced via a specific extraction method, effectively inhibits pathogens including Vibrio parahaemolyticus, addressing antibiotic resistance and restoring intestinal health.
Patent Information
- Application Number
- CN202411348676.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-26
- Publication Date
- 2024-12-13
AI Technical Summary
There is an urgent need for new antimicrobial agents to combat foodborne pathogens like Vibrio parahaemolyticus, as antibiotics are ineffective and contribute to resistance and environmental pollution.
A methanol-chloroform extraction method is used to produce a methanol phase extract from Buza leaves, which is then tested for its antibacterial and antifungal activities against various pathogens, including Vibrio parahaemolyticus, using methods like agar disc diffusion and minimum inhibitory concentration.
The extract effectively inhibits a range of pathogens in vitro and reduces Vibrio parahaemolyticus in vivo, restoring intestinal flora balance and reducing tissue damage in mice, with potential applications in foods and medicines.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a methanol phase extract of Buza leaves with antibacterial and antimicrobial activities and application thereof. Background Art
[0002] Foodborne pathogens pose a serious threat to global public health and human health. According to a report by the World Health Organization (WHO), about 600 million people worldwide become ill each year from eating food contaminated by foodborne pathogens, resulting in about 420,000 deaths. Among common foodborne pathogens, Vibrio parahaemolyticus can cause acute diarrhea, severe vomiting, gastric ulcers, sepsis, and even death in severe cases.
[0003] Antibiotics play a vital role in the treatment of infectious diseases. However, inappropriate use of antibiotics leads to antibiotic resistance in pathogenic bacteria, resulting in the death of approximately 700,000 people worldwide each year. Long-term overuse of antibiotics also creates food safety risks and environmental pollution problems. Therefore, it is urgent to screen and discover new antimicrobial agents.
[0004] Medicinal and edible plants have become a green natural compound library for antimicrobial screening due to their outstanding advantages of low toxicity and safety. Among them, the dried leaves of Microcos paniculata Linnaeus are often used as traditional Chinese medicinal materials and herbal tea ingredients, with the effects of clearing heat and dampness, digesting food and removing stagnation, analgesic and anti-inflammatory, and insecticidal. In recent years, studies have shown that a variety of compounds extracted from Microcos paniculata Linnaeus have anti-inflammatory effects, inhibit α-glucosidase activity, and have anti-tumor activity. However, there are few reports on the antibacterial effect of Microcos paniculata Linnaeus. Summary of the invention
[0005] The main purpose of the present invention is to provide a methanol-phase extract (MPE) of Buza leaves with antibacterial and antifungal activity, which can inhibit a variety of common pathogenic bacteria in vitro and has the effect of inhibiting pathogenic Vibrio parahaemolyticus in vivo.
[0006] Another object of the present invention is to provide the use of the methanol extract of Buzha leaves having antibacterial and antibacterial activities in the preparation of antibacterial and antibacterial foods and medicines.
[0007] To achieve the above object, the present invention adopts the following technical solution:
[0008] The present invention provides a methanol extract of Buza leaves having antibacterial and antifungal activities, which is prepared by a methanol-chloroform extraction method, specifically comprising the following steps:
[0009] (1) Wash the leaves and stems of fresh Buza leaves with running water, dry them, cut them into small pieces, pre-freeze them at -80°C for 2-4 hours, freeze-dry them at -80°C, grind them, and sieve them through a 300-mesh sieve to obtain Buza leaf powder;
[0010] (2) Weighing the bud leaf powder, first adding methanol and chloroform, vortex mixing under magnetic stirring for 4 to 6 hours, then adding sterile water, vortex mixing under magnetic stirring for 1 to 3 hours, to obtain a mixed solution;
[0011] (3) ultrasonically treating the mixed solution, filtering it with a 20-25 μm microporous filter membrane, and collecting the filtrate;
[0012] (4) After the methanol phase and the chloroform phase are separated, the concentrated sample is concentrated by rotary evaporation, and the methanol phase is extracted for the second time by a methanol-chloroform extraction method, and then stored at 4° C. in the dark.
[0013] Preferably, in step (2), the volume ratio of methanol to chloroform is 1:2, and the solid-liquid ratio of the shavings leaf powder to the mixture of methanol and chloroform is 1:10.
[0014] Preferably, the methanol extract of Buzha leaves has antibacterial and antibacterial activities, wherein the pathogens include Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Shigella dysenteriae, Vibrio alginolyticus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio merini and Vibrio fluvialis.
[0015] More preferably, the in vitro antibacterial and bacteriostatic pathogens include Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Shigella dysenteriae, Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Vibrio mesenchyme and Vibrio fluvialis; and / or the in vivo antibacterial and bacteriostatic pathogens are Vibrio parahaemolyticus.
[0016] More preferably, the in vivo antibacterial and antibacterial pathogen is pathogenic Vibrio parahaemolyticus ATCC17802.
[0017] The present invention also provides application of the methanol phase extract of Buza leaves having antibacterial and antibacterial activities in preparing antibacterial and antibacterial foods and medicines.
[0018] Preferably, the antibacterial and antibacterial pathogens include Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Shigella dysenteriae, Vibrio alginolyticus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio mesenchyme and Vibrio fluvialis.
[0019] More preferably, the foods and drugs have antibacterial and bacteriostatic pathogens in vitro including Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Shigella dysenteriae, Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Vibrio merschmidt and Vibrio fluvialis; and / or the foods and drugs have antibacterial and bacteriostatic pathogens in vivo including Vibrio parahaemolyticus.
[0020] Preferably, the types of food include ready-to-eat, fresh, refrigerated and frozen.
[0021] Preferably, the dosage form of the drug includes liquid, ointment, powder, tablet and capsule.
[0022] Compared with the prior art, the present invention has the following beneficial effects:
[0023] The methanol extract of Buzha leaves in the present invention can inhibit a variety of common pathogenic bacteria in vitro, such as Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Shigella dysenteriae, Vibrio alginolyticus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio merschmidt and Vibrio fluvii. At the same time, the methanol extract of Buzha leaves also has the effect of inhibiting pathogenic Vibrio parahaemolyticus in vivo. Experiments have shown that it can effectively remove Vibrio parahaemolyticus in the intestines of mice, eliminate the damage of the bacterial infection to the ileal tissue of mice, and reverse the imbalance of the diversity of the ileal flora of mice caused by the bacterial infection. It can be used to prepare antibacterial and antibacterial foods and drugs, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The following are the changes in the apparent morphological structure of the ileum of mice fed in different ways in the examples.
[0025] Figure 2 The results of microscopic observation of the microstructure of the ileum of mice fed in different ways in the example; the single-layer columnar epithelium (blue arrow), goblet cells (yellow arrow), Lieberkuhn glands (Lieberkuhn glands, green arrow) and muscle layer (orange arrow) of the ileum tissue were magnified 200× or 400×, respectively.
[0026] Figure 3 The β diversity changes of the intestinal flora of mice fed with different feeding methods in the examples.
[0027] Figure 4 The figure shows the changes in the intestinal flora of mice fed with different feeding methods at the phylum (A) and genus (B) levels in the examples, RA (Relative Abundance): relative abundance.
[0028] Figure 5 The changes in the ileal flora structure of mice fed with different feeding methods in the example; A: LefSe clade diagram; B: biomarkers (LDA>4.0, p<0.05). DETAILED DESCRIPTION
[0029] The technical scheme of the present invention is further described below by way of examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by those skilled in the art without creative work are within the scope of protection of the present invention.
[0030] The methods used in the following examples are all conventional methods unless otherwise specified, and the reagents are all conventional reagents unless otherwise specified.
[0031] Embodiment 1
[0032] In vitro inhibition test of methanol extract from Buzha leaves on various common pathogenic bacteria
[0033] (I) Preparation of methanol extract of Buza leaves
[0034] Methanol-chloroform extraction method: Rinse the leaf and stem tissue samples of fresh Buza leaves with running water, dry them, cut them into small pieces of about 1 cm, and pre-freeze them in a -80℃ refrigerator for 2 to 4 hours. Then place them in a freeze dryer and freeze-dry them at -80℃ for 72 hours. Use a multi-functional crusher to crush the freeze-dried plant samples, sieve them through a 300-mesh sieve, and set aside.
[0035] Weigh 10g of the above plant sample powder, add 33mL methanol (analytical grade) and 66mL chloroform (analytical grade) (1:2, v / v) at a solid-liquid ratio of 1:10 (m / v), use a magnetic stirrer to vortex mix for 5h, then add 60mL sterile water (analytical grade) and vortex mix for 2h. Ultrasonic cell disruptor is used to ultrasonically treat the above mixture for 20min (300W, ultrasonic probe diameter: 6mm), filter with a 20-25μm microporous filter membrane, and collect the filtrate. After the methanol phase and the chloroform phase are separated, use a rotary evaporator to evaporate and concentrate, and use the methanol-chloroform method to extract the concentrated sample for the second time to obtain MPE, which is stored at 4℃ in the dark for later use.
[0036] (II) In vitro antibacterial activity test of methanol extract of Buzha leaves
[0037] 2.1 Agar disc diffusion method
[0038] Vibrio test strains: The Vibrio test strains (Table 1) were inoculated into sterilized Tryptic Soy Broth (TSB, pH 8.4-8.5, 3.0% NaCl) liquid culture medium at 1% volume fraction, and cultured at 37°C, 180rpm for 16-18h. After two subcultures and activation, they were streaked onto TSB agar plates and cultured at 37°C for 16-18h. Single colonies were picked and inoculated into TSB liquid culture medium and cultured to the mid-logarithmic growth phase (mid-LGP, OD 600 =0.6~0.8), and the bacterial concentration was determined by conventional plate counting method for later use.
[0039] Non-Vibrio test strains: Similarly, non-Vibrio test strains (Table 1) were inoculated into corresponding culture media (pH 7.0-7.2, 0.5-1.0% NaCl) and cultured at 37°C until mid-LGP.
[0040] Table 1: Test strains and their culture media
[0041]
[0042]
[0043] ATCC: American Type Culture Collection, USA; GCCC: Guangdong Culture Collection Center, China; LS-SHOU: Laboratory stock, Shanghai Ocean University, China; LB: Luria-Bertani medium.
[0044] Take the culture medium of the above test strains (1×10 8 CFU / mL), evenly spread on sterile Mueller-Hinton Agar (M-HA) agar plates (100μL / plate), after the bacterial solution was absorbed, evenly paste sterile filter paper impregnated with the methanol phase extract of Buza leaves (10μL MPE (100mg / mL) / piece, diameter: 6mm), cultured at 37℃ for 12h, and measured the diameter of the inhibition zone (DIZ). Sterile water was used as the negative control group, gentamicin (10μg) test paper was used as the positive control group, and Escherichia coli K-12 was used as the quality control strain. Each group of experiments was repeated three times, and the results are shown in Table 2.
[0045] 2.2 Minimum inhibitory concentration method
[0046] In a 96-well sterilized bacterial culture plate, sterile MH Broth (M-HB) medium (100 μL / well) was added using an eight-channel pipette gun. In the first column of wells, MPE (100 μL / well, final concentration of 16.384 mg / mL) was added, mixed with the M-HB medium therein, and 100 μL / well was drawn and added to the second column of wells, and so on, to the 11th column of wells. Sterile water (100 μL / well) was added to the 12th column of wells as a negative control group. The culture solution of the above test strains (10 μL / well) was drawn using an eight-channel pipette gun and added to each well of the 96-well bacterial culture plate, and cultured at 37°C for 48 hours. The minimum inhibitory concentration (MIC) of the lowest drug concentration in the culture well that completely inhibited bacterial growth was taken as its minimum inhibitory concentration, and acceptable growth must appear in the growth control well (≥2 mm bottom covered or clearly turbid). Each group of experiments was repeated three times, and the results are shown in Table 2.
[0047] The extraction rate of the methanol phase extract of Buzha leaves was 15.50%. As shown in Table 2, the antibacterial rate of the methanol phase extract of Buzha leaves was 47.6%, which was able to inhibit 10 different test strains (n=20), including 2 Gram-positive bacteria: Staphylococcus aureus ATCC29213 and Bacillus cereus. cereus)Y1, and 8 Gram-negative bacteria: Pseudomonas aeruginosa ATCC27853, Shigella dysenteriae CMCC51252, Vibrio alginolyticus ATCC33787, Vibrio cholerae GIM1.449, Vibrio parahaemolyticus ATCC17802, Vibrio harveyi ATCC33842, Vibrio metschnikovii ATCC700040, and Vibrio fluvialis ATCC33809. The observed DIZ values ranged from 11.0±1.4 to 7.0±1.0 mm. In addition, there was no inhibition zone in the negative control, and the DIZ value of the positive control was 23.0 to 8.0 mm. As shown in Table 2, the MIC values of the methanol extract of Buzha leaves against the above 10 test strains ranged from 0.512 to 16.384 mg / mL.
[0048] Table 2: In vitro antibacterial activity test results of methanol extract of Buza leaves
[0049]
[0050]
[0051] Embodiment 2
[0052] In vivo antibacterial activity of methanol extract from Buzha leaves
[0053] Pathogenic Vibrio parahaemolyticus ATCC17802 (tdh - , trh + ) was used as the test strain, and the in vivo antibacterial activity of the methanol extract of Buzha leaves was evaluated using a mouse animal model experiment.
[0054] (I) Experiment on the removal of Vibrio parahaemolyticus ATCC17802 from the intestines of mice by methanol extract of Buza leaves
[0055] Strain and culture conditions: Vibrio parahaemolyticus ATCC17802 was inoculated into sterilized TSB (pH 8.5, 3% NaCl) medium at 1% volume fraction and cultured at 37°C and 180 rpm until OD 600 =0.8-0.9(4×10 9 -1×10 10 CFU / mL), centrifuged at 4°C and 5000 rpm for 3 min to collect the bacteria. Resuspend the bacterial precipitate with sterile saline and dispense into 1.5 mL sterilized centrifuge tubes (100 μL, 1×10 9 CFU) and stored at 4°C for future use.
[0056] Mouse animal model: 50 healthy male mice aged 4-8 weeks and weighing 30±2g were selected. The room temperature of the animal room was 20±2℃, the relative humidity was 60±5%, and the light-dark cycle was 12h. The first group was the normal control group (NTC group), and the mice were fed with normal feed; the second group was the methanol phase extract of Buzha leaves group (MPE group), and the mice were fed with the methanol phase extract of Buzha leaves on the basis of normal feed; the third group was the Vibrio parahaemolyticus ATCC17802 infection group (VP group), and the mice were fed with Vibrio parahaemolyticus ATCC17802 on the basis of normal feed; the fourth group was the MPE+VP treatment group, and the methanol phase extract of Buzha leaves was added on the basis of the feeding of the third group; the fifth group was the CIP+VP treatment group, and the antibiotic ciprofloxacin (CIP) was added on the basis of the feeding of the third group.
[0057] The experimental period was about 3 weeks. Before grouping, all mice were adapted to feeding for 5 days and had free access to distilled water and sterilized feed; one week thereafter, antibiotic cocktail (Abx: including 0.5g / L vancomycin, 1g / L neomycin sulfate, 1g / L metronidazole and 1g / L ampicillin) was added to the drinking water of mice, and all mice were free to drink Abx water and eat sterilized feed. One week later, all mice were randomly divided into groups, with 10 mice in each group, and the following tests were performed at fixed times:
[0058] On the first day of the experiment, mice in the NTC group and the MPE group were gavaged with 100 μL of sterile saline (0.86% NaCl); mice in the VP group, the MPE+VP treatment group, and the MPE+VP treatment group were given a bacterial solution of Vibrio parahaemolyticus ATCC17802 at a concentration of 1×10 9 The dose of CFU was induced by gavage of 100 μL to mice to induce the infection model. After the mice adapted for 2 hours, the MPE group and the MPE+VP treatment group: the methanol phase extract of Buzha leaves was taken at a dose of 200 mg / kg and gavage of 100 μL to mice; the CIP+VP treatment group: gavage of 100 μL CIP (100 mg / kg) to mice; the NTC group and the VP group: gavage of 100 μL sterile saline to mice. On the 2nd to 6th day of the experiment, the NTC group and the VP group: gavage of 100 μL sterile saline to mice every day; the MPE group and the MPE+VP treatment group: gavage of 100 μL Buzha leaf methanol phase extract (200 mg / kg) to mice every day; the CIP+VP treatment group: gavage of 100 μL CIP (100 mg / kg) to mice every day. All mice had free access to distilled water and sterilized feed.
[0059] On the 1st to 6th day of the group experiment, 10 fresh feces were collected from each group of mice, each was weighed and collected in a 1.5 mL sterilized centrifuge tube, and an appropriate amount of sterilized 1× phosphate buffered saline (PBS) was added and vortexed to mix. 100 μL of the suspension was spread on a sterilized thiosulfate-citrate-bile salt-sucrose medium (TCBS) agar plate (100 μL / plate), cultured at 37°C for 24 to 36 hours, and the number of colonies was counted. The results are shown in Table 3.
[0060] As shown in Table 3, no Vibrio parahaemolyticus ATCC17802 was detected in the fecal samples of mice in the NTC group and the MPE group on days 1 to 6 of the group experiment, indicating that the mice in each group were in an environment free of contamination by the pathogen and there was no cross-contamination between the groups. On day 1, the average content of Vibrio parahaemolyticus ATCC17802 in the feces of mice in the VP group was 2.0×10 4 CFU / mL; 5.7×10 3 ~1.8×10 2CFU / mL, indicating that this example successfully established a mouse model infected with Vibrio parahaemolyticus ATCC17802.
[0061] As shown in Table 2, on the first day, the average contents of Vibrio parahaemolyticus ATCC17802 in the feces of mice in the VP+CIP group and the VP+MPE group were 3.5×10 2 CFU / mL and 1.1×10 3 CFU / mL, compared with the VP group, dropped sharply by 57 times and 18 times, respectively (p<0.05). On the 3rd and 5th days, no Vibrio parahaemolyticus ATCC17802 was detected in the feces of mice in the VP+CIP group and the VP+MPE group. This shows that the intervention of the methanol extract of Buzha leaves (200 mg / kg) on the first day can effectively reduce the amount of bacteria in the intestinal feces of mice by 18 times; continuous intervention for 5 days can completely eliminate Vibrio parahaemolyticus ATCC17802 in the intestines of mice; and it has a similar therapeutic effect in vivo to the antibiotic CIP (100 mg / kg).
[0062] Table 3: Changes of Vibrio parahaemolyticus ATCC17802 content in intestinal feces of mice fed different ways over time
[0063]
[0064]
[0065] (II) Experiment on the effect of methanol extract of Buza leaves on eliminating ileal damage of mice infected with Vibrio parahaemolyticus ATCC17802
[0066] Observation of the apparent morphology and structure of mouse ileum tissue: On the 7th day of the group experiment, mice in each group were killed by cervical dislocation. Under sterile conditions, the mice were dissected along the midline of the abdomen, and the entire ileum tissue sample was taken out with sterilized instruments and placed in a sterilized culture dish for photographing and recording. Figure 1 shown.
[0067] like Figure 1 It can be seen that compared with the NTC group, there was no significant difference in the surface morphology of the ileum of mice in the MPE, VP+CIP and VP+MPE groups. Compared with the other four groups, the ileum surface color of mice in the VP group was reddish, which may be caused by infection with pathogenic Vibrio parahaemolyticus ATCC17802, leading to ileum inflammation or damage.
[0068] Microstructure observation of mouse ileum tissue: The collected mouse ileum tissue samples were immersed in 4% formaldehyde solution for 24 hours, and then dehydrated with 70% to 90% gradient alcohol. After that, the samples were embedded in paraffin and sliced (5μm / slice). Hematoxylin-eosin staining was used for staining. The microstructure of the ileum tissue samples of each group of mice was observed under a microscope at magnifications of 200× and 400×. The results are shown in Figure 2. Figure 2 shown.
[0069] like Figure 2 It can be seen that the surface of the ileum of mice in the NTC group, MPE, VP+CIP and VP+MPE groups was distributed with intestinal villi, and the surface was a single layer of columnar epithelium (blue arrows), with normal morphology and structure; goblet cells were distributed between epithelial cells (yellow arrows); the epithelial invagination at the base of the intestinal villi formed Lieberkuhn glands (green arrows); the mucosal muscular layer composed of double layers of smooth muscle cells separated the intestinal crypts from the submucosal layer, the submucosal layer was composed of connective tissue, and the rest of the intestinal wall included the muscular layer composed of smooth muscle cells (orange arrows) and the serosa layer; no obvious inflammatory cell infiltration was observed. However, goblet cells were distributed between the epithelial cells in the ileum of mice in the VP group, the number of intestinal glands was abundant, and the arrangement was irregular locally, and small focal aggregations of lymphocytes were occasionally seen in the lamina propria (red arrows). The results showed that intervening mice with the methanol extract of Buza leaves (200 mg / kg) for 6 consecutive days not only had no adverse effect on the ileal function of mice, but also could eliminate the damage to the ileum of mice caused by pathogenic Vibrio parahaemolyticus ATCC17802, and had a similar therapeutic effect to the antibiotic CIP (100 mg / kg).
[0070] (III) Experiment on the effect of methanol extract of Buza leaves on reversing the imbalance of ileal flora diversity in mice caused by Vibrio parahaemolyticus ATCC17802 infection
[0071] Analysis of the composition of mouse intestinal flora: The ileum tissue samples of each group of mice were rinsed with sterilized 1× PBS precooled at 4°C, and the ileum content samples were collected and placed in sterilized centrifuge tubes, stored in dry ice, and quickly transported to Shanghai Meiji Biomedical Technology Co., Ltd., China, to complete the analysis of the composition of mouse intestinal flora. The SPIN Kit for Soil (Qbigene, USA) was used to extract genomic DNA from the ileum content samples of each group of mice (n=5 / group), and the DNA concentration and purity of the samples were determined. The library was constructed using the Rapid DNA-Seq Kit (Bioo Scientific, USA), and the sequence of the amplicon was determined using the Miseq PE300 (Illumina, USA) high-throughput sequencing platform based on the V3-V4 region of the bacterial 16S rRNA gene (515F: 5′-GTGCCAGCMGCCGCGGTAA-3′, 806R: 5′-GGACTACHVGGGTWTCTAAT-3′).
[0072] QIIME (version 1.9.1) software was used to filter low-quality sequences from the original sequences. Using UPARSE software, the sequence alignment similarity threshold was set to 97%, and operational taxonomic units (OTUs) cluster analysis was performed on high-quality sequences to remove chimeras. The Ribosomal Database Project classifier was used to annotate each sequence for species classification. Mothur (version 1.30.2) software was used to calculate the alpha diversity index (Chao1, Ace, Simpson, and Shannon index) of OTUs. Principal component analysis (PCoA) and non-metric multidimensional scaling (NMDS) based on Bray-Curtis distance were used to perform beta diversity analysis of OTUs. The iconic bacterial populations of mouse ileum were identified based on the linear discriminant analysis effect size (LEfSe). Based on linear discriminant analysis (LDA), species with different abundances between groups of mouse ileum samples (scores > 4.0) were identified. The results are shown in Tables 4 and Figures 3 to 5 shown.
[0073] As shown in Table 4, the sequencing coverage of the ileum content samples of each group of mice was greater than 99.97%, indicating that the samples tested had sufficient sequencing depth.
[0074] As shown in Table 4, compared with the NTC group, the Ace index of the ileal flora of mice in the VP group increased by 1.42 times, which was significantly different (p<0.05). Compared with the NTC group, there were no significant differences in the richness (Chao1 and Ace index) and diversity (Shannon and Simpson index) of the ileal flora of mice in the MPE group and the VP+MPE group (p>0.05), indicating that the methanol extract of Buzha leaves not only had no significant effect on the α diversity index of the ileal flora of mice, but also eliminated the increase in the Ace index caused by Vibrio parahaemolyticus ATCC17802 infection. Compared with the NTC group, the Chao1, Ace, Shannon and Simpson indexes of the ileal flora of mice in the VP+CIP group increased by 1.55 times, 1.45 times, 0.67 times and 2.00 times, respectively, which were significantly different (p<0.05).
[0075] like Figure 3 As shown, the results of PCoA and NMDS analysis showed that compared with the NTC group, most of the ileum flora clusters of the VP group mice were far away from the NTC group; part of the ileum flora of the VP+MPE group mice were clustered with the NTC group; the ileum flora of the VP+CIP group mice were clustered far away from the NTC group, indicating that intervention with the methanol extract of Buzha leaves can eliminate the effect of Vibrio parahaemolyticus ATCC17802 infection on the α diversity of the ileum flora of mice, and partially reverse the changes in β diversity; treatment with the antibiotic CIP aggravates the changes in the β diversity of the ileum flora of mice.
[0076] like Figure 4As shown in A, at the phylum level, the ileal flora of the five groups of mice were mainly Firmicutes, Verrucomicrobiota, Bacteroidota, Proteobacteria and Actinobacteriota, accounting for more than 97% of the species of mouse ileal flora. Compared with the NTC group, the relative abundance of Verrucomicrobiota in the ileal flora of mice in the MPE group decreased by 11.34%, while Bacteroidota and Proteobacteria increased by 4.40% and 5.20%, respectively, but there was no significant difference (p>0.05); the relative abundance of Verrucomicrobiota and Proteobacteria in the ileal flora of mice in the VP group increased by 7.43% and 6.72%, respectively, while Firmicutes and Bacteroidota decreased by 6.76% and 8.03%, respectively, but there was no significant difference (p>0.05); the relative abundance of Firmicutes and Verrucomicrobiota in the ileal flora of mice in the VP+MPE group decreased by 24.73% and increased by 23.01%, respectively, but there was no significant difference (p>0.05); the relative abundance of Firmicutes and Verrucomicrobiota in the ileal flora of mice in the VP+CIP group decreased by 24.73% and increased by 23.01%, respectively, but there was no significant difference (p>0.05). The relative abundance of Firmicutes, Bacteroidota and Proteobacteria decreased significantly by 47.01%, 9.80% and 7.58% (p<0.05), while Verrucomicrobiota (60.69%, p<0.05) and Actinobacteriota (3.85%, p<0.01) increased significantly, indicating that the intervention with the methanol extract of Buzha leaves had no significant effect on the ileal microbiota of mice at the phylum level; while the antibiotic CIP treatment caused the relative abundance of Firmicutes, Bacteroidota and Proteobacteria in the ileal microbiota of mice to decrease significantly by 47.01%, 9.80% and 7.58% (p<0.05), while Verrucomicrobiota (60.69%, p<0.05) and Actinobacteriota (3.85%, p<0.01) increased significantly.
[0077] Table 4: Changes in α-diversity of intestinal flora in mice fed different diets
[0078]
[0079] Alpha diversity index: mean ± SEM (n = 5); *: p < 0.05, **: (p < 0.01), compared with the NTC group.
[0080] like Figure 4 As shown in B, at the genus level, the ileal microbiota of the five groups of mice were mainly composed of Akkermansia, Lactobacillus, Enterococcus, Hungatella, Parabacteroides, Bacteroides, Escherichia-Shigella, norank_f__Muribaculaceae, Parasutterella and Candidatus_Arthromitus, accounting for more than 95% of the ileal microbiota species. Compared with the NTC group, the relative abundance of Lactobacillus and Bacteroides in the ileal flora of mice in the VP group was significantly decreased by 37.14% and 6.18% (p<0.05), while that of Enterococcus was significantly increased by 22.73% (p<0.05); the relative abundance of Escherichia-Shigella in the ileal flora of mice in the MPE group was significantly reduced by 7.48% (p<0.05); the relative abundance of Lactobacillus and Escherichia-Shigella in the ileal flora of mice in the VP+MPE group was significantly decreased by 39.12% and 7.92%, respectively, while the relative abundance of norank_f__Muribaculaceae and Parasutterella was significantly increased by 5.3% and 2.71%, respectively (p<0.05); the relative abundance of Escherichia-Shigella in the ileal flora of mice in the VP+CIP group was significantly decreased by 39.12% and 7.92%, respectively, while the relative abundance of Escherichia-Shigella was significantly increased by 2.71% and 5.3%, respectively (p<0.05); the relative abundance of Escherichia-Shigella in the ileal flora of mice in the VP+MPE group was significantly decreased by 39.12% and 7.92%, respectively, while the relative abundance of Escherichia-Shigella was significantly increased by 2.71% and 5.3%, respectively (p<0.05); the relative abundance of Escherichia-Shigella in the ileal flora of mice in the VP+CIP group was significantly decreased by 39.12% and 7.92%, respectively, The relative abundance of Akkermansia in the intestinal flora increased significantly by 66.52% (p<0.05), while Lactobacillus, Hungatella, Parabacteroides, Bacteroides, Escherichia-Shigella and Parasutterella decreased significantly by 42.71%, 8.09%, 6.38%, 6.41%, 9.08% and 0.28% (p<0.05), respectively. This indicates that infection with Vibrio parahaemolyticus ATCC17802 significantly reduced the relative abundance of beneficial bacterial genera (such as Lactobacillus) in the ileal flora of mice; intervention with the methanol extract of Buzha leaves significantly reduced the proportion of harmful bacterial genera (such as Escherichia-Shigella); and antibiotic CIP treatment significantly changed the relative abundance of multiple genera of bacteria.
[0081] like Figure 5As shown, the markers of ileal flora of mice in the NTC group were Firmicutes and Lactobacillus; the marker of ileal flora of mice in the MPE group was g__unclassified_o__Lactobacillales; the relative abundance of Proteobacteria, Enterococcus and Parasutterella in the ileal flora of mice in the VP group increased significantly (p<0.05); Bacteroidota, Bacteroides and Parabacteroides were enriched in the ileal flora of mice in the VP+MPE group; Verrucomicrobiota and Akkermansia were enriched in the VP+CIP group, indicating that the methanol extract of Buzha leaves can enrich the beneficial bacteria genera (such as g__unclassified_o__Lactobacillales) in the ileum of mice.
[0082] In summary, the present invention provides a methanol phase extract of Buzha leaves and its antibacterial and antibacterial activity application. The methanol phase extract of Buzha leaves can inhibit a variety of common pathogens in vitro, has the effect of inhibiting pathogenic Vibrio parahaemolyticus in vivo, can eliminate Vibrio parahaemolyticus in the intestine of mice, eliminate the damage of the bacterial infection to the ileal tissue of mice, and reverse the imbalance of the diversity of the ileal flora of mice caused by the bacterial infection. The extract can be used for the preparation of food or medicine, and the prepared food and medicine also have antibacterial and antibacterial effects.
[0083] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A methanol extract of Buza leaves having antibacterial and antimicrobial activity, characterized in that: It is prepared by methanol-chloroform extraction method, comprising the following steps: (1) Wash the leaves and stems of fresh Buza leaves with running water, dry them, cut them into small pieces, pre-freeze them at -80°C for 2-4 hours, freeze-dry them at -80°C, grind them, and sieve them through a 300-mesh sieve to obtain Buza leaf powder; (2) Weighing the bud leaf powder, first adding methanol and chloroform, vortex mixing under magnetic stirring for 4 to 6 hours, then adding sterile water, vortex mixing under magnetic stirring for 1 to 3 hours, to obtain a mixed solution; (3) ultrasonically treating the mixed solution, filtering it with a 20-25 μm microporous filter membrane, and collecting the filtrate; (4) After the methanol phase and the chloroform phase are separated, the concentrated sample is concentrated by rotary evaporation, and the methanol phase is extracted for the second time by a methanol-chloroform extraction method, and then stored at 4° C. in the dark.
2. The methanol extract of Buza leaves according to claim 1, characterized in that In step (2), the volume ratio of methanol to chloroform is 1:2, and the solid-liquid ratio of the shavings leaf powder to the mixture of methanol and chloroform is 1:
10.
3. The methanol extract of Buza leaves according to claim 1, characterized in that The methanol extract of Buza leaves has antibacterial and antibacterial activities, wherein the pathogenic bacteria include Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Shigella dysenteriae, Vibrio alginolyticus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio mesneri and Vibrio fluvialis.
4. The methanol extract of Buza leaves according to claim 3, characterized in that The in vitro antibacterial and antibacterial pathogens of the methanol extract of Buzha leaves include Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Shigella dysenteriae, Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Vibrio mesenchyme and Vibrio fluvialis; the in vivo antibacterial and antibacterial pathogens of the methanol extract of Buzha leaves are Vibrio parahaemolyticus.
5. The methanol extract of Buza leaves according to claim 4, characterized in that The antibacterial and antibacterial pathogen is pathogenic Vibrio parahaemolyticus ATCC17802.
6. Use of the methanol extract of Buzha leaves according to any one of claims 1 to 5 in the preparation of antibacterial and antibacterial foods and medicines.
7. The use according to claim 6, characterized in that: Among them, the antibacterial and antibacterial pathogens include Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Shigella dysenteriae, Vibrio alginolyticus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio mesenchyme and Vibrio fluvialis.
8. The use according to claim 7, characterized in that: The pathogenic bacteria that the food and medicine can resist and inhibit in vitro include Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Shigella dysenteriae, Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Vibrio mesenchyme and Vibrio fluvialis; And / or the antibacterial and antibacterial pathogens in the food and medicine are Vibrio parahaemolyticus.
9. The use according to claim 6, characterized in that: The types of food include ready-to-eat, fresh, refrigerated and frozen.
10. The use according to claim 6, characterized in that: The dosage forms of the drug include liquid, ointment, powder, tablet and capsule.